侯晓敏,秦小江
(山西医科大学 1. 基础医学院、2. 公共卫生学院,山西 太原 030001)
槲皮素通过激活Kv1.5保护糖尿病大鼠冠脉损伤
侯晓敏1,秦小江2
(山西医科大学 1. 基础医学院、2. 公共卫生学院,山西 太原 030001)
槲皮素;Kv1.5;糖尿病;离子通道;冠状动脉;血管损伤
糖尿病可以引起心血管系统发生相应的结构和功能改变,其中对冠状动脉(coronary artery, CA)的损伤会引起糖尿病冠心病(coronary heart disease, CHD)。有研究报道,糖尿病病人患CHD的风险会比其他疾病明显增加[1],而且高血糖能够损伤CA血管平滑肌细胞(vascular smooth muscle cell, VSMC)电压依赖性钾通道(voltage-gated K+channels, Kv)[2]。在VSMC上主要表达的钾通道中,Kv是表达量最高的[3-4],而且最新研究发现,Kv1.5对调节CA供血是必需的[5]。
槲皮素(quercetin,quer)作用广泛,可对抗氧化反应[6-7]、抑制炎症[8]等。研究发现,如果在膳食中摄入比较多的槲皮素,能够大幅度降低心血管疾病的风险[9]。而且我们课题组前期离体实验发现,槲皮素可舒张大鼠CA,与其增大Kv电流有关[10]。有研究报道,饮食中较多摄入黄酮可降低糖尿病及相关心血管疾病发生风险[11-12],然而,关于槲皮素是否会对糖尿病引起的大鼠CA损伤产生影响,尚未见相关研究。
综上所述,糖尿病能够通过抑制CA VSMC Kv引起CA损伤,而槲皮素舒张大鼠CA与增大CA VSMC Kv电流有关,并且Kv1.5对调节CA供血至关重要。所以,我们提出实验假设,槲皮素是否能够通过增大Kv1.5,改善糖尿病大鼠CA损伤。因此在本课题中,我们综合应用离体心脏灌流、CA张力测定、膜片钳电生理和PCR实验,从Kv1.5角度研究槲皮素在保护糖尿病引起大鼠CA损伤中的作用。
1.1药品与试剂槲皮素购自Sigma公司,美国;其余试剂为分析纯,国产。
1.2实验动物30只 SD大鼠,♂,7~8周龄,体质量(220±20)g,购自山西医科大学动物中心。
1.3仪器血管张力测定仪:澳大利亚DMT公司;膜片钳放大器:美国AXON公司;离体心脏灌流装置:泰盟科技有限公司;PCR仪:美国Bio-Rad C1000 TM and CFX96TM Real-time system。
2.1糖尿病大鼠模型的制备与分组将大鼠(30只)随机分组,空白对照组(10只):生理盐水,i.p.;糖尿病组(20只):链脲佐菌素,i.p.,每日100 mg·kg-1,连续2 d给药。2周后,如果血糖 >0.0167 mol·L-1,认为糖尿病模型制备成功,成功造模20只。然后,将这部分大鼠继续分组:糖尿病组(10只,生理盐水,i.g.,8周)和槲皮素干预组(10只,每日槲皮素50 mg·kg-1,i.g.,连续8周)。
2.2离体心脏灌流大鼠麻醉后处死,开胸并快速将大鼠心脏连同主动脉根部剪下,利用主动脉根部将心脏固定于灌流装置上,记录每分钟的CA流量(coronary flow,CF)。
2.3CA张力测定参照本课题组前期方法[10, 13],把大鼠CA固定到张力测定仪上,正式实验前需要验证血管的活性,用KCl(60 mmol·L-1)收缩CA,当收缩幅度能达到3 mN以上,且坪台能够稳定时,可以继续实验。分别向浴槽内加入不同的药物:KCl(60 mmol·L-1)、Kv阻断剂4-氨基吡啶(4-aminopyridine , 4-AP, 10-4mol·L-1)、KCa阻断剂蝎毒素(iberiotoxin , Iber, 10-3mol·L-1)、KATP阻断剂格列本脲(glibenclamide , Glib, 10-4mol·L-1)和KIR阻断剂氯化钡(barium chloride , BaCl2, 10-3mol·L-1),观察CA的收缩反应并记录其张力数值。
2.4Kv电流记录方法采用分开两步进行酶解的方法得到CA VSMC,对细胞进行序列电压刺激(-80 mV~ +60 mV,间隔为10 mV),或单一给予+60 mV刺激),记录Kv电流。不论是序列电压刺激还是单一电压刺激,均选择最后50 ms的电流值,并且取其平均值,CA VSMC分离方法及Kv记录方法参照文献[10]。
