第十七届全国神经精神药理学学术会议论文摘要

2016年5月13-16日，安徽合肥

第十七届全国神经精神药理学学术会议论文摘要

2016年5月13-16日，安徽合肥

专题1　：精神分裂症与抑郁症

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急性及慢性应激诱导的小鼠中枢及外周神经递质谷氨酸、谷氨酰胺和γ-氨基丁酸的变化

陈亚萍1，王 闯2，程玉芳1，李亦文1，徐江平1

（1.南方医科大学药学院神经药理与新药发现课题组，广东广州 510515；2.宁波大学医学院，浙江宁波 315211）

目的 通过测定急性应激造模和慢性应激造模小鼠的血浆和脑组织内谷氨酸、谷氨酰胺、γ-氨基丁酸3种神经递质的含量，比较抑郁症不同阶段脑组织和血浆中神经递质的变化。方法 小鼠慢性应激造模8周，急性束缚应激3 h，用悬尾实验检查造模效果后取脑组织和血浆，以高丝氨酸作为内标，邻苯二甲醛柱前衍生后用高效液相色谱荧光检测器测定3种氨基酸的浓度。结果 小鼠在急性应激和慢性应激后悬尾的不动时间增加，且慢性应激模型与对照组相比具显著性差异。高效液相色谱中三种氨基酸在16 min完全分离，在0.01～6.40 μmol·L-1浓度范围内与峰面积有良好的线性关系。小鼠在急性应激造模后血浆谷氨酸浓度显著性上升，慢性应激造模小鼠血浆谷氨酰胺与对照组和急性应激造模组比明显较低；而脑组织中的谷氨酰胺在慢性应激模型中却略升高，谷氨酸没有明显变化，γ-氨基丁酸在造模过程中逐渐升高。结论 抑郁症的发生过程中，这3种神经递质氨基酸会相应发生变化，小鼠在受到急性应激后血浆中的谷氨酸会反射性增加，随着抑郁症的进程又会趋于平衡，而脑组织的γ-氨基丁酸在抑郁的发生发展过程不断升高，从而可以证实郁抑症不同阶段血浆中的氨基酸水平与大脑的氨基酸水平存在差异。

氨基酸；高效液相；急性应激；慢性应激

61647396

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突触可塑性在抗抑郁中的研究进展

钟秋萍，徐江平

（南方医科大学药学院神经药理与新药发现课题组，广州广东 510515）

摘要：抑郁症（depression）是一种以情绪低落为主的情感障碍性疾病，因其伴有自杀倾向，严重威胁人类健康和生存生活质量。且由于其致病机制未明，抗抑郁药物的发展受到很大的限制。现临床使用的抗抑郁药物因起效慢、副作用大等缺陷极大地影响了抑郁症的治疗。随着研究的深入，越来越多的数据表明，突触可塑性（synaptic plasticity）与抑郁症有着紧密的联系，且提高突触可塑性有利于改善抑郁症伴随的认知功能的下降。因此，本文将基于抑郁症发病基础对影响突触可塑性的神经营养因子、星形胶质细胞及促炎症因子的相关内容进行总结。

关键词：抑郁症；突触可塑性；神经营养因子；星形胶质细胞；促炎症因子

基金项目：国家自然科学基金（81301099；81373384；81503043）；广东省科技厅（粤科规财字［2015］150号）

通讯作者：徐江平，E-mail：jpx@smu.edu.cn，Tel：（020）61648236

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Determination of a novel phosphodiesterase-4 inhibitor chlorbipram in mouse plasma and brain by UFLC-MS/ MS：applicationinpharmacokineticstudiesafter intravenous administration

LI Yi-wen，XU Jiang-ping

（Group of Neuropharmacology and New Drug Discovery，School of Pharmaceutical Sciences，Southern Medical University，Guangzhou 510515，China）

Abstract：OBJECTlVE In this study，we evaluated a simple and sensitive method for determination of a novel phosphodiesterase-4（PDE4） inhibitor，chlorbipram，in mouse plasma and brain which wasapplied in a preclini⁃cal pharmacokinetic study ofintravenous administration of chlorbipram in Kunming mice.METHODS Ultra-fast liquidchromatography-tandem mass spectrometry（UFLCMS/MS）was used for the determination.Separation was achievedusinganAcquityUPLCBEHC18column （50 mm×2.1 mm，particle size 1.7 μm）with a gradient mobile phase consisting of water and methanol at a flow rate of 0.25 mL·min-1.Detection was performed in the multiplereaction monitoring（MRM）mode using electro⁃spray ionization（ESI）in the positive ion mode.Theliquidliquid extraction method with ethyl acetate was used for both pretreatment of plasma and brainhomogenates. RESULTS The calibration curves of chlorbipram showed good linearity over the concentration range of 0.5-200 μg·L-1（R2＞0.994）for mouse plasma and over the range of 0.25-100 μg·m-1（R2＞0.994）formouse brain homogenate.The extraction recovery was in the range of 78.3%-84.8%for chlorbipram andthe internal standard （IS）ZXI14 in two different biological matrices.The intraand inter-day precisionvalues were less than 13.0%and the accuracy ranged from 97.8%to 106.0%for quality control samples.No noteworthy matrix effects and instabil⁃ity were observed for chlorbipram.The developed meth⁃od was successfully applied to the pharmacokinetic study ofchlorbipramafterintravenousadministrationin Kunming mice.Results of the study show that chlorbip⁃ram（0.6 mg·kg-1）can bedetected in plasma within 5 min of intravenous administration，reaching a peak con⁃centration of 159.6 μg·L-1after approximately 2 min. Moreover，comparisons of the Cmax，tmaxand AUC0-tof mouseplasma and brain revealed that chlorbipram easily penetrates the blood-brain barrier and is transported to the brain.CONCLUSlON This validated method wassuc⁃cessfully applied to a pharmacokinetic study of chlorbip⁃ram in mice after intravenous administration.The results show that this novel drug crosses the blood-brain barrier and provides the basis for furtherstudies on chlorbipram.

Key words：phosphodiesterase-4 inhibitor；chlorbipram；UFLC-MS/MS；pharmacokinetics

Foundation item：The project supported by National NaturalScienceFoundationofChina（81503043；81373384）

Corresponding author：XU Jiang-ping，Tel：（020）61648236，E-mail：jpx@smu.edu.cn

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Allosteric modulation of sigma-1 receptors elicits rapid antidepressant activity

WANGYun1，2*，GUOLin1*， JIANGHua-feng1，ZHENG Long-tai1，ZHANG Ao3，ZHEN Xue-chu1

（1.Jiangsu Key laboratory for Translational Research for Neuropsychiatric diseases，The Collaborative Innovation Center for Brain Science，College of Pharmaceutical Sciences，Soochow University，Suzhou 215123，China；2.Jiangsu Key Laboratory of New Drug Research and ClinicalPharmacy， DepartmentofPharmacology，XuzhouMedicalCollege，Suzhou221002，China；3.Department of Medicinal Chemistry，Shanghai Insti⁃tute of Material Medica，Chinese Academy of Sciences，Shanghai 201203，China）

Abstract：OBJECTlVE Sigma-1 receptors are in⁃volved in the pathophysiological process of several neu⁃ropsychiatric diseases such as epilepsy，depression.Al⁃losteric modulation represents an important mechanism for receptor functional regulation.In the present study，we examined antidepressant activity of the latest identi⁃fied novel and selective allosteric modulator of sigma-1 receptor SOMCL-668（3-methyl-phenyl-2，3，4，5-tetra⁃hydro-1H-benzo［d］azepin-7-ol）.METHODSForced swimming test（FST），tail suspension test（TST）andchronic unpredicted mild stress（CUMS）model were used to evaluate the potential antidepressant effects of SOMCL-668.Western blotting assay was adopted to detect BDNF-GSK3β（glycogen synthase kinase 3β）pathway in the CUMS model.Immunostaining，immuno⁃precipitation，BDNF（brain-derived neurotrophic factor）secretion and neurite outgrowth were adopted to reveal the allosteric function of SOMCL-668 on sigma-1 recep⁃tor.RESULTS A single administration of SOMCL-668 decreased the immobility time in TST and FST，which were abolished by pretreatment of sigma-1 receptor an⁃tagonist BD1047.In the CUMS model，chronic applica⁃tion of SOMCL-668 rapidly ameliorated anhedonia-like behavior（within a week），accompanying with the en⁃hanced expression of BDNF and phosphorylation of GSK3β（Ser-9）in the hippocampus.SOMCL-668 also rapidly promoted the phosphorylation of GSK3β（Ser-9）in an allosteric manner in vitro.In the cultured primary neu⁃rons，SOMCL-668 enhanced the sigma-1 receptor ago⁃nist-induced neurite outgrowth and the secretion of BDNF.CONCLUSlON SOMCL-668，a novel allosteric modulatorofsigma-1 receptors，elicits a potent and rapid acting antidepressant effect.The present data provides the first evidence that allosteric modulation of sigma-1 re⁃ceptors may represent a new approach for antidepres⁃sant drug discovery.

Keywords： allostericmodulation； antidepressant；sigma-1 receptors；brain-derived neurotrophic factor；glycogen synthase kinase 3β

Corresponding author：ZHEN Xue-chu，E-mail：xuechuzhen@suda.edu.cn

*Co-first author.

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PDE4抑制剂FCPR03抗抑郁及改善神经炎症作用的研究

邹征强，陈佳佳，冯红芳，程玉芳，徐江平

（南方医科大学药学院神经药理与新药发现课题组，广东广州510515）

摘要：目的 研究化合物FCPR03对LPS诱导的神经炎症和小鼠抑郁症行为改善作用。方法 利用1 mg·L-1LPS诱导BV-2小胶质细胞活化的方法来模拟体外神经炎症模型，评价FCPR03的体外抗神经炎症作用；采用对小鼠腹腔注射1.2 mg·kg-1LPS 24 h后产生抑郁样行为的模型，来评价FCPR03的体内抗抑郁及改善神经炎症作用。结果 ①1 mg·L-1LPS刺激BV-2细胞24 h后，能显著地增加促炎性细胞因子（TNF-α，COX-2和iNOS）蛋白水平的产生，而经过FCPR03预处理后能浓度依赖性的降低细胞因子水平，提示FCPR03具有很好的抗神经炎症作用。②腹腔注射1.2 mg·kg-1LPS 24 h后，旷场实验结果显示，各组间水平得分和垂直得分均没有统计学差异。而在悬尾和强迫游泳实验中，和正常组相比，模型组不动时间显著升高，给药后能够剂量依赖性的降低小鼠的不动时间，表现出很好的抗抑郁作用。结论 PDE4抑制剂FCPR03能够改善LPS诱导的神经炎症，降低促炎性细胞因子的水平，同时还能够降低小鼠在悬尾和强迫游泳中不动时间，表明FCPR03同时具有抗抑郁和改善神经炎症的双重作用。

关键词：抑郁症；FCPR03；细胞因子；小胶质细胞；神经炎症

基金项目：国家自然科学基金（81503043；81373384）

通讯作者：徐江平，E-mail：jpx@smu.edu.cn，Tel：（020）61648236

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Effects of chronic mild stress on behavioral and neurobiological parameters-role of glucocorticoid

CHEN Jiao1，WANG Zhen-zhen1，ZUO Wei1，ZHANG Shuai1，CHU Shi-feng1，CHEN Nai-hong1，2

（1.State Key Laboratory of Bioactive Substances and Functions of Natural Medicines，Institute of Materia Medica& Neuroscience Center，Chinese Academy of Medical Sciences and Peking Union Medical College，Beijing 100050，China；2.HunanUniversityofChineseMedicine， Changsha 410208，China）

Abstract：Major depression is thought to originate from maladaptation to adverse events，particularly when impairments occur in mood-related brain regions.Hypo⁃thalamus-pituitary-adrenal（HPA）axis is one of the ma⁃jor systems involved in physiological stress response. HPA axis dysfunction and high glucocorticoid concentra⁃tions play an important role in the pathogenesis of de⁃pression.In addition，astrocytic disability and dysfunction of neurotrophin brain-derived neurotrophin factor（BDNF）greatly influence the development of depression and anxi⁃ety disorders.Therefore，we investigated whether de⁃pression-like and anxiety-like behaviors manifest in the absence of glucocorticoid production and circulation in adrenalectomized（ADX）rats after chronic mild stress （CMS）exposure and its potential molecular mecha⁃nisms.The results demonstrate that glucocorticoid-con⁃trolled rats showed anxiety-like behaviors but not de⁃pression-like behaviors after CMS.Molecular and cellular changes included the decreased BDNF in the hippocam⁃pus，astrocytic dysfunction with connexin43（cx43）de⁃creasing and abnormality in gap junction in prefrontal cor⁃tex（PFC）.Interestingly，we did not find any changes in glucocorticoid receptor（GR）or its chaperone protein FK506 binding protein 51（FKBP5）expression in the hip⁃pocampus or PFC in ADX rats subjected to CMS.In con⁃clusion，the production and circulation of glucocorticoids are one of the contributing factors in the development of depression-like behaviors in response to CMS.In con⁃trast，the effects of CMS on anxiety-like behaviors are in⁃dependent of the presence of circulating glucocorticoids. Meanwhile，stress decreased GR expression and en⁃hanced FKBP5 expression via higher glucocorticoid expo⁃sure.Gap junction dysfunction and changes in BDNF may be associated with anxiety-like behaviors.

Key words：depression；chronic mild stress；FKBP5；gap junction

Corresponding author：CHEN Nai-hong， Tel：（010）63165177，E-mail：chennh@imm.ac.cn

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Non-pharmacological management of children and ado⁃lescents with psychiatric disorders：psychopharmaco⁃logical perspectives and clinical evidence

ZHANG Zhang-jin

（School of Chinese Medicine，the University of Hong Kong，Hong Kong，China）

Abstract：Over the past three decades，morbidity in children and adolescents with various mental diseases has sharply risen in China due to the dramatic socioeco⁃nomic and demographic transformation，with an estimat⁃ed prevalence of 0.95%.Children are not little adults. Their psychological traits are under development and not yet matured.Young patients are more susceptible to ad⁃verse effects caused by pharmacotherapy，which even⁃poses a greater risk than disease itself in young patients. These shortcomings have led to a growing demand for the development of non-pharmacological management strategies.Brain stimulation and alternative medicine，in particular acupuncture and herbal medicine，are good candidates for such non-pharmacological management. We have evaluated efficacy and safety of electroconvul⁃sive therapy（ECT）for first-episode psychosis，acupunc⁃ture for obsessive compulsive disorder（OCD）and herb⁃al medicine for tic disorders in young patients.This talk will show findings obtained from these studies and eluci⁃date future research directions in non-pharmacological treatment of children and adolescent psychiatry based on clinical practice and psychopharmacological perspectives.

Key words：non-pharmacological management；children；adolescents；psychiatric disorders；brain stimulation；acupuncture；herbal medicine

Corresponding author：ZHANG Zhang-jin，Tel：00852-2589-0445，E-mail：zhangzj@hku.hk

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J147对慢性应激小鼠的抗抑郁样作用及机制

连乐竞，费 宁，陈 洁，张健波，朱娜萍，潘建春

（温州医科大学药学院脑科学研究所，浙江温州 325035）

摘要：目的 探讨J147对小鼠慢性应激模型抑郁样行为的影响及可能的作用机制。方法 连续3周交替采用多种不可预知的刺激方式，建立慢性不可预知性应激模型，同时灌胃给予不同剂量J147或腹腔给予丙咪嗪，采用强迫游泳、悬尾实验、穿梭箱实验，糖水消耗实验对J147抗抑郁作用进行行为学评价；行为学测试结束后杀鼠取脑组织，采用Western蛋白印迹法检测海马及前额叶中cAMP，pCREB以及BDNF的蛋白表达。结果 慢性灌胃给予J147的中高剂量组能明显提高应激小鼠糖水偏嗜度，改善由慢性应激造成的“快感缺失”，明显逆转应激小鼠在强迫游泳和悬尾实验中不动时间的延长，显著减少应激小鼠逃避失败次数。Western蛋白印迹法检测发现中高剂量组及阳性对照组显著逆转慢性应激小鼠海马及前额叶皮层中cAMP，pCREB 和BDNF蛋白表达低下。结论 J147可以改善由慢性应激所致的小鼠抑郁样行为，其机制可能涉及对cAMP/pCREB/ BDNF通路的调控。

关键词：J147；抑郁；不可预知慢性应激；信号转导通路

通讯作者：潘建春，E-mail：wenzhoupan2003@163.com，Tel：13857750765

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白藜芦醇对PTSD大鼠的作用及其机制

费 宁，连乐竞，朱娜萍，张健波，陈 洁，潘建春

（温州医科大学药学院脑科学研究所，浙江温州 325035）

摘要：目的 研究白藜芦醇对大鼠创伤后应激障碍（PTSD）引起的焦虑样症状的改善作用及其可能机制。方法 实验分为对照组、模型组和不同剂量的药物组，通过对大鼠进行时间依赖敏感性应激（TDS）建立PTSD模型，模型建立后连续18 d灌胃给予不同剂量的白藜芦醇治疗。然后进行行为学测试，通过环境恐惧测试和高架十字迷宫测试观测大鼠焦虑样行为的变化。行为学结束后杀鼠取脑组织，采用Western蛋白印迹法测定下丘脑、海马及杏仁核中的促糖皮质激素释放因子（CRF）和糖皮质激素受体（GR）的蛋白表达。结果 模型组大鼠表现出焦虑样症状，环境恐惧测试中僵立不动时间延长，高架十字迷宫测试中开臂时间和次数均降低，而白藜芦醇能有效逆转这种现象。Western蛋白印迹法检测发现，模型组下丘脑、海马和杏仁核中CRF表达较对照组升高，下丘脑中GR表达较对照组升高；而给予白藜芦醇后能改善这种现象。结论 白藜芦醇在一定程度上可以改善大鼠PTSD引起的焦虑样症状，其机制可能与下丘脑-垂体-肾上腺轴（HPA）的负反馈调节通路有关。

关键词：创伤后应激障碍；白藜芦醇；焦虑；HPA轴

通讯作者：潘建春，E-mail：wenzhoupan2003@163.com，Tel：13857750765

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淫羊藿苷对MK-801致精神分裂症模型小鼠的影响

陈 溪，谷洪顺，张 兰，李 林

（首都医科大学宣武医院药物研究室，北京市老年病医疗研究中心，北京市神经药物工程技术研究中心，北京100053）

摘要：目的 研究传统中药淫羊藿的主要有效成分淫羊藿苷对精神分裂症动物模型的影响。方法 腹腔注射N-甲基-D-天冬氨酸（NMDA）受体拮抗剂MK-801制备精神分裂症小鼠模型。应用旷场行为红外监测系统观察小鼠的活动性。结果 腹腔注射MK-801（0.6 mg·kg-1）能够明显延长小鼠旷场试验的总活动距离和中心区活动距离，表明出现精神分裂症的高活动性（阳性症状）和焦虑状态（阴性症状）。淫羊藿苷（50 mg·kg-1）灌胃给药能够显著缩短模型小鼠的总活动距离和中心区活动距离，表明能够减轻精神分裂症的高活动性和焦虑状态。结论 淫羊藿苷能够改善精神分裂症小鼠模型的阳性症状和阴性症状，提示可能有利于治疗精神分裂症。

关键词：淫羊藿苷；精神分裂症；NMDA受体；MK-801；动物模型；旷场试验

基金项目：国家自然科学基金（81273498；81473373）；国家科技重大专项（2015ZX09101016001）；北京市高层次卫生人才项目（2011-1-7；2014-2-014）

通讯作者：李 林，E-mail：linlixw@126.com，Tel：（010）83198886

T1-12

二氢黄酮类衍生物的合成及其抗精神分裂症研究

谷洪顺，陈 溪，张 兰，李 林

（首都医科大学宣武医院药物研究室，北京市神经药物工程技术研究中心，神经变性病教育部重点实验室，北京100053）

摘要：目的 设计合成一系列新型的具有哌啶和哌嗪基团的二氢黄酮类衍生物，并评价其抗精神分裂症作用。方法 以间苯三酚为原料，经傅克酰基化、缩合、氧化和水解等反应合成目标化合物。采用NMDA受体拮抗剂地卓西平（MK-801）诱导小鼠的高活动性精神分裂症模型，旷场试验检测合成的化合物对小鼠210 min内活动性的影响；采用多巴胺受体部分激动剂阿扑吗啡诱导小鼠的攀爬模型，攀爬试验检测合成的化合物对小鼠过度攀爬行为的影响。结果 合成了7个二氢黄酮类化合物，目标化合物的结构经核磁共振碳氢谱、高分辨质谱、红外和紫外光谱进行了确定。灌胃给予合成的二氢黄酮类化合物能够明显降低MK-801诱导小鼠的过度活动行为；在阿扑吗啡模型中，二氢黄酮类化合物能够显著降低阿扑吗啡诱导的小鼠过度攀爬时间。结论 合成的新型二氢黄酮衍生物能够改善精神分裂症小鼠模型行为学变化，具有潜在的抗精神分裂症作用。

关键词：二氢黄酮；合成；MK-801模型；阿扑吗啡模型；精神分裂症

基金项目：国家自然科学基金（81273498；81473373）；国家科技重大专项（2015ZX09101016001）；北京市高层次卫生人才项目（2011-1-7；2014-2-014）

通讯作者：李 林，E-mail：linlixw@126.com，Tel：（010）

83198886

T1-13

基于系统药理学思路的抑郁症发病机制研究进展

高 耀，田俊生，秦雪梅

（山西大学中医药现代研究中心，山西太原 030006）

摘要：抑郁症具有发病机制复杂，临床诊断客观性差等特点，急需基于“多靶点、多途径、多机制”的整体研究方法。采用系统药理学思路，对基因组学、转录组学、蛋白组学及代谢组学等系统生物学技术在抑郁症疾病研究中的应用进行综述，为抑郁症新型诊断生物标志物的发现与验证、抑郁症发病机制探讨、抗抑郁新药研发等提供依据。基因组学为从遗传学角度探讨抑郁症相关基因；转录组学为抑郁症的研究提供疾病发生的分子机理；蛋白质组学通过比较特定细胞、组织或器官蛋白质表达谱的变化，为阐明抑郁症的作用机制提供新的线索；代谢组学可以较全面地反应生物体的生理及代谢状态，有效地评估抑郁症疾病的病理状态。基于基因组学、转录组学、蛋白组学及代谢组学的系统生物学整体研究为阐明抑郁症复杂疾病的分子机制、分子生物标志物等方面提供了依据，而系统药理学涉及了系统生物学方法的应用，为系统的、整体的研究疾病的发病机制及药物作用机制提供研究思路。

关键词：抑郁症；系统药理学；系统生物学；组学

通讯作者：秦雪梅，E-mail：qinxm@sxu.edu.cn，Tel：（0351）7018379

T1-14

雷公藤内酯醇对应激小鼠抑郁样行为的改善作用

张健波，陈 洁，朱娜萍，费 宁，连乐竞，潘建春

（温州医科大学药学院脑科学研究所，浙江温州 325035）

摘要：目的 研究雷公藤内酯醇对应激小鼠抑郁样行为的改善以及对肿瘤坏死因子、核转录因子表达的影响。方法 48只ICR小鼠，随机分为6组，分别为对照组、模型组、不同剂量雷公藤内酯醇组。除对照组以外，各组小鼠均给与21 d慢性不可预知应激建立抑郁模型，并于第22天开始行为学检测，采用自发活动，糖水消耗以及强迫游泳测试观察小鼠抑郁样行为。Western蛋白印迹法检测各组小鼠海马和前额叶皮层中肿瘤坏死因子（TNF-α）和核转录因子（NF-κB）的表达。结果 旷场实验结果表明，各组小鼠活动能力之间无显著性差异。与正常组小鼠相比，慢性应激组小鼠糖水消耗量明显降低，强迫游泳时间明显增加，雷公藤内酯醇能够逆转此现象，同时使海马和前额叶皮层中TNF-α和NF-κB表达显著增加。结论 雷公藤内酯醇对慢性不可预知应激引起的小鼠抑郁样行为有一定程度的改善作用，其机制可能涉及对海马和额叶内TNF-α和NF-κB的调节。

关键词：雷公藤内酯醇；肿瘤坏死因子；核转录因子；凋亡因子

通讯作者：潘建春，E-mail：wenzhoupan2003@163.com，Tel：

13857750765

T1-15

胱天蛋白酶1对慢性应激所致小鼠抑郁样行为的作用及机制

王 芳，李明星，郑慧玲，徐骏峰，胡壮丽，陈建国

（华中科技大学同济医学院基础医学院药理学系，湖北武汉430030）

摘要：目的 研究胱天蛋白酶1对慢性应激所致小鼠抑郁样行为的影响及机制。方法 建立慢性社会挫败性应激小鼠抑郁模型，结合电生理记录和分子生物学技术，观察胱天蛋白酶1敲除小鼠抑郁样行为以及突触可塑性改变。结果 慢性社会挫败性应激减少小鼠社会接触行为，降低小鼠糖水偏好率，表现出抑郁样行为。抑郁小鼠海马LTP由（142.7±4.6）%显著降低至（116.6±3.7）%。AMPA受体GluA1和GluA2膜表达减少。胱天蛋白酶1敲除可减轻小鼠抑郁样行为，并增加AMPA受体膜表达，逆转突触传递损伤。给予白细胞介素1β可取消胱天蛋白酶1敲除所致的抗抑郁作用。结论 抑制胱天蛋白酶1活性可减轻小鼠抑郁样行为，是治疗抑郁症的新靶点。

关键词：抑郁症；胱天蛋白酶1；突触传递；慢性应激

基金项目：国家重点基础研究发展计划（973计划）资助项目（2013CB531300）

通讯作者：陈建国，E-mail：chenj@mails.tjmu.edu.cn，Tel：（027）83692636

T1-16

Leptin对小鼠焦虑样行为的作用及机制

徐骏峰，王 伟，吴鹏飞，王 芳，陈建国

（华中科技大学同济医学院基础医学院药理学系，湖北武汉 430030）

摘要：目的 探讨leptin对小鼠焦虑样行为的影响及机制。方法 采用急性束缚应激实验、急性强迫游泳应激实验、高架十字迷宫以及条件性恐惧行为学检测leptin（1 mg·kg-1，ip）对小鼠焦虑样行为及恐惧记忆消散的作用。场电位记录小鼠丘脑-外侧杏仁核的突触传递的变化。结果 整体给予leptin或者杏仁核脑区局部给予leptin均可显著促进条件恐惧学习记忆的消散。NMDA受体阻断剂MK-801可完全阻断瘦素促进恐惧记忆消散的效应。leptin易化NMDA受体介导的突触传递，该作用与NMDA受体和MAPK通路有关。Leptin不论是在基础状态下，还是在急性应激时均有很强的抗焦虑作用。结论 Leptin通过激活NMDA受体从而削弱杏仁核突触传递的易化现象，促进恐惧记忆消散来发挥抗焦虑作用。

关键词：leptin；焦虑；杏仁核；恐惧消散

基金项目：国家重点基础研究发展计划（973计划）（2013CB 531300）

通讯作者：陈建国，E-mail：chenj@mails.tjmu.edu.cn，Tel：（027）83692636

T1-17

Antidepressant effects and underlying mechanism of SKF83959ondepressive-likebehaviourinduced by chronic social defeat stress

ZHENG Hui-ling，JIANG Bo，WANG Fang，HU Zhuang-li，WU Peng-fei，CHEN Jian-guo

（Department of Pharmacology，School of Basic Medicine，Tongji Medical College，Huazhong University ofScience and Technology，Wuhan 430000，China）

Abstract：OBJECTlVEToinvestigatewhether SKF83959 could have anantide pressanteff ects in the chronic social defeat stress modeland the underlying mechanism.METHODSChronicsocialdefeat stress was used to induce depressive-like behaviour. RESULTS 14 d treatment of SKF83959（0.5 or 1 mg·kg-1，ip）in the depression model could produce significant anti⁃depressant effects by increasing the interaction time and reducing preference for sucrose solution and SKF83959 also rescued the stress-induced decrease in hippocampal⁃ dendritic spine density，neurogenesis and enhanced BDNF signaling pathway.By using specific inhibitors and siRNA/shRNA methods，we further demonstratedthat the SKF83959-mediated antidepressant-like effects required the activation of the D5 receptor and PLC signaling path⁃way.CONCLUSlONChronicadministrationof SKF83959 can have an antidepressant effectsvia the hippocampalD5/phospholipaseC/inositolphosphate3/ calmodulin-dependent kinaseⅡα/brain-derivedneurotrophic factor pathway.

Keywords：depression；SKF83959；neurogenesis；chronic social defeat stress

Foundation item：The project supported by National Basic Research Program of China（2013CB531303）

Corresponding author：CHEN Jian-guo，E-mail：chenj@ mails.tjmu.edu.cn

T1-18

小柴胡汤对断乳后孤养小鼠抑郁/焦虑样行为的调节作用

马 洁，杨静玉，王 芳，潘 星，周婷硕，吴春福

（沈阳药科大学药理学教研室，辽宁沈阳 110016）

摘要：目的 小柴胡汤首见于《伤寒杂病论》，是和解少阳剂的著名代表方。前期研究表明，小柴胡汤在多种行为绝望模型中表现为显著的抗抑郁作用，但是能否可以改善断乳后孤养小鼠的行为障碍，未有报导。本文旨在研究小柴胡汤对断乳后孤养小鼠抑郁/焦虑样行为的改善作用。方法 将C57/BL/6J雄性小鼠在断乳后进行6周的社会孤养应激，并同时给予小柴胡汤（0.8，2.3和7.0 g·kg-1）。采用强迫游泳实验、悬尾实验、开场实验以及攻击实验评价小柴胡汤对断乳后孤养小鼠的抑郁/焦虑样行为的影响。结果 6周的社会孤养应激导致断乳后孤养小鼠表现为显著的行为绝望、焦虑以及攻击行为。小柴胡汤能够显著降低断乳后孤养小鼠在强迫游泳和悬尾实验中的不动时间；小柴胡汤（0.8和7.0 g·kg-1）显著减弱断乳后孤养小鼠在开场实验中自主活动的增强，但是对在中央区域的停留时间无显著影响；小柴胡汤（2.3和7.0 g·kg-1）显著逆转孤养应激引起的断乳后小鼠攻击行为的增强，包括显著缩短攻击时间以及减少攻击次数。结论本文研究表明，小柴胡汤可以调节长期社会孤养应激导致的断乳后小鼠的焦虑及抑郁样行为表现。

关键词：小柴胡汤；断乳后孤养；行为绝望；开场实验；攻击实验

基金项目：国家自然科学基金重点项目（81130071）

通讯作者：吴春福，E-mail：chunfuw@gmail.com，Tel：（024）

23986339

T1-19

小柴胡汤抗抑郁作用成分及机制研究

张 阔1，王 芳1，熊志立2，杨静玉1，吴春福1

（沈阳药科大学1.药理学教研室，2.药物分析教研室，辽宁沈阳 110016）

摘要：目的 小柴胡汤首见于东汉中医名家张仲景的《伤寒杂病论》。课题组前期研究发现小柴胡汤在多种抑郁动物模型中均具有良好的抗抑郁作用，但是其明确的物质基础和作用靶点仍待阐明。方法 采用正交设计法寻找小柴胡汤抗抑郁核心组合；通过悬尾实验、强迫游泳实验、新奇抑制摄食实验、利血平拮抗实验对小柴胡汤及核心组合的抗抑郁作用进行评价；通过测定神经递质对抗抑郁机制进行研究；通过液相质谱联用检测核心组合的入血成分。结果 正交设计结果表明黄芩、人参、甘草可能是小柴胡汤抗抑郁作用的核心组合。灌胃核心组合（0.9，2.8和8.4 g·kg-1）和小柴胡汤（2.3，7.0和21 g·kg-1）15 d后，均能显著缩短悬尾和强迫游泳不动时间，缩短新奇抑制摄食潜伏期，拮抗利血平所致体温下降和眼睑下垂，增加下丘脑和纹状体5-HT和DOPAC水平。口服核心组合后血清中共检测到25个成分。结论黄芩、人参和甘草可能是小柴胡汤抗抑郁作用的核心组合。核心组合展现出与小柴胡汤相似的抗抑郁作用，且均能调节神经递质水平。血清中的成分可能是小柴胡汤抗抑郁作用的潜在活性成分。

关键词：小柴胡汤；核心组合；抗抑郁；活性成分

基金项目：国家自然科学基金重点项目（81130071）

通讯作者：吴春福，Email：wucf@syphu.edu.cn，Tel：（024）23986340；杨静玉，E-mail：yangjingyu2006@gmail.com，Tel：（024）23986340

T2　神经退行性疾病

T2-1

转录因子FoxO3A在β 淀粉样蛋白诱导的突触损伤中的作用

牛 波，王欣怡，汪海涛，程玉芳，徐江平

（南方医科大学药学院神经药理与新药发现课题组，广州广东510515）

摘要：目的 研究FoxO3A在β淀粉样蛋白诱导的突触损伤中的作用。方法 ①β淀粉样蛋白片段Aβ25～35处理PC12细胞造模，采用RT-PCR和Western蛋白印迹法分别检测FoxO3A和突触相关蛋白（synapsin 1和PSD95）转录和翻译水平的表达。②鉴定7月龄C57背景的APP/PS1转基因小鼠，分野生型和转基因型2组（n=10），进行经典的新物体识别实验和Morris水迷宫实验，确认转基因型小鼠出现认知障碍后取小鼠的大脑皮质和海马组织，检测FoxO3A和突触相关蛋白转录和翻译水平的表达。③利用FoxO3AsiRNA干扰PC12细胞中FoxO3A的表达，观察突触相关蛋白表达的变化；同时FoxO3AsiRNA干扰的情况下，Aβ25-35处理PC12细胞，观察突触相关蛋白有无变化。结果 ①β淀粉样蛋白片段Aβ25～35处理PC12细胞造模后，与对照组相比，模型组的突触相关蛋白的表达量均显著下降，显示突触损伤。而转录因子FoxO3A的转录和翻译水平的表达量均显著上升。②7月龄APP/PS1转基因小鼠行为学实验结果显示，转基因型有显著的学习记忆障碍（P＜0.05）。其海马组织和皮质组织中与野生型相比，转基因的突触相关蛋白的表达均显著下降，显示突触损伤。而转录因子FoxO3A的转录和翻译水平的表达均显著上升。③干扰FoxO3A表达，突触相关蛋白表达上升，而在干扰FoxO3A表达时，Aβ25～35造成的突触损伤有所减轻。结论 β淀粉样蛋白可导致体内和体外突触损伤，干扰FoxO3A表达，可减轻β淀粉样蛋白诱导的突触损伤。

关键词：FoxO3A；突触损伤；β淀粉样蛋白

基金项目：国家自然科学基金项目（81301099；81373384；81503043）

通讯作者：徐江平，E-mail：jpx@smu.edu.cn，Tel：（020）61648236

T2-2

FFPM，a PDE4 inhibitor，recues cognitive dysfunc⁃tion in APP/PS1 transgenic mice via cAMP/PKA/ CREB/BDNF signaling and anti-inflammatory effect

GUO Hai-biao，XU Jiang-ping

（Group of Neuropharmacology and New Drug Discovery，School of Pharmaceutical Sciences，Southern Medical University，Guangzhou 510515，China）

Abstract：OBJECTlVE In this study，we investigat⁃ed the effect of FFPM，a novel PDE4 inhibitor，on learn⁃ing and memory deficits as well as the underlying mecha⁃nism in APP/PS1 transgenic mouse model of Alzheimer disease（AD）.METHODS UPLC-MS/MS was used for pharmacokinetic study.Morris water maze and step down passive avoidance was used for evaluating cogni⁃tive function.Combination of xylazine and ketamine in⁃duced anesthesia was used to assessed the potential emesis of FFPM.ELISA analysis was used for cAMP mea⁃surement.Western blotting and immunofluorescence anal⁃ysis were used to examine the expression of p-PKA，CREB，p-CREB，BDNF，NeuN，NF-κB p65，iNOS，TNF-α and IL-1β.RESULTS Pharmacokinetic studies revealed that FFPM efficiently permeated into brain，which reached peak values of plasma and brain at 2 h after orally dosing.Chronic treatment with FFPM significantly improves the learning and memory ablility of APP/PS1 transgenic mice in Morris water maze and step down passive avoidance task.Interestingly，we found that while rolipram reduced the duration of α2 adrenergic receptor-mediated anesthesia induced by xylazine/ketamine，FFPM or vechicle did not have evident effect.FFPM increased the expression in cAMP，phosphorylation of PKA and CREB，BDNF，and reduced NF-κB p65，iNOS，TNF-α and IL-1β in the hippocampus of APP/PS1 trangenic mice，as observed by ELISA and western blotting analysis.Further，results of immunofluorescence analysis suggested that roflurpam enhanced the immunoreactivi⁃ties of BDNF and NeuN in hippocampus of APP/PS1 trangenic mice.CONCLUSlON Our data demonstrated that reversal effect of FFPM on cognitive deficits in APP/ PS1 transgenic mice might be related to stimulate cAMP/ PKA/CREB/BDNF pathway and anti-inflammatory effect，which further prevented the neuronal loss.Moreover，FFPM appears to be a option for PDE4 inhibitors in ADtherapy with little emetic potential.