2.5RT-PCR检测大鼠CAKv1.5mRNA表达大鼠处死后,显微镜下急性分离3组大鼠CA,并快速将CA放入液氮中保存,观察3组大鼠CA Kv1.5 mRNA变化,内参为β-actin,具体方法参照文献[14]。Kv1.5引物,正向: 5′-ATGCAGGGTCACTCCATC-3′,反向:5′-GGCTTCTCCTCTTCCTTG-3′;β-actin引物,正向:5′-AAGATCCTGACCGAGCGTGG-3′,反向: 5′-CAGCACTGTGTTGGCATAGAGG-3′。
3.1槲皮素对糖尿病大鼠CF的影响正常对照组CF为(7.81±0.62) mL·min-1,糖尿病组CF明显下降,其值为(6.13±0.45)mL·min-1,给糖尿病大鼠膳食补充槲皮素8周后,其CF有所增加,其值为(7.14±0.52)mL·min-1,见Tab 1。
3.2槲皮素对糖尿病大鼠CA环张力的影响KCl对空白组大鼠CA的最大收缩张力为(3.64±0.27)mN;糖尿病组大鼠的收缩幅度明显增强,达到(5.12±0.44)mN;膳食补充槲皮素可减弱糖尿病大鼠CA对KCl的收缩反应,其收缩幅度为(4.35±0.16)mN。与糖尿病组相比,“糖尿病+槲皮素”组CA对Kv通道阻断剂4-AP的收缩幅度明显低于单纯糖尿病组,证实槲皮素减轻糖尿病所致CA损伤的作用与激活Kv有关,见Tab 2。
Tab 1 Effects of quercetin on CF of diabetic rats (n=6)
*P<0.05vscontrol;#P<0.05vsdiabetes
3.3槲皮素对糖尿病大鼠CAVSMCKv电流的影响如Fig 1所示,空白对照组、糖尿病组、“糖尿病+槲皮素”组大鼠CA VSMC Kv最大电流密度分别为(54.52±4.15)pA·pF-1、(22.62±3.24)pA·pF-1和(41.40±2.45)pA·pF-1。与空白组相比,糖尿病组CA VSMC Kv电流明显减小,差异有统计学意义;糖尿病组补充槲皮素后,其CA VSMC Kv电流降低幅度有所减弱。
Fig 1 The Kv currents on isolated rat CA VSMC (n=6)
A: TheI-Vrelationship curve of Kv currents when the currents were evoked by a series of depolarizing pulses (from -80 mV to +60 mV); B: The Kv currents were recorded when the currents were evoked by +60 mV depolarizing pulses.*P<0.05vscontrol;#P<0.0.5vsdiabetes.
Tab 2 The contraction amplitude of CA to vasoconstrictor (mN, n=6)
*P<0.05vscontrol;#P<0.0.5vsdiabetes
3.4槲皮素对糖尿病大鼠CAKv1.5mRNA表达的影响如Fig 2所示,Kv1.5 mRNA在3组大鼠CA的相对表达量(与β-actin表达的比值)依次为:空白对照组表达量最多,“糖尿病+槲皮素”组次之,糖尿病组表达量最少。
Fig 2 The mRNA expression of Kv 1.5 on rat CA (n=6)
A: The photograph of electrophoresis of RT-PCR amplification of Kv1.5 mRNA on rat CA; B: The mRNA expression of Kv1.5 normalized to the amount of β-actin. 1: β-actin of control, 2: Kv1.5 of control, 3: β-actin of diabetes+quer, 4: Kv1.5 of diabetes+quer, 5:β-actin of diabetes, 6: Kv1.5 of diabetes.*P<0.05vscontrol;#P<0.05vsdiabetes.