Key words：FFPM；cognition；Alzheimer disease；phos⁃phodiesterase-4

Foundation iterm：The project supported by National Natural Science Foundation of China（81503043；81373384）

Correspondence author：XU Jiang-ping，Tel：（020）61648236，E-mail：jpx@smu.edu.cn

T2-3

Down-regulation ofprogranulinimprove learning and memory in mouse model of fragile X syndrome through TNFR2/NFκ B pathway

ZHANG Kun1*，LI Yu-jiao1*，ZHENG Kai-yin1*，YANG Qi1，YANG Le1，ZHANG Chao1，SONG Qian2，LIU Shui-bing1，WU Yu-mei1，ZHAO Ming-gao1

（1.Department of Pharmacology，School of Pharmacy，FourthMilitaryMedicalUniversity，Xi′an 710032，China；2.Center for Neuron and Disease，Frontier Insti⁃tutes of Life Science and of Science and Technology，Xi′an Jiaotong University，Xi′an 710032，China）

Abstract：OBJECTlVE To exhibit the role of PGRN in fragile X syndrome（FXS）cognition disability develop⁃ment.METHODS Expression of PGRN was detected in FMR1 WT and KO brain and plasma by western blotting and elisa.The role of PGRN in LTP and LTD induction was detected by MED64.PGRN binding receptors were detected by co-immunoprecipitation.We analyzed chang⁃es of KO mice after down regulating PGRN by fear mem⁃ory test and spine morphology analysis.RESULTS We found higher levels of PGRN in FMR1 knock out（KO）mice than in wild type（WT）mice.Moreover excessive PGRN will lead defects in dendritic spine pruning and LTP expression without affecting LTD.As PGRN Be⁃cause PGRN can directly bind to two TNFα receptors，it important to detect the roles of two TNFRs in LTP.We proved activation of TNFR2 but not TNFR1 is necessary for LTP expression.In further research we found that PGRN activating TNFR2，and then increased NFκB and p-GluR1（ser845/831）levels which couldn′t further be el⁃evated after chemical LTP induction，uncovering a ceil⁃ing LTP disability produced by excessive PGRN.In addi⁃tion，knocking down PGRN correct abnormal signal path⁃ways，immature dendritic spine morphology and multiple behavioral phenotypes，including fear memory deficits，hyperactivity，and behavioral inflexibility through interfer⁃ing TNFR2/NFκB pathway.Basing on our research we found that partially blocking TNFR2/NFκB pathway using JSH-23，a small chemical molecule，was a good treat⁃ment for FXS.CONCLUSlON Our results support the PGRN as a novel treatment target for FXS，and broaden our under⁃standing of neurotrophins role in neurodevelopment.

Key words：fragile X syndrome； progranulin； TNF-α，TNFR2/NFκB；pathway

Foundation item：The projectsupportedby National NaturalScienceFoundationofChina（31070923；31271144）；and Program for New Century Excellent Talents in University

Correspondence author： ZHAO Ming-gao，E-mail：minggao@fmmu.edu.cn；ZHANG Kun，E-mail：kunzhang1900@163.com

*Co-first author

T2-4

TRPV4 channel activation mediated excitatory effects of hAmylin on rat primary hippocampus neurons

ZHANG Nan1*，YANG Sheng-chang1，2*， BAIHui3，ZHANG Jiang-hua1， CHENLing1， JIA Zhan-feng4，REN Lei-ming1，KANG Lin5，ZHANG Wei1

（1.Department of Pharmacology，Institution of Chinese Integrative Medicine，Hebei Medical University，Shijiazhuang 050017，China；2.Department of physiology，Hebei University of Chinese Medicine，Shijiazhuang050020，China；3.Department of Cardiac Ultrasonic Diagnosis，the Second Hospital of Hebei Medical University，Shijiazhuang 050000，China；4.Department of Pharmacology，Hebei MedicalUniversity， Shijiazhuang050017，China；5.Department of Anatomy，Hebei Medical University，Shijiazhuang 050017，China）

Abstract：OBJECTlVE Alzheimer disease（AD）and type II diabetes mellitus（DM2）are the most common dis⁃eases in aging people，with β-amyloid and amylin accu⁃mulation respectively.Studies have shown a strong corre⁃lation between these two diseases，and amylin oligomer⁃ization in the brain provides a novel risk target for devel⁃oping AD.Although cumulative studies reported that amy⁃lin aggregation induced cytotoxicity in neuron by altering Ca2+homeostasis，but the underlying mechanisms are not fully understood.METHODS To address this ques⁃tion we performed calcium imaging and whole-cell patch clamp recordings on primary cultured rat hippocampal neu⁃rons to investigate the effects of amylin.RESULTS We revealed that human amylin（hAmylin），but not rat amylin or pramlintide（hAmylin analgue），evoked a rapid in⁃crease in intracellular calcium in a dose dependent man⁃ner.This effect relied on extracellular calcium but not abolished by hAmylin receptor antagonist AC187.Addi⁃tionally，blockade of L-type Ca2+channel partially reduced hAmylin-induced Ca2+response in presence of extracellu⁃lar Na+.In whole-cell recordings，hAmylin depolarized membrane potential and increased the cell excitability. Moreover，application of transient receptor potential vanil⁃loid（TRPV）antagonist ruthenium red attenuated hAmy⁃lin-induced Ca2+increase.Single-cell RT-PCR revealed that TRPV4 mRNA was expressed in most of hAmylin-re⁃sponsive neurons.In addition，selective TRPV4 antago⁃nist HC067047 inhibited hAmylin-evoked Ca2+response.CONCLUSlON These results indicated that hAmylin aggre⁃gation precipitating on the neuron membrane activated TRPV4 channels and then triggered membrane voltage gated calcium channel opening following membrane depolariza⁃tion.We suggest that TRPV4 is a key molecular target for the cytotoxic effect of hAmylin on hippocampal neurons.

Key words：amylin；calcium；hippocampus neurons；TRPV4

Foundation item：The project supported by National Natural Science Foundation of China（31200808；81573416）

Corresponding author：REN Lei-ming，E-mail：ren-leiming@ 263.net；KANG Lin，E-mail：jiepouky@163.com；ZHANG Wei，E-mail：weizhang@hebmu.edu.cn

*Co-first author

T2-5

新型促智药物的研究

章 正，罗焕敏

（暨南大学医学院药理学系，广东广州 510632）

摘要：目的 研究、发现具有神经营养因子样作用的小分子促智药物，并探讨其作用机制。方法 根据我国《药品注册管理办法》及其他相关的技术指导原则，结合现代分子生物学技术、基因组技术、蛋白质组技术等，筛选获得有“苗头”的化合物。完成候选化合物的药学、药理毒理学研究，并申请临床批件，为临床试验做准备。结果 本研究从能够进入脑内的小分子化合物库中筛选、获得了候选化合物MDHB，它可明显拮抗Aβ毒性、保护神经元，同时可促进神经元突起再生和神经网络重建；能够促进血糖利用、改善糖代谢、抑制CA3区tau的磷酸化及齿状回神经元死亡，改善地塞米松模型小鼠的学习记忆能力。进一步研究发现，MDHB的作用主要是通过激活A2A受体进而激活PI3K/Akt信号通路、抑制ERK1/2磷酸化、诱导Akt磷酸化而实现的；MDHB一方面可以拮抗Aβ诱导的线粒体损伤，减少ROS产生，提高MMP水平，上调Bcl-2表达，下调Bax表达，抑制cleaved胱天蛋白酶9和cleaved胱天蛋白酶3蛋白的产生，从而对神经元起保护作用；另一方面它还可上调内源性BDNF mRNA和

MAP2 mRNA的表达，促进神经元突起再生，使神经网络重建。MDHB的药学研究、急性毒性研究、非临床药代动力学研究也已基本完成，数据正在整理之中。MDHB已获得国家新药发明专利授权。结论 MDHB具体神经营养因子和促智药的双重作用，它既可以修复损伤的神经网络、使其得到重建，又可改善糖代谢、促进ATP产生和利用。MDHB有望成为一类新型的、理想的、具有广阔市场前景的记忆增强剂。

关键词：MDHB；阿尔茨海默病；β淀粉样蛋白；细胞凋亡

基金项目：国家自然科学基金项目（81173037）；广东省科技计划项目（2012B050300018）

通讯作者：罗焕敏，E-mail：tlhm@jnu.edu.cn

T2-6

KCNQ3上磷酸酰肌醇4，5-二磷酸的结合位点及调控机制

金雅康*，曹栎雯*，崔建民，镇学初

（苏州大学药学院，江苏苏州 215123）

摘要：目的 明确KCNQ3通道上磷酸酰肌醇4，5-二磷酸（PIP2）的结合位点，并阐明PIP2如何通过与这些位点的结合来调节KCNQ3的活性，从而揭示PIP2调节神经元KCNQ3离子通道的分子机制。方法 通过对KCNQ家族进行同源对比，筛选出潜在的PIP2结合位点。对相关点突变进行点突变，构建载体并在HEK293t细胞系中瞬时表达。通过全细胞记录（whole-cell recording），分析比较序列突变前后的电生理特性，进一步筛选潜在结合位点，在此基础上，通过内面向外记录（Inside-out recording）来研究结合位点与PIP2浓度依赖性关系，以此确认PIP2的结合位点。结果 通过同源比对，筛选出22个带正电的氨基酸残基为潜在的PIP2结合位点，分别位于电压感受区、孔道区域和C末端。在这些位点中，我们发现了8个潜在的PIP2结合位点突变为中性氨基酸后，会导致全细胞电流显著降低；其余位点，虽然也带正电荷，但突变后并不能影响全细胞电流。另外，我们还发现一些位点的突变反而会增加全细胞电流，如R146和K148等，原因还不明确。结论 R153，R158和R205等为潜在的PIP2结合位点，这些位点突变后，可能影响了PIP2与KCNQ3的结合，从而影响了KCNQ3的全细胞电流。具体的作用机制有待下一步研究。

关键词：KCNQ3通道；磷酸酰肌醇4，5-二磷酸；膜片钳；结合位点；门控机制

基金项目：国家自然科学基金（31271143；81373382）

通讯作者：崔建民，E-mail：jcui@wustl.edu；镇学初，E-mail：zhenxuechu@suda.edu.cn

*共同第一作者

T2-7

人参皂苷Rg1改善MPTP/p诱导的慢性帕金森病模型小鼠神经炎症的机制研究

衡 洋1，2，张秋双1，2，牟 正1，2，胡金凤1，2，苑玉和1，2，陈乃宏1，2，3（1.中国医学科学院神经科学中心，北京 100730；2.中国医学科学院北京协和医学院药物研究所天然药物活性物质与功能国家重点实验室，北京 100050；3.湖南中医药大学药学院，湖南长沙 410208）

摘要：目的 在1-甲基-4-苯基-1，2，3，6-四氢吡啶（MPTP）和丙磺舒（probenecid）（MPTP/p）联合诱导的慢性帕金森病模型小鼠中评价Rg1的药效，并且尝试阐明其中的机制。方法 运用行为学测试如爬竿、转棒，来评价小鼠的行为学改变；通过western蛋白印迹法来检测小鼠黑质部位α-突触核蛋白（α-synuclein）的水平及存在状态；运用酶联免疫吸附实验（ELISA）检测小鼠黑质部位炎症因子的改变；运用电子透射显微镜对小鼠黑质部位超微结构改变进行检测；运用免疫组化方法对上述结果进行验证。结果 口服Rg1能够明显的改善MPTP所诱导的小鼠的死亡率，行为学障碍，黑质部位的多巴胺神经元的丢失以及超微结构的破化。Rg1的神经保护作用可能是通过其抗炎作用来实现的，Rg1能够改善MPTP所诱导的黑质部位星形胶质细胞和小胶质细胞的激活，炎症因子的增多，如TNF-α和IL-1β。此外，Rg1还能够改善模型动物中黑质部位α-突触核蛋白的寡聚体，磷酸化以及疾病相关的形式。结论 人参皂苷Rg1具有神经保护作用，而这种神经保护作用可能是通过改善异常α-突触核蛋白在黑质部位引起的神经炎症实现的，Rg1具有作为PD治疗药物的前景。

关键词：帕金森氏病；1-甲基-4-苯基-1，2，3，6-四氢吡啶；神经炎症；α-突触核蛋白；人参皂苷Rg1

基金项目：国家自然科学基金（81274122；81373997；U1402221；81573640；81273629）；北京市自然科学基金（7131013）；教育部博士点基金（20121106130001）；新药作用机制研究与药效评价北京市重点实验室资助（BZ0150）

通讯作者：苑玉和，E-mail：yuanyuhe@imm.ac.cn，Tel：（010）63165182；陈乃宏，E-mail：chennh@imm.ac.cn，Tel：（010）63165177

T2-8

LW-AFC对阿尔茨海默病动物模型肠道菌群的调节作用

程肖蕊1，王建辉1，叶福强2，倪 铭2，周文霞1，张永祥1

（军事医学科学院1.毒物药物研究所，2.放射与辐射医学研究所，北京 100850）

摘要：目的 研究LW-AFC对阿尔茨海默病（AD）动物模型肠道菌群的调节作用。方法 给予AD动物模型快速老化模型小鼠SAMP8 LW-AFC 3个月后，使用Morris水迷宫实验、穿梭箱实验和新异物体识别实验分别测试各受试动物的空间学习记忆能力、主动回避反应能力和物体识别记忆能力，然后收集各动物粪便提取总DNA，使用Illumina Miseq platform进行宏基因组测序。结果 与对照组SAMR1相比，SAMP8的空间学习记忆能力、主动回避反应能力和物体识别记忆能力发生退化，而给予LW-AFC可以逆转SAMP8这些学习记忆能力的损害。与SAMR1相比，SAMP8的肠道中有8个菌群显著上高、12个菌群显著下降。SAMP8口服LW-AFC后，其肠道有22个菌群发生了变化，其中有16个上调、6个下调，且LW-AFC可使SAMP8肠道中的15个菌群的丰度达到SAMR1的水平。进一步分析发现，有7个菌群皆与空间学习记忆能力、主动回避反应能力和物体识别记忆能力显著相关，其中3个正相关、4个负相关，包括拟杆菌目（Bacteroidales）、梭菌目（Clostridiales）、脱硫弧菌目（Desulfovibrionales）、CW040和3个未能鉴定的菌群，而给予SAMP8 LW-AFC可使这7个菌群回复到SAMR1的水平。结论 LW-AFC可改善AD动物模型SAMP8的学习记忆能力，且可调节其肠道菌群。

关键词：LW-AFC；菌群组；快速老化模型小鼠；阿尔茨海

默病

基金项目：国家科技部重大专项（2013ZX09508104；2012ZX09301003-002-001）

通讯作者：程肖蕊，E-mail：cxr916@163.com，Tel：（010）66930624

T2-9

Modulatingbalanceofsynapticandextrasynaptic NMDA receptors（NMDARs）shows priority over nonselective NMDAR antagonists against beta-amyloid induced neurotoxicity

HUANG Yan，SHEN Wei，LIU Gang，ZHOU Wen-xia，ZHANG Yong-xiang

（Beijing Institute of Pharmacology and Toxicology；State Key Laboratory of Toxicology and Medical Countermea⁃sures，Beijing 100850，China）

Abstract：OBJECTlVE Alzheimer disease（AD）pa⁃tients suffer a disturbance in the balance between synap⁃tic NMDARs（NR2A，mediating the protective pathway）and extrasynaptic NMDARs（NR2B，mediating the excito⁃toxic pathway），and this study aim to find out whether re⁃storing the balance of NR2A and NR2B is beneficial for AD.METHODS Aβ treated hippocampal slices and Aβ icv injection animals were used as AD models in this study.Long-term potentiation（LTP）and behavior test were used to evaluate the effects of drugs and signal pathways were analyzed by Western blotting.RESULTS Specific activation of NR2A and inhibition of NR2B showed the best protective effect against Aβ-induced LTP deficits.In Aβ icv injection animals，the combination of ifenprodil and D-cycloserine（a co-activator of NMDRs similar to D-serine）showed better protective effects against Aβ-induced cognitive deficits（nest building，novel object recognition and Morris water maze）than ifenprodil or D-cycloserine alone.The ratio of NR2A to NR2B，TORC dephosphorylation and ERK1/2 activation（which could be initiated by NR2A）decreased in the hippocam⁃pal tissues of Aβ-treated animals.As a result，the activa⁃tion of CREB and the content of brain-derived BDNF de⁃creased.The combination of ifenprodil and D-cycloserine showed the best protective effects，indicating that Aβ-in⁃duced toxicology was mediated both by inhibiting NR2A and enhancing NR2B.CONCLUSlONS These results in⁃dicate that enhancing synaptic NMDARs and inhibiting extrasynaptic NMDARs concurrently showed good ef⁃fects against Aβ-induced neurotoxicity，suggesting that modulation of the balance between NR2A and NR2B could be a potential strategy for AD drug development and therapy.

Key words：beta-amyloid； NR2A； NR2B；balance；Alzheimer disease

基金项目：国家自然科学基金（81202505）

通讯作者：ZHOU Wen-xia，E-mail：zhouwx@bmi.ac.cn，Tel：（010）66931625

T2-10

肠道菌群在CA-30改善皮质酮致突触可塑性损伤中的作用

李 栋，黄 晏，周文霞，张永祥

（军事医学科学院毒物药物研究所，北京 100850）

摘要：目的 CA-30是六味地黄汤中提取的寡糖成分，本研究拟考察肠道菌群对其改善皮质酮导致海马突触可塑性损伤作用的影响。方法 采用皮质酮皮下注射复制长时程增强（long-term potentiation，LTP）损伤模型；采用灌胃给予复合抗生素（新霉素200 mg·kg-1；杆菌肽200 mg·kg-1；那他霉素500 μg·kg-1）复制肠道菌耗竭模型；SPF级BALB/c小鼠随机分为5组（每组10只）：正常组，皮质酮模型组，抗生素组，皮质酮+CA-30（1.0 g·kg-1），皮质酮+抗生素+CA-30。BALB/c小鼠灌胃给予混合抗生素和CA-30，第7天灌胃结束1 h后，皮下注射皮质酮（50 mg·kg-1）造模，然后进行LTP实验。结果 结果表明，与正常对照组比较，皮下注射皮质酮（50 mg·kg-1）后LTP增幅明显减小，抗生素对正常LTP无明显影响。与模型组相比，CA-30可明显改善皮质酮所致的LTP损伤，而同时给予抗生素后CA-30的改善作用消失。

结论 CA-30能够显著改善皮质酮导致的小鼠海马突触可塑性的损伤，肠道菌群耗竭后CA-30的改善作用消失，提示CA-30通过作用于肠道菌群而发挥其改善LTP的作用。

关键词：肠道菌群；长时程增强；CA-30；皮质酮

基金项目：国家自然科学基金（81202505；81302759）

通讯作者：周文霞，E-mail：zhouwx@bmi.ac.cn

T2-11

基于快速老化模型小鼠的LW-AFC防治阿尔茨海默病的研究

王健辉，程肖蕊，张小锐，刘 峰，程军平，周文霞，张永祥（军事医学科学院毒物药物研究所抗毒药物与毒理学国家重点实验室，北京 100850）

摘要：目的 基于快速老化模型小鼠（SAMP8），研究六味地黄糖苷（LW-AFC）防治阿尔茨海默病（Alzheimer disease，AD）的作用及其机制。方法 采用新异物体识别实验、Morris水迷宫实验、跳台实验、穿梭箱实验分别检测LW-AFC对AD模型SAMP8小鼠物体识别记忆能力、空间学习记忆能力、被动回避反应能力和主动回避反应能力的影响，采用液相蛋白芯片技术检测受试动物血浆中细胞因子的含量，放射免疫法检测HPA轴和HPG轴的变化。结果SAMP8小鼠的物体识别记忆能力、空间学习记忆能力、被动回避反应能力和主动回避反应能力明显降低，血浆中促炎因子明显升高，而抗炎因子明显下降。LW-AFC能显著改善SAMP8小鼠的物体识别记忆能力、空间学习记忆能力、被动回避反应能力和主动回避反应能力，明显降低血浆中促炎因子IL-1β，IL-2，IL-6，IL-17，IL-23，GM-CSF，INF-γ，TNF-α，TNF-β，MCP-1，MIP-1β，RANTES和Eotaxin的含量，提高抗炎因子IL-4，IL-10，IL-5和G-CSF的水平，显著降低下丘脑中CRH和GnRH、垂体中ACTH和LH及FSH、血浆中CORT的水平，明显提高血浆Testosterone水平。结论LW-AFC通过调节SAMP8小鼠的NIM网络功能紊乱来发挥其增强认知功能的作用，有可能成为防治AD的潜在药物。

关键词：LW-AFC；阿尔茨海默病；神经内分泌免疫调节

基金项目：国家科技重大专项（2012ZX09301003-002-001；2013ZX09508104）

通讯作者：周文霞，E-mail：zhouwx@bmi.ac.cn；张永祥，E-mail：zhangyx@bmi.ac.cn

T2-12

基于APP/PS1双转基因小鼠的LW-AFC及其组分抗阿尔茨海默病作用的初步研究

雷曦1，2，3，程肖蕊2，3，周文霞1，2，3

（1.广西医科大学，广西南宁 530021；2.军事医学科学院毒物药物研究所，北京 100850；3.抗毒药物与毒理学国家重点实验室，北京 100850）

摘要：目的 基于阿尔茨海默病（Alzheimer disease， AD）动物模型APP/PS1双转基因小鼠，研究六味地黄糖苷（LW-AFC）及其活性组分多糖（LWB-B）、糖苷（LWD-b）和寡糖（CA-30）防治AD的作用。方法 应用老化度评分小鼠的老化程度，筑巢试验考察小鼠的日常活动的执行能力，微弱光线旷场检测小鼠的自主活动度，并用3H-TdR掺入法检测小鼠的脾细胞增殖能力以及用流式细胞术检测脾淋巴细胞亚型。结果 与野生型相比，APP/PS1双转基因小鼠的老化度评分显著升高、筑巢能力和自主活动性均显著降低，分别灌胃给予LW，LW-AFC，LWB-B，LWD-b和CA-30均能够明显改善APP/PS1小鼠的老化征象，提高其自主活动能力，改善其筑巢功能，且效果均好于阳性药乙酰胆碱酯酶抑制剂多奈哌齐和NMDA受体拮抗剂美金刚；与野生型相比，APP/PS1双转基因小鼠的免疫功能出现缺陷，而LW，LW-AFC及其组分对其免疫缺陷具有恢复的作用，具体为：LW，LW-AFC，LWB-B，LWD-b和CA-30能够显著提高Con A刺激的T淋巴细胞增殖能力，LW-AFC，LWB-B和LWD-b能够显著提高LPS刺激的B淋巴细胞增殖能力，效果均强于多奈哌齐与美金刚；LW，LW-AFC，LWB-B，LWD-b 和CA-30能够显著升高APP/PS1转基因小鼠成熟T细胞的水平，降低中性粒细胞的水平，LWB-B和LWD-b能够显著升高巨噬细胞的水平。结论 LW-AFC及其组分能够显著改善APP/PS1双转基因小鼠老化征象和社会认知能力，其作用机制可能是通过调节免疫功能实现的。

关键词：LW-AFC；APP；PS1；转基因小鼠；学习记忆；筑巢；免疫

基金项目：国家科技重大专项（2012ZX09301003-002-001；2013ZX09508104）

通讯作者：周文霞，E-mail：zhouwx@bmi.ac.cn，Tel：（010）66931625

T2-13

基于LPS诱导BALB/c小鼠炎症模型的LW-AFC抗炎作用研究

曾菊1，2，程斌1，2，程肖蕊1，2，周文霞1，2，张永祥1，2

（1.军事医学科学院毒物药物研究所，北京 100850；2.抗毒药物与毒理学国家重点实验室，北京 100850）

摘要：目的 神经炎症是阿尔兹海默病（Alzheiemr dis⁃ease，AD）的重要发病机制之一，本研究主要考察中药五类新药六味地黄苷糖（LW-AFC）的抗炎活性。方法 灌胃给予雄性BALB/c小鼠LW-AFC两周，在体记录兴奋性突触后电位、外周血淋巴细胞亚群以及外周血和脑组织中炎性细胞因子检测观察前4 h单次腹腔注射脂多糖（LPS）（250 μg·kg-1，ip）建立炎症模型，观察LW-AFC的作用。结果 腹腔注射LPS 4 h后小鼠的兴奋性突触后电位降低，即长时程增强减弱，预防给予LW-AFC可明显改善炎症所导致小鼠神经突触可塑性的降低；结果显示LW-AFC可显著调高炎症模型小鼠脾脏淋巴细胞中CD19+细胞的比例，调低CD4+/CD3+细胞比例。同时，LW-AFC能明显升高外周血中CD3+CD4+细胞比例，降低CD4+/CD3+细胞比例。LW-AFC并可显著抑制炎症小鼠外周血、海马和大脑皮质中炎性细胞因子TNFα，IL-6，IL-12和INFγ的分泌。结论 LW-AFC可通过抗炎作用保护神经突触可塑性免受炎症的损害，有可能成为防治AD的潜在药物。

关键词：LW-AFC；突触可塑性；炎症因子；淋巴细胞亚群

基金项目：国家科技重大专项（2012ZX09301003-002-001；2013ZX09508104）

通讯作者：周文霞，E-mail：zhouwx@bmi.ac.cn，Tel：（010）66931625

T2-14

lnhibition of sPLA2-ⅡA prevents LPS-induced neuro⁃inflammation by suppressing ERK1/2-cPLA2a pathway in mice cerebral cortex

XIANG Yan-xiao1，CHEN Lin2，LIU Hui-qing2，LIU Xiao-qian2，WEI Xin-bing2，SUN Bao-zhu1，WANGTian3，ZHANG Xiu-mei2

（1.Qilu Hospital，Shandong University，Jinan 250012，China；2.DepartmentofPharmacology，Shandong University，Jinan250012，China；3.Department of Pharmacology，Yantai University，Yantai 264100，China）

Abstract：OBJECTlVENeuroinflammation is in⁃volved in various central nervous system（CNS）disor⁃ders，includingbraininfections，ischemia，trauma，stroke，and degenerative CNS diseases.In the CNS in⁃flammation，secretory phospholipase A2-ⅡA（sPLA2-ⅡA）acts as a mediator，resulting in the generation of the pre⁃cursors of pro-inflammatory lipid mediators，such as prostaglandins（PGs）and leukotrienes（LTs）.However，the role of sPLA2-ⅡA in neuroinflammation is more com⁃plicated and remains unclear yet.In the present study，we investigated the effect of sPLA2-ⅡA inhibition by spe⁃cific inhibitor SC-215 on the inflammation in LPS-induced mice cerebral cortex and primary astrocytes.METHODS ① We used primary rat astrocytes to studythe role of sPLA2-ⅡA in LPS-induced neuroinflammation.RT-PCR analysis was performed to examine the mRNA level of sPLA2-ⅡA in primary rat astrocytes and microglia.ELISA-basedassay was used to measure the PGE2 concentra⁃tion of the supernatant of astrocytes.Western blot deter⁃mined the protein expression related to AA signaling.②In vivo induced by the intracerebroventricle microinjec⁃tion（icv）of LPS was employed to explore the regulation of MAPK-cPLA2α-PGE2 pathways by sPLA2-ⅡA in mice cerebral cortex.ELISA-based assay was used to determine the PGE2 concentration of the supernatant of mice cerebral cortex.Protein expression of sPLA2-ⅡA，phosphor-ERK1/2，phosphor-p38，phosphor-cPLA2a，COX-2 and mPGES-1 were evaluatedby Western blot.③We used primary rat astrocytes to simulate inflammatory situation to study the role of acetylpuerarin in LPS-stimu⁃lated route of eicosanoid biosynthetic pathway.We used MTT solution to examine the cytotoxicity of acetylpuera⁃rin in primary rat astrocytes.Western blot and immunoflu⁃orescence staining were used to determine the protein level of group V sPLA2.PGE2 and LTC4 concentration was measured by using ELISA-based assay kit.Western blot examined the activation of ERK1/2，cPLA2a，NF-kB and protein expression of COX-2 and LOX-5.RESULTS Our results showed that the inhibition of sPLA2-ⅡA alle⁃viated the release of PGE2 by suppressing the activation of ERK1/2，cPLA2a，COX-2 and mPGES-1.CONCLU⁃SlONThese findings demonstrated that sPLA2-Ⅱ A showed the potential to regulate the neuroinflammation in vivo and in vitro，indicating that sPLA2-ⅡA might be a novel target for the treatment of acute neuroinflammation.

Key words：primary rat astrocytes；acetylpuerarin

Foundation item：The project supported by Natural Sci⁃ence Foundation of Shandong Province（ZR2010HM132；ZR2010HM082）；and Graduate Independent Innovation Foundation of Shandong University（21300072613091）

Corresponding author：ZHANG Xiu-mei，E-mail：zhangxm@sdu.edu.cn

T2-15

PDE4 inhibitor Roflupram suppresses neuroinflam⁃mation through autophagy induction in BV2 microglial cells

YOU Ting-ting，ZENG Bing-qing，LI Yi-wen，CHENG Yu-fang，XU Jiang-ping

（Guangdong Provincial Key Laboratory of New Drug Screening，School of Pharmaceutical Sciences，Southern Medical University，Guangzhou 510515，China）

Abstract：OBJECTlVENeuroinflammation involes in the pathogenesis of mental disorders and neurodegen⁃erative disorders of central nerve system（CNS）.Studies have shown that cAMP induced autophagy whereas au⁃tophagy suppressed inflammatory activity.Moreover，it was indicated that phosphodiesterase-4（PDE4）inhibitor Roflupram（ROF）suppressed neuroinflammation and im⁃proved behavior of APP/PS1 transgenic Alzheimer dis⁃ease（AD）mice in our previous experiments.However，mechanisms of these effect has not been established un⁃equivocally.Here，we examined the effects of ROF on autophagy against neuroinflammation caused by BV2 mi⁃croglial cells activation.METHODSWestern blotting analysis，fluorescent labeling and enzyme linked immu⁃nosorbent assay was used to assess the effects of ROF on autophagy and the mechanisms of anti-neuroinflam⁃mation in LPS puls ATP-induced or Aβ25-35-activated BV2 microglial cells.RESULTSThe level of endogenous LC3-Ⅱwas significantly increased and p62 decreased in BV2 microglial cell line by ROF in a concentration-and time-dependent manner.Enhancement fluorescence-sig⁃nal was observed in BV2 cells treated with ROF using ac⁃ridine orang stain and lyso-tracker red.In addition，immu⁃nofluorescence for LC3 indicated a significant increase in punctate LC3.Either LPS puls ATP or Aβ25-35enhanced conversion of pro-caspase 1 to cleaved-caspase-1，in⁃creased production of mature IL-1β in stimulated BV2 cells.These effects were reduced by ROF.Moreover，hoechst staining shown that ROF decreased apoptosis of mouse neuroblastoma N2a cells in conditioned medi⁃um from activated-BV2 microglia.But these effects were reversed after autophagy inhibition with 3-Methylade⁃nine.CONCLUSlONTogether these results indicated that PDE4 inhibitor ROF reducing release of IL-1β and limited neuroinflammation caused by activated BV2 mi⁃croglia through inducing autophagy；the pharmacological manipulation of microglia autophagy modifed neuroin⁃flammatory response and ROF could potentially be used for the therapeutic intervention of inflammation-associated disease in CNS.

Key words：phosphodiesterase-4；roflupram；autophagy；neuroinflammation

Foundation item：The project supported by National Natural Science Foundation of China（81503043）

Correspondingauthor： XUJiang-ping，E-mail：jpx@smu.edu.cn

T2-16

Dihydromyricetin protects neurons in MPTP-induced Parkinson disease via suppressing activity of glyco⁃gen synthase kinase-3 beta

REN Zhao-xiang*，ZHAO Ya-fei*，CAO Ting，ZHEN Xue-chu （Jiangsu Key laboratory for Translational Research and Therapy for Neuropsychiatricdisorders&the Collabora⁃tive Innovation Center for Brain Science，College of Pharmaceutical Sciences，Soochow University，Suzhou 215123，China）

Abstract：OBJECTlVE Parkinson disease（PD）is a neurodegenerative disease characterized by the selec⁃tive loss of dopaminergic neurons of the substantia nigra pars compacta（SNc）.Although the precise etiology of PD remains unclear，it is general believed that mitochondrial dysfunction and oxidative stress（OS）play critical roles in the pathology of PD.Dihydromyricetin（DHM），a flavo⁃noids natural product extracted from Ampelopsis gros⁃sedentata has been recently found to elicit a potent antioxidative effect.In the present study，we explored the role of DHM in dopaminergic neuronal protection.METH⁃ODS Male C57BL/6 mice were intraperitoneally injected with MPTP（25 mg·kg-1）for 7 d to induce PD.For the groups with DHM and MPTP treatment，mice were given 5 or 10 mg·kg-1DHM three days before and during MPTP administration and were continuously given DHM for additional three days（totally 13 d DHM treatment）. For the group with saline or DHM alone，mice were in⁃jected continuously with saline or DHM for 13 d.At day 14，locomotor activity，rotarod test and pole test were conducted.After behavioral tests，mice were sacrificed and brain tissue was collected for immunofluorescence staining and western blotting.MES23.5 cells were cul⁃tured in DMEM containing 10%fetal bovine serum，1% penicillin/streptomycin and insulin-transferrin-selenium.Cells were treated with MPP+and DHM，and collected forcell vi⁃ability，ROS test，apoptosis analysis and Western blotting. RESULTS In this study，we found that DHM significantly attenuatedMPTPlesion-inducedmouse behaviour impairments and loss of dopaminergic neuron.In MES 23.5 cells，DHM dose-dependently attenuated MPP+-in⁃duced cell injury and inhibited the production of ROS in⁃duced by MPP+.We further found，at the first time，that DHM increased Glycogen synthase kinase 3 beta（GSK-3β）phosphorylation in dose-and time-dependent man⁃ners that may associate with the drug-elicited dopaminer⁃gic neuronal protection.CONCLUSlONThe present study demonstrated that DHM is a potent neuroprotec⁃tive agent of DA neuron via modulating Akt/GSK-3β path⁃wayand indicates that DHM may be a promising thera⁃peutic candidate for Parkinson′s disease.

Keywords：dihydromyricetin；tyrosinehydroxylase；GSK-3β；Akt；Parkinson disease

Foundation item：The project supported by National NaturalScienceFoundationofChina（81130023；30825042）；National Basic Research Plan（973）of the MinistryofScienceandTechnologyofChina （2011CB504403）；the Priority Academic Program Devel⁃opment of Jiangsu Higher Education Institutes（PAPD）；and Jiangsu Key Laboratory Grant（BM2013003）

Corresponding author：ZHEN Xue-chu，E-mail：zhenxuechu@ suda.edu.cn

*Co-first author

T2-17

Oncometabolite 2-hydroxyglutarate inhibits microglial activation via AMPK pathway

HAN Chao-jun，ZHENG Long-tai，ZHEN Xue-chu

（Department of Pharmacology，Soochow University College of Pharmaceutical Sciences，Suzhou 215123，China）

Abstract：OBJECTlVE To investigate the anti-in⁃flammatory effects of oncometabolite 2-hydroxyglutarate （2HG）on microglia-mediated inflammatory response. METHODS This study used LPS-induced BV2 cells to in⁃vestigate the anti-inflammatory properties of 2HG.The production of NO and TNF-α in supernatants were mea⁃sured by Griess and ELISA assay.MTT assay was used to measure cell viability.IL-1β，TNF-α，IL-6，iNOS and COX-2 mRNA levels were quantified by Quantitative real-time PCR（Q-PCR）.The phosphorylation of AMP-activated protein kinase（AMPK），glycogen synthase kinase 3β（GSK3β）and p65 protein expression levels were examined by Western blot.RESULTS 2HG sup⁃presses NO generation and pro-inflammatory cytokines expression in LPS-induced BV2 cells.2HG inhibited LPS-induced GSK3β phosphorylation by activating AMPK in BV-2 microglia cells.The GSK3β induced phosphoryla⁃tion（Ser 536）subunit of NF-κB was also inhibited by presence of 2HG in LPS activated BV-2 microglia cells. CONCLUSlON 2HG inhibits the microglial activation through activation of AMPK and suppression of GSK3β signaling pathway.

Key words：2-hydroxyglutarate；microglia；inflammation

Foundation item：The project supported by National Natural Science Foundation of China（81372688；81373382）；and National Basic Research Plan（973）of Ministry of Science and Technology of China（2011CB5C4403）

Corresponding author：ZHEN Xue-chu，E-mail：zhenxuechu@suda.edu.cn； ZHENG Long-tai，E-mail：zhenglongtai@suda.edu.cn

T2-18

lnvolvement of HCN channel in muscarinic inhibitory action on tonic firing of dorsolateral striatal cholinergic interneurons

ZHAO Zhe，ZHANG Kang，YAN Hai-tao，WANG Li-yun，WEI Xiao-li，ZHENG Jian-quan

（Beijing Institute of Pharmacology and Toxicology，Beijing 100850，China）

Abstract：OBJECTlVE To clarify the type of musca⁃rinic（M）receptors and hyperpolarization activated cyclic nucleotide gated（HCN）channel expressed on choliner⁃gic interneurons（ChIs），to demonstrate whether the extrinsic application of muscarinic agonist could affect the function of the striatum，and to disclose the regulatory mechanism.METHODSWesuckedthecytoplasm through pipette with negative pressure，and analyzed the mRNA transcribed in the intracellular with single cell re⁃verse transcription polymerase chain reaction（scRTPCR）.Also，we checked the expression level through histochemical staining.We detected the regulatory effect on spiking by extrinsic application of M receptor agonist and HCN channel blocker through electrophysiological recording.Then we demonstrate the regulatory effect of M agonist involved HCN channel.Finally we disclosed the regulatory mechanism of the M agonist on HCN chan⁃nel current（Ih）.RESULTS scRT-PCR revealed that all the five subtypes of M receptors and four subtypes of HCN channels were expressed on ChIs.Among them，M2 receptor and HCN2 channel were the most dominant ones and expressed in every single studied ChI.The extrinsic application of M receptor agonist and HCN chan⁃nel blocker could inhibit the spiking of ChIs.The regulatory effect of M receptor agonist on the spiking involved the HCN channel.The muscarinic inhibition of Ihwas generat⁃ ed dominantly through M2-like receptor and HCN2 chan⁃nel.It was mainly mediated by the PKA-independent Gi/ocAMP signaling pathway.CONCLUSlONOur data sug⁃gest that ACh regulates not only the output of striatal pro⁃jection neurons，but also the firing activity of ChIs them⁃selves by activating presynaptic M receptors in the dorsal striatum.The activation of M2 receptors and blockage of HCN2 channels may play an important role in ACh inhibi⁃tion on the excitability of ChIs.This finding adds a new G-protein coupled receptor mediated regulation on ChIs and provides a cellular mechanism for control of choliner⁃gic activity and ACh release in the dorsal striatum.

Key words：cholinergic interneurons；muscarinic recep⁃tor；HCN channel；scRT-PCR；muscarinic Inhibitory

Foundation item：The project supported by National Inte⁃grated Drug Discovery Technology Platform Foundation of China（2012ZX09301003-001）；and National Major Scientific and Technological Special Project for“Signifi⁃cant New Drug Development”（2014ZX09507-003）

Corresponding author：ZHENG Jian-quan，E-mail：zhengjq@bmi.ac.cn

T2-19

Ubiquitin ligase Hrd1 promotes tau degradation through autophagy

FENG Li-jie1，2，DING Qian1，2，ZHANG Jing2，MA Yu-yang1，2，ZHU Na1，2，SHEN Yu-jun1，2，FANG Sheng-yun1，2，3，SHEN Yu-xian1，2

（1.School of Basic Medical Sciences，Anhui Medical Univer⁃sity，Hefei 230032，China；2.Institute of Biopharmaceuti⁃cals，Anhui Medical University，Hefei 230032，China；3. Center for Biomedical Engineering and Technology，Univer⁃sity of Maryland，Baltimore，MD，USA）

Abstract：OBJECTlVEThis study aims to explore whether ER membrane-spanning ubiquitin ligase（E3）Hrd1 enhances tau degradation through autophagy-lyso⁃some pathway.METHODSN2a cells were transfected with tau，Hrd1 plasmids and treated with lysosome inhibi⁃tor NH4Cl，autophagy inhibitor Baf A1 or autophagy in⁃ducer EBSS，respectively.Hrd1-siRNA was used to knockdown the endogenous Hrd1.Immunoblot was used to observe the levels of tau.Cycloheximide（CHX）chase was used to observe the half-life of tau protein.Immu⁃nofluorescence staining was used to observe the colocal⁃ization of tau，Hrd1 and autophagy related protein LC3，or lysosome marker LAMP1.Co-immunoprecipitation was used to identify the type of ubiquitin chain linked to tau mediated by Hrd1 under autophagy inhibition.Im⁃munocytochemistry，MTT，TUNEL assay were used to detected the cellular morphology，cell proliferation and apoptosis，respectively.RESULTSHrd1 significantly decreased tau levels while NH4Cl or Baf A1 stabilizedtau.NH4Cl treatment can postpone the degradation of tau in Hrd1-expressing cells，while EBSS enhanced Hrd1-mediated tau degradation.Hrd1 facilitates the for⁃mation of autophagosome in tau-expressing cells and enhances the colocalization of tau and lysosome.Further⁃more，Hrd1 mainly mediated lysine 63-linked polyubiqui⁃tin chains of tau under autophagy inhibition，which is re⁃quired for autophagic tau degradation.Hrd1 significantly alleviated tau cytotoxicity while autophagy inhibition re⁃verses the cytoprotective effect of Hrd1 on tau ex⁃pressing cells.CONCLUSlONHrd1 enhances tau deg⁃radation through autophagy and attenuates intracellular tau accumulation and toxicity.

Key words：tauopathies；tau；Hrd1；ubiquitin；autophagy

Foundation item：The project supported by National Natural Science Foundation of China（81302755）

Corresponding author：SHEN Yu-xian， Tel：（0551）65161121，E-mail：shenyx@ustc.edu.cn

T2-20

N-硬脂酰酪氨酸通过CB2受体减轻Tau及氧应激造成的细胞老化

胡 悦，殷 明，王泽剑

（上海交通大学药学院，上海 200240）

摘要：阿尔茨海默病的2个主要病理特点分别是β-淀粉样蛋白沉积引起的老年斑和Tau蛋白异常修饰引起的神经纤维缠结。在APP-PS1-Tau三转AD小鼠，减少Aβ生成无明显改善认知等作用，而减少Tau蛋白引起的神经纤维缠结可明显改善小鼠的认知能力。根据本实验室以往研究，在氧应激下会引起Tau蛋白异常从而引起神经纤维缠结；合成的内源性大麻素相似物N-硬脂酰酪氨酸（NsTyr）具有神经保护作用。目的研究NsTyr对抗Tau和氧应激造成细胞老化的保护作用及其机制。方法 对照组：正常HEK293细胞。模型组：以50 μM H2O2孵育未转染和转染tau441的细胞模拟氧应激；给药组：在单纯氧应激和Tau及氧应激共同作用模型的基础上分别给予0.3～3 μm的Nstyr；在3 μmol·L-1Nstyr样本中分别加入CB1受体阻断剂AM251或CB2受体阻断剂AM630，通过细胞衰老β-半乳糖苷酶染色观察细胞衰老情况和western蛋白印迹法检测衰老相关通路的蛋白表达。结果模型组中H2O2可引起Tau蛋白异常修饰，伴随细胞衰老；Nstyr能抑制由Tau蛋白异常修饰和氧应激造成的神经细胞老化；而加入AM-630可显著削弱Nstyr的细胞保护效果。结论Nstyr通过大麻素受体CB2对抗Tau蛋白异常修饰和氧应激引起的神经细胞衰老。

关键词：Tau蛋白；大麻素受体CB2；衰老；N-硬脂酰酪氨酸

基金项目：国家自然基金项目（81270432）

通讯作者：王泽剑，E-mail：wangzejian@sjtu.edu.cn

T2-21

PDE2抑制剂Bay 60-7550对Aβ1-42所致的学习记忆功能损伤的改善作用

陈 洁，朱娜萍，张健波，费 宁，连乐竞，许笑笑，潘建春

（温州医科大学药学院脑科学研究所，浙江 温州 325000）

摘要：目的探讨PDE2抑制剂Bay 60-7550对Aβ1-42所致小鼠学习记忆功能损伤模型的改善作用及其可能的作用机制。方法实验分为假手术组，模型组，不同剂量的给药组，采用脑内微量注射Aβ1-42的方法建立学习记忆功能障碍的小鼠模型。术后连续14 d脑微量注射不同剂量的Bay 60-7550治疗，之后开始行为学测试，通过跳台实验和水迷宫实验观测各组小鼠的学习记忆功能的变化，行为学测试结束后处死小鼠取脑组织，采用Western蛋白印迹法测定海马和额叶中第二信使通路上蛋白激酶A（PKA），蛋白激酶G （PKG）以及脑源性神经营养因子（BDNF）的蛋白表达。结果模型组小鼠表现出明显的学习记忆能力损伤症状，跳台实验中较假手术组潜伏期明显减少，水迷宫实验中较假手术组潜伏期明显增加，而Bay 60-7550能够显著逆转这种现象。Western蛋白印迹法检测发现模型组较假手术组海马及额叶中的PKG表达明显减少，BDNF表达明显降低，药物组的PKG及BDNF表达有显著升高，并且Bay 60-7550的这种作用可以被PKG的拮抗剂所拮抗。结论 PDE2抑制剂Bay 60-7550可以改善Aβ1-42所致小鼠学习记忆功能损伤，其机制可能涉及到对cGMP通路及脑源性神经营养因子的调控。

关键词：PDE2抑制剂；Bay 60-7550；Aβ1-42；学习记忆

通讯作者：潘建春，E-mail：wenzhoupan2003@163.com

T2-22

PDE4抑制剂对Aβ1-42诱导的学习记忆功能损伤的改善作用

朱娜萍，张键波，陈 洁，吴飞燕，费 宁，连乐竞，潘建春

（温州医科大学药学院脑科学研究所，浙江温州 325000）

摘要：目的研究PDE4抑制剂罗利普兰（Rol）对Aβ1-42诱导的学习记忆功能损伤的改善作用及其可能涉及的机制。方法实验分为对照组，模型组，Rol 3个剂量组，采用小鼠脑内注射Aβ1-42的方法建立学习记忆功能损伤模型，术后连续15 d给予不同剂量Rol治疗。药物处理24 h后进行行为学检测，通过跳台实验和水迷宫实验观测小鼠学习记忆功能的改变。行为学结束后杀鼠取组织。对肾上腺称重，采用Western蛋白印迹法检测各组海马及前额叶内促肾上腺皮质激素释放因子（CRF）的表达，采用ELISA检测各组血清皮质酮（CORT）的含量。结果与对照组相比，模型组小鼠学习记忆能力明显下降，跳台实验潜伏期明显减少，水迷宫实验潜伏期明显增加，Rol能逆转这些现象。ELISA结果显示模型组CORT水平显著上升，Western蛋白印迹法结果显示CRF水平明显升高，BDNF水平明显降低，并且肾上腺与体重比值明显上升，Rol给药能明显改善这些指标。

结论PDE4抑制剂Rol可以改善Aβ1-42诱导的学习记忆功能损伤，其机制可能涉及HPA轴。

关键词：PDE4抑制剂；Aβ1-42；学习记忆；HPA轴

通讯作者：潘建春，E-mail：wenzhoupan2003@163.com

T2-23

高频电磁辐射对小鼠精神-认知行为损伤的效应

孙立君，张黎明，李云峰

（军事医学科学院毒物药物研究所，北京 100850）

摘要：目的探讨高频率电磁辐射对小鼠精神-认知行为可能造成的影响。方法ICR小鼠暴露于电磁混响室（频率为3GHz、SAR值4 W·kg-1，4 h·d-1），连续暴露7 d，之后进行焦虑样（开场实验、高架十字迷宫实验、孔板实验）、抑郁样（小鼠强迫游泳）以及认知功能（新物体识别）行为学检测。结果①自主活动检测：电磁辐射不会影响小鼠的自主活动。②焦虑样行为模型：开场实验中，辐射组小鼠中央区域停留时间显著低于正常对照组；高架十字迷宫实验中，辐射组小鼠进入开臂的总次数和总时间百分比与正常组相比显著性减少；孔板实验中，辐射组小鼠钻孔探索的时间比正常组显著性减少。③抑郁样行为模型：辐射组小鼠在强迫游泳实验中不动时间比正常组显著性延长。④新物体识别实验：辐射组小鼠的识别指数（RI）显著性低于正常组。结论首次发现小鼠在高频率电磁辐射中进行连续短时期的暴露，不会影响自发活动，但能导致焦虑样、抑郁样行为以及学习记忆功能的损伤，为辐射机制的研究和药物发现提供了潜在的模型。

关键词：电磁辐射；焦虑样行为；抑郁样行为；认知损伤

基金项目：国家自然科学基金项目（81173036）

通讯作者：李云峰，E-mail：lfy619@aliyun.com，Tel：（010）930650

T2-24

小胶质细胞的酸敏感离子通道介导炎症反应

高双骑，胡壮丽，王 芳，陈建国

（华中科技大学同济医学院基础医学院药理学系，湖北武汉430030）

摘要：目的小胶质细胞是神经胶质细胞中的一种，也是中枢神经系统中最重要的免疫细胞，参与中枢的免疫应答和炎症反应，清除脑中损伤的神经和感染性物质。已有研究显示酸敏感离子通道（ASICs）与炎症关系密切，然而ASICs在小胶质炎症反应中的作用未见报道。方法首先应用分子生物学和电生理技术验证ASICs的功能性表达，再通过LPS刺激和细胞划痕实验探讨ASICs的功能。结果通过实时荧光定量PCR，蛋白免疫印记，免疫荧光等多种手段检测到培养大鼠小胶质细胞上表达有ASIC1，ASIC2a和ASIC3。应用脂多糖（LPS）激活培养的大鼠小胶质细胞，不但上调细胞上ASIC1和ASIC2a的蛋白表达，也增加酸诱导的细胞内Ca2+浓度和ASICs样电流幅度。在给予ASICs非特异性阻断剂阿米洛利或ASIC1a特异性阻断剂PcTx1时，上述现象被阻断。在LPS诱导的炎症反应中，阿米洛利和PcTx1同样可以减少小胶质细胞iNOS和COX-2等细胞因子的表达。在划痕实验中小胶质细胞ASIC1和ASIC2a表达明显上调，而划痕引起的迁移也可以被阿米洛利和PcTx-1阻断，进一步研究证实ASICs促进小胶质细胞迁移功能的调节可能是通过影响ERK磷酸化水平实现。结论小胶质细胞上表达的ASICs参与神经炎症反应，这或许可以为神经炎症相关的疾病提供一种新的治疗策略。

关键词：ASICs；小胶质细胞；炎症反应；细胞迁移

基金项目：国家自然科学基金（81473199）

通讯作者：陈建国，E-mail：chenj@mails.tjmu.edu.cn，Tel：（027）83692636

T2-25

山茱萸环烯醚萜苷对3xTg拟阿尔茨海默病小鼠学习记忆的影响及其作用机制

包训杰，张 兰

（首都医科大学宣武医院药物研究室，神经变性病教育部重点实验室，北京100053）

摘要：阿尔茨海默病（Alzheimer disease，AD）是最常见的神经系统退行性疾病之一，临床表现为进行性的认知功能损害，且十分缺乏有效的药物。AD主要病理特征包括神经元变性、丢失，细胞外β淀粉样蛋白（β-amyloid protein，Aβ）沉积以及神经元纤维缠结（NFT）等，因此传统单一靶点的药物往往难以起到明显的效果。山茱萸环烯醚萜苷（CIG）是我室从传统补肾中药山茱萸的果实中提取的有效成分，主要成分包括马钱苷和莫诺苷。目的 验证CIG治疗AD的效果。方法 将老年3xTg小鼠（18月龄）每天灌胃给予CIG100和200 mg·kg-1持续2个月后进行了Morris水迷宫和物体识别试验。使用ELISA试剂盒检测小鼠脑内可溶性与不可溶性Aβ水平，使用硫磺素染色、免疫组化的方法检测脑内Aβ沉积情况，使用Western蛋白印迹法检测APP及其代谢酶和tau蛋白多个位点的磷酸化水平。结果 CIG可以明显改善18月龄3xTg小鼠的学习认知能力，降低脑内可溶与不可溶性Aβ，减少Aβ斑块面积，降低APP水平，提高α-分泌酶（ADAM10）、胰岛素降解酶（IDE）、中性肽链内切酶（NEP）表达水平，降低tau蛋白多个位点（Ser396，Ser404 和Thr217）的磷酸化水平，提高mBDNF转录水平，增加脑源性神经营养因子表达量（BDNF）。结论 CIG通过多个作用途径显示出明显的抗AD效果，其进一步机制有待深入研究，有望成为治疗AD药物的潜在候选者。

关键词：阿尔茨海默病；山茱萸环烯醚萜苷；β-淀粉样蛋白；tau蛋白；认知

通讯作者：张 兰，Tel：（010）83198855，Fax：（010）83154745，E-mail：lanizhg@hotmail.com

T2-26

人参皂苷Rg1对慢性糖皮质激素处理小鼠海马神经元NLRP-1炎症小体激活的调节作用

胡 文，张耀东，张碧琼，尹艳艳，李维祖

（安徽医科大学基础医学院药理学教研室，安徽合肥 230031）

摘要：目的研究人参皂苷Rg1对慢性糖皮质激素处理导致小鼠海马神经元NLRP1炎症小体激活的调节作用及其机制。方法雄性小鼠分为对照组、地塞米松（DEX，5 mg·kg-1）模型组、RU486（5 mg/kg）+DEX组和人参皂苷Rg1（1，2和4 mg·kg-1）+DEX组，作用28 d。旷场实验记录小鼠自发活动情况；新体识别实验检测小鼠的识别记忆功能；HE染色观察小鼠大脑病理形态学变化；免疫组化检测MAP-2蛋白表达的变化；Western蛋白印迹法检测GR，NLRP-1，ASC胱天蛋白酶1，胱天蛋白酶5，IL-1β和IL-18蛋白表达的变化。结果旷场实验结果显示，与对照组比较，模型组小鼠运动距离、运动速度、穿线次数和站立次数均有所降低；与模型组比较，RU486和人参皂苷Rg1（2和4 mg·kg-1）组小鼠运动距离、运动速度、穿越格子数和站立次数均明显增加（P＜0.05）。新物体识别实验结果显示，与对照组比较，DEX组小鼠识别指数（DI）明显降低，RU486组（P＜0.05）和人参皂苷Rg1（2和4 mg·kg-1）组（P＜0.01）小鼠识别指数均有所提高。HE染色结果显示，对照组小鼠皮层和海马神经元排列整齐，无明显变化，DEX处理模型组病理显示排列紊乱、嗜酸性变、核浓缩等变化，RU486及人参皂苷Rg1（2和4 mg·kg-1）组均有所改善。免疫组化结果显示，Rg1（2和4 mg·kg-1）组（P＜0.01）和RU486组（P＜0.01）均可以提高皮层和海马区神经元MAP2蛋白表达。Western蛋白印迹法结果显示，RU486和人参皂苷（2和4 mg·kg-1）组可以上调GR蛋白表达以及下调NLRP1，胱天蛋白酶1、胱天蛋白酶5，ASC，IL-1β和IL-18等蛋白的表达（P＜0.05）。结论人参皂苷Rg1可以通过抑制NLRP-1炎症小体的激活改善糖皮质激素长期作用引起的神经元炎症损伤。

关键词：糖皮质激素；人参皂苷Rg1；NLRP1炎症小体

基金项目：国家自然科学基金（81371329）

通讯作者：李维祖，E-mail：liweizu@126.com，Tel：（0551）65161053

T2-27

神经元内Aβ1-42积聚降低CX3CL1表达并诱发小胶质细胞炎症反应

陈培清，赵文娟，殷 明

（上海交通大学药学院，上海 200240）

摘要：目的阿尔茨海默病（AD）早期Aβ1-42神经元内积聚及其引起的小胶质细胞活化以及相关炎症反应是AD最主要的病理机制之一。趋化因子CX3CL1（也称为fractal⁃kine）对于神经元与小胶质细胞间的信号传递和随后小胶质细胞的活化及炎症因子的释放均起到了重要作用。本研究旨在观察Aβ1-42神经元内积聚引起的CX3CL1、小胶质细胞活化以及相关炎症反应的改变。方法混合培养神经元和小胶质细胞，应用特异性表达人Aβ1-42片段的lentiviral Aβ1-42感染原代培养的神经元，模拟AD早期Aβ1-42神经元内沉积病理改变。分别在3，5，7和9 d检测Aβ1-42在神经元和小胶质细胞内沉积，CX3CL1，CX3CR1，TNF-α，IL-1β和IL-6水平变化，以及小胶质细胞状态的改变。结果lentiviral Aβ1-42感染神经元后，Aβ1-42主要在神经元内沉积，CX3CL1及CX3CR1水平下降，小胶质细胞处于活化状态，炎症因子TNF-α，IL-1β和IL-6水平显著增加。结论神经元内Aβ1-42积聚可能通过CX3CL1影响神经元、小胶质细胞间信息传递，引起小胶质细胞活化及相关炎症反应，参与AD发病。

关键词：阿尔茨海默病；Aβ1-42；CX3CL1；小胶质细胞；

基金项目：国家自然科学基金（81471232）

通讯作者：赵文娟， Tel：（021）34206836， E-mail：zhaowj@sjtu.edu.cn；殷 明，Tel：（021）34206836，E-mail：myin@sjtu.edu.cn

T2-28

糖尿病脑病的神经病理改变及潜在药物靶点研究

王晓良，李 江，师 思，尹华静，于文雯

（中国医学科学院药物研究所，北京 100050）

摘要：糖尿病已成为我国发病人数最多，发展速度最快的慢性疾病。至2015年发病人数已超过1亿人，而糖耐量异常，即未来有可能发展为糖尿病的人数则超过1.5亿人之多。除了多年来已熟知的并发症以外，糖尿病引起的脑结构和认知功能改变，即糖尿病脑病越来越引起人们的重视。糖尿病相关的认知功能改变是其它人群的一倍以上。但对其发病机理和防治策略仍不清楚。我们在2型糖尿病模型KK-Ay小鼠上系统研究了脑微结构的改变，中枢糖脂代谢异常对神经元和胶质细胞的影响，脑突触可塑性的改变及机制，以及借助蛋白质组学研究了潜在的药物靶点等。

研究发现，KK-Ay小鼠与对照小鼠相比在3，5和7月龄时出现明显的血糖，血脂及胰岛素水平的升高，且呈进展性变化，同时在Morris水迷宫试验中出现明显的学习，记忆障碍，脑组织者中Aβ沉积和磷酸化Tau显著增加，类似AD样改变。脑组织病理学研究发现，在糖尿病早期即出现神经胶质细胞的病理性改变，炎症介质释放增加，星形胶质细胞的形态异常和GFAP表达水平降低等。高频刺激引起的长时程增强（LTP）在3月龄时即显著降低，并始终处于低水平，LTP的变化主要与神经元NMDA受体亚型的表达变化相关。抗AD药物，多奈哌齐，美金刚和PHPB治疗后，可改变糖尿病鼠的认知能力，同时蛋白组学研究证实与AD相关的一些蛋白被改变并能够被药物回调。与AD不同的是，大多数糖尿病脑病的病理改变在一定时期内可通过控制动物饮食和药物治疗得到改善。此研究对糖尿病脑病的机理和干预手段提出了新的研究思路。

关键词：糖尿病脑病；认知障碍；星形胶质细胞；突触可塑性；蛋白组学

通讯作者：王晓良，E-mail：wangxl@imm.ac.cn

T2-29

Effects and underlying mechanisms of cannabinoid re⁃ceptor 2 on neuroinflammation and cognitive perfor⁃mance in Alzheimer disease model mice

SHI Jing-pu1，2，SHI Yuan-yuan1，2，SUN De-jia1，WANG Bo1，JIA Hui-qun2，LI Jin1

（1.State Key Laboratory of Toxicology and Medical Counter⁃measures，Beijing Key Laboratory of Neuropsychopharma⁃cology，Beijing Institute of Pharmacology and Toxicology，Beijing 100850，China；2.The Fourth Hospital of Hebei Medi⁃cal University，Shijiazhuang 050000，China）

Abstract：OBJECTlVEThe endogenous cannabi⁃noid system has been considered as a potential thera⁃peutic target to ameliorate neuroinflammation in Alzheimer disease（AD）.The aim of the study was to investigate thespecificcontributionofcannabinoidreceptor2 （CB2R），one of the main components of the endoge⁃nous cannabinoid system，to the neuroinflammation and cognitive impairment induced by β-amyloid protein（Aβ）in AD model mice.METHODSWe explored the effects of selective CB2R agonist JWH-015 on cognitive perfor⁃mance and inflammatory parameters in 8-month APP/ PS1 transgenic mice.Then CB2RKO mice with intrahip⁃pocampal injection of Aβ1-42（CB2RKO/AD model）was used to confirm whether these effects of JWH-015 wasmediated by CB2R and explore the effects of CB2R defi⁃ciency on non-spatial cognitive performance in AD model mice.In addition，the effects of JWH-015 on dendritic complexity in affected brain regions of APP/PS1 mice were observed through Golgi-Cox staining.Cognitive func⁃tion was assessed using the Novel object recognition test（NOR）and Morris water maze（MWM）test.Levels of amyloid Aβ deposits was examined by Congo red staining and the immunoreactivity of microglia and as⁃trocyte were evaluated by immunofluorescence.The lev⁃els of proinflammatory cytokines were examined by RT-qPCR.RESUlTSAdministration of JWH-015 could re⁃verse the non-spatial cognitive impairment in novel ob⁃ject recognition test of APP/PS1 mice，but it was ineffec⁃tive for CB2RKO/AD model.And CB2R deficiency dete⁃riorated this cognitive deficit.APP/PS1 mice showed a dramatic increasing of microglia marker Iba1 immunore⁃activity in cortex region，which was attenuated by JWH-015 treatment.Deletion of CB2R abated the beneficial ef⁃fect of JWH-015 completely，and enhanced the immuno⁃fluorescence intensity of Iba1 in CB2RKO/AD model.In addition，microglia in CB2R/AD model showed a striking different morphology，which was relative bigger round so⁃ma with numerous thicker and shorter branches.Accom⁃panying with microglia morphological changes，CB2R de⁃ficiency notably altered microglia functional phenotype in prefrontal cortex（PFC），up-regulating mRNA expres⁃sion levels of M1 microglia phenotype marker IL-6，TNF-α and iNOS.And JWH-015 could contribute M1/M2 mi⁃croglia phenotype transformation in APP/PS1 mice but not CB2RKO/AD model.Lastly，JWH-015 could restore the dendritic complexity in PFC of APP/PS1，which may be related with the regulation of microglia-mediated neu⁃roinflamaion.CONCLUSlONSpecificactivationof CB2R can reverse non-spatial cognitive impairment of AD model mice，which is related with the regulation of microglia-mediated neuroinflammation and dendrite com⁃plexity in affected brain regions.Deletion of CB2R can deteriorate non-spatial cognitive deficit and notably alter microglia morphological and functional phenotype in AD model mice.These findings suggest that CB2R takes a significant effect on AD progressive neuoinflammation and cognitive competence and support a potential role of CB2R as a pharmacologic target.

Key words：cannabinoid receptor 2；alzheimer disease；microglia；neuroinflammation；neuroplasticity；learning and memory

Foundation item：The project supported by National Natural Science Foundation of China（81471233）；and Science and Technology Nova Plan of Beijing Grant （Z121102002512046）

Corresponding author：LI Jin，E-mail：jinli9802@163.com；WANG Bo，E-mail：wbcmx@sina.com

T2-30

二苯乙烯苷（泰思胶囊）治疗阿尔茨海默病的研究与开发

李 林，张 兰，张如意

（首都医科大学宣武医院，北京市神经药物工程研究中心，北京100053）

摘要：阿尔茨海默病（AD）是一种多因素相关的复杂性疾病。近年来研发的一些AD治疗药物在临床试验中不成功，重要原因之一是药物仅针对单靶点或单致病途径，不易取得好的疗效。因此目前国际上许多学者提出，应当应用多靶点、多途径的治疗策略来研发治疗AD的药物。二苯乙烯苷（泰思胶囊）是我室自行研制的治疗AD的中药创新药物。在临床前药效学研究中，二苯乙烯苷灌胃给药在8种拟老年痴呆动物模型（包括APP转基因AD小鼠、Aβ侧脑室注射AD小鼠、IBO基底前脑注射致胆碱能损伤致痴呆大鼠、线粒体复合体Ⅳ抑制剂致痴呆大鼠、老年大鼠、D-半乳糖致痴呆小鼠、慢性脑缺血致痴呆大鼠、高胆固醇血症致痴呆大鼠）能够明显改善学习记忆功能，减少脑内淀粉样斑块和Aβ含量，减低β-分泌酶和早老素-1表达，抑制α-突触核蛋白过表达和聚集，抑制tau蛋白过度磷酸化，保护细胞骨架结构，增高胆碱乙酰基转移酶/乙酰胆碱酯酶的比值，抗神经炎症，抗氧化应激，增高线粒体活性和能量代谢，增高神经营养因子NGF和BDNF及其受体表达，保护神经突触的结构和功能，减少神经元死亡。其突出特点和优势是作用在AD发病机制的多靶点多途径，尤其具有神经保护和神经营养作用，可阻止及延缓神经元死亡。泰思胶囊获得国家食药监局新药临床研究批件，已完成治疗AD的随机、双盲、安慰剂对照、阳性药（多奈哌齐）对照、多中心2期临床研究，显示良好的有效性和安全性，目前已进入治疗AD的3期临床研究。

关键词：二苯乙烯苷；阿尔茨海默病；β-淀粉样肽；α-突触核蛋白；tau蛋白过度磷酸化；神经营养因子；突触

基金项目：国家自然科学基金（81273498；81341087）；国家科技部“重大新药创制”科技重大专项（2015ZX09101016001）

通讯作者：李 林，E-mail：linlixw@126.com

T2-31

硫化氢对大鼠杏仁核区依赖的恐惧记忆的作用及机制

夏芷萱，王灿明，吴鹏飞，王 芳，陈建国

（华中科技大学同济医学院药理学系，湖北 武汉 430030）

摘要：目的探讨内源性气体信号分子H2S在杏仁核介导的情感记忆行为和突触可塑性中的调控作用。方法采用亚甲基蓝法检测大鼠脑组织H2S的含量；条件性恐惧记忆观察H2S对大鼠情感记忆行为的影响；电生理记录大鼠杏仁核区NMDA受体介导的电流及LTP。结果外源性给予H2S供体NaHS可明显增强杏仁核介导的线索型恐惧记忆，H2S可减缓恐惧记忆的消散，促进恐惧记忆自发性的恢复和复发；NaHS能选择性上调含NR2B亚基的NMDA受体的功能，进而增强杏仁核的LTP和条件性恐惧记忆。结论H2S通过增强含NR2B亚基的NMDA受体功能，增强情感记忆。

关键词：硫化氢；杏仁核；线索型恐惧记忆；NMDA受体

基金项目：国家重点基础研究发展计划（973计划）资助项目（2013CB531300）

通讯作者：陈建国，E-mail：chenj@mails.tjmu.edu.cn，Tel：（027）83692636

T2-32

新疆发酵驼乳益生菌对Balb/c小鼠的免疫调节作用及机制

肖雪筠，新华·那比

（新疆医科大学药理学教研室，新疆 乌鲁木齐 830054）

摘要：目的研究新疆驼乳和乳酪乳清中分离出的四种益生菌对免疫低下Balb/c小鼠的免疫调节作用及作用机制。方法 小鼠利用环磷酰胺制造免疫低下模型，成模后灌胃高浓度（1.5×107cfu·L-1）及低浓度（1.5×109cfu·L-1）单菌悬液，干预4周，观察对小鼠体重、脏器指数、脾淋巴细胞数目、肠道菌群及相关细胞因子的影响。结果 除东方伊萨酵母菌低浓度组外，其余益生菌组与模型组相比体重均有增加（P＜0.05），其中马乳酒样乳杆菌组体重增加最明显（P＜0.01）。与正常组相比，除东方伊萨酵母菌高浓度组和马乳酒样乳杆菌高浓度组外，脾淋巴细胞增殖能力均有增强（P＜0.05）；与模型组相比，4种益生菌高浓度组和低浓度组均能提高小鼠脾淋巴细胞增殖能力（P＜0.05）。与正常组相比，戊糖乳杆菌高/低浓度组以及高加索乳杆菌高浓度组脾系数显著增加（P＜0.05）。与模型组相比，4种益生菌组脾脏系数显著提高（P＜0.01），结肠及胸腺指数没有显著差异（P＞0.05）。结论 4种益生菌组均能不同程度提高免疫低下小鼠的免疫力。

关键词：益生菌；免疫；细胞因子；机制

基金项目：国家自然科学基金（81160344）

通讯作者：新华·那比，E-mail：xinhua99@hotmail.com，Tel：13579882816

T2-33

Dopamine D2 receptors modulate spatial cognition in medial entorhinal cortex

JIN Xue-qin，HUANG Zhuo

（Department of Molecular and Cellular Pharmacology，School of Pharmaceutical Sciences，Peking University⁃Health Science Center，Beijing 100191，China）

Abstract：OBJECTlVETo figure out how the re⁃duction ofdopamineD2 receptors（D2R）influences the progression of Alzheimer disease（AD）.METHODS AND RESULTSHere，using siRNA technique，selec⁃tively knocking-down D2R in medial entorhinal cortex （mEC）impaired spatial memory.Consistently，mice in⁃tracranially injected with 5 μmol·L-1D2 antagonist L-741，626 failed to detect the spatial changes of object when moved the object to a new location.To investigate the cellular mechanism by whichD2R regulates spatial cogni⁃tion，we made horizontal brain slices containing entorhi⁃nal cortex area from 5-7 weeks-old mice.Whole-cell patch-clamp recordings were obtained from mEC layerⅡstellate cells，which have been shown to be critically involved in spatial navigation.Bath application of 10 μmol·L-1D2R agonist，bromocriptine，significantly increased the action potential threshold，thus reduced neuronal intrin⁃sic excitability.This is due to sustained reduction of axo⁃nal intracellular Ca2+via T-type Ca2+channels.CONCLU⁃SlON D2R play a crucial role in modulating stellate cell excitability through suppressing axonal T-type calcium channels activities.This mechanism may contribute to spatial information processing and impairment of D2R function might be involved in the progression of AD.

Key words：Alzheimer disease spatial cognition；D2R；T-type calcium channel

Foundationitem：Theprojectsupportedby973 program（2015CB559200）；Ministry of Science and Technology of China（2014ZX09507003-006-004）；and National Natural Science Foundation of China（81371432）

Corresponding author：HUANG Zhuo，Tel：18610108083，E-mail：huangz@hsc.pku.edu.cn

T2-34

以微管相关蛋白tau为靶点的抗阿尔茨海默病药物研究

张 兰，杨翠翠，祝艳秋，包训杰，李 林

（首都医科大学宣武医院药物研究室，神经变性病教育部重点实验室，北京 100053）

阿尔茨海默病（Alzheimer disease，AD）是老年人中发病率最高的神经退行性疾病。细胞内神经原纤维缠结（neu⁃rofibrillary tangles，NFT）是AD患者典型的病理变化之一。异常过度磷酸化的微管相关蛋白tau是组成NFT的主要成分。Tau蛋白常见的病理改变是Tau发生异常过度磷酸化，结合微管的能力下降，从结合状态变成游离单体，进而发生异常聚集，最终形成神经纤维缠结。目前针对Tau蛋白的药物研发主要包括：抑制Tau蛋白过度磷酸化、抑制Tau蛋白聚集、促进Tau蛋白降解和微管稳定剂。其中，抑制Tau蛋白过度磷酸化对于治疗AD有最为重要的意义，成为目前最具潜力的药物治疗靶点。Tau蛋白磷酸化受蛋白磷酸酶PP2A和蛋白磷酸激酶GSK-3β等的共同调节。开发PP2A激活剂和GSK-3β抑制剂成为目前AD药物研究的新策略。

山茱萸环烯醚萜苷（Cornel iridoid glycoside，CIG）是我室从山茱萸中提取的主要有效部位。我们的研究发现，在tau蛋白过度磷酸化的4个细胞模型（GSK-3β转染、siPP2A、WT-GFX致GSK-3β激活、OA致PP2A抑制）和4个动物模型（3*Tg拟AD小鼠、SAMP8快速老化小鼠、WTGFX致tau蛋白过度磷酸化大鼠、OA致tau蛋白过度磷酸化大鼠）上，CIG能够降低tau蛋白在多个AD发病相关位点的过度磷酸化，从而维持微管完整保护细胞结构，其作用机制包括：①化）提高PP2A活性，从而促进tau蛋白的去磷酸化；②通过减低GSK-3β的含量，并且增高GSK-3β-ser9的磷酸化而抑制GSK-3β的活性，从而减少tau蛋白的磷酸化。山茱萸环烯醚萜苷具有治疗AD的应用前景。

莫诺甘（Morroniside）是山茱萸环烯醚萜苷的主要有效单体成分，在tau蛋白过度磷酸化的细胞模型上，我们也观察到莫诺甘对tau多个位点过度磷酸化的抑制作用，并对PP2A催化亚基C的翻译后修饰（甲基化和磷酸化）有明显的调节作用。莫诺甘可能是山茱萸环烯醚萜苷发挥药效作用的重要物质基础，作为潜在的PP2A激活剂，对AD治疗具有开发前景。

关键词：阿尔茨海默病；Tau蛋白；山茱萸环烯醚萜苷；莫诺甘

通讯作者：张兰，Tel：（010）83198855；E-mail：lanizhg@ hotmail.com

T2-35

基于组胺H3受体的抗缺氧化合物筛选及其作用机制

廉靖靖，颜玲娣，董华进，李玉蕾，周培岚，苏瑞斌，宫泽辉

（抗毒药物与毒理学国家重点实验室，军事医学科学院毒物药物研究所，北京100850）

高原地区特有的低氧环境会使人因供氧不足而产生急、慢性高原疾病。组胺H3受体广泛分布于中枢，与缺氧脑损伤及呼吸调节相关。前期研究结果表明急进高原模型下，相关脑区组胺H3受体表达发生改变；组胺H3受体反相激动剂Pitolisant在密闭缺氧模型下可提升动物的耐缺氧氧能力。我所七室以Pitolisant为先导化合物经过结构优化得到26个衍生物。目的系统评价Pitolisant抗缺氧效应，评筛Pitolisant衍生物的抗缺氧作用，以期获得具有自主知识产权的靶向组胺H3受体的抗缺氧化合物。方法运用CHOPKAcat-HRH3细胞系，检测化合物对PKA再分布的影响，确证化合物的体外活性；采用常压密闭缺氧模型、急性低压低氧致死性模型评价化合物静态抗缺氧能力；采用步入式低压氧舱模拟高原（5000 m）缺氧状态，结合小动物跑轮仪，评价化合物抗运动缺氧作用；采用实验动物低压氧舱模拟急进高原（8000 m）6 h，检测化合物对急进高原造成脑损伤的改善作用。结果①组胺H3受体反相激动剂Pitolisant可提升静止状态下动物的耐缺氧能力，增强缺氧环境下动物的跑轮耐力；②Pitolisant降低急进高原所造成的脑组织乳酸累积，改善血管源性脑水肿；③筛选得到对组胺H3受体具有拮抗活性的2个化合物，即WH-1-42和WH-3-15，仅WH-3-15具有一定的抗缺氧效应，但存在较强的中枢镇静作用。结论 Pitolisant特异性靶向组胺H3受体，可通过改善缺氧造成的脑损伤并提升动物耐缺氧能力。

关键词：缺氧；组胺H3受体；运动耐力；脑水肿

基金项目：国家科技重大专项“重大新药创制”综合性新药研究开发技术大平台资助项目（2012ZX09301003-003）

通讯作者：苏瑞斌，E-mail：ruibinsu@126.com，Tel：（010）66931607；周培岚，E-mail：zhoupeilan0502@sina.com，Tel：（010）66931621