CA给心肌细胞提供养分,其管壁主要是VSMC。在本实验中我们发现,糖尿病组的CF明显低于正常组,在糖尿病大鼠的饮食中补充8周槲皮素后,其CF有所回升,说明糖尿病削弱了大鼠CA的舒张能力。而且该结论在离体CA张力测定中也得到了进一步证实,糖尿病组大鼠CA对KCl的收缩反应更敏感,而补充槲皮素后得到了好转。此外我们发现,糖尿病补充槲皮素组CA对Kv通道阻断剂4-AP的收缩反应明显低于糖尿病组,而对其他几种阻断剂没有明显差异,这提示槲皮素减轻糖尿病所致CA损伤与激活Kv有关。
课题组前期研究发现,槲皮素可舒张正常SD大鼠CA,该作用与激活大鼠CA VSMC Kv有关[10]。因此,为了进一步证实槲皮素对糖尿病所引起CA损伤的改善作用是否也与Kv相关,我们记录了3组大鼠CA VSMC Kv电流,其最大电流密度分别为:空白对照组(54.52±4.15)pA·pF-1,糖尿病组(22.62±3.24)pA·pF-1,糖尿病+槲皮素组(41.40±2.45)pA·pF-1。有研究报道,高浓度葡萄糖可损伤CA VSMC Kv[2],在本实验中我们也发现了与之相一致的现象,糖尿病组Kv电流较空白对照组明显降低,膳食补充槲皮素可以得到改善。
在目前已经明确的钾离子通道中,VSMC上表达最多的是Kv,Kv 在调节CA张力方面起着关键性的作用[15],而且最新研究发现Kv1.5对调节CA供血是必需的[5]。在本实验中,我们进一步通过RT-PCR实验测定CA VSMC Kv1.5 mRNA表达变化,得出了与前述实验相吻合的结论,同时也证明槲皮素是通过激活Kv1.5起到了对糖尿病所导致的大鼠CA损伤的保护作用。本课题的实验结果将为临床使用槲皮素预防和治疗糖尿病CA损伤提供基础依据。
[1] Wang R X, Shi H F, Chai Q, et al. Molecular mechanisms of diabetic coronary dysfunction due to large conductance Ca2+-activated K+channel impairment[J].ChinMedJ, 2012,125(14): 2548-55.
[2] Liu Y, Terata K, Rusch N J, et al. High glucose impairs voltage-gated K(+) channel current in rat small coronary arteries[J].CircRes, 2001,89(2): 146-52.
[3] Ko E A, Han J, Jung I D, et al. Physiological roles of K+channels in vascular smooth muscle cells[J].JSmoothMuscleRes, 2008,44(2): 65-81.
[4] Cox R H. Molecular determinants of voltage-gated potassium currents in vascular smooth muscle[J].CellBiochemBiophys, 2005,42(2): 167-95.
[5] Ohanyan V, Yin L, Bardakjian R, et al. Requisite role of Kv1.5 channels in coronary metabolic dilation[J].CircRes, 2015,117(7): 612-21.
[6] 卢 宁, 韩吉春, 任博雪, 等. 二氢槲皮素预处理对心肌缺血/再灌注损伤抗氧化作用的影响[J]. 中国药理学通报, 2017,33(4): 487-92.
[6] Lu N, Han J C, Ren B X, et al. Antioxidation effect of dihydroquercetin pretreatment in isolated rat hearts during myocardial ischemia reperfusion injury[J].ChinPharmacolBull, 2017,33(4): 487-92.
[7] 刘红亮, 胡 磊, 王靖凯, 等. 槲皮素对H2O2损伤PCI2细胞的保护效果与机制[J]. 中国药理学通报, 2014,30(3): 373-7.
[7] Liu H L, Hu L, Wang J K, et al. Protective effects of quercetin on PCI2cells with H2O2-induced oxidative damage[J].ChinPharmacolBull, 2014,30(3): 373-7.
[8] Mahmoud M F, Hassan N A, El Bassossy H M, et al. Quercetin protects against diabetes-induced exaggerated vasoconstriction in rats: effect on low grade inflammation[J].PLoSOne, 2013,8(5): e63784.
[9] Zamora-Ros R, Jimenez C, Cleries R, et al. Dietary flavonoid and lignan intake and mortality in a Spanish cohort[J].Epidemiology, 2013,24(5): 726-33.
[10] Hou X, Liu Y, Niu L, et al. Enhancement of voltage-gated K+channels and depression of voltage-gated Ca2+channels are involved in quercetin-induced vasorelaxation in rat coronary artery[J].PlantaMed, 2014,80(6): 465-72.
[11] Jacques P F, Cassidy A, Rogers G, et al. Higher dietary flavonol intake is associated with lower incidence of type 2 diabetes[J].JNutr, 2013,143(9): 1474-80.
[12] Kim J H, Kang M J, Choi H N, et al. Quercetin attenuates fasting and postprandial hyperglycemia in animal models of diabetes mellitus[J].NutrResPract, 2011,5(2): 107-11.
[13] 房龙梅, 侯晓敏, 杨 蓉, 等. 阿魏酸对离体大鼠冠状动脉的舒张作用及机制探讨[J]. 中国药理学通报, 2016,32(4): 554-8.
[13] Fang L M, Hou X M, Yang R, et al. Vasodilatory effect of Ferulic acid oninvitrorat coronary arter[J].ChinPharmacolBull, 2016,32(4): 554-8.