T2-36

金钗石斛多糖对LPS诱导的大鼠海马GSK-3β和p-S396蛋白的影响

王丽娜，杨 林，李 菲，吴 芹，石京山

（遵义医学院基础药理省部共建教育部重点实验室，贵州遵义563000）

摘要：目的观察金钗石斛多糖（dendrobium nobile polysaccharides，DNP）对LPS诱导的大鼠海马GSK-3β，p-GSK-3β（Ser9）和p-S396异常磷酸化的影响。方法20只雄性SD大鼠随机分为5组，假手术组、模型组、DNP给药组（40，80和160 mg·kg-1）。DNP给药组ig分别给予DNP，假手术组和模型组ig给予等体积的蒸馏水。连续给药7 d，每天2次。预防性给药7 d后，侧脑室注射LPS（5 g·L-1）模拟大鼠急性炎症模型，制模后2 h麻醉固定，取双侧海马，采用western蛋白印迹法检测大鼠海马tau蛋白异常磷酸相关蛋白激酶p-GSK-3β（Ser9）及tau磷酸化位点P-S396的蛋白表达。结果 模型组与假手术组相比，总GSK-3β的蛋白表达无明显差异，P-GSK-3β（Ser9）与GSK-3β的比值减少（P＜0.05），说明GSK-3β在Ser 9位点的磷酸化水平下降；p-S396的蛋白表达明显升高（P＜0.05），tau蛋白Ser 396异常磷酸化；预防性给予DNP（40，80和160 mg·kg-1）明显增加GSK-3β在Ser 9位点的磷酸化水平，降低p-S396的蛋白表达。结论 DNP预防性给药对LPS诱导的大鼠海马tau异常磷酸化有保护作用，其机制可能与其增加大鼠海马GSK-3β在Ser 9位点的磷酸化水平以及降低tau蛋白S396位点的磷酸化水平有关。

关键词：金钗石斛多糖；脂多糖；tau蛋白；异常磷酸化；

基金项目：贵州省科技厅联合基金（黔科合J字LKZ［2012］43号）

通讯作者：石京山；E-mail：shijs@zmc.edu.cn

T2-37

山茱萸环烯醚萜苷及其物质基础莫诺苷提高PP2A活性、拮抗tau蛋白过度磷酸化机制研究

杨翠翠，李 林，张 兰

（首都医科大学宣武医院药物研究室，神经变性病教育部重点实验室，北京100053）

目的：老年性痴呆（Alzheimer disease，AD）病理特征之一为脑内神经元纤维缠结（neurofibrillary tangles，NFT），高度磷酸化的微管相关蛋白Tau蛋白是NFT的主要成分。蛋白磷酸酯酶和蛋白激酶与Tau蛋白磷酸化密切相关。山茱萸环烯醚萜苷（Cornel iridoid glycoside，CIG）是本室从中药山茱萸中提取的有效部位，莫诺苷为其主要单体成分。本研究探讨CIG及莫诺苷对微管相关蛋白tau过度磷酸化的抑制作用及其作用机制。方法：①采用冈田酸OA或者PP2Ac siRNA特异性抑制PP2A活性致tau蛋白过度磷酸化，观察CIG对tau蛋白磷酸化水平及PP2A通路的影响；为证明CIG对PP2A通路作用是否与GSK-3β途径存在交互作用，本研究采用磷脂酰肌醇3激酶（PI3K）抑制剂wortmannin及激酶A（PKA）抑制剂GF-109203X（WT/GFX）与人神经母细胞瘤细胞SK-N-SH共孵育，建立微管相关蛋白tau过度磷酸化拟AD细胞模型，观察CIG对tau蛋白磷酸化水平、微管结构、细胞形态及激酶通路的影响。②为进一步揭示CIG拮抗tau蛋白磷酸化的物质基础，在以上研究的基础上，再次采用冈田酸OA或者PP2Ac siRNA特异性抑制PP2A活性致tau蛋白过度磷酸化，观察MOR对tau蛋白磷酸化水平及PP2A通路的影响。结果 ①CIG能够抑制OA模型细胞tau蛋白Thr205，Thr212，Ser214和Thr217位点的磷酸化，并且通过降低PME-1/LCMT-1的比值从而抑制PP2A催化亚基C的去甲基化，提高PP2A活性，从而促进tau蛋白的去磷酸化。②为了进一步验证CIG增高PP2A活性是通过调节PP2A催化亚基C翻译后修饰，本研究将PP2AcsiRNA转染入HEK293细胞。结果发现CIG不能降低模型细胞tau蛋白在Ser396位点的磷酸化。但是，CIG仍能降低模型细胞tau蛋白在Thr205，Thr212和Ser214位点的磷酸化，提示CIG的作用机制除了PP2A途径以外，可能还有其他靶点或途径。③为了证明CIG作用与PP2A不依赖于GSK-3β的作用，将WT（PI3K抑制剂）和GFX（PKA抑制剂）与SK-N-SH细胞进行孵育。结果显示，CIG能够使模型细胞损伤的微管形态基本恢复正常，保护细胞形态完整；能够明显抑制模型细胞tau蛋白多个位点的过度磷酸化，但是不能增高p-GSK-3β-ser9的表达，CIG能够增高模型细胞的PP2A活性，其机制与抑制PP2A催化亚基C的磷酸化和去甲基化有关。④MOR能够通过调节PP2A催化亚基C提高PP2A活性降低tau蛋白在多个位点的磷酸化，并维持微管结构保护细胞形态。转染PP2Ac siRNA后，MOR不能抑制tau蛋白的磷酸化水平，且对PP2A无影响。结论 CIG降低tau蛋白过度磷酸化修饰可能是通过调节PME1和LCMT的表达，从而降低PP2A的非甲基化提高PP2A活性使tau蛋白去磷酸化，其物质基础MOR的降低tau蛋白磷酸化的主要作用靶点为PP2A。

关键词：山茱萸环烯醚萜苷；莫诺苷；PP2A；tau蛋白

通讯作者：李 林，Tel：（010）83198886，E-mail：linli97@ hotmail.com；张 兰，Tel：（010）83198855；E-mail：lanizhg@ hotmail.com

T2-38

非受体型Src酪氨酸激酶介导神经炎症以及帕金森病病理过程

张丹

（中国医学科学院药物研究所，北京 100050）

摘要：目的研究非受体型Src酪氨酸激酶介导神经炎症作用机制以及在帕金森病病理过程中的作用。方法体外实验：LPS刺激小胶质细胞，采用荧光共振能量传递（FRET）技术、酶活性检测以及ELISA方法，实时观察Src的激活、炎症因子生成。体内实验：观察不同月龄α-synuclei⁃nA53T转基因小鼠脑内Src酪氨酸激酶活性、脑内神经炎症反应以及小鼠行为学损伤。结果FRET结果显示，LPS可引起小胶质细胞上Src酪氨酸激酶活性的明显上升，且与氧自由基以及多种炎症因子的产生呈正相关，干扰Src酪氨酸激酶的表达可抑制炎症因子生成，提示其与神经炎症的密切关系。机制研究结果表明，Src酪氨酸激酶对神经炎症的调控并不完全依赖于NADPH氧化酶。在α-synucleinA53T转基因小鼠脑内可观察到激活的Src酪氨酸激酶。荧光免疫双染发现，激活的Src主要存在于小胶质细胞。随着小鼠年龄增长，Src活性明显上升，同时小鼠脑内炎症反应以及小鼠行为学障碍均逐渐加重。小鼠连续1个月口服给予Src抑制剂Bafetinib 10 mg·kg-1，脑内炎症反应明显减轻，小鼠行为学损伤明显减弱，证明Src激活与神经炎症以及帕金森病病理改变均有密切的关系。结论Src酪氨酸激酶参与小胶质细胞激活及炎症因子的产生，进而参与神经炎症反应以及与神经炎症密切相关的帕金森病病理改变，可能成为药物干预新靶标。

关键词：Src酪氨酸激酶；小胶质细胞；NADPH氧化酶；神经炎症；帕金森病

基金项目：国家重点基础研究发展计划（973计划）（2011 CB504103）；教育部新世纪优秀人才项目（3332013128）

通讯作者：张 丹，E-mail：danzhang@imm.ac.cn，Tel：（010）63165178

T2-39

β-胍丙酸通过激活AMPK依赖的自噬信号通路延长果蝇寿命

张 海，杨 思，龙利红，李 笛，张建康，王 芳，陈建国

（华中科技大学同济医学院基础医学院药理学系，湖北武汉430030）

摘要：目的研究β-胍丙酸（GPA）能否通过激活AMPK介导的自噬信号通路而延长果蝇寿命。方法在果蝇培养基中加入β-GPA（0，300，900和2700 mmol·L-1）喂养，观察对果蝇寿命的影响。用Western蛋白印迹法检测自噬相关信号分子AMPK，p-AMPK，Atg1，Atg5和Atg8的表达水平。结果β-GPA 900和2700 mmol·L-1培养基喂养，可显著延长果蝇的寿命。β-GPA喂养后，果蝇中自噬标志物Atg1和Atg8蛋白表达水平显著升高。Atg5 siRNA可取消β-GPA的作用。采用RNAi或compound C抑制AMPK蛋白表达后，自噬相关蛋白Atg1及Atg8表达显著降低，且显著抑制β-GPA延长寿命的作用。结论β-GPA通过激活AMPK-Atg1介导的自噬信号通路而延长果蝇寿命。

关键词：自噬；β-胍丙酸；果蝇；寿命

基金项目：国际合作项目（2011DFA32670）；PCSIRT （IRT13016）

通讯作者：陈建国，E-mail：chenj@mails.tjmu.edu.cn，Tel：（027）83692636

T2-40

胍丁胺-Ⅰ型咪唑啉受体系统对认知功能的调节作用

赵太云，吴 宁，李 锦

（军事医学科学院毒物药物研究所，北京 100850）

摘要：认知功能是机体认识和获取知识的智能加工过程的能力，涉及学习、记忆、语言、思维、精神和情感等一系列随意、心理和社会行为。认知功能障碍指与上述学习记忆以及思维判断有关的大脑高级智能加工过程出现异常，从而引起严重学习、记忆障碍，同时伴有失语、失用、失认或失行等改变的病理过程。近年来的研究结果表明，Ⅰ型咪唑啉受体内源性配体胍丁胺具有促进正常状态下认知功能和改善阿尔茨海默症等病理状态下认知功能障碍的作用，且本课题组近期利用非组织特异性敲除和前脑特异性敲除Ⅰ型咪唑啉受体的小鼠发现其学习记忆功能显著降低。本文结合本课题研究结果，就国内外对胍丁胺与Ⅰ型咪唑啉受体对认知功能调节作用的研究进展进行综述，并提出内源性胍丁胺与Ⅰ型咪唑啉受体可能是一新的认知功能调节系统的科学假说，以期为进一步发现增强认知功能和改善病理性认知功能障碍的干预措施提供有益的线索。

关键词：胍丁胺；咪唑啉受体；认知功能；认知功能障碍

基金项目：国家973课题（2015CB553504）；国家自然科学基金（81373385）

通讯作者：李 锦，E-mail：jinli9802@163.com，Tel：（010）66932681

T2-41

人载脂蛋白E基因多态性影响胶质细胞活化和神经元内Aβ沉积

赵文娟

（上海交通大学药学院，上海 200240）

摘要：人类载脂蛋白E（apolipoprotein E，ApoE）主要有ApoE2，ApoE3和ApoE4三种异构体，分别由等位基因APOE ε2，ε3和ε4编码。APOE ε4（APOE4）是目前唯一得到公认的阿尔茨海默病（Alzheimer disease，AD）高风险因素。神经元内Aβ42沉积是AD早期神经元损伤、引起AD发病的主要因素。我们应用特异表达不同人APOE基因型的APOE-TR小鼠及特异性表达人Aβ1-42片段的Aβ1-42lentivirus，研究APOE基因多态性在AD发病早期对神经元内Aβ沉积及AD发病的影响。研究结果表明，APOE基因多态性影响Aβ42在神经元内沉积，与APOE2-和APOE3-TR小鼠相比，脑内微量Aβ1-42lentivirus注射2周后APOE4-TR小鼠大脑皮质神经元内Aβ42沉积增多。免疫荧光染色显示，Aβ42主要存在于神经元内，小胶质细胞内也可观察到部分Aβ42免疫阳性染色，但星形胶质细胞内未检测到Aβ42免疫阳性染色。与APOE2-和APOE3-TR小鼠相比，APOE4-TR小鼠脑内神经元内Aβ42沉积明显增加，而Aβ42免疫阳性小胶质细胞减少。此外，与老年APOE3-及年轻APOE4-TR小鼠相比，脑内微量注射Aβ1-42lentivirus引起的神经元内Aβ42沉积更易在老年APOE4-TR小鼠引起细胞外Aβ积聚、星形胶质细胞活化、tau蛋白磷酸化及细胞凋亡等AD病理改变。提示，ApoE4可能通过影响小胶质细胞-神经元内Aβ42代谢平衡，促进神经元内Aβ42沉积，并进而引起细胞外Aβ积聚、tau蛋白磷酸化等AD病理学改变，促进AD发病。

关键词：载脂蛋白E；β-淀粉样蛋白；小胶质细胞；阿尔茨海默病

基金项目：国家自然科学基金（81471232）；上海市自然科学基金（1443190140）；美国国立卫生研究院基金（P01 AG030128；R01 AG035379）

通讯作者：赵文娟，E-mail：zhaowj@sjtu.edu.cn，Tel：（021）34206836

T2-42

lcariside ll ameliorates spatial memory impairment in transgenic mice model of Alzheimer disease via sup⁃pressing β-amyloid production

YAN Ling-li，DENG Yuan-yuan，LIU Yuan-gui，GONG Qi-hai

（Department of Pharmacology and Key Laboratory for Basic Pharmacology of Ministry of Education，Zunyi Medical Uni⁃versity，Zunyi 563099，China）

Abstract：OBJECTlVEAbnormally increased pro⁃ duction and accumulation of β-amyloid（Aβ）is the most prominent hallmark of Alzheimer disease（AD），target⁃ing with Aβ production for AD treatment is gain attention. In the present study，the effects of IcarisideⅡ（ICSⅡ），one of the active ingredients extracted from Herba Epime⁃dii，on learning and memory as well as the underlying mechanisms in amyloid precursor protein（APP）/PS1 transgenic AD mice were investigated.METHODS Ninemonth-old APP/PS1 transgenic mice（APP/PS1）and wild type mice littermates（WT）were randomly divided into six groups，APP/PS1 and WT treated groups were administrated with ICSⅡ（10 and30 mg·kg-1）orally daily for three months，and control groups were received the volume-matchednormalsaline.Morriswatermaze （MWM）was used to examine the learning and memory functions.Senile plaques（SPs）in the hippocampus and cortex of APP/PS1 transgenic mice were observed by thioflavine S staining.The protein expression of APP，βsite APP cleavage enzyme 1（BACE1），a disintegrin and metalloproteinase domain 10（ADAM10），the phos⁃phorylationofthetranslationinitiationfactoreIF2α （eIF2α-P）and phosphorylated PERK（PERK-P），and the levels of Aβ1-40and Aβ1-42in the hippocampus and cor⁃tex were detected by western boltting.RESULTS ICSⅡcould effectively ameliorated spatial memory impairment and decreased the number of senile plaques.In addi⁃tion，ICS II significantly inhibited Aβ production by upreg⁃ulating ADAM10 and downregulating BACE1，and result⁃ed in beneficial effects such as inhibition of eIF2α phos⁃phorylation and PERK activation in APP/PS1 transgenic mice model.CONCLUSlON ICSⅡcould improve cogni⁃tive function in transgenic mice model of Alzheimer dis⁃ease via modulating Aβ production.

Key words：lcarisideⅡ；Alzheimer disease；spatial memory impairment；β-amyloid；β-site APP cleavage en⁃zyme 1

Foundation item：The project supported by National Natural Science Foundation of China（81560585）；Sci⁃ence and Technology Innovation Talent Team of Guizhou Province（20154023）；and Outstanding Youth Science and Technology Talent Capital of Guizhou Province （201326）.

Corresponding author：GONG Qi-hai，E-mail：gqh@ zmc.edu.cn

T2-43

镉染毒暴露对大鼠血管性痴呆模型学习记忆的影响

邵亚杰1，2，曾贵荣1，周仕达1，俞利林1，贾昀涛1，袁湘中1，姜德建1，2

（1.湖南省药物安全评价研究中心，湖南长沙 410331；2.湖南师范大学生命科学学院，湖南长沙 410006）

摘要：目的 研究重金属镉染毒对大鼠血管性痴呆模型学习记忆的影响。方法 雄性SD大鼠80只，随机分为空白对照组和模型对照组，模型组大鼠连续灌胃给予不同浓度的镉溶液（1，5和25 mg·L-1），每日1次，连续1个月，染毒后大鼠采用双侧颈总动脉血管结扎法建立血管性痴呆模型，随后将动物随机分为4组，分别为空白+VD组、氯化镉低浓度+ VD组、氯化镉中浓度+VD组和氯化镉高浓度+VD组。分别于模型建立后1和2个月对各组大鼠进行水迷宫定位航行和空间探索，2月后解剖取材，检测大鼠血浆、肝、肾和脑组织中镉的含量。结果 镉染毒5和25 mg·L-1组在模型建立2个月水迷宫检测登台潜伏期明显延长、总路程明显增加，经平台次数明显减少，大鼠血浆、肝和脑组织中镉的含量明显增高。镉染毒低浓度在模型建立1和2个月进行水迷宫检测，其中登台潜伏期、总路程、经平台次数与血管性痴呆模型比较无显著性差异，血浆、肝和脑组织中镉的含量明显与5 和25 mg·L-1组比较明显降低。结论 长期镉染毒能加重大鼠血管性痴呆学习记忆的降低，有效暴露浓度为5 mg·L-1。

关键词：镉染毒；血管性痴呆；水迷宫；学习记忆

基金项目：国家重大科技专项（2011ZX09401-301-1）

通讯作者：姜德建，Tel/Fax：（0731）83285166，E-mail：dejianjiang@yahoo.com

T2-44

防治乙醇中枢毒性损伤新靶点——抗坏血酸转运体2

田 华1，2，叶小霞1，侯晓节1，杨晓伟1，杨静玉1，吴春福1

（1.沈阳药科大学药理教研室，辽宁沈阳 110016；2.齐齐哈尔医学院药理教研室，黑龙江齐齐哈尔 161006）

摘要：目的 研究抗坏血酸转运体2（SVCT2）在乙醇神经毒性损伤中的神经保护作用及其调节机制。方法 首先建立4 d狂饮大鼠模型，采用高效液相色谱、激光共聚焦显微成像、基因芯片、ELISA、RT-PCR和Western蛋白印迹等实验技术，从神经行为学和分子生物学等角度研究乙醇神经损伤与氧化应激的关系，深入研究SVCT2在乙醇致中枢神经系统损伤过程中的保护作用及其调节机制。结果 4 d狂饮乙醇致大鼠中枢神经系统氧化应激损伤，大鼠脑脊液中AA水平显著增加，而细胞内AA显著降低，诱导神经元MAPKs，NF-κB信号途径的活化，并下调rno-miR-125a-5p的表达。抑制的SVCT2表达，可显著加剧乙醇诱导的大鼠皮质神经元损伤，提示SVCT2在乙醇诱导的氧化应激损伤中发挥一定的神经保护作用。乙醇神经毒性损伤中受到JNK/p38 MAPKs，NF-κB信号途径及miR-125a-5p多种途径机制。结论 在乙醇神经毒性损伤过程中，SVCT2在自我防御机制及代偿性恢复过程中发挥重要神经保护作用，可能成为一种防治乙醇诱导神经退行性病变的新型治疗策略。

关键词：抗坏血酸；乙醇；抗坏血酸转运体2；氧化应激；rno-miR-125a-5p；神经退行性病变

通讯作者：吴春福，Tel：（024）3986339，E-mail：wucf@ syphu.edu.cn；杨静玉，Tel：（024）23986340，E-mail：yangjingyu2006@gmail.com

T2-45

TRPC5通道和磷脂爬行酶1相互作用调节神经细胞磷脂酰丝氨酸暴露和凋亡

李 杰1*，郭继政1*，杜 鹃1，姚晓强3，汪 凯2，沈 兵1（1.安徽医科大学基础医学院生理教研室，安徽 合肥230032；2.安徽医科大学第一附属医院神经内科，安徽合肥 230022；3.香港中文大学医学院生物医学学院，香港新界）

摘要：目的 TRPC5通道是瞬时受体电位通道TRPC家族成员之一，通透钙离子，可以被溶血卵磷脂、镧离子、Genistein、钙库耗竭和机械牵拉等激活，与Na+-H+交换体调节蛋白、钙调蛋白和微管不稳定蛋白等相作用，参与细胞的多种钙信号反应。磷脂爬行酶1（PLSCR1）是Ca2+结合的棕榈酰化Ⅱ型膜蛋白，可与EGFR、c-Src和onzin等多种蛋白相互作用，介导细胞膜的磷脂酰丝氨酸暴露，参与细胞的生长、分化、凋亡等过程。本研究试图阐明TRPC5和PLSCR1是否存在相互作用及其在神经细胞凋亡中的可能作用。方法 采用免疫共沉淀、荧光共振能量转移、原位接近等实验检测TRPC5和PLSCR1的相互作用关系；用流式细胞仪和共聚焦显微镜检测过表达TRPC5和PLSCR1、空载体等的HEK293细胞、TRPC5敲除和对照等的小鼠脑片神经细胞磷脂酰丝氨酸暴露情况；用原位末端标记法检测小鼠脑片神经细胞凋亡情况。结果 免疫共沉淀、荧光共振能量转移、原位接近等实验结果显示，过表达TRPC5和PLSCR1的HEK293细胞和小鼠神经细胞中TRPC5和PLSCR1都存在物理上的相互作用，而且二者是通过C末端相互作用。流式细胞仪和共聚焦显微镜检测结果显示，与空载体转染对照相比，低渗缓冲液、LaCl3和Genistein类似物Daidzein都可以显著引起TRPC5和PLSCR1过表达细胞的磷脂酰丝氨酸暴露。小鼠脑片结果也显示，低渗缓冲液、LaCl3和Daid⁃zein显著引起脑片神经细胞的磷脂酰丝氨酸暴露，TRPC5通道阻断抗体T5E3可以显著抑制这一效应，TRPC5敲除小鼠脑片这一作用也显著减弱。脑缺血再灌注小鼠的脑片上神经细胞出现显著的磷脂酰丝氨酸暴露和神经细胞凋亡，而TRPC5敲除小鼠这一效应也显著减弱。结论 TRPC5和PLSCR1形成钙信号复合物，参与介导低渗环境引起的神经细胞磷脂酰丝氨酸暴露和凋亡，在脑缺血再灌注神经系统损伤中有重要作用。

关键词：TRPC5；磷脂爬行酶1；磷脂酰丝氨酸暴露；脑缺血再灌注；凋亡

基金项目：安徽省杰出青年基金（1108085J11）

通讯作者：沈 兵，Tel：（0551）65161132，E-mail：shenbing@ ahmu.edu.cn

*共同第一作者

T3　疼痛与成瘾医学

T3-1

新型镇痛候选新药α O-芋螺毒素GeXlVA的研究

罗素兰1，长孙东亭1，李晓丹1，朱晓鹏1，吴 勇1，胡远艳1，CherylDowell2， MelissaMcIntyre2， VictorTsetlin3，James C.Baxter4，Keith S Elmslie4，Peta J.Harvey5，Quentin Kaas5，David J.Craik5，J.Michael McIntosh2

（1.海南大学热带生物资源教育部重点实验室、海口市海洋药物重点实验室，海南 海口570228；2.George E.Wahlen Veterans Affairs Medical Center and Depart⁃ments of Biology and Psychiatry，University of Utah，Salt Lake City，UT 84112，USA；3.Shemyakin-Ovchin⁃nikov Institute of Bioorganic Chemistry，Russian Acade⁃my of Sciences，Miklukho-Maklaya Street，16/10 Moscow 117997，Russia；4.Department of Pharmacology，Kirks⁃ville College of Osteopathic Medicine，AT Still University，800 W Jefferson Street，Kirksville，MO 63501，USA；5. Institute for Molecular Bioscience，The University of Queensland，Brisbane，QLD 4072，Australia）

摘要：目的 神经痛是中枢或外周神经系统损伤或功能异常导致的疼痛综合征，折磨着全球10%以上的人口。我国神经痛患者至少有1亿人，不仅严重影响患者的身心健康和生活质量，而且消耗了数以百亿美元计的医疗资源。为此，本研究的目的是从大量的芋螺毒素分子中发现能治疗神经痛的新型镇痛候选海洋药物。方法 以神经型α9α10乙酰胆碱受体（nAChR）为靶点，筛选鉴定该受体的芋螺毒素特异阻断剂，并对其结构与功能、药理药效进行系统深入的研究。结果 经过多年的努力，筛选鉴定出1个特异阻断α9α10 nAChR的αO-新家族芋螺毒素GeXIVA，其半阻断剂量（IC50）仅为3.8 nmol·L-1，是迄今发现的α9α10 nAChR亚型活性最强的阻断剂。GeXIVA是一个全新的化合物，由28个氨基酸组成的多肽，含有4个半胱氨酸（Cys），形成2对分子内二硫键。人工合成了GeXIVA三种可能的二硫键异构体，分别称为bead异构体（GeXIVA［1，2］），ribbon异构体（GeXIVA［1，4］）和globular异构体（GeXIVA［1，3］）。其中的bead异构体对α9α10 nAChR的活性最强。GeXIVA与α9α10 nAChR的结合特异性很高，而对其他多种乙酰胆碱受体亚型活性很弱或完全没有活性，选择性都在近百倍以上。利用900兆的核磁共振仪解析出了GeXIVA的三维空间结构。发现GeXIVA是国际上第一个电压依赖性的α9α 10 nAChR强阻断剂。阐明了GeXIVA与α9α10 nAChR相互作用的分子机制，发现GeXIVA与受体结合的部位是两个新位点。GeXIVA对钙离子通道没有影响。在CCI神经痛模型上，肌肉注射GeXIVA的镇痛活性比吗啡强～1000倍，不成瘾，且不会影响机体的运动功能，安全性好。在整体动物实验中，肌肉注射有效剂量可低至1 nmol以下。连续给药7 d后停药和连续给药14 d后停药，停药后的第7天和第14天内，还有很好的镇痛效果。GeXIVA不但能镇痛，还能预防神经受伤，使受伤的神经得到修复，具有很好的神经保护功效。结论 αO-芋螺毒素GeXIVA是作用靶点清楚的、结构新颖的、给药途径方便的、很难得的治疗神经痛的原创候选新药，海南大学拥有该芋螺毒素的全部自主知识产权，蕴含着巨大的经济价值和社会效益，具有极好的开发应用前景。

关键词：镇痛候选新药；αO-芋螺毒素GeXIVA；α9α10乙酰胆碱受体；神经痛；结构与功能；药效学。

基金项目：国家自然科学基金（81420108028；41366002）；长江学者和创新团队发展计划（IRT1123；IRT_15R15）；国家863计划（2012AA021706）

通讯作者：罗素兰，E-mail：luosulan2003@163.com，Tel：（0898）66289538

T3-2

脊髓TLR4参与眶下神经部分切断引起的慢性广泛性疼痛

胡婷婷1，王冉冉1，汤莹莹2，于捷3，张世红1，陈忠1

（1.浙江大学医学院药理学系，浙江杭州 310058；2.浙江大学附属妇产科医院药学部，浙江杭州 310006；3.浙江中医药大学基础部，浙江杭州 310053）

摘要：目的 慢性广泛性疼痛的发病机制尚不清楚，缺少有效的治疗手段。Toll样受体4（Toll like receptor 4，TLR4）在神经病理性疼痛的发展过程中起着重要作用。最近我们发现，部分切断小鼠眶下神经可引起中枢敏化在脊髓内的扩散以及后爪疼痛敏感性增强。本实验旨在研究TLR4是否参与眶下神经部分切断引起的慢性广泛性疼痛。方法异氟烷吸入麻醉小鼠，经口腔切口部分切断眶下神经，诱导面部和后爪的疼痛增敏。比较TLR4基因突变和敲除小鼠与相对应的野生型小鼠的面部和后爪疼痛行为学差异，以及脊髓不同节段TLR4及下游信号蛋白MyD88的表达变化，并评价分别在延髓小脑池和腰髓（L4/L5）节段给予TLR4抑制剂Rs-LPS对疼痛行为学的影响。结果 神经损伤后第3天，所有基因型小鼠的面部都出现机械痛觉超敏和热痛觉过敏，并至少维持到术后14 d。野生型小鼠后爪在术后7 d开始出现机械痛觉超敏和热痛过敏，但是TLR4基因突变和敲除的小鼠后爪出现机械痛觉过敏的时间延迟，并且严重强度减弱，但热痛过敏未受影响。眶下神经部分切断后，野生型小鼠延髓中TLR4及下游信号分子MyD88没有显著性变化，L4/L5脊髓中TLR4表达变化不明显，但MyD88表达增加。在野生型小鼠延髓小脑池和L4/L5节段鞘内注射Rs-LPS可减缓后爪机械痛觉过敏，但不影响热痛过敏。结论 TLR4-MyD88通路参与眶下神经部分切断引起的慢性广泛性疼痛的发生，提示TLR4可能作为临床治疗慢性广泛性疼痛的靶点。

关键词：慢性广泛性疼痛；眶下神经部分切断；中枢敏化；TLR4；MyD88；

基金项目：国家自然科学基金面上项目（81473187）

通讯作者：陈 忠，E-mail：chenzhong@zju.edu.cn，Tel：（0571）88208228

T3-3

R-Modafinil attenuates nicotine-taking and nicotineseeking behavior in rats

WANGXiao-fei1，2， BIGuo-hua1， YANGHong-ju1，Eliot L.Gardner1，XI Zheng-xiong1

（1.NeuropsychopharmacologySection，NationalInsti⁃tute on Drug Abuse-Intramural Research Program，Na⁃tional Institutes of Health，Baltimore，MD 21224，USA；2.军事医学科学院毒物药物研究所，北京 100850）

Abstract：OBJECTlVE（±）Modafinil（MOD）is a mild psychostimulant used in humans for treatment of sleep disorders and has been investigated as a potential medication for the treatment of psychostimulant addic⁃tion.However，the therapeutic efficacy of（±）MOD for addiction has been inconclusive.METHODSHerein we used animal models of self-administration and in vivo mi⁃crodialysis to study the pharmacological action and the mechanisms of the R-enantiomer of modafinil（R-MOD）and its S-enantiomer（S-MOD）on nicotine-taking and nicotine-seeking behavior.RESULTSWe found that systemic injections of R-MOD（10-30 mg·kg-1，ip）inhibit⁃ed nicotine self-administration in Long-Evans rats，while a higher dose（100 mg·kg-1）of S-MOD was required.As alcohol-preferring rats（P-rats）display more robust nico⁃tine taking and nicotine seeking than Long-Evans rats，we used P-rats to further study the effects of R-MOD on nicotine-seeking behavior.We found that R-MOD（30-100 mg·kg-1）not only inhibited intravenous nicotine selfadministration，but also inhibited nicotine-induced rein⁃statement of drug seeking after extinction and cue-in⁃duced nicotine seeking（assessed in an extinction test）after 3 weeks of withdrawal.Further，in vivo brain micro⁃dialysis assays demonstrated that pretreatment with RMOD（30 and 100 mg·kg-1）increased basal extracellu⁃lar dopamine（DA）levels in the nucleus accumbens （NAc），but decreased nicotine-induced increases in ex⁃tracellular DA，suggesting that a DA-dependent mecha⁃nism may in part underlie mitigation of nicotine′s effects produced by R-MOD.CONCLUSlONThese data sug⁃gest that R-MOD is more potent than S-MOD in attenuat⁃ing nicotine-taking and nicotine-seeking behavior in rats，and warrants further investigation for treatment of nico⁃tine dependence in humans.

Key words：R-（-）-modafinil；nicotine；dopamine；selfadministration；reinstatement；alcohol-preferring rats

Corresponding author：XI Zheng-xiong，（443）740-2517，E-mail：zxi@mail.nih.gov

T3-4

Essential role of NO signaling pathway in hippocam⁃pal CA1 in morphine-associated memory depends on glutaminergic receptors

SHEN Fang*，WANG Xue-wei*，GE Fei-fei，LI Yi-jing，CUI Cai-lian

（Neuroscience Research Institute，Department of Neuro⁃biology，School of Basic Medical Sciences，Key Labora⁃tory of Neuroscience，The Ministry of Education and the Ministry of Health，Peking University，Beijing 100191，China）

Abstract：OBJECTlVEThe nitric oxide（NO）/solu⁃ble guanylyl cyclase（sGC）/cGMP-dependent protein kinase（PKG）signaling pathway has been reported to play a key role in memory processing.Here，we intend⁃ed to investigate its role in drug-associated reward mem⁃ory.METHODSFirst，rats were received intra-CA1 ad⁃ministration of 7-NI，ODQ or Rp-8-Br-PET-cGMPS to as⁃sess the role of the NO/sGC/PKG signaling pathway in the retrieval of morphine-conditioned memory.Then，Western blotting was used to evaluate the nNOS，sGC， PKG，NR2B and p-Akt protein levels in the CA1 after the morphine CPP expression test.Co-immunoprecipitation was used to detect the interaction between PKG and GluR1 in the CA1.RESULTS① The NO pathway in the CA1 is critical for the retrieval of morphine-associat⁃ed reward memory.Specifically，the nNOS，sGC and PKG protein levels in the CA1 were increased after the expression of morphine conditioned place preference （CPP）.Intra-CA1 injection of an NOS，sGC or PKG in⁃hibitor prevented morphine CPP expression.②The in⁃volvement of the NO pathway in morphine CPP requires NR2B-containingNMDAreceptors（NR2BNMDARs）. NR2B-NMDAR expression was elevated in the CA1 fol⁃lowing morphine CPP expression，and intra-CA1 injec⁃tion of the NR2B-NMDAR antagonist Ro25-6981 not only blocked morphine CPP expression but also inhibited the up-regulation of nNOS，sGC and PKG.Moreover，the Ro25-6981-induced blockade of morphine CPP was abol⁃ished by intra-CA1 injection of a NOS substrate or an sGC activator.③The NR2B-NMDAR stimulated the NO pathway by up-regulating the phosphorylation of AktS⁃er473.Morphine CPP expression enhanced the pAktS⁃er473 level，which has been corroborated to regulate nNOS activity，and this effect was reversed by intra-CA1 injection of Ro25-6981.④GluR1 acted downstream of the NO pathway.The membrane level of GluR1 in the CA1 was increased after morphine CPP expression，and this effect was prevented by pre-injection of a PKG inhibi⁃tor into the CA1.Additionally，coimmunoprecipitation re⁃vealed an interaction between PKG and GluR1；this re⁃sult further indicated a role of PKG in regulating GluR1 trafficking.CONCLUSlONThe results of our study dem⁃onstrated that the activation of the NR2B-NMDAR/NO/ sGC/PKG signaling pathway is necessary for the retriev⁃al of morphine-associated reward memory.

Key words：nitric oxide signaling pathway；morphine；re⁃ward memory；NMDAR；AMPAR；hippocampal CA1 region

Foundation item：The project supported by National Natural Science Foundation of China（31271163）；National Basic Research Program（2015CB553500）；and Science Fund for Creative Research Groups from National Natural Science Foundation of China（81221002）

Corresponding author：CUI Cai-lian，E-mail：clcui@ bjmu.edu.cn

*Co-first author

T3-5

“Two-hit”affected motor behavior and sensorimotor gating differentially in male and female rats

WU Jing-min，SHEN Hao-wei

（National Institute On Drug Dependence，Peking Univer⁃sity，Beijing 100191，China）

Abstract：OBJECTlVEThe″two-hit″hypothesis ofschizophrenia proposes that a genetic or neurodevelop⁃mental insultmay sensitize an individual for a subsequent adverse event during adolescence that ultimately leads to onset of the clinical syndrome.In present study，we in⁃vestigated the differential effect of“two-hit”on motor be⁃havior and sensorimotor gating on male and female rats. METHODSSprague-Dawley rats received received theN-methyl-D-aspartate（NMDA）receptor antagonist MK-801（1.0 mg·kg-1sc）or salinedaily during postnatal day（PND）7-13，and then were assigned to isolation rearing or group post weaning（PND 21）.Locomotor ac⁃tivityin novel environment andprepulse inhibition of acous⁃ticstartle（PPI）wereexamined on PND 35-37 and PND 75-80，respectively.RESULTSBoth male and female rats treated by“two-hit”exhibited higher activities in nov⁃el environment.Interestingly，only male rats treated by “two-hit”showedhigher locomotor activity in the central area of locomotor chamber than control group.Further⁃more，“two-hit”treatmentcaused significant anomalies in PPI on both sexes，while female rats displayed more⁃severedisruption in PPI compared with male.CONCLU⁃SlONOur data indicate that“two-hit”produced psy⁃chotic-like insults in both sexes of rats，and the PPI im⁃pairment are more severe in female comparing to male.

Key words：″Two-hit″treatment；sex differences；locomo⁃tor activity；repulse inhibition

Foundation item：The project supported by Notronal Natural Secience Foundotion of China（81221002）

Corresponding author：SHEN Hao-wei，Tel：（010）82802471，E-mail：shenhaowei@gmail.com

T3-6

Brain hyperpolarization-activated，cyclic nucleotidegated（HCN）channels modulate methamphetamine′s actions in rats：involvement of dopamine in nucleus accumbens

CAO Dan-ni，SONG Rui，ZHANG Shu-zhuo，WU Ning，LI Jin

（State Key Laboratory of Toxicology and Medical Coun⁃termeasures，Beijing Key Laboratory of Neuropsycho⁃pharmacology，Beijing Institute of Pharmacology and Toxicology，Beijing 100850，China）

Abstract：OBJECTlVEMethamphetamine addic⁃tion is believed to result primarily from increasing dopa⁃mine release and inhibiting dopamine uptake.Recently，someevidencessuggestthatthehyperpolarizationactivated，cyclic nucleotide-gated（HCN）channels play important roles in the functional modulation of dopaminer⁃gic neurons and the pathophysiology of related diseases. However，nothing is known about the effects of HCN channels on methamphetamine addiction.In the present study，we investigated the role of the brain HCN channels inmethamphetamineaddiction.METHODSand RESULTSAcute intracerebroventricular（i.c.v.）injec⁃tion or bilateral nucleus accumbens microinjection of nonselective HCN channel blocker ZD7288（0.3125 and 0.625 μg）significantly reduced methamphetamine（infu⁃sion 0.0125 or 0.05 mg·kg-1）-induced self-administration under fixed ratio 2 reinforcement，as well as the break⁃point of methamphetamine（infusion 0.05 mg·kg-1）un⁃der progressive ratio reinforcement in rats.And more，compared with the i.c.v.injection，bilateral nucleus ac⁃cumbens microinjection of ZD2788 exerted stronger in⁃hibitory effects，which suggested that blockade of HCN channels in the NAc reduced the reinforcing effects and rewarding efficacy of methamphetamine.We also found that ZD7288（0.625 and 1.25 μg，i.c.v.）significantly in⁃hibited methamphetamine（1 mg·kg-1，ip）-induced hy⁃peractivity with no effect on spontaneous activity in rats. At last，by the microdialysis experiments in vivo，block⁃ade of HCN channels with ZD7288（0.625 and 1.25 μg，i. c.v.）decreased methamphetamine（1 mg·kg-1，ip）-in⁃duced elevation of extracellular dopamine levels in the nucleus accumbens.CONCLUSlON These results dem⁃onstrate that HCN channels in the NAc are involved in the reinforcing properties of methamphetamine and high⁃light the importance of HCN channels in the regulation of dopamineneurotransmissionunderlyingmethamphet⁃amine addiction.

Key words：methamphetamine；dopamine；hyperpolar⁃ization-activated cyclic nucleotide-gated channels；In vivo microdialysis；nucleus accumbens；self-administration

Foundation item：The project supported by National Basic Research Program of China（″973″）（2015CB553504）；Beijing Nova Program（xx2014A014）；and National Natural Science Foundation of China（81573405）

Corresponding author：LI Jin，Tel：（010）66932681，E-mail：jinli9802@163.com

T3-7

Alterations of prefrontal cortical microRNAs in meth⁃amphetamine self-administering rats：from controlled drug intake to escalated drug intake

DU Hao-yue*，CAO Dan-ni*，SHI Jing-jing，CHEN Ying，WANG Lv，LU Guan-yi，WU Ning，LI Jin

（State Key Laboratory of Toxicology and Medical Coun⁃termeasures，Beijing Key Laboratory of Neuropsycho⁃pharmacology，Beijing Institute of Pharmacology and Toxicology，Beijing 100850，China）

Abstract：OBJECTlVEDrug addiction is a pro⁃cess that transits from recreative and regular drug use in⁃to compulsive drug use.The two patterns of drug use，controlled drug intake and escalated drug intake，repre⁃sent different stages in the development of drug addic⁃tion；and escalation of drug use is a hallmark of addic⁃tion.AccumulatingstudiesindicatethatmicroRNAs（miRNAs）play key regulatory roles in drug addiction. However，the molecular adaptations in escalation of drug use，as well as the difference in the adaptations be⁃tween escalated and controlled drug use，remain un⁃clear.In the present study，therefore，the alterations of miRNAs expression in PFC were investigated in rats with escalated and controlled methamphetamine use，and the function of putative targets was analyzed using bioin⁃formatic method.METHODSmiRNA microarray assay and RT-qPCR were used to screen differential expressed miRNAs and validate the differential expression，respec⁃tively.Bioinformatic analysis was performed.RESULTS we established the escalated methamphetamine self-ad⁃ministration model in rats by permitting animals extended access to drug（6 h/session，LgA group），whereas ani⁃mals that were restricted to access to drug（1 h/session，ShA group）remained low and stable self-administration，suggesting the controlled drug intake.A total of 28 altered miRNAs in the prefrontal cortex（PFC）were found in the groups of controlled methamphetamine selfadministration（1 h/session）and escalated self-adminis⁃tration（6 h/session），and some of them were validated. Compared with saline control group，miR-186 was veri⁃fied to be up-regulated while miR-195 and miR-329 were down-regulated in the rats with controlled methamphet⁃amine use.In the rats with escalated drug use，miR-127，miR-186，miR-222 and miR-24 were verified to be up-regulated while miR-329 was down-regulated com⁃pared with controls.Furthermore，bioinformatic analysis indicated that the predicted targets of these verified miR⁃NAs involved in the processes of neuronal apoptosis and synaptic plasticity.However，the putative regulated mole⁃cules may be different between controlled and escalated drug use groups.CONCLUSlONThe alterations of miR⁃NAs expression in rat PFC under the conditions of con⁃trolled methamphetamine use and escalated use were detected respectively，which may be involved in neuro⁃nal apoptosis and synaptic plasticity.These findings may extend our understanding of the molecular adaptations underlying the transition from controlled methamphet⁃amine use to addiction.

Keywords：methamphetamineaddiction；escalated drug use；controlled drug use；prefrontal cortex；microRNAs

Foundation item：The project supported by National Basic Research Program of China（2015CB553504）；National Natural Science Foundation of China（81571302）；and China Postdoctoral Science Foundation Funded Project（2015M572688）

Corresponding authors：LI Jin，Tel：（010）66932681，E-mail：jinli9802@163.com；WUNing，Tel：（010）66930635，E-mail：wuning7671@126.com

*Co-first authors

T3-8

lnvolvement of dorsal hippocampal CaMKⅡ activa⁃tion in aversive memory formation induced by condi⁃tioned morphine withdrawal

LONG Jian-dong，WU Wei-wei，WANG Yu-jun，LIU Yao，LIU Jing-gen

（Key Laboratory of Receptor Research，Shanghai Insti⁃tute of Materia Medica and Collaborative Innovation Center for Brain Science，Chinese Academy of Sciences，Shanghai 201203，China）

Abstract：OBJECTlVETo investigate the role of calcium/calmodulin（CaM）-dependent protein kinaseⅡ（CaMK-Ⅱ）in the formation of aversive memory induced by naloxone-precipitated morphine withdrawal.METH⁃ODSMale SD rats were trained in a classic conditioned place aversion model.Immunoblotting assay was used to quantify CaMKⅡexpression and CaMKⅡ phosphoryla⁃tion.The ability of intra-dorsal hippocampus microinjec⁃tion of CaMKⅡinhibitor KN-93（10 nmol·L-1，1.2 μL per⁃side） to inhibit the aversive memory formation was detected.RESULTSCaMKⅡwas activated in the dor⁃sal hippocampus after naloxone-precipitated withdrawal. Microinjection of CaMKⅡinhibitor KN-93 significantly dis⁃rupted the formation of naloxone-precipitated morphine withdrawal.CONCLUSlONActivation of CaMKⅡin the dorsal hippocampus is responsible for the formation of aversive memory induced by naloxone-precipitated with⁃drawal in morphine-treated rats.

Key words：aversive memory；dorsal hippocampus；CaMKⅡ；KN-93

Foundation item：The project supported by Ministry of Science and Technology of China（2013CB835100）；and NationalNaturalScienceFoundationofChina （81130087；91232716）；and Committee of Science and Technology of Shanghai（13JC140680）

Corresponding author：LIU Jing-gen，E-mail：jgliu@ mail.shcnc.ac.cn

T3-9

ΚOpioid receptor activation in different brain regions dif⁃ferentially modulates anxiety-related behaviors in mice

WANG Yu-jun，HANG Ai，LU Yu-chen，LONG Yu，ZAN Gui-ying，LI Xue-ping，WANG Qian，LIU Jing-gen

（Key Laboratory of Receptor Research，Shanghai Insti⁃tute of Materia Medica and Collaborative Innovation Center for Brain Science，Chinese Academy of Science，Shanghai 201203，China）

Abstract：OBJECTlVEDynorphin/κ opioid recep⁃tor system is implicated in the modulation of emotion. The anxiety-related effects of κ receptor remain contro⁃versial as agonists show opposite effects in different stud⁃ies.Here we investigated the role of κ agonist U50，488H in anxiety over a wide range of doses and identified the neuroanatomical substrates that mediated the behavioral responses.METHODSWe first evaluated the effects of U50，488H on anxiety-related behaviors in different ani⁃mal paradigms.We then used phosphorylation of extra⁃cellular signal-related kinase 1/2（pERK1/2）to map neu⁃ral circuits and find the candidate brain regions involved. Finally，we examined the behavioral consequences with local microinjection of U50，488H into these brain re⁃gions.RESULTSWe found that κ agonist U50，488H produced biphasic effects in anxiety，with low dose being anxiogenic and high dose being anxiolytic.The anxiogenic effect of U50，488H was paralleled by an increase of pERK1/2 activation in the nucleus accumbens，whereas the anxiolytic effect was paralleled by an increase in the lateral septal nucleus and dorsomedial hypothalamic nucleus.Microinjection of U50，488H into the nucleus accumbens exerted anxiogenic effects，whereas microin⁃jection of U50，488H into the lateral septal nucleus and dorsomedialhypothalamicnucleusexertedanxiolytic effects.A κ opioid receptor-dependent mechanism was involved in the nucleus accumbens and lateral septal nucleus in anxiety-related behaviors，but not in the dorso⁃medial hypothalamic nucleus.CONCLUSlONThe pres⁃ent study demonstrated that activation of κ opioid receptor produced biphasic effects in anxiety in a dose-dependent manner，and provided the neuroanatomical substrates un⁃derlying the biphasic behavioral effects of κ agonist.

Key words：κ opioid receptor；pERK1/2；region-specific；dual effects；anxiety

Foundation item：The project supported by Ministry of Science and Technology of China（2013CB835100）；NationalNaturalScienceFoundationofChina （81130087；91232716；81401107）；and Committee of Science and Technology of Shanghai（13JC140680）

Corresponding author：LIU Jing-gen，E-mail：jgliu@ mail.shcnc.ac.cn

T3-10

P38 mitogen-activated protein kinase activation in amygdala mediates κ opioid receptor agonist U50，488H-induced conditioned place aversion

ZAN Gui-ying，WANG Qian，WANG Yu-jun，HU Xiao-wu，SHU Xiao-hong，LIU Jing-gen

（Key Laboratory of Receptor Research，Shanghai Insti⁃tute of Materia Medica，Chinese Academy of Sciences and Collaborative Innovation Center for Brain Science，Shanghai 201203，China）

Abstract：OBJECTlVEΚ Opioid receptor agonists produce aversive effects in rodents，however the underly⁃ing mechanisms remain unclear.Activation of p38 mito⁃gen-activated protein kinase（MAPK）has been discov⁃ ered to play a critical role in the modulation of affective behaviors.The present study was undertaken to detect the possible involvement of p38 MAPK in the aversive effects induced by κ opioid receptor activation.METHODS We utilized a U50，488H conditioned place aversion mice model to study the mechanism of κ opioid receptor medi⁃ated aversive behavior.We then quantified phospho-p38 （P-p38）MAPK protein levels in the amygdala after U50，488H pairing.Finally，the ability of intra-amygdala micro⁃injection with p38 MAPK inhibitor SB203580 to block the acquisition of aversive behavior was detected.RESULTS We found that the κ opioid receptor agonist trans-（±）-3，4-dichloro-N-methyl-N-［2-（1-pyrrolidinyl）-cyclohexyl］benzenacetamide methanesulfonate salt（U50，488H）produced significant place aversion in mice as measured by the conditioned place preference procedure，accom⁃panied with significant p38 MAPK activation in the amyg⁃dala，but not in the nucleus accumbens and hippocam⁃pus.Stereotaxic microinjection of the p38 MAPK inhibitor 4-（4-fluorophenyl）-2-（4-methylsulfonylphenyl）-5-（4-pyr⁃idy-l）-1H-imidazole（SB203580）into amygdala signifi⁃cantly inhibited p38 MAPK activation and completely blocked the conditioned place aversion in mice.CON⁃CLUSlON The present study demonstrated that κ opioid receptor activation by U50，488H significantly induced conditioned place aversion in mice.After U50，488H pair⁃ing，P-p38 MAPK level significantly increased in the amygdala，but not in the hippocampus and nucleus ac⁃cumbens.Intra-amygdala administration of p38 MAPK in⁃hibitor SB203580 significantly decreased p38 MAPK acti⁃vation and completely blocked the acquisition of condi⁃tioned place aversion.These findings demonstrated that p38 MAPK activation in the amygdala was required for U50，488H-induced aversive behavior in mice.

Key words：κ opioid receptor；U50，488H；conditioned place aversion；p38 MAPK

Foundation item：The project supported by Ministry of ScienceandTechnologyofChina（2013CB835100；2015CB553500）；National Natural Science Foundation of China（81171296；81130087；91232716）；and Commit⁃tee of Science and Technology of Shanghai（13JC140680）Corresponding author：LIU Jing-gen，E-mail：jgliu@ mail.shcnc.ac.cn

T3-11

lmidazoline receptor antisera-selected protein（lRAS）modulatesopioidtoleranceanddependenceby regulating μ opioid receptor trafficking

LI Fei，MA Hao，ZHAO Tai-yun，ZHANG Ling，WU Ning，LI Jin

（State Key Laboratory of Toxicology and Medical Coun⁃termeasures，Beijing Key Laboratory of Neuropsycho⁃pharmacology，Beijing Institute of Pharmacology and Toxicology，Beijing 100850，China）

Abstract：OBJECTlVE Our previous studies showed that imidazoline receptor antisera-selected protein（IRAS）had a potential role in modulating opioid tolerance and dependence in vivo，while the underlying mechanism remained unknown.Regarding the property of IRAS and the trafficking processes of μ opioid receptor（MOR）being involved in the development of opioids-induced tolerance and dependence，we investigated whether IRAS and MOR interacted with each other，and the role of IRAS in MOR trafficking，including internalization，recycling，and downregulation，as well as opioid-in⁃duced desensitization and resensitization.METHODS and RESULTSIn the CHO or HEK293 cells co-express⁃ing MOR and IRAS，we found that IRAS，through its PX domain，interacted with MOR.By interacting with MOR，IRAS facilitates the recycling of internalized MOR and prevents its downregulation，and hence functionally at⁃tenuates opioid-induced desensitization and accelerates resensitization of MOR，which is believed as one of mechanismsmediatingopioidtoleranceanddepen⁃dence.Finally，using IRAS knockout mice we generat⁃ed，it was found that IRAS deficiency exacerbated the analgesic tolerance and physical dependence caused by opioids.CONCLUSlONWe demonstrate for the first time that IRAS is a new MOR interacting protein and reg⁃ulates agonist-induced trafficking of MOR via sorting in⁃ternalized MOR to the recycling pathway，which may be a molecular mechanism underlying IRAS modulating opi⁃ oid tolerance and dependence.

Keywords： μopioidreceptor； opioidtolerance；dependence；imidazoline receptor antisera-selected pro⁃tein；interaction；receptor trafficking

Foundation item：The project supported by National Basic Research Program of China（2015CB553504）；the Key Project of National Natural Science Foundation of China（30930040）；and Beijing Natural Science Foun⁃dation of China（7122137）

Corresponding authors：LI Jin，E-mail：jinli9802@163. com；WU Ning，E-mail：wuning7671@126.com

T3-12

A novel rodent model of impulsivity induced with prami⁃pexole in a Y-maze avoidance task

MA Wen-tao，LIU Ke，GONG Ze-hui，YONG Zheng，SU Rui-bin

（State Key Laboratory of Toxicology and Medical Counter⁃measures，Institute of Pharmacology and Toxicology，Acad⁃emy of Military Medical Science，Beijing 100850，China）

Abstract：Impulse control disorders have been grad⁃ually associated with pramipexole therapy of Parkinson disease，and this has been confirmed in various rodent models of impulsivity，such as the delay-discounting task and a gambling-like task.OBJECTlVESTo evaluate whether the pathologic behaviors induced by pramipex⁃ole in the Y-maze avoidance task qualify as depicting a novel model of impulsivity.METHODSThe present study measured premature shuttle numbers and exces⁃sive shuttle numbes of rats between the safe arm and the other two arms of Y maze before the next Y maze avoidance task following acute administration of saline or pramipexole（0.1，1.0 and 10 mg·kg-1）in Sprague-Dawley rats.The correct response numbers to choose the safe arm following a light clue in avoidance task and shuttle numbers in the 3 minutes both before and after the avoid⁃ance task were recorded as well.Then nalmefene（5 mg· kg-1）and fluoxetine（10 mg·kg-1），which are clinical ther⁃apies for Impulse control disorders，were applied in Y maze task separately before pramipexole administration using the same parameters.Cocaine，as a positive con⁃trol drug，was tested in this model as well.Finally，C57 mice were administrated with pramipexole（0.3，1.0，3.0 and 10 mg·kg-1）to test if mouse could apply to this mod⁃el.RESULTSPramipexole produced a dose-depen⁃dent increase in premature and excessive shuttle num⁃bers in both SD rats（P＜0.05，P＜0.01）and C57 mice （P＜0.01），while had little influence on the correct response numbers（P＞0.05）.Shuttle numbers in the 3 min before the task，as a parameter for locomotor activ⁃ity，were not significantly changed in both SD rats and C57 mice after administration of pramipexole.But shuttle numbers in the 3 min after were significant increased in high dosage of pramipexole in SD rats（P＜0.05），but not in C57 mice.Premature and excessive shuttle num⁃bers induced with pramipexole were decreased about 40%-60%with nalmefene and fluoxetine administration in SD rats.Excessive shuttle numbers in 3 min after Y maze task were decreased as well.Cocaine increased premature and excessive shuttle numbers only in a high dose of 30 mg·kg-1with decreased correct response numbers.CONCLUSlONPramipexole induced signifi⁃cant impulsive behavior in both SD rats and C57 mice in the Y-maze avoidance task，suggesting for the first time that this task may be useful as a rodent model of impul⁃sivity with high efficiency.

Key words：impulse control disorders；pramipexole；Y-maze；behavioral addiction；rodent model；impulsivity

Foundation item：The project supported by National Natural Science and Technology Major Project of China （2012ZX09301003-003）

Corresponding author：SU Rui-bin，E-mail：ruibinsu@ 126.com；YONG Zheng，E-mail：yongzhabc@126.com.

T3-13

关联小胶质细胞的神经元信号分子在可卡因成瘾中的作用

胡壮丽，高双骑，王 芳，陈建国

（华中科技大学同济医学院基础医学院药理学系，湖北武汉 430030）

摘要：药物成瘾是一个日益严重的医学和社会问题，但目前的治疗效果并不理想。小胶质细胞作为中枢神经系统具有免疫和炎症调节功能的细胞，在成瘾中的作用也越来越受到重视。但小胶质细胞的激活情况复杂不易调节，如果能找到一种信号分子来调节小胶质细胞，从而起到干预成瘾的作用，则能为治疗成瘾提供新的思路和靶点。TLR4是小胶质细胞表面特异性表达的模式识别受体（PRR），已报道在可卡因成瘾中发挥重要作用，注射TLR4的阻断药纳洛酮或纳曲酮抑制可卡因诱导的大鼠奖赏记忆行为。但另一方面，临床使用纳曲酮却并不能对可卡因滥用者起到理想的治疗效果，这也提示仅仅阻断TLR4受体还不够。HMGB1是一种危险相关分子模式分子（DAMP），其作用的发挥是通过与PRR结合，而HMGB1分子结构上有TLR4的结合位点，那么，其是否能够作为一种关联小胶质细胞的信号分子在可卡因成瘾中发挥重要作用？我们在研究中发现，可卡因腹腔注射后激活小胶质细胞的同时，也引起NAc脑区HMGB1表达上调，而上调的HMGB1主要是来自神经元的释放；阻断HMGB1的合成和释放则抑制小胶质细胞的激活，并使可卡因诱导的大鼠位置偏好行为得到很好的改善；进一步用干扰肽BoxB S106阻断HMGB1-TLR4通路，也能改善可卡因诱导的大鼠位置偏好行为。提示在可卡因成瘾过程中，应激会诱导神经元释放大量HMGB1，后者作为信号源与TLR4受体结合，进而将损伤信号传递给小胶质细胞，从而介导成瘾的奖赏记忆行为。综上，HMGB1有可能作为激活小胶质细胞的一种关键信号分子，在可卡因成瘾中发挥重要作用。

关键词：小胶质细胞；神经元；可卡因成瘾

基金项目：国家自然科学基金（81473198）

通讯作者：陈建国，E-mail：chenj@mails.timu.edu.cn

T3-14

Dissociative role for dorsal hippocampus in mediating heroin craving and relapse through CDK5 and RhoB sig⁃naling revealed by proteomic analysis

CHEN Zhong-guo1， LIU Xing1， WANG Wei-sheng1，GENG Fan2，GAO Jing1，GAN Chen-ling3，CHAI Jing-rui1，HE Ling3，HU Gang2，ZHOU Hu1，4，LIU Jing-gen1

（1.Key Laboratory of Receptor Research，Shanghai Insti⁃tute of Materia Medica and Collaborative Innovation Center for Brain Science，Chinese Academy of Scienc⁃es，Shanghai 201203，China；2.Jiangsu Key Laboratory of Neurodegeneration，Department of Pharmacology，Nanjing Medical University，Nanjing 210029，China；3. Department of Pharmacology，China Pharmaceutical University，Nanjing 210009，China；4.SIMMUOMICS Laboratory，Joint Research Laboratory of Translational “OMICS”between Shanghai Institute of Materia Medica，Chinese Academy of Sciences，China and University of Ottawa，Canada）

Abstract：OBJECTlVEAddiction is characterized by drug craving，compulsive drug taking and relapse，which is attributed to aberrant neuroadaptation in brain regions implicated in drug addiction，induced by chang⁃ing gene and protein expression in these regions after chronic drug exposure.Accumulating evidence suggests that the dorsal hippocampus（DH）plays an important role in mediating drug-seeking and taking behavior and relapse.However，the molecular mechanisms underly⁃ingtheseeffectsoftheDHareunclear.Hereweusedlabelfree proteomics to analyze proteome changes in the DH following chronic exposure to heroin and further explored their roles in heroin addiction.METHODSThe rats were trained to intravenously self-administer heroin，then the DH were processed in a centrifugal proteomic reactor and quantified by LC-MS/MS based label-free quantitation.The results of quantitative proteomic analy⁃sis were verified by western blotting.Subsequently，we explored the role of CDK5 and RhoB，which were in⁃duced by heroin self-administration，in heroin craving and context-induced relapse.RESULTSA total of 4015 proteins were quantified with high confidence and 361 proteins were significantly changed，compared to saline control groups.Among them，CDK5（cyclin-dependent kinase 5）and RhoB（ras homolog family member B）were up-regulated in rats with a history of extended access to heroin.Functionally，inhibition of CDK5 in the DH en⁃hanced heroin-seeking and craving，indicating that CDK5 signaling in the DH acts as homeostatic compen⁃satory mechanism to limit heroin-seeking and craving，whereas blockade of the Rho-ROCK pathway attenuated context-induced heroin relapse，indicating that RhoB sig⁃naling in the DH is required for the retrieval（recall）of ad⁃diction memory.CONCLUSlONOur findings suggest that manipulation of CDK5 signaling in the DH may have key roles in determining vulnerability to opiate craving，whereas manipulation of RhoB signaling in the DH may have critical roles in determining vulnerability to relapse. Overall，the present study suggests that the DH can ex⁃ert dissociative effects on heroin addiction through CDK5 and RhoB signaling.

Key words：CDK5；dorsal hippocampus；heroin self-ad⁃ministration；label-free proteomic analysis；RhoB；cen⁃trifugal proteomic reactor

Foundation item：The project supported by Ministry of ScienceandTechnologyofChina（2013CB835100；2015CB553502；2013ZX09507001）；National Natural Science Foundation of China（81130087；91232716；21375138）；Committee of Science and Technology of Shanghai（13JC140680）；and″One Hundred Talent Pro⁃gram″of Chinese Academy of Sciences

Corresponding author：LIU Jing-gen，E-mail：jgliu@ mail.shcnc.ac.cn；ZHOU Hu，E-mail：zhouhu@simm.ac.cn

T3-15

Blockade of HCN channels in nucleus accumbens cho⁃linergic neurons preventscue-inducedrelapse of co⁃caine-seeking

ZHANG Jing-liang，HUANG Zhuo（Department of Molecular and Cellular Pharmacology，State Key Laboratory of Nature and Biomimetic Drugs，Peking University School of Pharmaceutical Sciences，Beijing 100191，China）

Abstract：OBJECTlVETo study the relationship between HCN channels of nucleus accumbens（NAc）cholinergic interneurons（CINs）and addiction relapse. METHODSThe SD rats were used to establish cue-in⁃duced addiction relapse models.The cell-attached re⁃cordings were used to study the spontaneous firing fre⁃quency of CINs in nucleus accumbens（NAc）on coronal accumbens brain slices（350 μm），and the whole-cell voltage-clamp recordings were used to evaluate the func⁃tional current of CINs hyperpolarization activated，cyclic nucleotide-gated（HCN） channels（Ih） and AMPA/ NMDA ratio of medium spiny neurons（MSNs），which are the principal neurons of NAc.RESULTSCellattached recordings revealed that there was a（96.70± 31.23）%increase in spontaneous firing frequency of NAc CINs from（1.96±0.33）HZ（Naïve group，n=17 cells）to（3.85±0.61）HZ（relapse group，n=12 cells，P＜0.01）.Actually，HCN channel is the major contributor to spontaneous firing of CINs and specific HCN channels blocker，ZD7288（15 μmol·L-1），restored the increased fir⁃ing frequency〔（1.89±0.46）HZ，n=12 cells，P＜0.01〕of CINs in vitro.The functional HCN current from NAc CINs was significantly increased when compared to control group，with the maximal activation current density in⁃creasing from（2.87±0.62）pA/pF（naïve group，n=4 cells）to（6.57±1.07）pA/pF（relapse group，n=4 cells，P＜0.05）.Furthermore，microinfusion ZD7288 to NAc core markedly decreased the AMPA/NMDA ratio（1.74±0.18，n=21 cells）of MSNs compared with NS-treated group （2.77±0.42，n=18 cells）in relapse rats（T=15 min），and prevented cue-induced relapse of cocaine-seeking in vivo. CONCLUSlONEnhancedfunctionalHCNchannelmediated currents increased spontaneous firing frequency in NAc CINs，thus enhanced AMPA/NMDA ratio in MSNs，thereby facilitated cue-induced cocaine relapse invivo.Therefore， ourresultsindicatethatHCN channels in NAc CINs play crucial role in addiction relapse and could be apotential target for addiction treatment.

Key words：HCN channels；nucleus accumbens；cholin⁃ergic interneurons；cocaine；relapse；AMPA/NMDA ratio；medium spiny neurons

Foundation item：The project supported by 973 program （2015CB559200）；Ministry of Science and Technology of China（2014ZX09507003- 006- 004）； and National Natural Science Foundation of China（81371432）

Correspondingauthor： HUANGZhuo， E-mail：huangz@hsc.pku.edu.cn

T3-16

催产素调节甲基苯丙胺所致成瘾性记忆改变的表观遗传学研究

范鑫雨，侯 颖，孙 超，许栊文，杨静玉，吴春福

（沈阳药科大学药理学教研室，辽宁沈阳 110016）

摘要：目的 在甲基苯丙胺（METH）诱导的条件位置偏爱（CPP）形成阶段，从表观遗传学角度出发，探索催产素是否通过调节METH诱导的学习记忆改变进而发挥抑制METH成瘾的作用。方法 行为学检测包括条件位置偏爱和水迷宫，Day1-2小鼠适应CPP环境，Day3小鼠进行CPP前测并适应水迷宫环境，Day4-9进行CPP匹配与定位航行实验，Day10进行CPP检测以及空间探索实验。行为学结束后1 h，取脑并分离海马，采用Western蛋白质印迹法检测突出囊泡蛋白表达情况，采用液质联用技术检测基因组DNA甲基化程度。结果 METH能够诱导小鼠CPP的形成，改变小鼠在平台所在象限停留的时间，并影响突出囊泡蛋白表达量和基因组DNA甲基化的程度（P＜0.05，P＜0.01）；催产素能够显著抑制METH诱导的CPP形成，平台所在象限的停留时间、突出囊泡蛋白表达量和DNA甲基化的改变。结论催产素通过抑制METH诱导的基因组DNA甲基化改变，进而影响与学习记忆相关蛋白-突出囊泡蛋白的变化，并抑制METH诱导的小鼠空间记忆能力改变，最终抑制METH诱导的CPP形成。

关键词：催产素；甲基苯丙胺；学习记忆；表观遗传学

通讯作者：吴春福，E-mail：chunfuwu@gmail.com，Tel：（024）23986339

T4　脑中风与脑损伤

T4-1

MicroRNA-195 inhibits tau hyperphosphorylation induced by chronic brain hypoperfusion in rats

AI Jing

（Department of Pharmacology，Harbin Medical University，Harbin 150086，China）

Abstract：Chronic brain hypoperfusion（CBH）is considered as a preclinical condition of mild cognitive im⁃pairment and is thought to precede dementia.However，how CBH promotes dementia pathological feature is unclear.In our series studies，using a CBH rat model generated by bilateral common carotid artery occlusion （2VO），we explored the potential molecular mechanism at microRNA level.We observed that 2VO decreased the learning and memory ability in rats，as assessed by Morris water maze，and leads to tau hyperphosphorylation at Ser202/Thr205，Ser262，Thr231 and Ser422，upregulat⁃ed expression of APP and BACE1 proteins，increased Aβ generation，increased activation of Cdk5/p25，as well as PP2A inactivation in rat hippocampi.In recipro⁃cal，qRT-PCR analysis showed that microRNA-195（miR-195）was down-regulated in the hippocampi of rats follow⁃ing CBH，and in the plasma of dementia patients.Further study showed thatwhenendogenousmiR-195 was knocked down using overexpression of its antisense mole⁃cule（pre-AMO-miR-195）via a lentivirus（lenti-pre-AMO-miR-195），this knockdown elicited dementia in rats and in⁃creasedthetauphosphorylationatSer202/Thr205，Ser262，Thr231，Ser422，expression of APP and BACE1 proteins，the Cdk5/p25 activation as well as PP2A inactiva⁃tion.But over-expression of miR-195 using lenti-pre-miR-195 effectively prevents these phenomena.By dual Lucifer⁃ase reporter assay，we found that miR-195 could post-tran⁃scriptional regulates the expression of APP，BACE1，Cdk5/ p35 and methylesterase（PME-1）.Importantly，over-ex⁃pression of miR-195 using lenti-pre-miR-195 reduces de⁃mentiavulnerability and prevents tau hyperphosphorylation，amyloidogenesis，and activation of Cdk5/p25 and PP2A in⁃activation.Additionally， chromatinimmunoprecipitation analysis showed that NFκB was bound to the promoter re⁃gion of miR-195 and inhibited its expression.We conclude that miR-195 may play a key role in determining dementiasusceptibility in 2VO rats by inhibiting tau hyperphosphory⁃lation through the regulation of APP/BACE1/Aβ/Cdk5/p35 and APP/BACE1/Aβ/PME-1/PP2Apathway.Thus，exog⁃enous complement of miR-195 may be a potentially valu⁃able anti-dementia approach.

Key words：chronic brain hypoperfusion；dementia；miR-195；PP2A；tauhyperphosphorylation；Cdk5；APP；BACE1

Foundation item：The project supported by National NaturalScienceFoundationofChina（81471115；81271207；81070882）

Corresponding author：AI Jing，E-mail：aijing_86@aliyun.com

T4-2

PDE4抑制剂FCPR16对氧糖剥夺损伤后SH-SY5Y细胞的保护作用

陈佳佳，冯红芳，邹征强，徐江平

（南方医科大学药学院神经药理与新药发现课题组，广东广州510515）

摘要：目的 研究PDE4抑制剂FCPR16对氧糖剥夺（OGD）损伤后SH-SY5Y细胞的作用及可能的机制。方法预先用化合物FCPR16处理SHSY5Y细胞30 min，进行OGD损伤4 h后，复氧24 h。然后测定细胞存活率（CCK-8法）、细胞凋亡（TUNEL染色）；蛋白质免疫印迹检测细胞BCL-2，BAX，Cyt C和胱天蛋白酶3等凋亡相关蛋白表达，以及检测p-PKA和p-CREB蛋白表达。结果 OGD造模能显著减低SH-SY5Y细胞的存活率，增加细胞的凋亡。PDE4抑制剂FCPR16能明显提高OGD后细胞的存活率，减少细胞的凋亡。蛋白质免疫印迹检测证实，FCPR16能增加OGD后BCL-2/BAX蛋白的比值，减少Cyt c的释放，减少活化型胱天蛋白酶3蛋白的产生，上调p-PKA和p-CREB的表达水平。结论 PDE4抑制剂FCPR16对OGD损伤后SHSY5Y细胞具有明显的保护作用，其保护机制可能与激活p-PKA/p-CREB通路，调节相关凋亡蛋白表达，发挥抗凋亡作用有关。

关键词：PDE4抑制剂；FCPR16；氧糖剥夺损伤；细胞凋亡

基金项目：国家自然科学基金项目（81503043；81373384）；粤科规财字［2015］150号

通讯作者：徐江平，E-mail：jpx@smu.edu.cn

T4-3

Complement C3 induces ischemic neuronal injury mediated by toll-like receptor in diabetic mice

HE Ping*， LIN Zheng*， LI Wen-lu， LIN Hao-ran，HUANG Yu-wen，DAI Hai-bin

（The Second Affiliated Hospital，Zhejiang University School of Medicine，Hangzhou 310009，China）

Abstract：OBJECTlVE Complement component C3 （C3），a key factor in the complement system，is heavily involved in various inflammation-associated diseases. However，it remains obscure for its role in the pathogenesis of ischemic brain injury in diabetes.METHODS Middle cerebral artery occlusion （MCAO）-induced ischemic stroke in diabetic mice and cultured cortical cerebral neuron were used this study.RESULTS Increased C3 activity was associated with ischemic cortical neuronal injury in diabetes，while deficiency of complement C3 attenuated ischemic cortical neuronal injury.Furthermore，C3 pro⁃moted ischemia-induced neuronal cell death through acti⁃vation of TLR2 and TLR4.Notably，complement C3/C3a recruited and bound to TLR2 and 4 complexes after brain ischemia.In addition，C3 directly induced the ex⁃pression of TLR2 and TLR4 in vivo and in vitro through NF-κB signaling pathways as evaluated by the phosphor⁃ylation of key pathway components p65.Finally，system⁃ic administration of a complement C3 inhibitor protected against MCAO-induced stroke in diabetic condition.CON⁃CLUSlON Complement C3 promotes ischemic brain injury in diabetes，and warrants further exploration as a novel target for therapeutic intervention in diabetic stroke.