[14] Chai Q, Xu X, Jia Q, et al. Molecular basis of dysfunctional Kv channels in small coronary artery smooth muscle cells of streptozotocin-induced diabetic rats[J].ChinJPhysiol, 2007,50(4): 171-7.
[15] Dick G M, Tune J D. Role of potassium channels in coronary vasodilation[J].ExpBiolMed, 2010,235(1): 10-22.
QuercetinprotectscoronaryarteryfrominjuryinducedbydiabetesinratsbyactivatingKv1.5
HOU Xiao-min1, QIN Xiao-jiang2
(1.SchoolofBasicMedicalSciences; 2.SchoolofPublicHealth,ShanxiMedicalUniversity,Taiyuan030001,China)
AimTo research into the effect of quercetin on coronary artery(CA) damage in diabetic rats and its relationship with Kv1.5.MethodsThirty male rats were randomly divided into blank control group, diabetes group and diabetes group + quercetin group. The effects of quercetin on heart coronary flow (CF) in diabetes rat were observed by CF measurement; the effects of quercetin on CA tension in diabetes rat were detected by CA tension measurement. To investigate the mechanism of quercetin improving CA lesions caused by diabetes, Kv currents of CA VSMC in rats were recorded using whole cell patch clamp, and Kv1.5 mRNA of CA VSMC was determined.ResultsCompared with normal group, CF of diabetic rats dropped significantly, and CF could increase with the supplement of quercetin in rat diet; the maximum contraction amplitude of CA in response to the contraction of KCl could be reduced with supplement of quercetin in diabetic rat dietary; compared with diabetes group, the contraction of CA from “diabetes + quercetin” to 4-AP significantly decreased; compared with blank control group, CA VSMC Kv currents of diabetes group had a significant decrease(P<0.05), and dietary supplement of quercetin could improve the above changes; RT-PCR results indicated that the expression of Kv1.5 mRNA on rat CA was the highest in control group, then in diabetes group and the lowest in “diabetes+quercetin” group.ConclusionQuercetin has protective effect on coronary muscle damage caused by diabetes, which maybe related to Kv1.5 channel.
quercetin; Kv1.5; diabetes; ion channel; coronary artery; vascular injury
:目的研究槲皮素对糖尿病大鼠CA损伤的改善作用及该作用与Kv1.5的关系。方法30只 ♂ SD大鼠,随机分为3组:空白对照组、糖尿病组、“糖尿病+槲皮素”组。通过大鼠冠脉流量(coronary flow,CF)测定,观察槲皮素对糖尿病所致CF变化的影响;利用冠状动脉(coronary artery,CA)张力测定,观察槲皮素对糖尿病所致CA张力变化的影响;应用膜片钳记录CA血管平滑肌细胞(vascular smooth muscle cell, VSMC)电压依赖性钾通道(voltage gated potassium channel,Kv)电流及测定CA VSMC Kv1.5 mRNA表达水平,探讨槲皮素改善糖尿病所致CA损伤的机制。结果糖尿病组CF较空白组明显下降,糖尿病大鼠饮食中补充槲皮素,可使得其CF有所增加;膳食补充槲皮素可减弱糖尿病大鼠CA对KCl的收缩反应(P<0.05);与糖尿病组相比,“糖尿病+槲皮素”组CA对Kv阻断剂4-AP的收缩幅度明显降低;糖尿病组大鼠CA VSMC Kv电流较空白组明显降低(P<0.05),膳食补充槲皮素可减小其降低幅度;RT-PCR结果表明,Kv1.5 mRNA相对表达量空白组最高,“糖尿病+槲皮素”组次之,糖尿病组最少。结论槲皮素对糖尿病CA损伤有保护作用,该作用与激活Kv1.5存在一定相关性。
时间:2017-9-5 9:26 网络出版地址:http://kns.cnki.net/kcms/detail/34.1086.R.20170905.0925.044.html
2017-05-19,
2017-08-10
山西省高等学校科技创新项目(No 2017146,2017147);山西省青年科技研究基金(No 201701D221247,201701D221 259);山西医科大学博士启动基金(No 03201510,032015 21);山西医科大学青年基金(No 02201604,02201613)
侯晓敏(1983-),女,博士,讲师,研究方向:天然产物的心血管药理作用,E-mail:houxiaominyaoli@163.com; 秦小江(1986-),男,博士,讲师,研究方向:环境污染物致血管损伤的作用机制及其防治,通讯作者,E-mail:sxykdxyxy@163.com
10.3969/j.issn.1001-1978.2017.10.022
A
:1001-1978(2017)10-1442-04
R-332;R284.1;R322.12;R322.74;R587.201.31