Key words：complement C3；ischemia；MCAO；Tolllike receptors；NF-κB；diabetic mice

Foudation item：The project supported by National NaturalScienceFoundationofChina（81173040；81302747；81373391；81471395；81573402）

Corresponding author：DAI Hai-bin，E-mail：haibindai@ zju.edu.cn *Co-first author

T4-4

长时程增强在脑卒中的研究进展

余 汇，程玉芳，徐江平

（广东省新药筛选重点实验室，南方医科大学药学院，广东广州510515）

摘要：脑卒中（stroke）是一种以猝然昏倒、不省人事，伴发口角歪斜、语言不利而出现半身不遂为主要临床症状的脑血管疾病。医学界把它同冠心病、癌症并列为威胁人类生命健康的三大疾病，具有高发病率、高致残率、高死亡率的特点，给社会和家庭带来了沉重的负担。2012年我国卫生部公布的引起死亡的因素中，脑卒中在城市中居于第三位，在农村中居于第二。目前针对脑卒中的治疗仍然以挽救卒中周边未死亡的神经细胞为主。突触可塑性在脑卒中病人自我恢复中起重要的作用，突触传递的长时程增强（long term potentiation，LTP）是突触可塑性的功能性指标之一，也是研究学习记忆的理想模型。LTP的形成与突触前递质的释放和突触后离子通道以及相应的蛋白激酶等密切相关。通过脑卒中后LTP的变化研究突触可塑性产生的机制，并采取相应的措施调节突触的可塑性，可能会对脑卒中的治疗和预后产生极大的作用。本文就全脑缺血损伤机制以及LTP在脑卒中形成的可能机制，包括脑卒中后相应的受体、蛋白激酶、逆行信使等的变化以及突触和树突结构变化与LTP形成的关系作一综述，还涉及通过调节LTP而影响脑卒中预后的相关研究进展。

关键词：脑卒中；突触可塑性；长时程增强

基金项目：国家自然科学基金项目（81301099；81373384；81503043）

通讯作者：徐江平，E-mail：jpx@smu.edu.cn，Tel：（020）61648236

T4-5

星形胶质细胞对话神经元代谢

陆佳彤1，顾新华2，谢 娴1，卢韵碧1，唐 淳2，张纬萍1

（1.浙江大学医学院药理系，浙江杭州 310058；2.中国科学院武汉物理与数学研究所，湖北武汉 430071）

摘要：目的 探索神经元内代谢水平降低时，星形胶质细胞对神经元代谢的补偿。方法 ①小鼠侧脑室注射尼克酰胺磷酸核糖转移酶（NAMPT）特异性抑制剂FK866（1 nmol·L-1，1 μmol·L-1，7 mL），以抑制神经元代谢水平。给药不同时间后，采用ELISA检测脑内尼克酰胺腺嘌呤二核苷酸（NAD）水平的变化，利用Western蛋白印迹法及GFAP免疫荧光染色，观察星形胶质细胞的激活情况，以自发活动观察小鼠活动量以及旷场探索的能力。测脑室给药后7 d，腹腔注射稳定同位素13C标记的乳酸或醋酸，采集核磁共振氢谱和碳谱检测分别测定神经元、星形胶质细胞的代谢变化，以及细胞间谷氨酸-谷氨酰胺循环变化。结果 FK866 1 nmol·L-1和1 μmol·L-1的均能够抑制小鼠脑内NAD水平，侧脑室注射1 nmol·L-1FK866后2 d，小鼠脑内NAD水平恢复正常，而注射1 mmol·L-1FK866的小鼠，NAD水平降低可持续10 d以上；注射后7 d，Western蛋白印迹法及免疫荧光显示，1 nmol·L-1及1 μmol·L-1FK866注射均可导致GFAP表达量上升，显示星形胶质细胞激活。合用烟酰胺单核苷酸（NMN）可以逆转FK866激活星形胶质细胞的作用。核磁共振氢谱结果显示，1 nmol·L-1FK866注射7 d后，脑内谷氨酸、谷氨酰胺等代谢产物水平增加，碳谱结果星形胶质细胞和神经元代谢均增强，神经元内谷氨酰胺向谷氨酸的转变增加，星形胶质细胞内谷氨酸向谷氨酰胺的转变降低；而1 mmol·L-1FK866对脑内的代谢物水平无明显影响，但使神经元利用乳酸的水平增加。注射1 nmol·L-1FK866可降低小鼠的活动度，但增加小鼠对旷场中心区的探究行为。

结论 神经元代谢受到一过性抑制，诱导星形胶质细胞激活，可补偿神经元代谢，这种代谢可引起小鼠神经行为变化。

关键词：尼克酰胺磷酸核糖转移酶；尼克酰胺腺嘌呤二核苷酸；神经代谢；谷氨酸-谷氨酰胺循环

基金项目：国家重点基础研究发展计划（973计划）项目（2013CB910200）；国家自然科学基金（81573400；31225007）；浙江省自然科学基金（LY14H310005）

通讯作者：张纬萍，E-mail：weiping601@zju.edu.cn，Tel：13757179548

T4-6

细胞水平研究G蛋白偶联受体GPR17同源二聚体相互作用

蒋文学2，姜静2，阳雨虹2，陆佳彤1，卢韵碧1，魏尔清1，唐淳2，张纬萍1

（1.浙江大学医学院药理系，浙江杭州 310058；2.中国科学院武汉物理与数学研究所，湖北武汉 430071）

摘要：目的 建立活细胞标记G蛋白偶联受体（GPCR）的方法，采用荧光寿命成像（FLIM）结合荧光共振能量转移（FRET）分析GPR17（一种P2Y/CysLT受体）同源二聚体相互作用。方法 建立基于切割绿色荧光蛋白（split GFP，sGFP）的GPCR标记方法，以sGFP分别标记到GPR17细胞外3个loop，或以GFP融合到GPR17的N端或细胞内第3个loop，以mcherry融合到GPR17的N端或细胞内第3个loop，以sGFP或GFP作为FRET的供体，以mcherry作为FRET的受体，采用FLIM-FRET方法分析不同标记供体和受体之间的绝对距离，基于距离约束，使用结构预测软件ITASSER预测获得GPR17单体的结构，然后使用Xplor-NIH基于距离约束和单体结构，进一步计算研究GPR17同源二聚体之间的相互作用。结果 将GFP的第10～11 β片段（GFP10～11）作为标签，采用基因工程的方法插入到GPR17的细胞外不同loop上，合成并纯化GFP，采用酶切的方法获得带成熟荧光基团的含1～9 β片段（GFP1～9），将携带GFP10～11标签的GPR17转到HEK293细胞表达，在活细胞水平获得快速的（＜5 min）、高度特异的细胞表面标记。FLIM-FRET检测显示，GPR17-N-GFP单转时GFP的寿命为2.143 ns，与GPR17-N-mcherry共转时为2.033 ns，GPR17-K255GFP单转时GFP的寿命为2.096 ns，与GPR17-K255mcherry共转时为2.047ns，GPR17-R291sGFP单转时sGFP的寿命为2.076 ns，与GPR17-N-mcherry共转时为1.744 ns，GPR17-R214sGFP单转时sGFP的寿命为1.923 ns，与GPR17-N-mcherry共转时为1.761 ns。根据GFP或sGFP荧光寿命的变化，我们获得了距离的信息，采用距离约束，构建了GPR17的同源二聚模型。结论GPR17能够形成同源二聚体，构建的模型可以指导突变，以促进或抑制二聚体形成，来研究同源二聚体的生物学功能，同时类似标记及FLIM-FRET方法可以用于其他GPCRs同源、异源二聚体的研究。

关键词：GPR17；切割绿色荧光蛋白；GPCR；同源二聚体

基金项目：国家重点基础研究发展计划（973计划）项目（2013CB910200）；国家自然科学基金（31225007；81573400；81173041；81273491）

通讯作者：张纬萍，E-mail：weiping601@zju.edu.cn，Tel：13757179548

T4-7

NAMPT介导脑缺血后小胶质细胞激活的作用及机制

陈晨祥，田雨鑫，陆佳彤，谢 娴，张纬萍，卢韵碧

（浙江大学医学院药理学系，浙江杭州 310058）

摘要：目的 观察小胶质细胞诱导表达尼克酰胺磷酸核糖转移酶（NAMPT）及NAMPT对小胶质细胞激活的影响，并探讨NAMPT介导小胶质细胞激活的机制。方法 ①大鼠局灶性脑缺血再灌注处理（MCAO 1h/R 14 d）后，脑片免疫荧光染色观察NAMPT在小胶质细胞的表达；②以BV2细胞替代小胶质细胞，以缺糖缺氧再恢复（OGD/R）模拟脑缺血处理后，以PCR和ELISA分别检测细胞表达和释放NAMPT；③以重组人NAMPT蛋白模拟细胞释放的NAMPT，NAMPT蛋白单独或者合并TNF-α蛋白处理BV2细胞后，光镜下观察细胞形态变化；以ELISA检测细胞释放TNF-α，IL-6和NAMPT的量；以Western蛋白印迹法检测胞浆与核内NF-κB的水平变化；④以OGD 1 h/R 24 h诱导BV2细胞上调表达NAMPT，以NAMPT酶活性特异性抑制剂FK866处理细胞，观察BV2细胞的形态变化，以ELISA和Western蛋白印迹法分别检测TNF-α，NAMPT，IL-6释放量及胞浆与核内NF-κB的表达变化；⑤以FK866侧脑室注射预给药，大鼠局灶性脑缺血再灌注处理（MCAO 1 h/R 14 d）后，以行为学检测反映大鼠神经功能损伤/恢复情况，脑片免疫组化、免疫荧光染色观察神经元存活、星形胶质细胞及小胶质细胞激活情况。结果 ①大鼠MCAO 1 h/R 14 d后，缺血中心区及周边区小胶质细胞大量诱导表达NAMPT，而星形胶质细胞始终不表达NAMPT；②OGD 1 h/R 12-24 h使BV2小胶质细胞上调表达和释放NAMPT；③重组人NAMPT蛋白（450 μg·L-1）处理，导致BV2细胞形态变圆，细胞释放TNF-α和IL-6显著增多；NAMPT抗体（500 μg·L-1）明显抑制NAMPT诱导的BV2细胞释放TNF-α和IL-6；NAMPT蛋白（450 μg·L-1）合并TNF-α（10 μg·L-1）处理可诱导BV2细胞释放更多的IL-6；且NAMPT蛋白单独或者合并TNF-α处理后，细胞NF-κB上调表达并发生核移位；④FK866（0.001～1 nmol·L-1）可抑制OGD 1 h/R 24 h诱导的BV2细胞形态变圆及TNF-α，NAMPT和IL-6的释放；FK866 1 nmol·L-1可抑制OGD1 h/R 24 h诱导BV2细胞NF-κB的表达上调和核移位；⑤FK866预给药可改善局灶性脑缺血（MCAO 1 h/R 14 d）所致的大鼠神经功能下降，减小脑梗死体积，提高神经元存活率，抑制胶质疤痕形成，抑制小胶质细胞激活和增殖。结论 脑缺血后期小胶质细胞诱导表达并释放NAMPT，上调表达和释放的NAMPT均参与小胶质细胞的激活和炎症因子释放，其机制与NAMPT促进NF-κB活化有关；抑制NAMPT有抗脑缺血作用，机制与其抑制小胶质细胞激活相关。

关键词：脑缺血；神经炎症；小胶质细胞；NAMPT；NF-κB

基金项目：国家自然科学基金（81573400；81173041）；浙江省自然科学基金（LY14H310005）

通讯作者：卢韵碧，E-mail：yunbi@zju.edu.cn

T4-8

缺糖缺氧诱导G蛋白偶联受体17上调表达后介导BV2小胶质细胞激活

陈晨祥，田雨鑫，陆佳彤，谢 娴，魏尔清，张纬萍，卢韵碧

（浙江大学医学院药理学系，浙江杭州 310058）

摘要：目的 探讨缺糖缺氧再灌注处理（OGD/R）后BV2小胶质细胞上调表达GPR17对细胞形态及功能的影响。方法 以GPR17siRNA或GPR17全长cDNA质粒敲入干预GPR17基因表达，以5-脂氧酶抑制剂抑制LTD4的合成和UDP分解酶促进UDP分解；以OGD/R模拟体外脑缺血处理BV2小胶质细胞后，以PCR和Western蛋白质印迹法检测BV2小胶质细胞表达GPR17的情况，光镜下观察细胞形态变化，MTT检测细胞活性变化及以ELISA检测细胞释放TNF-α，IL-1β和NO的变化。结果 ①在无OGD/R处理的条件下，以GPR17的配体LTD4和UDP处理BV2小胶质细胞，细胞活性和细胞释放炎症因子均无明显变化；②以OGD 1 h/R 48 h处理BV2细胞，GPR17siRNA预处理下调表达GPR17或抑制LTD4的合成、促进UDP分解预处理后，可抑制OGD/R诱导的细胞活性增高和细胞释放TNF-α，IL-1β和NO增多；③以OGD 1 h/R 48 h处理BV2细胞或直接以GPR17全长cDNA质粒敲入BV2细胞导致GPR17过表达后，再加用GPR17的配体LTD4和UDP，则可促进细胞释放上述炎症因子。结论 缺糖缺氧诱导G蛋白偶联受体17上调表达后可介导BV2小胶质细胞激活。

关键词：缺糖缺氧；BV2小胶质细胞；GPR17

基金项目：国家自然科学基金（81273491；81173041）；浙江省自然科学基金（LY14H310005）

通讯作者：卢韵碧，E-mail：yunbi@zju.edu.cn，Tel：（0571）88208223

T4-9

Neuroprotectiveeffectsofmitophagyagainst cerebral ischemia and its regulatory mechanisms

ZHANGXiang-nan，YUANYang，SHENZhe，ZHENG Yan-rong，WU Xiao-li，HU Wei-wei，CHEN Zhong （Department of Pharmacology，Key Laboratory of Medical Neurobiology of The Ministry of Health of China，College ofPharmaceuticalSciences， ZhejiangUniversity，Hangzhou 310058，China）

Abstract：Mitochondria homeostasis is critical for neuronal survival.In ischemic brains，damaged mito⁃chondria lead to neuronal cell death.Mitochondrial au⁃tophagy，mitophagy，is an intracellular process by deliv⁃ering damaged or aged mitochondria to lysosomes for degradation.Mitophagy deficiency has been linked with several neurodegenerative disorders.However the contri⁃butions of mitophagy in cerebral ischemia were not fully understood.We previously found that mitophagy was ac⁃tivated in reperfusion but not ischemia phase in ischemic stroke models.We further found that ischemia-reperfu⁃sion（I-R）-induced mitophagy cleared the damaged mito⁃chondria in a PARK2-involved manner，and thus re⁃duced mitochondria-dependent cell apoptosis.Interest⁃ingly，we found that two endoplasmic reticulum（ER）stressors，tunicamycin（TM）and thapsigargin（TG），showed significant protection against ischemia-induced brain injury in their relative low dosages.In addition，both TM and TG promoted PARK2 recruitment to mito⁃chondria and reinforced I-R-induced mitophagy.The neu⁃roprotection of TM and TG was reversed by Park2 silenc⁃ing.To further identify the molecular mechanisms under⁃lying I-R-induced mitophagy，we focused on the mitopha⁃gy receptor Nix.Nix-/-mice showed mitophagy deficien⁃cy and aggravated brain infarct after I-R injury，which can be rescued by AAVs expressing Nix.Interestingly，the Park2 deletion did not diminish Nix-mediated mitopha⁃gy in ischemic brains.Both Park2 and Nix knockout showed synergetic mitophagy deficiency in ischemic neu⁃rons，suggesting Nix may execute mitophagy in a dis⁃tinct biochemical pathway from Park2.Taken together，our investigations suggested that reinforcing mitophagy can be a novel strategy to rescue ischemic brain injury.

Key words：cerebral ischemia；mitophagy；ER stress；Park2；Nix

Foundation items：The project supported by National NaturalScienceFoundationofChina（81102429；81373393）；and Zhejiang Leading Team of S&T Innova⁃tion（2011R50014）

Correspondingauthor：CHEN Zhong，E-mail：chenzhong@ zju.edu.cn

T4-10

MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ 2 protein coding region in rat with chronic brain hypoperfusion

YAN Mei-ling，SUN Li-hua，AI Jing

（Department of Pharmacology，Harbin Medical University，Harbin 150086，China）

Abstract：OBJECTlVETo examine the role of microRNA-9（miR-9）in regulating Nav1.1/Nav1.2 traffick⁃ingunder chronic brain hypoperfusion（CBH）generated bybilateralcommoncarotidarteryocclusion（2VO）. METHODS ①Bilateral common carotid artery occlusion （2VO）was used to establish the animal model ofCBH；②qRT-PCR was used to detect the level of miR-9 and SCN2B in hippocampi and cortices of rats following CBH or other treatments；③Western blot and immunofluores⁃cence were chosen to detect the expression of Nav1.1，Nav1.2 and Navβ2.④Transfecting miR-9 mimics and/or its inhibitor（AMO-miR-9）into the cultured primary neuron cells to detect whether miR-9 can regulate Nav1.1/Nav1.2 trafficking by targeting Navβ2 in vitro；⑤Luciferase report⁃er assay and/or miR-maskingtechnology were performed to identify the specific regulational effect of miR-9 on Navβ 2；⑥Injecting lenti-pre-miR-9 and/or lenti-pre-AMO-miR-9 were stereotaxic injected into the hippocampi of rats de⁃tect whether miR-9 can regulate Nav1.1/Nav1.2 traffick⁃ing by targeting Navβ2 in vivo.RESULTS The impair⁃ment of Nav1.1/Nav1.2 trafficking and decreased expres⁃sion of Navβ2 was found in the hippocampi and cortices of rats following CBH generated by bilateral 2VO.MiR-9 was increased in both the hippocampi and cortices of rats following CBH by qRT-PCR.Intriguingly，miR-9 sup⁃pressed，while AMO-miR-9 enhanced，the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane.Fur⁃ther study showed that overexpression of miR-9 inhibited the Navβ2 expression by targeting on its coding se⁃quence（CDS）domain by dual luciferase assay.Howev⁃er，binding-site mutation or miR-masks failed to influ⁃ence Navβ2 expression as well as Nav1.1/Nav1.2 traffick⁃ing process，indicating that Navβ2 is a potential target for miR-9.Lentivirus-mediated miR-9 overexpression al⁃so inhibited Navβ2 expression and elicited translocation deficits to cell membrane of Nav1.1/Nav1.2 in rats，whereas injection of lentivirus-mediated miR-9 knock⁃down could reverse the impaired trafficking of Nav1.1/ Nav1.2 triggered by 2VO.CONCLUSlON We conclude that miR-9 may play a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Navβ2 pro⁃tein in 2VO rats at post-transcriptional level，and inhibi⁃tion of miR-9 may be a potentially valuable approach to prevent Nav1.1/Nav1.2 trafficking disturbance induced by CBH.

Key words：microRNA-9；chronic brain hypoperfusion；sodium channel

Foundation item：The project supported by National NaturalScienceFoundationofChina（81121003；81371211；81000499；81271207；81471115）；Postdoc⁃toral Science Foundation of China（2012 T50376）；and Postdoctoral Science Foundation of Heilongjiang prov⁃ince（LBH-Q13108；LBH-z13155）

Corresponding author：AI Jing，E-mail：aijing_86@ aliyun.com

T4-11

miRNA let-7c-5p通过抑制小胶质细胞的激活对缺血性脑损伤的保护作用及其机制研究

倪敬书*，王小玉*，陈霜霜，贾佳，镇学初

（苏州大学药学院，江苏苏州 215021）

摘要：目的 研究脑内高表达的microRNAlet-7c-5p对脑缺血再灌注损伤的保护作用，探究其保护作用的机制。方法 用线栓法建立小鼠局暂性大脑中动脉栓塞模型，侧脑室定位注射let-7c-5p类似物实现其过表达。体外培养原代小胶质细胞和BV-2细胞，建立体外缺糖缺氧（OGD）再灌注模型，以LPS和原代神经元OGD上清刺激原代小胶质细胞和BV-2诱导炎症反应。分别采用Western蛋白印迹法和实时qPCR检测炎症因子和let-7c-5p的表达；免疫组化法检测小胶质细胞的激活情况；采用荧光素酶报告基因检测let-7c-5p的直接靶点。结果 和阴性对照组相比，过表达let-7c-5p组，显著减少梗死体积；抑制损伤区小胶质细胞的激活；降低半暗带iNOS，TNF-α和IL-6的mRNA和蛋白表达水平。体外实验结果近一步表明，过表达let-7c-5p可以抑制各种炎症介质的mRNA和蛋白的表达；降低胱天蛋白酶3 的mRNA和蛋白表达水平；可以降低共转染野生型胱天蛋白酶3 HEK 293细胞的荧光强度。结论 过表达let-7c-5p对缺血再灌注损伤具有保护作用，直接靶向胱天蛋白酶3抑制小胶质细胞激活可能是这种保护作用的机制之一。

关键词：miRNA；let-7c-5p；脑缺血；小胶质细胞

基金项目：国家自然科学基金（81130023；81373382；81371278；81471336）；国家“973”项目

通讯作者：贾 佳，E-mail：jiajia@suda.edu.cn，镇学初，E-mail：zhenxuechu@suda.edu.cn

*共同第一作者

T4-12

3′-大豆苷元磺酸钠对脑缺血/再灌注损伤损伤大鼠线粒体和血脑屏障的影响

袁 娲*，陈 琴*，肖 海，曾靖

（赣南医学院，江西赣州 341000）

摘要：目的 研究3′-大豆苷元磺酸钠（DSS）对脑缺血再灌注损伤大鼠线粒体和血脑屏障的影响。方法 采用MCAO方法制备脑缺血再灌注损伤模型，观察DSS对脑缺血再灌注损伤后大鼠线粒体肿胀度；线粒体膜电位；线粒体抗氧化；以及血脑屏障结构及其通透性的影响。结果 DSS能显著降低脑缺血再灌注损伤后大鼠线粒体的肿胀度（P＜0.05，P＜0.01）；能显著升高脑缺血再灌注损伤后大鼠线粒体膜电位（P＜0.05，P＜0.01）；能显著升高脑缺血再灌注损伤后线粒体组织中SOD和GSH-Px的活性（P＜0.05，P＜0.01）；能显著降低脑缺血再灌注损伤后线粒体MDA含量（P＜0.05，P＜0.01）；可显著提高血脑屏障的完整性及改善血脑屏障通透性。结论 DSS对大鼠脑缺血再灌注损伤后线粒体功能具有显著的保护作用，能降低线粒体肿胀程度和升高线粒体膜电位有，同时DSS还可显著保护脑缺血/再灌注损伤大鼠引发的血脑屏障损伤作用。其对线粒体功能的保护作用可能与改善缺血脑组织能量代谢和抗氧化作用有关。

关键词：3′-大豆苷元磺酸钠；脑缺血再灌注损伤；线粒体；抗氧化；血脑屏障

基金项目：国家自然科学基金（81160399；81560583）；江西省高等学校科技落地计划项目（KJLD13085）；江西省教育厅科技项目（GJJ12560）

通讯作者：曾 靖，E-mail：zengjing61@hotmail.com，Tel：13879769873；肖 海，E-mail：xh669168@sina.com，Tel：13576675898

*共同第一作者

T4-13

以炎症为靶点治疗慢性缺血后的脑白质损伤

胡薇薇，周怡亭，张 菁，马 婧，陈 忠

（浙江大学医药学部药理系，神经科学研究中心，浙江杭州310058）

摘要：大脑长期慢性缺血将可能导致脑白质损伤及认知功能损害，造成皮层下缺血性血管性痴呆。目前临床上缺乏针对性的有效药物，因此急需针对其发病机制寻找减轻髓鞘丢失或促进髓鞘再生修复的治疗新靶点。课题组研究发现慢性缺血后髓鞘再生过程受到抑制，主要是因为脑室下区增殖的前体细胞OPC向白质区迁移过程受到阻碍，因而OPC迁移障碍是慢性缺血后髓鞘再生和白质修复的关键限速步骤。进一步发现，慢性缺血早期胶质细胞高表达炎症因子IL-1β，其受体拮抗剂（IL-1Ra）或基因敲除其受体（IL-1R1 KO）可以促进脑室下区的OPC（逆转录病毒标记）向脑白质区迁移的过程，但对OPC的增殖没有影响；而在IL-1R1 KO小鼠上，通过腺病毒介导的IL-1R1过表达可以逆转OPC迁移的增多。并且发现阻断IL-1β受体IL-1R1后可以促进髓鞘再生、神经传导功能恢复及改善认知功能。该工作提示控制炎症反应、促进OPC的迁移可能是减轻慢性缺血引起的白质损伤的重要途径，而IL-1β及其受体IL-1R1是重要的药物干预靶点。同时IL-1β的多肽类似物KdPT可以在慢性缺血后进入脑内促进OPC的迁移，从而促进白质修复和改善认知功能。此外，小胶质细胞抑制剂米诺环素在慢性缺血早期应用可显著减轻白质损伤和认知功能损害，其机制主要是通过减少成熟少突胶质细胞凋亡、促进脑室下区OPC的增殖从而促进髓鞘再生过程，为二次开发“老药”米诺环素提供了重要的实验依据。以上研究通过针对胶质细胞介导的炎症反应，发现多个促进慢性缺血后白质修复的新靶点，为皮层下缺血性血管性痴呆治疗提供了新途径。

关键词：慢性缺血；白质损伤；少突胶质前体细胞；髓鞘再生；炎症

通讯作者：陈 忠，E-mail：chenzhong@zju.edu.cn，Tel：（0571）88208228

T4-14

组胺H3受体拮抗剂通过促进神经再生发挥脑外伤后神经保护作用

廖儒佳1，王 露1，周怡亭1，蒋 磊2，胡薇薇2，陈 忠1

（浙江大学1.药学院，2.医学院，浙江杭州 310058）

摘要：目的 目前针对脑外伤后的神经损伤缺乏针对性的治疗手段，已有研究发现组胺具有神经保护作用，而组胺H3受体拮抗剂可以提高脑内组胺水平，因此本课题将研究H3受体拮抗剂对脑外伤的作用及与神经再生相关的作用机制。方法 C57BL6小鼠、H3受体敲除小鼠（H3RKO）、组氨酸脱羧酶基因敲除小鼠（HDCKO）进行液氮局部冻伤或控制性皮质撞击的脑外伤手术，术后给予H3受体拮抗剂Thiop⁃eramide或A331440、H3受体激动剂Immepip，组氨酸脱羧酶抑制剂αFMH和H1受体拮抗剂Pyrilamine或H2受体拮抗剂Cimetidine。术后进行运动功能评价，检测梗死体积、SVZ和SGZ区域神经前体细胞的数量及细胞增殖情况。采用逆转录病毒标记SVZ区和SGZ区的新生细胞，检测迁移到损伤周边的新生细胞的数量及分化情况。结果 在两种脑外伤模型中，H3受体拮抗剂Thioperamide或A331440显著减少脑外伤后期的梗死面积，改善其运动功能，该作用可被Immepip逆转，并且在H3RKO小鼠上不存在。Thioper⁃amide能显著增加神经前体细胞数量，此作用可被组氨酸脱羧酶抑制剂αFMH和H1受体拮抗剂Pyrilamine逆转，但不能被H2受体拮抗剂Cimetidine逆转，同时在组胺合成酶基因敲除小鼠HDCKO上Thioperamide不能促进神经前体细胞数量的增加，表明H3受体拮抗剂可以通过提高组胺水平作用于H1受体促进神经再生。进一步发现Thioperamide不影响神经干细胞的增殖而可促进神经前体细胞的分化。并且，Thioperamide还能增加损伤周边区的逆转录病毒标记的新生细胞数量，这些新生细胞可进一步分化为有功能的神经元。结论 组胺H3受体拮抗剂在脑外伤后期通过促进神经再生发挥神经保护作用，为脑外伤的治疗提供了新的治疗途径。

关键词：组胺H3受体拮抗剂；组胺；脑外伤；神经再生；神经保护

基金项目：国家自然科学基金（81273490；81473186）

通讯作者：陈 忠，E-mail：chenzhong@zju.edu.cn，Tel：（0571）88208228

T4-15

高或低血糖在急性灌注早期通过增加梗死区脑糖水平加重脑卒中损伤

张 帅，陈乃宏

（天然药物活性物质与功能国家重点实验室，中国医学科学院北京协和医学院药物研究所，北京 100050）

摘要：目的 从脑糖水平的变化解释再灌注早期血糖的波动对脑卒中损伤的影响。方法 采用线栓栓塞大脑中动脉诱发大脑局灶性脑缺血模型（MCAO）模拟缺血性脑卒中，在再灌注前5 min单次腹腔注射0，0.5，1，1.5或2 g·kg-1葡萄糖剂量诱导再灌注早期血糖的波动。通过TTC染色、神经功能学评分及升高肢体摆动测试对再灌注24 h和28 d后大鼠的脑损伤进行评价，并检测再灌注24 h和28 d后不同脑区脑糖水平的变化。结果 给予各剂量葡萄糖后，再灌注时血糖波动在5～15 mmol·L-1，并持续2 h。大鼠梗死面积的统计及行为学评价结果表明：血糖波动在6～10 mmol·L-1时可使再灌注24 h和28 d后脑损伤显著低于血糖大于10 mmol·L-1和血糖小于6 mmol·L-1的情况。在高血糖（＞10 mmol·L-1）和低血糖（＜6 mmol·L-1）的影响下，缺血侧皮层及纹状体的脑糖水平显著增加。高葡萄糖水平会增加氧糖剥夺后脑片组织的损伤。结论 再灌注早期血糖的升高或降低会加重梗死区葡萄糖的聚积，葡萄糖水平的升高会进一步加重卒中后损伤。

关键词：血糖；脑糖；缺血性脑卒中

通讯作者：陈乃宏，E-mail：chennh@imm.ac.cn，Tel：（010）63165177

T4-16

Neuroprotective role and its mechanism of lMM-H004 in transient global cerebral ischemia/reperfusion

ZHANG Zhao1，CHU Shi-feng2，CHEN Nai-hong1，2

（1.Institute of Materia Medica，Chinese Academy of Medical Sciences and Peking Union Medical College，Beijing 100050，China；2.Hunan University of Chinese Medicine，Changsha 410208，China）

Abstract：OBJECTlVE To investigate the neuropro⁃tective effect and its mechanism of IMM-H004，a 3-piper⁃azinylcoumarin compound derived from coumarin，after transient global ischemia/reperfusion（TGCI/R）.METHODS Adult male SD rats were treated with IMM-H004 immedi⁃ately after TGCI/R.Nissl staining and Fluoro-Jade B stainingwere used to test the delayed neuronal death（DND）in the CA1 of hippocampus and cortex.RT-PCR，western blot were used to test the expression of Survivin and HBXIP.Dual luciferase reporter assay was used to test the activition mechanism of Survivin.Cell viability，mitochondrial membrane potential and Co-IP assay were employed to test the role of HBXIP and Survivin interac⁃tion after I/R.RESULTS Here we confirmed that treat⁃ment of rats with IMM-H004 immediately after TGCI/R ameliorated delayed neuronal death（DND）in the CA1 of hippocampus and cortex.Further study suggested that IMM-H004 contributed to the expression of anti-apoptotic protein Survivin through the activation of PI3K dependent Protein Kinase B（PKB/Akt），which led to the phosphory⁃lation of forkhead box O1（FoxO1），then relieved the in⁃hibiting effect on Survivin promoter.Additionally，IMMH004 also enhanced the expression of hepatitis B X-inter⁃acting protein（HBXIP），which formed a complex with Survivin to prevent the activation of Caspase death cas⁃cade，thereby halting apoptotic cell death.Finally，injec⁃tion of HBXIP siRNA into hippocampus followed by micro⁃electroporationbeforeischemia/reperfusionabolished the protective effect of IMM-H004.Further study revealed that HBXIP maintained the high expression of Akt and Survivin.CONCLUSlON Our findings demonstrated that DND after TGCI/R was alleviated by IMM-H004 through promoting the formation of Survivin-HBXIP complex，which further emphasized the importance of endogenous protein involved in self-repair after stroke.

Key words：IMM-H004；transient global cerebral isch⁃emia/reperfusion；self-repair after stroke；survivin；HBXIP

Foundation item：The project supported by National NaturalScienceFoundationofChina（81274122）；National Key Sci-Tech Major Special Item（2012ZX09301002-004）；Beijing Natural Science Foundation（7131013；7142115）；and Beijing Key Laboratory of New Drug MechanismsandPharmacologicalEvaluationStudy （BZ0150）

Corresponding author：CHEN Nai-hong，E-mail：chennh@imm.ac.cn

T4-17

泛素连接酶RNF2通过与MANF相互作用改善脑缺血诱导的神经元损伤

沈玉君1，2，陈 滢2，杨 文2，沈玉先1，2

（安徽医科大学1.基础医学院，2.生物药物研究所，安徽合肥230032）

摘要：目的证明泛素连接酶RNF2与中脑星形胶质细胞源性神经营养因子（MANF）的相互作用及其对缺血性脑损失的保护作用。方法 用酵母双杂交系统筛选与MANF相互作用的蛋白；用Co-IP、Pull-down及免疫荧光双标等方法验证MANF与RNF2的相互作用；采用闭塞大脑中动脉（MCAO）建立大鼠局灶性脑缺血再灌注模型，用抗NeuN和抗GFAP抗体作为神经元和星形胶质细胞的标志蛋白与RNF2进行免疫荧光双标，检测RNF2在缺血再灌注损伤的大鼠脑组织中的表达和细胞分布，并将MANF与RNF2进行免疫荧光双标以分析它们的相关性；体外培养原代神经元，并用衣霉素（TM）处理，DAPI及PI染色检测神经细胞总数及死细胞数量，western蛋白印迹法检测MANF、RNF2、胱天蛋白酶3和CHOP等蛋白的水平；将MANF和RNF2的质粒或siRNA分别转染Neu2A细胞，观察MANF和RNF2表达的相互调节。结果 MANF和RNF2在缺血脑组织中的神经元和星形胶质细胞中表达均上调，且表达量和阳性细胞的类型及分布相似；RNF2与MANF存在相互作用，体外过表达RNF2可上调MANF的表达，沉默RNF2可降低MANF的表达；过表达RNF2可减少TM诱导的PI阳性神经细胞的数目，沉默RNF2增加PI阳性细胞数目；RNF2可抑制TM诱导的Neu2A细胞中胱天蛋白酶3的激活。结论 RNF2在缺血脑组织中的表达上调可能是适应性调节，其神经保护作用可能与MANF的相互作用有关。

关键词：泛素连接酶RNF2；神经营养因子；脑缺血；ER应激

基金项目：国家自然科学基金（81301060）

通讯作者：沈玉先，E-mail：shenyx@ahmu.edu.cn，Tel：（0551）65161121

T4-18

lL-4敲除可能是通过增加神经元兴奋性加重小鼠脑损伤

陈晓玲，王克威

（北京大学药学院，北京 100191）

摘要：目的研究白细胞介素4（IL-4）对小鼠局灶性脑缺血再灌注损伤（MCAO/R）的保护作用和可能的机制。方法动物水平采用小鼠大脑中动脉阻塞1.5 h，再灌24 h造模来研究野生型和IL-4敲除小鼠的差异，测定脑梗死体积，行为学评分和脑水肿。细胞水平采用氧糖剥夺30 min，复氧复糖24 h（OGD/R）模型来研究野生型和IL-4敲除小鼠的原代皮层神经元对缺氧缺糖损伤的敏感程度。机制研究采用Real-Time PCR检测MCAO/R后不同脑区IL-4和IL-4受体（IL-4R）的mRNA的表达情况；运用膜片钳记录野生型和IL-4敲除小鼠皮层神经元的兴奋性。结果①雄鼠中，与IL-4+/+相比，IL-4+/-组脑梗死体积增加，脑含水量升高；IL-4-/-组脑梗死体积增加，神经功能缺损评分加重（P＜0.01）；且无性别差异。②通过CCK-8检测细胞活力和测定细胞乳酸脱氢酶（LDH）的释放量反映神经元的损伤程度，与IL-4+/+相比，IL-4+/-组细胞活力下降27.3%，LDH释放增加25.9%；IL-4-/-组细胞活力下降19.5%，LDH释放增加28.0%（P＜0.05）。③与假手术组相比，模型组IL-4R显著增加，与对侧未缺血部位相比，缺血半暗带和缺血核心区脑组织的IL-4R的mRNA表达量分别增加3.9倍和6.3倍（P＜0.01）。④与IL-4+/+相比，IL-4-/-小鼠皮层神经元阈值下降（P＜0.05），兴奋性增加。结论IL-4敲除可能是通过增加神经元兴奋性加重小鼠脑损伤。

关键词：IL-4；IL-4R；OGD；MCAO；阈值

通讯作者：王克威，E-mail：wangkw@bjmu.edu.cn，Tel：（010）82805065

T4-19

Calpain依赖的ErbB4切割降解在脑缺血性神经元损伤中的作用

高银萍1，刘秀秀1，龚冬梅1，廖美华2，韩 峰2，卢应梅1

（1.浙江大学城市学院，浙江杭州 310015；2.浙江大学药学院药理毒理学研究所，浙江杭州 310058）

摘要：目的Neuregulin-1β/ErbB4信号通路在脑缺血过程中发挥重要作用，但其具体机制尚不明确，本研究详细探讨Neuregulin-1β/ErbB4信号通路在脑缺血引起的神经元损伤过程中的作用及其调节机制。方法体外培养小鼠皮层原代神经元，采用FACS法、TUNEL染色等观察糖氧剥脱（oxygen-glucose deprivation，OGD）造模后Neuregulin-1β （NRG-1β）对神经元凋亡率的影响。免疫荧光观察OGD造模后ErbB4表达量变化。利用ErbB4中间神经元特异性敲除鼠（PV-Cre；ErbB4loxP/loxP）和对照小鼠（ErbB4loxP/loxP），考察大脑中动脉梗塞（tMCAO）模型下ErbB4的变化，采用NRG-1β进行调节，观察缺血面积、神经元存活率。利用HEK293细胞考察Calpain抑制剂ALLN和ALLM对OGD模型下ErbB4降解片段的调控。HEK293细胞转染ErbB4质粒和ErbB4（E872K）突变体质粒，WB和FACS观察OGD造模后细胞凋亡损伤变化。结果OGD损伤刺激下的皮层原代神经元ErbB4表达下降，凋亡率升高，NRG-1β对该损伤具有保护作用。随MCAO造模时间增加，ErbB4表达量下降，缺血面积增加，外源性给予NRG-1β对此具有保护作用。但NRG-1β对ErbB4条件敲除小鼠无明显保护作用，提示NRG-1β是经由ErbB4信号通路减轻神经元缺血损伤。另外，ErbB2在体内MCAO模型和体外OGD刺激下无明显变化。而OGD条件下，HEK293细胞凋亡损伤伴随有ErbB4表达量下降，可被Calpain抑制剂ALLN的调控所抑制。转染ErbB4（E872K）突变体质粒相比转染野生型ErbB4，缺血损伤的凋亡率下降，提示Calpain参与介导的ErbB4切割降解参与缺血损伤。结论Neuregulin-1β/ErbB4信号通路在脑缺血病理过程中发挥保护作用，本研究发现Calpain抑制剂通过减少ErbB4的降解来保护神经元，为进一步开发治疗脑缺血相关疾病药物提供了实验依据和思路。

关键词：脑缺血；ErbB4；Neuregulin-1β；Calpain；切割降解

基金项目：国家自然科学基金（81202533）

通讯作者：卢应梅，E-mail：lufx@zju.edu.cn，Tel：（0571）88284325

T4-20

TlGAR contributes to ischemic tolerance induced by cerebral preconditioning through scavenging of reac⁃tive oxygen species and inhibition of apoptosis

ZHOU Jun-hao，ZHANG Tong-tong，SONG Dan-tan，XIA Yun-fei，QIN Zheng-hong，SHENG Rui

（Department of Pharmacology and Laboratory of Aging and Nervous Diseases，Soochow University School of Pharmaceutical Science，Suzhou 312000，China）

Abstract：OBJECTlVEPreviousstudyshowedthat TIGAR（TP53-induced glycolysis and apoptosis reg⁃ulator）protected ischemic brain injury via enhancing pen⁃tose phosphate pathway（PPP）flux and preserving mito⁃chondria function.This study was aimed to study the role of TIGAR in cerebral preconditioning.METHODSThe ischemic preconditioning（IPC）and isoflurane precondi⁃tioning（ISO）models were established in primary cul⁃tured cortical neurons and in mice.For OGD model，the cultured neurons were rinsed three times with Hepes bal⁃anced salt solution and placed in a chamber which filled with 95%N2 and 5%CO2at 37°C for 4 h.The neurons were incubated with NBM containing 10%B27，0.5 μmol·L-1glutamine and LV-shTIGAR（MOI=10）or LV-sh-scram⁃ble（LV-negative control，LV-NC）for 24 h，and then changed back to the regular medium.The shNC and shTIGAR groups contains con，ISO，OGD and ISO+ OGD groups.Cell Counting Kit-8 and LDH assay were used to determine cell viability.The neuronal NADPH and GSH were measured 3 h after reperfusion with the EnzyChrom NADP/NADPH assay kit and GSH Kit.Immu⁃noblotting was used to examine the protein expression of TIGAR，Bcl-2 and caspase-3.RESULTS Both IPC and ISO increased TIGAR expression in cortical neurons. Preconditioning might upregaulte TIGAR through SP1 transcription factor.Lentivirus mediated knockdown of TIGAR significantly abolished the ischemic tolerance induced by IPC and ISO.ISO also increased TIGAR in mice cortex and hippocampus and alleviated subsequent brain ischemia-reperfusion injury，while the ischemic tolerance induced by ISO was eliminated with TIGAR knockdown in mouse brain.ISO increased the produc⁃tion of NADPH and glutathione（GSH），and scavenged reactive oxygen species（ROS），while TIGAR knock⁃downdecreasedGSHandNADPHproductionand increased the level of ROS.Supplementation of ROS scavenger NAC and PPP product NADPH effectively rescue the neuronal injury caused by TIGAR deficiency. Notably， TIGARknockdowninhibitedISO-induced anti-apoptotic effects in cortical neurons.These results suggestthatTIGARparticipatesinthecerebral preconditioningthroughreductionofROSand subsequent cell apoptosis.CONCLUSlONour results showed that TIGAR participated in the cerebral precondi⁃tioning through PPP pathway mediated clearance of ROS and inhibition of neuronal apoptosis.The combina⁃tion of TIGAR and cerebral preconditioning may be a new therapeutic target for stroke prevention and treatment.

Key words：TIGAR；ischemic preconditioning；isoflu⁃rane；cortical neurons；NADPH；middle cerebral artery occlusion；reactive oxygen species

Foundation item：The project supported by National NaturalScienceFoundationofChina（81173057；81373402）

Corresponding author：SHENG Rui，E-mail：sheng_rui@ 163.com，Tel：（0512）65882071

T4-21

蒙药嘎日迪-13对脑缺血大鼠不同时相脑组织中P-选择素和E-选择素mRNA表达的影响

何静波，张浩楠，田彩云，郑延泽，武海军，曹丽霞，杨玉梅（包头医学院，内蒙古包头024000）

摘要：目的探讨脑缺血损伤后不同时相P-选择素和E-选择素mRNA表达的变化以及蒙药嘎日迪-13对其的影响。方法取雄性SD大鼠300只，将大鼠随机分为假手术组、脑缺血模型组和嘎日迪-13子3个剂量组，每组又随机分为缺血1 h组、6 h组、12 h组、24 h组、72 h组与120 h组6个时相组，嘎日迪-13组分别以3，6和12 g·L-1浓度20 mL·kg-1灌胃，每天1次，连续27 d后，采用改良Zea Longa线栓法制备大鼠大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）模型，进行神经功能评分，在各时相规定时间点麻醉大鼠后取脑，半定量RT-PCR检测脑组织中P-选择素和E-选择素的mRNA在1，6，12，24，72和120 h的表达。结果与假手术组比较，模型组P-选择素及E-选择素mRNA表达显著升高（P＜0.01）。与模型组比较，嘎日迪-13给药组可明显降低P-选择素和E-选择素mRNA的表达（P＜0.01，P＜0.05）。结论嘎日迪-13可明显降低大鼠脑缺血后脑组织中P-选择素和E-选择素mRNA的基因表达，对大鼠脑缺血损伤具有明显的保护作用，提示抑制P-选择素和E-选择素mRNA表达可能是其保护脑缺血损伤的重要机制之一。

关键词：嘎日迪-13；脑缺血大鼠；P-选择素；E-选择素

基金项目：国家自然科学基金（81160560）

通讯作者：杨玉梅，E-mail：yym457@aliyun.com，Tel：13847230457

T4-22

蒙药嘎日迪-13对脑缺血损伤大鼠不同时相血清、脑组织lL-1β的影响

张浩楠，何静波，郑延泽，田彩云，武海军，曹丽霞，杨玉梅（包头医学院，内蒙古包头014040）

摘要：目的研究蒙药嘎日迪-13对脑缺血损伤大鼠不同时相血清中IL-1β含量、不同脑区IL-1β表达量的影响。方法360只雄性SD大鼠随机分为脑缺血模型组、假手术组、嘎日迪-13 0.06，0.12和0.24 g·kg-1剂量组，每组又随机分为6个时相组，灌胃给药，每1次，连续27 d后，线栓法制备大鼠大脑中动脉栓塞模型，进行神经功能评分后取材测定血清中IL-1β含量，HE染色观察脑组织病理学改变，免疫组化观察不同脑区IL-1β的表达。结果与假手术组比较，模型组大鼠神经功能评分、血清中IL-1β含量、脑组织各区IL-1β表达量明显升高（P＜0.01）；与模型组比较，嘎日迪-13给药组可明显降低脑缺血大鼠神经功能评分、血清中IL-1β含量、脑组织各区IL-1β表达量（P＜0.01或P＜0.05）。结论蒙药嘎日迪-13可明显降低血清中IL-1β含量、脑组织各区IL-1β表达量，提示蒙药嘎日迪-13抑制炎症因子IL-1β表达可能是其对脑缺血保护作用的机制之一。

关键词：蒙药；嘎日迪-13；脑缺血；大鼠；IL-1β

基金项目：国家自然科学基金（81160560）

通讯作者：杨玉梅，E-mail：yym457@aliyun.com，Tel：13847230457

T4-23

蒙药嘎日迪-13保护脑缺血的物质基础初步研究

赵云山，何静波，赵冬冬，邬国栋，刘全礼，杨玉梅（包头医学院，内蒙古包头014040）

摘要：目的挥发油、矿物质、减毒物质是嘎日迪-13中的主要组分。通过观察嘎日迪-13全方及其主要组分对脑缺血大鼠的影响，初步研究其保护脑缺血的物质基础。方法120只雄性大鼠随机分为假手术组、模型组、嘎日迪-13全方组、挥发油大剂量和小剂量组、矿物质大剂量和小剂量组、减毒物质大剂量和小剂量组。灌胃给药，每天1次，连续27 d后，线栓法建立大鼠局灶性脑缺血模型，24 h后进行神经功能评分、脑梗死指数测定和血清中IL-1，IL-6和MCP-1的含量测定。结果 与假手术组比较，模型组大鼠神经功能评分明显增加（P＜0.05），脑梗死指数明显升高（P＜0.05），血清中IL-1，IL-6和MCP-1含量明显升高（P＜0.05）。与模型组比较，嘎日迪-13全方组神经功能评分明显降低（P＜0.05），脑梗死指数明显降低（P＜0.05），其它各组对神经功能评分和脑梗死指数均无显著影响；嘎日迪-13全方组、挥发油大剂量组、矿物质大剂量组IL-1，IL-6和MCP-1含量明显降低（P＜0.05），其余各组IL-1，IL-6和MCP-1含量变化不明显。结论 嘎日迪-13中的挥发油、矿物质可抑制血清炎症因子的表达，但单独应用任一组分对脑缺血没有明显保护作用，可能须与其他组分共同作用才能达到保护脑缺血损伤的目的；减毒物质在嘎日迪-13中的作用不确定。

关键词：蒙药；嘎日迪-13；脑缺血；大鼠；物质基础

基金项目：国家自然科学基金（81160560）；内蒙古科学技术协会（2015123120）

通讯作者：杨玉梅，E-mail：yym457@aliyun.com

T4-24

Purinergic signaling sensitizes leukocyte-endothelial adhesion and promotes neurovascular injury

WANG Huan1*，HONG Ling-juan1*，TAN Chao1，LU Nan-nan1，JIANG Quan1，TAO Rong-rong1，WANG Cheng-kun1，LIU Zhi-rong2，HAN Feng1

（1.College of Pharmaceutical Sciences，Zhejiang Univer⁃sity，Hangzhou 310058，China；2.Department of Neurology，the Second Affiliated Hospital of Zhejiang University，School of Medicine，Hangzhou 310058，China）

Abstract：OBJECTlVEThe brain endothelial/micro⁃veselinjury couldlead to an increase in blood-brain barrier dysfunction and neuronal damage，but the molecular events remain unclear.The goal of this study is to deter⁃mine the potential link between leukocyte adhesion and microglia overactivation during vascular inflammation. METHODSIn vivo two-photon laser scanning microsco⁃py were used to examine leukocyte adhesion on in⁃flamed cerebral microvessels and activation of microglia in cecal ligation and puncture（CLP）mice.CXCL1-neu⁃tralizing antibody，P2RX7antagonist A438079 and shRNA transfection were used to address the potential signaling involved in the process.RESULTSTime-lapse in vivo two-photon imaging of GFP+cells in the brain of Cx3cr1GFP/+mice demonstrated a close association be⁃tween dynamic changes of microglial process outgrowth and leukocytes adhering to adjacent brain vessels in CLP mice.P2RX7knockdown effectively suppressed ele⁃vation of L-selectin，ICAM-1 and chemokine CXCL1 in brain vessels after CLP.Furthermore，we demonstrate functional channel activity of P2RX7by whole-cell patch clamp recording，P2RX7inhibition blocked the ATP-initiat⁃ed P2RX7cation channel activationand Et+efflux in brain endothelial cells.Pharmacological inhibition of the P2RX7pathway not only decreased endothelial ICAM-1 expression and leukocyte adhesion but also prevented microglia overactivation and reduced brain injury.CON⁃CLUSlON These results highlight the potential for neuro⁃vascular protection using pharmacological blockadedur⁃ing vascular inflammation.

Key words：vascular inflammation；leukocyte adhesion；P2RX7；ICAM-1；two-photon imaging；microglia；neuro⁃vascular

Foundation item：The project supported by National Natural Science Foundation of China（81120108023；81302748；81300991）

Corresponding author：HAN Feng，E-mail：changhuahan@ zju.edu.cn

*Co-first author

T4-25

具有高PPARγ激动活性的新型血管紧张素ll受体拮抗剂Tek-2的药理学作用研究

孙德佳，刘 越，史敬璞，史媛媛，王 勃，李 锦

（军事医学科学院毒物药物研究所，北京 100850）

摘要：目的研究新结构的替米沙坦衍生物Tek-2是否兼具阻断血管紧张素Ⅱ1型受体（angiotensinⅡreceptor-1，AT1受体）和激动过氧化物酶增殖激活物受体γ（peroxi⁃some proliferator-activated receptor-γ，PPARγ）的双重作用，观察Tek-2对高血压及脑卒中的影响。方法采用［125I］-AngⅡ放射性受体配体结合实验，研究Tek-2与大鼠原代血管平滑肌细胞的AT1受体的亲和力；采用荧光素酶报告基因检测方法，研究Tek-2对PPARγ的激动活性及选择性；采用细胞内钙流动检测方法，研究Tek-2对AngII激动AT1受体的拮抗效应；采用自发性高血压大鼠（spontaneously hypertensiverat rat，SHR）模型，评价Tek-2的体内降压效应；采用短暂性大脑中动脉栓塞（transcient middle cere⁃bral occlusion，tMCAO）小鼠模型，评价Tek-2对脑卒中的改善作用。结果离体研究显示，Tek-2对AT1受体具有较高亲和力，Ki值与替米沙坦接近，分别为9.25±1.08和（3.3± 0.7）nmol·L-1；Tek-2（0.01～1 μmol·L-1）能浓度依赖的拮抗AngII诱导的细胞内钙流增加，IC50值为（0.7±0.11）nmol·L-1，是AT1受体拮抗剂。Tek-2还具有激动PPARγ的活性，在0.1～10 μmol·L-1浓度下Tek-2对PPARγ的激活强度有高于等浓度替米沙坦的趋势，而对PPARα无激活作用。SHR模型研究显示，Tek-2（5和10 mg·kg-1，po）在给药第2，3和4周时均能显著降低SHR收缩压和舒张压，其降压作用弱于等剂量的替米沙坦。tMCAO小鼠模型研究显示，Tek-2（5 和10 mg·kg-1，po）能够显著降低tMCAO小鼠的大脑梗死体积、神经功能损伤评分以及脑内TNF-α、IL-6和iNOS的mRNA表达水平，其改善脑卒中的作用优于等剂量的替米沙坦。结论 设计合成了兼具阻断AT1受体和高活性激动PPARγ的双重作用的新型替米沙坦衍生物Tek-2，该化合物具有降低血压、改善脑卒中的作用。

关键词：替米沙坦衍生物；血管紧张素Ⅱ1型受体；过氧化物酶增殖激活物受体γ；高血压；脑卒中

基金项目：北京市科委科技新星项目（Z121102002512046）

通讯作者：李 锦，E-mail：jinli9802@163.com；王 勃，E-mail：wbcmx@sina.com

T4-26

Toll样受体-4血红素氧化酶-1同工酶cross-talk参与脑缺血过程中的炎性机制

张艳丽1，潘 越1，刘怡洁1，王 芮2，羊红玉3，卢应梅2

（浙江大学1.药学院，2.城市学院，3.医学院附属第一人民医院，浙江 杭州 310015）

摘要：目的在我国，血管性痴呆（vascular dementia）为痴呆发病的首位。血管性痴呆是由慢性脑低灌注造成，但是具体的发病机制不明。我们探讨TLR4与HO-1信号参与慢性脑低灌注导致的小鼠认知功能障碍的机制。方法C57BL/6J小鼠右侧颈总动脉结扎后4周，观察其学习记忆相关行为学变化；免疫印迹和免疫荧光染色分析小鼠的皮质和海马区的TLR4/HO-1表达变化。通过沉默TLR4、HO-1激动剂或抑制剂进一步探索TLR4/HO-1与神经元损伤的关联性。结果慢性脑低灌注损伤4周，小鼠认知功能下降。同时皮层和海马区TLR4、HO-1蛋白水平上调。体外TLR4的诱导剂脂多糖（LPS）刺激原代培养皮层神经元，导致HO-1的蛋白水平上调，该现象被TLR4 siRNA部分逆转。HO-1抑制剂加重LPS诱导的神经元凋亡现象；HO-1激动剂改善慢性脑低灌注导致的小鼠认知功能障碍。结论HO-1参与了慢性脑缺血发展过程中由TLR4诱导的炎症反应。基于TLR4/HO-1的信号调控可能为血管性痴呆的防止提供靶点。

关键词：脑低灌注损伤；认知功能障碍；TLR4；HO-1；神经细胞损伤

基金项目：国家自然科学基金（81473202；81302765）；杭州市“131”及“521”人才项目

通讯作者：卢应梅，E-mail：lufx@zju.edu.cn，Tel：（0571）88284325

T4-27

山茱萸环烯醚萜苷对创伤性脑损伤模型的药理作用及机制研究

马登磊，杨翠翠，张 丽，李雅莉，张 兰，李 林

（首都医科大学宣武医院药物研究室，神经变性病教育部重点实验室，北京脑重大疾病研究院，北京100053）

摘要：目的探究山茱萸环烯醚萜苷（Cornel iridoid glycoside，CIG）对于及创伤性脑损伤的是否有治疗作用，并进一步探讨CIG的药理作用机制。方法采用Feeney′s自由落体打击模型造成SD大鼠创伤性脑损伤模型。实验分为急性期实验和慢性期实验，设置对照组，模型组，CIG不同剂量给药组（30，60和120 mg·kg-1），阳性药组（单唾液酸神经节苷脂4 mg·kg-1）。急性期实验在术前7 d给药，恢复期实验在术后3 h灌胃给药，直至实验结束。造模后进行行为学和相关指标检测。结果mNSS评分结果显示，CIG在急性期和慢性期给药均能够明显降低模型大鼠神经功能损伤评分（P＜0.05）；新物体识别实验结果显示，CIG给药能够增加TBI大鼠的分辨指数（P＜0.05）；尼氏染色的结果显示，CIG对于TBI大鼠模型脑损伤灶周围的脑组织细胞形态有明显的保护作用；免疫组化的结果显示，CIG可以明显的降低炎症指标TNF-α，IL-1β，以及凋亡指标Bax和胱天蛋白酶3的表达，增加IL-1ra和Bcl-2的表达。Western蛋白印迹法结果显示，TBI大鼠在给予CIG后可以明显增加神经营养因子BDNF、NGF以及突触相关蛋白Synaptophysin和Syn⁃apsin-I的表达。结论CIG能够改善创伤性脑损伤大鼠神经功能，减轻认知障碍程度，改善组织形态，保护神经细胞，降低生物标志物S100β含量，显示出一定的脑损伤保护作用。CIG能够降低创伤性脑损伤大鼠的可能机制为降低炎症反应，抑制细胞凋亡，调节神经营养因子以及突触相关蛋白的表达。

关键词：创伤性脑损伤；山茱萸环烯醚萜苷；炎症；细胞凋亡；神经营养；

通讯作者：李 林，E-mail：linli97@hotmail.com，Tel：（010）83198855

T4-28

山茱萸环烯醚萜苷对大鼠局灶性脑缺血损伤后细胞色素c和胱天蛋白酶3表达的影响

张 丽，马登磊，陈 晨，包训杰，张 兰，李 林

（首都医科大学宣武医院药物研究室，北京市神经药物工程技术研究中心，神经变性病教育部重点实验室，北京100053）

摘要：目的观察山茱萸环烯醚萜苷（Cornel iridoid glycoside，CIG）对大鼠局灶性脑缺血损伤后细胞色素c和胱天蛋白酶3表达的影响。方法线栓法制备大鼠局灶性脑缺血大鼠模型，缺血90 min，再灌24 h，用免疫组织化学方法检测胱天蛋白酶3和细胞色素c表达，用western蛋白印迹方法检测细胞色素c含量。结果在免疫组化检测中，与模型组相比，CIG各剂量组均可降低细胞色素c阳性细胞数量；CIG大剂量组也可显著降低胱天蛋白酶3阳性细胞表达数量。在western蛋白印迹法检测中，CIG中剂量组和大剂量组可明显降低细胞色素c蛋白表达，CIG中剂量组可以降低胱天蛋白酶3蛋白表达。结论CIG对大鼠局灶性脑缺血损伤有明显改善作用，其机制可能与降低细胞色素c和胱天蛋白酶3表达有关。

关键词：山茱萸环烯醚萜苷；局灶性脑缺血；细胞色素c；胱天蛋白酶3

通讯作者：李林，E-mail：linli97@hotmail.com，Tel：（010）83198855

T4-29

Protective effects of icariin against cerebral hypoper⁃fusion in rats

CAI Rui，LIU Bo，GONG Qi-hai，WU Qin，SHI Jing-shan，LI Fei

（Department of Pharmacology and Key Laboratory of Basic Pharmacology of Ministry of Education，Zunyi Med⁃ical University，Guizhou 563009，China）

Abstract：OBJECTlVETo investigate effect of icar⁃iin on cerebral hypoperfusionrat model and explore the potential mechanism.METHODSMale Sprague Dawley rats were divided into five groups：sham，model，icariin 15，30 and 60 mg·kg-1groups.Through bilateral com⁃mon carotid artery occlusion（BCCAO）to induce rat ce⁃rebral hypoperfusion model，administrated with different doses of icariin from the 10th day after BCCAO to the 24th day.Spatial learning and memory function was test⁃ed by Morris water maze，cerebralmicrovessels was la⁃beled by CD31 antibody，nitric oxide（NO）content in cortex was tested by Griess method，the protein expres⁃sion of vascular endothelial growth factor A（VEGFA），vascular endothelial growth factor receptor 2（VEGFR2），extracellular signal-regulated kinase1/2（ERK1/2）and endothelial nitric oxide synthase（eNOS），as well as the phosphorylation level of ERK1/2 and eNOS were ob⁃served by Western blotting.RESULTSIcariin treat⁃ment significantly improved the behavior function of BCCAO rats，increased the cerebral microvessels density and the level of NO content，and elevated the protein expression of VEGFA，VEGFR2，as well as the phos⁃phorylation level ofERK1/2 and eNOS.CONCLUSlON Icariin improves the function of learning and memory of BCCAO rats via promoting angiogenesis in cerebral hypoperfusion rats.

Key words：icariin；cerebral hypoperfusion；angiogene⁃sis；vascular endothelial growth factor；bilateral common carotid artery occlusion

Foundation item：The project supported by Joint Fund of Science and Technology Department of Guizhou Prov⁃ince（LKZ［2013］12）

Correspondent author：LI Fei，E-mail：lifei@zmc.edu.cn

T4-30

拟人参皂苷-F11抗大鼠脑缺血的自噬性相关机制研究

刘月阳，张天宇，刘迎璐，袁琳琳，杨静玉

（沈阳药科大学药理学教研室，辽宁沈阳 110016）

摘要：目的前期研究表明，拟人参皂苷-F11（PF11）具有显著的大鼠脑缺血保护作用，而诸多文献表明，自噬参与了脑缺血的损伤过程，因此本文将探讨PF11对大鼠永久性脑缺血的保护作用是否与脑缺血的自噬性机制相关。方法 采用线栓法制备大鼠永久性脑缺血模型，在造模后0.5 h尾静脉给予3，6和12 mg·kg-1的PF11，在脑缺血后24 h运用免疫荧光方法及Western蛋白印迹方法检测神经元的损伤情况，以及自噬相关蛋白的表达。结果 在大鼠永久性脑缺血后给予PF11能够显著的增加由于缺血损伤而导致的神经细胞的死亡，表现为PF11能够剂量依赖性的增加

NeuN的数量，以及降低FJB染色的阳性细胞数；同时，给予

PF11后24 h检测自噬相关蛋白的表达发现，PF11能够剂量依赖性的降低永久性脑缺血模型所介导的自噬小体LC3

的显著性增多，降低自噬底物SQSTM1的表达，以及增加溶酶体膜相关蛋白LAMP2的表达；同时，PF11能够增加脑缺血所致的缺血后期溶酶体和组织蛋白酶cathepsin B的共定位水平。结论 PF11可以通过缓解脑缺血后导致的自噬流的阻滞而起到缺血保护作用。

关键词：大鼠永久性脑缺血；自噬；PF11

基金项目：辽宁省百千万人才工程培养经费资助（2014921036）

通讯作者：杨静玉，E-mail：yangjingyu2006@gmail.com，Tel：（024）23986340

T5　睡眠与癫痫

T5-1

Modulation of abnormal neuronal circuit in temporal lobe epilepsy

CHEN Zhong1，WANG Yi1，YING Xiao-ying2，CHEN Bin1，XU Ceng-lin1

（1.Department of Pharmacology，Key Laboratory of Medical Neurobiology of the Ministry of Health of China，2.Institute of Pharmaceutics，College of Pharmaceutical Sciences，SchoolofMedicine，ZhejiangUniversity，Hangzhou 310058，China）

Abstract：OBJECTlVETemporallobeepilepsy （TLE）is one of the most common types of human epilep⁃sy，and they are often resistant to current treatments. METHODS By using optogenetic，electrophysiological，imaging and pharmacology strategies，we aimed toinves⁃tigate the underlying circuit mechanism of TLE and tried to developthe novel and efficient approach to control epi⁃lepsy.RESULTS①Using microPET and multi-channel EEG recording，we found an abnormal neural network，characterizedbyearlyhypometabolismandafterdis⁃charge spread，during the epileptogenensis of TLE.②Deep brain stimulation，especially low frequency stimula⁃tion，targeted the epileptic focus and the areas outside of the focus（critical regions for seizure spread），such as the piriform cortex，cerebellum，entorhinal cortex or su⁃biculum，reduced seizure severity in TLE.Its anti-epilep⁃tic effect is time-window dependent and polarity depen⁃dent，which shows a promising strategy for treating epi⁃leptic seizures.③ Using an optogenetic strategy，we demonstrated that excitatory projection from entorhinal cortex to hippocampus instructs the brain-stimulation treatments of epilepsy.④Our Data from both the clinical and experimental studies further demonstrated that a dis⁃inhibitoryGABAergic neuron-mediated microcircuit in the subiculum contributes to secondary generalized seizures in TLE.⑤Finally，based on abnormal synchronization of the electrical activity in epileptic circuit，we developed electro-responsive hydrogel nanoparticles modified with angiopep-2 to facilitate the delivery of the antiepileptic drug phenytoin sodium，which greatly improves the thera⁃peutic index.CONCLUSlONOur findings may update the current view of epileptic neuronal networks and sug⁃gest possible promising ways for epilepsy treatment.

Key words：epilepsy；neural circuits；brain stimulation；optogenetics；electro-responsive

Foundation item：The project supported by National NaturalScienceFoundationofChina（91332202；81221003）；and Program for Zhejiang Leading Team of S&T Innovation（2011R50014）

Corresponding author：CHEN Zhong，E-mail：chenzhong@ zju.edu.cn

T5-2

Electro-responsive nanoparticles improve antiepileptic effect of phenytoin in generalized tonic-clonic seizures

WANG Yi， Ying Xiao-ying， CHEN Li-ying，LIU Yao，WANG Ying，XU Ceng-lin，CHEN Zhong

（Department of Pharmacology，Key Laboratory of Medical Neurobiology of the Ministry of Health of China，College of Pharmaceutical Sciences，School of Medicine，Zhejiang University，Hangzhou 310058，China）

AbstractOBJECTlVEPreviously we developed electro-responsivehydrogelnanoparticles（ERHNPs）modified with angiopep-2（ANG）to facilitate the delivery of the antiepileptic drug phenytoin sodium（PHT）.However，the electro-responsive characteristics were not verified directly in epileptic mice and the optimal preparation formula for electro-responsive ability is still unclear. METHODS In the present study we synthesized PHT-loaded ANG-ERHNPs（ANG-PHT-HNPs）and PHT-loaded non-electro-responsive hydrogel nanoparticles（ANGPHT-HNPs）by changing the content of sodium 4-vinyl⁃ benzene sulfonate（NaSS）in the preparation formulae and further evaluated the anti-epileptic effect in in vivo ep⁃ilepsy models.RESULTSIn vivo microdialysis analysis showed that ANG-PHT-ERHNPs not only have the char⁃acteristics of a higher distribution in the central nervous system，but also have electro-responsive ability，which resulted in a strong release of non-protein bound PHT during seizures.In both electrical-（maximal electrical shock）and chemical-induced（pentylenetetrazole and pi⁃locarpine）epilpesy models，ANG-PHT-ERHNPslowered the effective therapeutic doses of PHT and demonstrated the improved antiepileptic effects compared with ANGPHT-HNPs or PHT solution.CONCLUSlON ANG-ERHNPs are able to transport PHT into the brain efficiently and release them when epileptiform activity occurred，which is due to the content of NaSS in formula.This may change the therapeutic paradigm of existing drug treat⁃ment for epilepsy into a type of on-demand control for epilepsy in the future.

Key words：electro-responsive；nanoparticle；sodium 4-vinylbenzene sulfonate；epilepsy；microdialysis

Foundation item：The project supported National Natural Science Foundation of China（91332202；81273492；81471316）；and Program for Zhejiang Leading Team of S&T Innovation（2011R50014）

Corresponding author：CHEN Zhong，E-mail：chenzhong@ zju.edu.cn

T5-3

Low-frequency stimulation of primary site retards positive transfer of secondary sites in epilepsy

WANG Ying，KUANG Yi-fang，WANG Yi，XU Ceng-lin，CHEN Zhong

（DepartmentofPharmacology， KeyLaboratoryof Medical Neurobiology of the Ministry of Health of China，College of Pharmaceutical Sciences，School of Medicine，Zhejiang University，Hangzhou 310058，China）

Abstract：OBJECTlVE Positive transfer of secondary focus（PTS）refers to new epileptogenesis from the outside of primary seizure focus，which is only minimally controlled by existing medications and surgery.The present study was aimed to investigate whether low frequency stimulation （LFS）would inhibit PTS in epilepsy.METHODSWe compared theepileptogenesis of PTS when LFS was applied during different stage of primary epileptogenesis in a rat-kindling model，and we also analyzed the surgical re⁃sectionfrom epilepsy patients who experienced the PTS. RESULTS We found that PTS at both the contralateral amygdalaandtheipsilateralhippocampuswere facilitated after the primary site（right amygdala）was fully kindled.LFS of the primary site during primary epilepto⁃genesis retarded both types of PTS，while LFS deliveredduringthelaterstagesofprimaryepileptogenesis showed slight anti-epileptogenic effects on PTS.We also noted that LFS blocked the decrease in potassium chlo⁃ride cotransporter 2（KCC2）levels that typically accom⁃panies PTS.Further，we verified that patients who had experienced PTS showed lower expression levels of KCC2 than did patients with only one seizure focus. CONCLUSlON LFS of the primary site retarded PTS in a kindling model，a process that may result from modu⁃latd expression of KCC2.These findings suggest LFS as a potential clinical therapeutic approach for PTS.

Key words：low-frequency stimulation；epilepsy；posi⁃tive transfer of secondary focus；KCC2

Foundation item：The project supported National Natural Science Foundation of China（91332202；81221003）；and Program for the Zhejiang Leading Team of S&T Inno⁃vation（2011R50014）

Correspondingauthor：CHENZhong，E-mail：chenzhong@ zju.edu.cn

T5-4

Blocking GluN2B subunits reverses enhanced seizure susceptibility after prolonged febrile seizures with a wide therapeutictime-window

CHEN Bin1，FENG Bo1，TANG Yang-shun1，WANG Yi1，HU Wei-wei1，CHEN Zhong1，2

（1.Department of Pharmacology，Key Laboratory of Medical Neurobiology of the Ministry of Health of China，School of Basic Medical Sciences，College of Pharma⁃ceutical Sciences，School of Medicine，Zhejiang Univer⁃sity，Hangzhou 310058，China；2.Epilepsy Center，De⁃partment of Neurology，the Second Affiliated Hospital，Zhejiang University，Hangzhou 310008，China）

Abstract：OBJECTlVE Febrile seizures（FSs），the most common type of convulsive events in infants，are closely associated with adult temporal lobe epilepsy （TLE）.It is urgent to investigatethe underlying mecha⁃nismson how FSs promote TLE andfind the potential ther⁃apeutic targets.METHODSIn the present study，the expression of GluN2B Tyr-1472 phosphorylation was tested at different times after prolonged FSs or IL-1β treatment. MES and KA induced seizure models were used to test the seizure susceptibility in the adult rats with a history ofearly-life prolonged FSs with or without GluN2B antago⁃nist ifenpeodil treatment.RESULTS We found that the phosphorylation of GluN2B Tyr-1472 gradually increased to a peak at 24 h and remained high expression within 7 d after prolonged FSs.IL-1β treatment alone，which could mimic the effect of prolonged FSs on adult seizure susceptibility in previous study，increased GluN2B Tyr-1472 phosphorylation.Both IL-1 receptor antagonist（IL-1Ra）treatment and IL-1R1 deletion were sufficient to re⁃ verse the hyper-phosphorylation of GluN2B Tyr-1472 in⁃duced by prolonged FSs.GluN2B antagonis tifenprodil showed a wide therapeutic time-window（3 days）to re⁃verse the enhanced seizure susceptibility after prolonged FSsor IL-1β treatment.CONCLUSlON Our study demon⁃strated that GluN2B phosphorylation at Tyr-1472 siteme⁃diated by the transient increase of IL-1β was involved in the enhanced adult seizure susceptibility after prolonged FSs，implicating GluN2B-containing NMDARs is a new attractive target with awide therapeutic time window to prevente pileptogenesis in patients with infantile FSs.

Key words：febrile seizures；GluN2B；IL-1β；seizure sus⁃ceptibility；ifenprodil

Foundation item：The project supported National Natural Science Foundation of China（91332202；81503045）；and Program for Zhejiang Leading Team of S&T Innova⁃tion（2011R50014）

Correspondingauthor：CHENZhong，E-mail：chenzhong@ zju.edu.cn

T5-5

Arousal control by dopamine D1 receptor-expressing neurons in nucleus accumbens

LUO Yan-jia1，WANG Lu1，LI Ya-dong1，YUAN Xian-shan1，QU Wei-min1，HUANG Zhi-li1，2，3

（1.Department of Pharmacology，2.State Key Laboratory of Medical Neurobiology，3.The Institutes of Brain Science and Collaborative Innovation Center for Brain Science，Fudan University，Shanghai 200032，China）

Abstract：OBJECTlVEThe nucleus accumbens （NAc），a part of basal ganglia（BG），has been reported to play a role in motivation and reward.Clinical and basic data indicate that dysfunction of NAc causes numerous neurological disorders associated with sleep-wake distur⁃bances.There are two different subpopulations of principle projection neurons in the NAc，dopamine D1 receptor （R）-expressing medium spinyneurons（D1R-MSNs）and D2R-MSNs，both of which are GABAergic MSNs. However，whether NAc D1R-MSNs are involved in sleepwake regulation and the underlying neural circuit remain poorly understood.METHODS We used designer recep⁃tors exclusively activated by designer drugs（DREADD）method to selectively manipulatethe activity of D1RMSNs.We injected adeno-associated virus（AAV）con⁃taining the double floxed Gq-coupled designer receptor hM3Dq（for excitation）or Gi-coupled designer receptor hM4Di（for silencing）into the NAc of D1R-Cre mice.After AAV injection for 3-4 weeks，we implanted mice with electrodesforrecordingEEGandelectromyogram （EMG）activity.A single intraperitoneal（ip）injection of CNO was given into the mice to modulate NAc D1RMSNs activity and assess the effects on sleep-wake be⁃havior.Meanwhile，we expressed ChR2-mCherry selec⁃tively in NAc D1R-MSNs of freely moving mice，and ex⁃amined the effect of optical stimulation of ChR2-mCherry terminals in the VP，SNc/VTA on sleep-wake pattern.In addition，we used ChR2-mCherry coupled with patchclamp recording and optogenetic stimulatonor hrGFP as a tracer to map the projections of NAc D1R-MSNs ana⁃tomically and functionally.RESULTS We found that acti⁃vation of NAc D1R-MSNs pharmacogenetically or optoge⁃netically increased wakefulness or induced immediatet⁃ransitions from non-rapid eye movement（non-REM，NREM）sleep to wakefulness，respectively.Conversely，inhibition of NAc D1R-MSNs increased NREM sleep dur⁃ing both inactive and active periods.Anatomical mapping revealed dense mCherry NAc D1R-MSNs terminals in the ventral pallidum（VP），lateral hypothalamus（LH），sub⁃stantia nigra pars compacta（SNc）and ventral tegmen⁃tal area（VTA）.In vitro patch-clamp study showed that NAc D1R-MSNs sent inhibitory inputs to the VP，prefer⁃entially innervated GABAergicinterneuronsin the SNc，andprojected toboth NAc-projecting and non-NAc-project⁃ing neurons in the VTA.Similar to optical activation of D1R-MSN cell bodies in the NAc，optogenetic stimulation of the terminals in the downstream nucleus，VP，or SNc/ VTA also induced immediatetransitionsfrom NREM sleep to wakefulness.CONCLUSlONThese results indicat⁃edthat activation of NAc D1R-MSNs induces wakeful⁃ness，possibly through multiple pathways，including inhi⁃bition of VP neurons and GABAergic interneurons in the SNc/VTA.

Key words：dopamine D1receptor；nucleus accum⁃bens；optogenetics；pharmacogenetics；sleep-wake cycle

Foundation item：The project supported by National Natural Science Foundation of China（81420108015）

Corresponding author：HUANG Zhi-li，E-mail：huangzl@ fudan.edu.cn

T5-6

Asubsetofmedialparabrachialnucleusneurons induced wakefulness in rats

XU Qi1，2*，WANG Diran-ru1*，DONG Hui1，CHEN Li1，QU Wei-min1，LU Jun1，3，HUANG Zhi-li1

（1.Department of Pharmacology，State Key Laboratory of Medical Neurobiology，Institutes of Brain Science and the Collaborative Innovation Center for Brain Science，Shanghai Medical College of Fudan University，Shanghai 200032，China；2.Department of Physiology，School of Basic Medical Sciences，Anhui Medical University，Heifei 230032，China；3Department of Neurology，Beth Israel DeaconessMedicalCenter，HarvardMedicalSchool，Boston，USA）

Abstract OBJECTlVE To elucidate the role and brain circuits of PB neurons in the regulation of wakeful⁃ness.METHODS A chemogenetic approach was em⁃ployed to clarify the electroencephalographic outcome of activating PB neurons，which was targeted by adeno-as⁃sociatedviralvectors（AAV） carryinghM3Dq.The hM3Dq were stereotaxically injected bilaterally into the PB，and the sleep-wake cycle was recorded after intra⁃peritoneal injections of vehicle or clozapine-N-oxide （CNO），a hM3Dq ligand to evoke neuronal excitation.In addition，the stainings against c-Fos and mCherry were performed to confirm the activation of PB neurons and the expression to hM3Dq in the brain.Next，the optoge⁃netic stimulation of channelrhodopsin-2（ChR2），a blue light-gated cation channel，expressed in PB neurons was used to further explore the temporal properties and brain circuit of wake responses evoked by activating PB neurons in rat.RESULTS Bath application of CNO （500 nmol·L-1）to PB slices isolated from PB-hM3Dq rats resulted in increased firing frequency and recurrent bursting.EEG recording showed that CNO（0.3 mg·kg-1）injected intraperitoneally induced continuous wakeful⁃ness for 15 h，while CNO has no effect on the control rats.Only activation of the median part of PB（MPB），but not the lateral part of PB induced wakefulness.Sur⁃prisingly，after long period of wakefulness after CNO treatment，all rats did not exhibit any sleep rebound in both amount of sleep，and the power spectrum for each stage.Then the optogenetics was used to clarify the neu⁃ronal pathway of MPB in sleep-wake regulation，brief pulses of light（5 ms）evoked single action potentials with high frequency fidelity between 1 Hz and 20 Hz in PB neurons.Blue light pulses at 5-ms in the frequency range of 5-20 Hz when the rats were sleepy，the latency from to wakefulness was dependent on the pulse fre⁃quency with the shortest sleep latency when stimulated bilaterally at 20 Hz in rats with the ChR2 expressed in MPB.photostimulation of ChR2-expressing MPB termi⁃nals in the basal forebrain（BF）or parasubthalamic nu⁃cleus（PSTN）also induced an immediate transition from sleep to wake.Light stimulation for 1 h in the BF/PSTN portion containing ChR2-expressing MPB terminals re⁃markably increased wakefulness.CONCLUSlON The MPB neurons have a crucial role in controlling initiation and maintenance of wakefulness through the MPB-BF/ MPB-PSTN pathways.

Key words：channelrhodopsin-2；hM3Dq；wake；para⁃brachial nucleus

Foundation item：The project supported by National NaturalScienceFoundationofChina（31271164；81420108015；31530035；31421091；81401100）

Corresponding author：HUANG Zhi-li，E-mail：huangzl@ fudan.edu.cn

*Co-first author

T5-7

纹状体-黑质网状部在睡眠觉醒调控中的作用

董 辉1，陈泽卡1，袁向山1，王典茹1，方 腾1，曲卫敏1，黄志力1，2

（1.复旦大学基础医学院药理学系，上海 200032；2.医学神经生物学国家重点实验室，上海 200032）

摘要：目的 探索基底神经节的主要信息入口背侧纹状体和信息出口黑质网状部参与睡眠觉醒调节的作用和机制。方法 脑立体定位方法将携带Cre酶依赖表达的药理遗传学兴奋型元件hM3Dq、抑制型元件hM4Di或光遗传学元件ChR2的AAV病毒分别注射到腺苷A2A受体-，多巴胺D1受体-cre小鼠背侧纹状体，小清蛋白（parvalbumin）-cre小鼠黑质网状部。通过腹腔给予氧化氯氮平（clozapine N-ox⁃ide，CNO）激活表达hM3Dq的神经元或抑制表达hM4Di的神经元；通过在黑质埋植套管外源给予470 nm波长蓝光激活表达ChR2的纹状体黑质神经元末梢，记录小鼠脑电和肌电，线下解析睡眠和觉醒状态。结果 在活跃期或非活跃期，腹腔给予CNO特异性抑制背侧纹状体表达hM4Di的直接通路D1-MSNs均可促进小鼠睡眠，相反激活间接通路D2-MSNs增加睡眠，同时黑质网状部c-Fos表达。在纹状体注射光遗传学元件ChR2载体病毒，在黑质用分别以5，10，15，20和30 Hz频率470 nm波长蓝光激活来自纹状体的神经元末梢可诱导NREM睡眠向觉醒的快速转换，随着刺激频率增加转换概率增加且潜伏期缩短。激活黑质表达hM3Dq的小清蛋白阳性GABA能神经元，引起动物睡眠，相反利用hM4Di抑制黑质小清蛋白阳性GABA能神经元引起动物觉醒。结论 背侧纹状体两种投射神经元在睡眠觉醒中作用相反，即D1-MSNs是觉醒相关神经元，D2-MSNs是睡眠相关神经元。纹状体通过黑质网状部GABA能神经元作调控睡眠觉醒行为。

关键词：纹状体；黑质网状部；睡眠；药理遗传学；光遗传学

基金项目：国家自然科学基金重点国际（地区）合作与交流项目（81420108015）

通讯作者：黄志力，E-mail：huangzl@fudan.edu.cn，Tel：（021）54237043

T5-8

表观遗传因子CDYL在颞叶癫痫发病中的作用及其机制研究

赖世荣，黄 卓

（北京大学医学部药学院分子与细胞药理学系，北京 100191）

摘要：目的 研究表观遗传因子CDYL对神经元兴奋性的影响及其在癫痫发病过程中所发挥的作用。方法 ①利用卡英酸颞叶癫痫动物模型结合脑电记录的方法，评估特异性增加或降低海马体中CDYL表达对癫痫易感性的影响。②利用RNA干扰、转基因技术结合膜片钳记录方法，评估敲低或增加CDYL对海马齿状回DG神经元的兴奋性影响及其机制。③利用ChIP-seq高通量方法，分析CDYL在海马体神经元中调控的下游靶基因。结果 ①CDYL敲低或过表达明显缩短或延长癫痫发作的潜伏期。②CDYL敲低导致DG神经元动作电位阈值降低，从而增加神经元兴奋性。CDYL过表达的神经元兴奋性降低。③CDYL表达抑制SCN8A转录，从而导致轴突起始段Nav1.6钠离子通道表达降低。结论 CDYL是调节神经元兴奋性的重要因子，它通过控制SCN8A的转录水平从而调控神经元的阈值，进而影响神经元的兴奋性。因此，在颞叶癫痫的发病过程中起到了重要的作用。

关键词：表观遗传；CDYL；中枢神经系统兴奋性；颞叶癫痫；SCN8A

基金项目：青年973项目（2015CB559200）；国家自然科学基金（81371432）；国家科技重大专项（2014ZX09507003-006-004）

通讯作者：黄 卓，E-mail：huangz@hsc.pku.edu.cn，Tel：18610108083

T5-9

Mechanism of role of α2-adrenoceptor in sedative action

YANG Yi，ZHOU Pei-lan，SU Rui-bin，GONG Ze-hui

（State Key Laboratory of Toxicology and Medical Counter⁃measures，Institute of Pharmacology and Toxicology，Acade⁃my of Military Medical Sciences，Beijing 100850，China）

Abstract：OBJECTlVE To investigate the subtype （s）of α2-adrenoceptor（α2-AR）that mediated the seda⁃tion of mice.And demonstrate the influence of cAMP and G protein gated inwardly rectifying K+channels on the se⁃dation of animals.METHODSCHO-PKAcat-EGFP cells were stably transfected with pCDNA3.1/hygro-hα2A/α2Cto express the human α2A-or α2C-adrenoceptor.PKA redistri⁃bution assay was used to compare the effect and selec⁃tivity of different agonists and antagonists of α2-AR on the two subtypes.The loss of righting reflex and locomo⁃tor activity of mice induced by α2-AR agonist dexmedeto⁃midine were studied as the sedative aniaml models.Ati⁃pamezole（antagonistwithnosubtypeselectivity），BRL44408（antagonist of α2A-AR），JP-1302（antagonist of α2C-AR），ARC239（antagonist of α2B-and α2C-AR），dbcAMP（cAMP analogue）and Tertiapin-Q trifluoroace⁃tate salt（T-Q，G protein gated inwardly rectifying K+channels blocker）were microinjected in lateral ventricles respectivly before the dexmedetomidine（iv）administra⁃tion.The loss of righting reflex and locomotor activity of mice were detected after the agonist，nonselective or se⁃lective antagonist of α2-AR administration.RESULTS There were no significant selectivity on the α2A-AR or α2CAR of the agonists dexmedetomidine，medetomidine，noradrenaline，clonidine，guanfacine，xylazine，moxoni⁃dine by PKA redistribution assay in CHO-PKAcat-α2A-AR or CHO-PKAcat-α2C-AR cells.α2A-AR was selectively blocked by BRL44408.α2C-AR were blocked by JP-1302 and ARC239.The loss of righting reflex induced by the dexmedetomidine（0.25 mg·kg-1）was antagonized by ati⁃pamezole（4 μg）and BRL44408（20 μg）.However，neithor JP-1302（88 μg）nor ARC239（48 μg）can re⁃versed the sedative action of dexmedetomidine.The loss ofrightingreflexinducedbythedexmedetomidine （0.20 mg·kg-1）was inhibited by T-Q and dbcAMP dosedependently.CONCLUSlON α2A-AR might be the main subtype of α2-AR which mediated sedation.cAMP and G protein gated inwardly rectifying K+channels might be in⁃volved in the mechanism of sedation of α2-AR agonists。

Key words：α2-adrenoceptor；sedation；subtype；dex⁃medetomidine；signal pathway

Foundation item：The project supported by National ScienceandTechnologyMajorProjectofChina （2012ZX09301003-003）

Corresponding author：SU Rui-bin，E-mail：ruibinsu@ 126.com；ZHOU Pei-lan，E-mail：zhoupeilan0502@sina.com

T6　神经药理学评价方法与新技术及其他领域

T6-1

罗氟司特对脓毒血症小鼠的作用及其机制研究

冯红芳，陈佳佳，邹征强，徐江平

（南方医科大学药学院神经药理与新药发现课题组，广东广州 510515）

摘要：目的 研究罗氟司特对脓毒血症小鼠的保护作用以及相关的分子作用机制。方法 连续7 d，小鼠灌胃给予罗氟司特（0.3，1.0和3.0 mg·kg-1），第7天，采用盲肠结扎穿刺的方法（CLP）建立小鼠脓毒血症模型。造模后连续7 d，记录小鼠死亡时间，每隔24 h观察小鼠生存状态，按照评分标准评分；造模后24 h，收集血液、腹腔灌洗液和肺、肝、脾，进行细菌计数；检测TNF-a和IL-6的含量；检测乳酸脱氢酶（LDH），谷草转氨酶（GOT）和谷丙转氨酶（GPT）的含量；肺、肝、脾组织病理切片，经HE染色后于光学显微镜下观察病理变化，并对病理图片进行半定量分析。结果 假手术组、模型组和罗氟司特0.3，1.0和3.0 mg·kg-1组存活率分别为100%，0%，8.3%，20.0%和31.2%，组间具有显著差异（P＜0.01）；临床观察得分组间具有显著差异（P＜0.01）；与假手术组相比，模型组细菌密度显著升高（P＜0.01），药物组细菌密度下降（P＜0.05，P＜0.01）；与假手术组相比，模型组TNF-a，IL-6，LDH，GOT和GPT含量显著升高（P＜0.05，P＜0.01），药物组明显下降（P＜0.05，P＜0.01）；对各组肺、肝、脾进行病理检查，正常对照组未见明显组织损伤，模型组炎性细胞浸润、细胞变形坏死等变化显著，药物组损伤减轻。结论 罗氟司特对脓毒血症小鼠具有明显的保护作用。

关键词：罗氟司特；脓毒血症；盲肠结扎穿刺；全身炎症反应

基金项目：国家自然科学基金（81503043；81373384）

通讯作者：徐江平，E-mail：jpx@smu.edu.cn，Tel：（020）61648236

T6-2

基于褪黑激素受体相关信号通路的自闭症发病机制研究

洪玲娟1，田 允1，王慧娟2，王 欢1，邵世怡1，陈 琳1，韩峰1（1.浙江大学药学院药理毒理学研究所，浙江杭州 310058；2.浙江大学医学院附属儿童医院，浙江 杭州 310052）

摘要：目的 自闭症（Autism）的病因尚不清楚，临床治疗缺乏有效治疗药物。孕期服用抗癫痫药物丙戊酸（valpro⁃ic acid，VPA）增加后代患自闭症风险。本项目研究VPA影响褪黑激素受体（melatonin receptor type 1，MT1）内吞转运的分子机制。方法 采用VPA制作自闭症动物模型，检测其行为学及神经生化指标。在体外细胞系HEK293和Neu⁃ro-2a中过表达MT1和不同时期内含体标记蛋白（Rab5，Rab7，Rab11），转盘式共聚焦显微镜活细胞Time-lapse记录MT1的内吞转运过程。HEK293过表达Flag-MT1，不同浓度褪黑激素刺激细胞60 min后，使用ELISA法检测细胞膜表面MT1的相对含量。使用上述方法检测VPA （0.5 mmol·L-1，24 h）预处理后，MT1的内吞转运过程和细胞膜表面MT1的相对含量。利用膜片钳技术记录VPA对Neuro-2a的全细胞电流的影响，并且使用Western蛋白印迹法检测下游信号蛋白磷酸化水平变化。在HEK293细胞中过表达β-arrestin 2-YFP和MT1-CFP，荧光能量共振转移（fluorescence resonance energy transfer，FRET）技术解析VPA对β-arrestin 2和MT1相互作用的影响。结果 动物实验结果显示，褪黑激素口服治疗可以减轻实验动物社会交往行为学异常，改善海马触CaMKII及其突触前与突触后底物磷酸化水平。采用细胞生物学技术进一步开展了基于褪黑激素受体相关信号通路的自闭症发病机制研究。我们发现，HEK293和Neuro-2a中MT1在褪黑激素处理后内吞首先进入早期内含体，经由晚期内含体，一部分受体进入溶酶体降解，一部分受体通过循环内含体回到细胞膜上，VPA预处理减弱了受体内吞。ELISA结果显示，VPA预处理后细胞膜表面MT1相对含量显著升高，但是由膜片钳技术检测发现VPA并不影响Neuro-2a的全细胞电流。Western蛋白印迹法结果发现，VPA降低了β-arrestin 2下游蛋白ERK1/2和G蛋白下游蛋白PKA的磷酸化水平。并且通过FRET技术发现VPA处理降低了MT1与β-arrestin 2之间相互作用能力。结论 褪黑激素/MT1/β-arrestin 2参与自闭症病理过程中神经元信号转导，其稳态失衡最终诱发了一系列自闭症关联的神经生物化学及功能改变。

关键词：自闭症；褪黑激素受体；丙戊酸；β-arrestin 2；荧光能量共振转移

基金项目：国家自然科学基金（81402908；81403024）

通讯作者：韩 峰，E-mail：changhuahan@zju.edu.cn，Tel：（0571）88208402

T6-3

PEG-胆固醇双重修饰PBCA纳米粒脑靶向制剂的研究

胡 晓，李 林，张 兰

（首都医科大学宣武医院药物研究室，北京市神经药物工程技术研究中心，神经变性病教育部重点实验室，北京100053）

摘要：目的 中枢神经系统疾病目前存在的主要问题是血脑屏障通透性的问题，本实验期望通过制备PEG-胆固醇双重修饰PBCA纳米粒，提高难透过BBB的中枢神经系统疾病的治疗及早期诊断药物的治疗作用。方法 ①采用乳化聚合法制备双重修饰PBCA纳米粒并对其理化特性进行评价。②双重修饰PBCA纳米粒体内药动学及脑组织分布研究实验前12 h大鼠进行颈静脉插管手术。实验时于插管处给予原药溶液，胆固醇单修饰及PEG-胆固醇双重修饰PBCA纳米粒。给药后不同时间点取血200 μL，化学/发光分析仪测定血液中香豆素-6的含量。大鼠尾静脉分别给予原药溶液、胆固醇单修饰及PEG-胆固醇双重修饰PBCA纳米粒。给药后不同时间点处死大鼠，取脑组织测定香豆素-6的含量。结果①制备的纳米粒粒径为（191.1±1.22）nm、载药量约为1.97%，包封率大于98%。同时双重修饰PBCA纳米粒在体外具有更明显的缓释效果。②双重修饰PBCA纳米粒在体内具有更高的血药浓度及缓释效果，能持续释放12 h，而胆固醇单修饰纳米粒仅释放8 h，原药溶液仅4 h内能检测到药物。脑组织分布研究结果表明，双重修饰PBCA纳米粒静脉给药后脑组织中药物含量显著增高且成缓慢释放的过程，说明具有成为脑靶向制剂的研究潜力。结论 本实验制备的PEG20000-胆固醇双重修饰PBCA纳米粒，可解决中枢神经系统疾病的治疗及诊断药物体内BBB通透率低、安全性差、缓释性差等瓶颈问题，具有重要应用前景。

关键词：中枢系统疾病；纳米粒；血脑屏障；缓释性；脑靶向

通讯作者：李 林，Tel：（010）83198886；E-mail：linli97@ hotmail.com；张 兰，Tel：（010）83198855；E-mail：lanizhg@ hotmail.com

T6-4

双光子成像技术在神经药理学研究中的应用

高银萍1，黄继云2，刘双双2，卢应梅1

（浙江大学1.城市学院，2.医药学部 浙江 杭州 315000）

摘要：目的 近年来，随着荧光基因探针技术和显微成像技术的发展，使不同细胞、亚细胞和分子基团同时成像成为了可能。借助于共聚焦激光扫描显微术和双光子显微术，可开展神经系统疾病的药理学调控研究。方法 双光子激发成像与传统的共聚焦显微镜成像方法比较，具有容易穿透标本、纵向分辨率高、光毒性小、可观察厚标本和活体组织的优点。①采用荧光工具小鼠，如Tie2 GFP/+血管内皮荧光标记、Thy1-YFP锥体神经元荧光标记、Cx3cr1GFP/+小胶质细胞荧光标记小鼠，制作脑缺血、外伤等疾病模型。可以考察作用于神经系统的药物是否影响关联指标动态变化，包括皮层脑区的脑微血管密度、血流灌注程度、微血管流速和白细胞黏附等。②采用特异性小分子化合物荧光探针用来标记分子基团或细胞，可以考察药物调控前后神经元、星型胶质细胞等Ca2+信号、氧化还原损伤信号等的动态变化。③活体双光子成像技术也可结合光遗传学等技术，用于探讨药物对神经环路病理变化的影响。结论 活体双光子成像技术能为药物调控提供功能性指标，可以作为新型技术手段用于神经药理学分子机制解析研究。

关键词：双光子成像技术；荧光；探针；神经元；神经药理

基金项目：国家自然科学基金（81473202）

通讯作者：卢应梅，E-mail：lufx@zju.edu.cn，Tel：（0571）88284325

T6-5

建立斑马鱼组群模型评价精神类化合物对于社会交互行为的影响

颜 慧，宫泽辉，苏瑞斌

（抗毒药物与毒理学国家重点实验室，军事医学科学院毒物药物研究所，北京100850）

摘要：目的 斑马鱼的群集性（shoaling）是一种天生偏好行为，可被药物或外来威胁所干扰。利用这一特性建立组群行为模型，并评价几种精神类化合物急性处理对该类行为的影响。方法 利用Zebralab斑马鱼行为视频跟踪分析系统自动记录动物行为参数，以鱼群内个体间平均距离（IID）、个体与其最近的鱼的距离（NND）、鱼群平均矢量大小等作为评测指标，每8条鱼为一群，甲基苯丙胺（METH）浓度2 mg·L-1，氯胺酮（KET）浓度20 mg·L-1，地卓西平马来酸盐（MK-801）浓度0.25 mg·L-1分别急性暴露30 min后进行行为测试。结果 正常的斑马鱼表现为成群游动，与正常组相比，20 mg·L-1氯胺酮急性暴露后使得鱼群的个体间距离显著减少，个体与其最近同伴的距离显著降低，而鱼群平均矢量显著增加，表明氯胺酮可以使得斑马鱼的群集性显著提高，提示斑马鱼的警觉性异常增加；MK-801处理后使得鱼群的IID显著提高，而鱼群平均矢量显著减小，表明MK-801处理后改变了斑马鱼的天性，其组群性社交活动受到抑制；METH 2 mg·L-1处理后除了使得IID有一定程度降低外，对于斑马鱼的组群行为没有明显影响。结论 正常的斑马鱼以群集方式运动，这种方式是小型鱼类的保护行为，使得鱼群中的个体少些警觉，更不易成为捕食对象，成群行为也更易找到食物、更易交配等。利用这一特性建立的组群模型对于精神类化合物很敏感，且在行为表现上具有特征性，可以作为精神活性物质的行为评价体系中的一个评测环节，成为抗精神障碍药物研发中快速筛选的有力补充。

关键词：斑马鱼；组群行为模型；群集性；甲基苯丙胺；氯胺酮；MK-801

基金项目：国家科技重大专项（2012ZX09301003-003；2015ZX0951003）

通讯作者：苏瑞斌，E-mail：ruibinsu@126.com

T6-6

Tumor necrosis factor-α regulates signaling of EP4 through association of TRAF2 and GRK2 in fibro⁃blast-like synoviocytes

WANG Qing-tong1， HUANG Bei1， LIU Kang-kang1，YANG Si-min1， WU Cheng-yi1， YAN Shang-xue1，ZHANGLing-ling1， CHEN Jing-yu1， WU Hua-xun1，SUNWu-yi1， HUANG Qiong1， HAN Yong-sheng2，HU Yong3，WEI Wei1

（1.Institute of Clinical Pharmacology，Anhui Medical Uni⁃versity，Key Laboratory of Anti-inflammatory and Im⁃mune Medicine，Ministry of Education，Hefei 230032，China；2.Department of Emergency Internal Medicine，Af⁃filiated Anhui Provincial Hospital，Anhui Medical University，Hefei 230001，China；3.Department of Orthopedics，the First Affiliated Hospital of Anhui Medical University，Hefei 230022，China）

Abstract：We previously found that after tumor ne⁃crosis factor-α（TNF-α）treatment，cyclic adenosine mo⁃nophosphate（cAMP）concentration of fibroblast-like syn⁃oviocytes（FLS）in collagen-induced arthritis（CIA）rats was decreased.Here，we show that after TNF-α treat⁃ment，the prostaglandin E2 receptor 4（EP4）signaling of FLS in CIA rats was attenuated.Similarly，EP4 mem⁃brane expression was reduced by TNF-α stimulation in primary human FLS（hFLS）.TNF receptor 2（TNFR2）was predominant in TNF-α induced hFLS proliferation via recruiting TNF receptor associated factor 2（TRAF2）to membrane.More interestingly，we noticed that TRAF2 in⁃teractedwithGproteincoupledreceptorkinase2 （GRK2）in the cytoplasm of hFLS and helped to bring GRK2 to cell membrane in response of TNF-α.Subse⁃quently，the complex of TRAF2 and GRK2 dismissed，GRK2 was then phosphorylated，contributing to the de⁃sensitization and internalization of EP4，leading to a downregulated intracellular cAMP.Silencing of TRAF2 by siRNA failed to induce the translocation of GRK2，and resulted in an upregulated expression of membrane EP4 and intracellular cAMP.These results elucidate a novel form of cross-talk between TNF-α and EP4 signaling. The interaction between TRAF2 and GRK2 may become a potential new drug target for the treatment of autoim⁃mune diseases.

Key words：tumor necrosis factor-α；prostaglandin E2 receptor；TNF receptor associated factor 2；G protein coupled receptor kinase；fibroblast-like synoviocytes

Corresponding author：WEI Wei，wwei@ahmu.edu.cn

T6-7

Regulation of PGE2 signaling pathways and TNF-alpha signaling pathways on function of bone mar⁃row-derived dendritic cells and effect of CP-25

ZHANGLing-ling*， LIYing， SHENGKang-liang，CHEN Jing-yu，WU Yu-jing，ZHANG Feng，CHANG Yan，WU Hua-xun，FU Jing-jing，WEI Wei

（Institute of Clinical Pharmacology，Anhui Medical Uni⁃versity，Key Laboratory of Anti-inflammatory and Im⁃mune Medicine，Ministry of Education，Anhui Collabora⁃tive Innovation Center of Anti-inflammatory and Immune Medicine，Hefei 230032，China）

Abstract：This study was to investigate PGE2 and TNF-α signaling pathway involving in the maturation and activation of bone marrow dendritic cells（DCs）and the effect of CP-25.Bone marrow DCs were isolated and stimulated by PGE2 and TNF-α respectively.The mark⁃ers of maturation and activation expressed on DCs，such as CD40，CD80，CD83，CD86，MHC-II，and the ability of antigen uptake of DCs were analyzed by flow cytome⁃try.The proliferation of T cells co-cultured with DCs，the signaling pathways of PGE2-EP4-cAMP and TNF-α-TRADD-TRAF2-NF-κB in DCs were analyzed.The re⁃sults showed that both PGE2 and TNF-α up-regulated the expressions of CD40，CD80，CD83，CD86，and MHC-II，decreased the antigen uptake of DCs，and DCs stimulated by PGE2 or TNF-α could increase T cell prolif⁃eration.CP-25（10-5，10-6and 10-7mol·L-1）decreased significantly the expressions of CD40，CD80，CD83，CD86 and MHC-Ⅱ，increased the antigen uptake of DCs，and suppressed T cell proliferation induced by DCs.PGE2 increased the expressions of EP4，NF-κB and down-regulated cAMP level of DCs.TNF-α could also up-regulate TNFR1，TRADD，TRAF2，and NF-κB expression of DCs.CP-25（10-5，10-6and 10-7mol·L-1）de⁃creased the expressions of EP4 and NF-κB，increased cAMP level in DCs stimulated by PGE2.CP-25（10-5，10-6and 10-7mol·L-1）also could down-regulate significantly TNFR1，TRADD，TRAF2，and NF-κB expression in DCs stimulated by TNF-α.These results demonstrate that PGE2 and TNF-α could enhance DCs functions by mediating PGE2-EP4-cAMP pathway，TNF-α-TNFR1-TRADD-TRAF2-NF-κB pathway respectively.CP-25 might inhibit the function of DCs through regulating PGE2-EP4-cAMP and TNF-α-TNFR1-TRADD-TRAF2-NF-κB pathways.

Key words：PGE2；TNF-α；EP4；TRAF2；CP-25；den⁃dritic cells

Foundation item：The project supported by National NaturalScienceFoundationofChina（81173075；31100640；81330081；81473223）；and China Postdoc⁃toral Science Foundation（2013M540509）.

Corresponding author：ZHANG Ling-ling，llzhang@ ahmu.edu.cn；WEI Wei，wwei@ahmu.edu.cn

T6-8

Ginsenoside metabolite compound K exerts its antiinflammatory activity by downregulating dendritic cells function in collagen-induced arthritis

CHEN Jing-yu， WUHua-xun， WANGQing-tong，LIU Kang-kang，WEI Wei

（Clinical Pharmacology Institute of Anhui Medical Univer⁃sity，key Laboratory of Antiinflammatory and Immune Medicine，Ministry of Education，Hefei 230032，China）

Abstract：OBJECTlVEA vitro study has shown joint-protective effects of ginsenoside metabolite com⁃pound K（CK）in RA，but the effects of CK in RA animal model and the mechanisms are still unknown.In this study，the effects of CK on collagen-induced arthritis （CIA）and the underlying mechanisms were investigated. METHODSAfter the onset of arthritis，mice were given CK（7，28 and 112 mg·kg-1）and MTX（2 mg·kg-1）.To evaluate the severity of arthritis，arthritis global assess⁃ment and swollen joint count were evaluated every 3 d. Levels of serum antibodies were detected by ELISA.Pro⁃liferation of T cells was measured by CCK.Subset of T cells，phagocytosis and the expression of co-stimulators CD80，CD86 and MHCⅡon dendritic cells（DC）were assayed by flowcytometry.Ability of DC in activation of T cell was measured by mixed lymphocyte reaction.Levels of CCL19 and CCL21 in lymph nodes were assayed by immunology and histology chemistry.The expression of CCR7 on DC was assayed by western blotting.RE⁃SULTS CK attenuated CIA clinical signs，regulated the levels of antibodies in serum，alleviated the histopatholo⁃gy of spleen and joint in CIA mice.CK inhibited T cell pro⁃liferation，up-regulated the percentage of naïve T cell，down-regulated the percentage of activated T cells.In ad⁃dition，CK inhibited levels of chemokines CCL19 and CCL21 in lymph nodes，suppressed DC activation and the expression of CCR7 on DC.CONCLUSlON CK at⁃tenuation CIA and the mechanisms were likely due to suppressing immune response by inhibiting DC activa⁃tion.

Key words：ginsenoside metabolite compound K；den⁃dritic cells；adjuvant-induced arthritis；immune response

Foundation item：The project supported byNational Natural Science Foundation of China（81173075）；Anhui Province Nature Science Foundation in University （KJ2011Z180；KJ2011Z181）.

Corresponding author：WEI Wei，E-mail：wwei@ ahmu.edu.cn

T6-9

GRK2 overexpression inhibits lGF1-induced prolifera⁃tion and migration of human hepatocellular carcino⁃ma cells by down-regulating EGR1

MA Yang1，HAN Chen-chen1，HUANG Qiong1，SUN Wu-yi1，WEI Wei1

（Institute of Clinical Pharmacology，Anhui Medical Uni⁃versity，Key Laboratory of Anti-inflammatory and Im⁃mune Medicine，Ministry of Education，Hefei 230032，China）

Abstract：Gprotein-coupledreceptorkinase2 （GRK2）is a serine/threonine kinase that involves in a va⁃riety of important signaling pathways and alternation of GRK2 protein level or activity causes diseases such as heart failure，rheumatoid arthritis，and obesity.However，the role and mechanism of GRK2 involving in hepatocel⁃lular carcinoma（HCC）progression is not fully investigat⁃ed.In this study we find that GRK2 plays an inhibition role in IGF1-induced HCC cell proliferation and migra⁃tion.Overexpression of GRK2 causes a decrease in early growth response-1（EGR1）expression，while knock⁃down of GRK2 leads to the dramatically increase in EGR1 expression in the treatment of IGF1.Through coimmunoprecipitation and Western blot assay，we confirm that GRK2 can interact with insulin-like growth factor 1 re⁃ceptor（IGF-1R）and inhibiting IGF1-induced activation of IGF-1R signaling pathway.Silencing EGR1 attenuates GRK2 overexpression-caused inhibition of cell prolifera⁃tion，tumor colony number and migration activity，while overexpressing of EGR1 restores the anti-proliferative and migratory effect by GRK2 overexpression in HCCLM3 cells.Taken together，these results suggest that GRK2 may inhibit IGF1-induced HCC cell growth and migration through down-regulation of EGR1 and indicate that en⁃forced GRK2 may offer a potential therapeutic approach against HCC.

Key words：GRK2；EGR1；IGF1R signaling pathway；cell proliferation；cell migration

Foundation item：The project supported byNational Natural Science Foundation of China（81173075）；Anhui Province Nature Science Foundation in University （KJ2011Z180；KJ2011Z181）.

Corresponding author：WEI Wei，E-mail：wwei@ ahmu.edu.cn

T6-10

JAK1-STAT3 blockade by JAK inhibitor SHR0302 at⁃tenuates inflammatory responses of adjuvant-induced arthritis rats and decreases Th17 and total B cells

WU Hua-xun*，YAN Shang-xue*，CHEN Jing-yu，LUO Xue-xia，LI Pei-pei， JIA Xiao-yi，DAI Xing，WANG Chun，HUANG Qiong，LIU Li-hua，ZHANG Yun-fang，ZHOU Ai-wu，CHANG Yan，ZHANG Ling-ling，WEI Wei （Institute of Clinical Pharmacology of Anhui Medical Uni⁃versity，key Laboratory of Anti-inflammatory and Immune Medicine，Ministry of Education，Anhui Collaborative In⁃novation Center of Anti-inflammatory and Immune Medi⁃cine，Hefei 230032，China）

Abstract：OBJECTlVE Toinvestigatethe effects of JAK inhibitor（SHR0302）on adjuvant-induced arthritis （AA）ratsand the partial mechanisms focused on T，B lymphocyte subsetsthrough JAK1-STAT3 pathway，in⁃cluding Th17，Treg，total B cells and memory B cells. METHODSAnimals were divided randomly intonormal control，AA，SHR0302（0.3，1.0 and 3.0 mg·kg-1）and MTX.The effects of SHR0302 on AA rats by evaluating arthritisindex，arthritis global assessmentand paw swell⁃ing degree，histopathology of joint and spleen.We exam⁃ined the proliferation of T，B and FLS；Th17，Treg，total B and memory B cell proportion was measured by flow cytometry；Cytokines TNF-α，IL-1β，IL-10，IL-17 and antibody IgG1，IgG2a levels in serum were measured by ELISA；The expression of p-JAK1 and p-STAT3 was measuredby Western blotting.RESULTS SHR0302 sup⁃pressed the severity of AA rats by attenuating the arthri⁃tisindex，arthritis global assessmentand pawswellingdegree，and alleviated histopathology of spleen and joint of AA rats.SHR0302 can inhibit the proliferation of T，B and FLS，and down-regulated cytokines TNF-α，IL-1β，IL-17 and antibody IgG1，IgG2a levels，and suppressed the proportion of Th17 and total B，and inhibited JAK1-STAT3 phosphorylation；There was no significant effect onTregfunctionandmemoryBcellproportion. CONCLUSlON SHR0302 may attenuate the severity of AA rats，partially through reducing Th17 function and to⁃tal B cell proportion by inhibiting JAK1-STAT3 phosphory⁃lation.

Key words：JAK inhibitor；adjuvant-induced arthritis；Th17；Treg；memory B cells；STAT3

Corresponding author：WEI Wei， E-mail：wwei@ ahmu.edu.cn

*Co-first author

T6-11

Effect of secreted immunoglobulin D on function of peripheral blood mononuclear cells through immuno⁃globulin D receptor in rheumatoid arthritis

WU Yu-jing， CHEN Wen-sheng， CHEN Heng-shi，ZHANG Ling-ling， CHANG Yan， YAN Shang-xue，DAI Xing，MA Yang，HUANG Qiong，WEI Wei

（Institute of Clinical Pharmacology，Anhui Medical Uni⁃versity，Key Laboratory of Anti-inflammatory and Immune Medicine，Ministry of Education，Anhui Collaborative In⁃novation Center of Anti-inflammatory and Immune Medi⁃cine，Hefei 230032，China）

Abstract：OBJECTlVE Immunoglobulin D（IgD）is a surface immunoglobulin that is expressed as either mem⁃brane IgD（mIgD）or secreted IgD（sIgD）.Researchers have shown that sIgD is often elevated in patients with autoimmune diseases.The possible roles of sIgD on the function of peripheral blood mononuclear cells（PBMCs）in rheumatoid arthritis（RA）are still unclear and few stud⁃ies have been performed.The objective of this study was to investigate the abnormal level of immunoglobulin D （IgD）and the effects of it by binding its receptor（IgDR） on peripheral blood mononuclear cells（PBMCs）in rheu⁃matoid arthritis（RA）.METHODS Blood samples were obtained from 54 RA patients and 42 healthy controls. The levels of sIgD，human soluble receptor activator of nuclear factor-κB ligand（sRANKL），anti-cyclic citrullinat⁃ed peptide（anti-CCP），C-reactive protein（CRP）were determined in serum samples by ELISA.Rheumatoid fac⁃tor（RF）was detected by quantitative nephelometry. Erythrocytes sedimentation rate（ESR）was tested by Westergren method.IgDR and mIgD were detected by using flow cytometry.After PBMCs were cultured and treatedwithdifferentconcentrationsofhumanIgD. PBMCs proliferation were measured by CCK-8，inflam⁃matory cytokine production were assessed by inflamma⁃tion antibody array，T-/B-cell subsets and IgDR expres⁃sion were tested by flow cytometry.RESULTS A signifi⁃cantly higher level of sIgD，mIgD and IgDR were detect⁃ed in RA patients compared with healthy controls.The concentrations of sIgD were positively correlated with sRANKL，rheumatoid factor and C-reactive protein in RA patients.Strikingly，IgD could enhance the proliferation of PBMCs and induce IL-1α，IL-1β，TNF-α，IL-6 and pro⁃duction from PBMCs.Moreover，the percentage of acti⁃vated T cell subsets（CD4+CD69+，CD4+CD154+）and ac⁃tivated B cell subsets（CD19+CD23+，CD19+CD21+，CD19+IgD+and CD19-CD138+）were increased by IgD. The percentage of unactivated T cell subset（CD4+CD62L+）and immature B cell subset（CD19+IgM+IgD-）were de⁃creased by IgD in PBMCs.Furthermore，the expressions of IgDR on T and B cells were significantly increased by treatment with IgD.CONCLUSlONIgD enhanced the activation of PBMCs through stimulation of IgDR，which may contribute to RA pathogenesis.IgD represents a po⁃tentially novel immunotherapeutic target for the manage⁃ment of RA.

Key words：rheumatoid arthritis；immunoglobulin D；immunoglobulin D receptor；T cells；B cells；PBMCs

Corresponding author：WEI Wei， E-mail：wwei@ ahmu.edu.cn

T1-3
lnvolvementofPl3K/Akt/FoxO3aandPKA/CREB signaling pathways in protective effect of fluoxetine against corticosterone-induced cytotoxicity in PC12 cells

ZENG Bing-qing，WANG Hai-tao，XU Jiang-ping

（Group of Neuropharmacogy and New Drug Discovery，School of Pharmaceutical Sciences，Sourthern Medical University，Guangzhou 510515，China）

OBJECTlVE Phosphatidylinositol-3-kinase/ protein kinase B/forkhead box O3a（PI3K/Akt/FoxO3a）signaling pathway plays a critical role in mediating neuro⁃nal cell survival.And it has been proved that antidepres⁃sants can protect neuron from brain insults.However，the involvement of PI3K/Akt/FoxO3a in the protective ef⁃fect of fluoxetine has not been fully clarified.The present study is design to investigate the effect of fluoxetine on corticosterone（CORT）-induced injury model in PC12 and explorethe underlying mechanism.METHODS MTT assay was used to evaluate cell viability.Hoechst 33342 staining was performed to measure apoptosis rate of cells.Western blot was applied to detect expression of Akt，FoxO3a and CREB and other proteins.RT-PCR was performed to detect the mRNA level of Bim，Bax，Puma and BDNF.RESULTS Firsty，CORT induced cel⁃lular toxicity in a concentration-dependent manner in PC12 cells，which was significant at 250 μmol·L-1，while fluoxetineincreased cell viability and prevented cell apop⁃tosis significantly at 3 μmol·L-1，and the survival promot⁃ing effect reached maximal at 10 μmol·L-1.Secondly，the protective effects of fluoxetine against cell death inducedbyCORTwasattenuatedbythepretreatmentof LY294002，KRX-0401 and H89，suggesting the involve⁃ment of both PKA pathway and PI3K/Akt pathways in the protective effect of fluoxetine.Furthermore，fluoxetine time and concentration-dependently reversed the CORT-induced attenuate phosphorylation of Akt，FoxO3a as well as CREB，and blockage of PKA and PI3K/Akt result⁃ed in decreased levels of p-CREB，p-Akt and p-FoxO3a in the presence of fluoxetine.In addition，treatment of fluoxetine attenuated the mRNA levels of Bim，Bax，Puma and enhanced the mRNA level of BDNF.CONCLUSlON The results above implied that fluoxetine has a significant neuroprotectiveeffectagainstCORTviaPI3K/Akt/ FoxO3a and PKA/CREB pathways，respectively.

depression；FoxO3a；CREB；corticosterone

The project supported by National NaturalScienceFoundationofChina（81301099；81373384）；Natural Science Foundation of Guangdong Province（S2013040014202）；and China Postdoctoral Science Foundation（2013M542192）

WANG Hai-tao，E-mail：wht821@ smu.edu.cn；XU Jiang-ping，E-mail：jpx@smu.edu.cn

中央财政支持地方高校发展专项资金-重大疾病模型构建与创新药物研制平台

徐江平，E-mail：jpx@smu.edu.cn，Tel：（020）