The 12th China-Japan Joint Seminar on Histochemistry and Cytochemistry

Sponsored by

Branch of Histology and Embryology, Chinese Society for Anatomical Sciences

The Japanese Society of Histochemistry and Cytochemistry

Organized by

Hebei Society for Anatomical Sciences Hebei North University

The 12th China-Japan Joint Seminar on Histochemistry and Cytochemistry

Sponsored by

Branch of Histology and Embryology, Chinese Society for Anatomical Sciences

The Japanese Society of Histochemistry and Cytochemistry

Organized by

Hebei Society for Anatomical Sciences Hebei North University

Proceedings

Aug. 26-29, 2017

Zhangjiakou, Hebei, China

01  Differentiation-dependent changes in DNA methylation and histone H3 acetylation: possible roles of these epigenetic factors in mouse spermatogenesis

Takehiko Koji

Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan Mammalian spermatogenesis is a well-ordered process, consisting of spermatogonial mitosis, spermatocytic meiosis and spermiogenesis. The process is also characterized by successive chromatin condensation and a high frequency of spermatogenic cell apoptosis. Therefore, epigenetic factors can be implicated in the regulation of spermatogenesis and any epigenetic disorder may lead to the induction of apoptosis. When we analyzed global level of DNA methylation by immunohistochemistry with anti-5-methylcytosine in paraffin-embedded sections of mouse testis, there was no difference among any stages of germ cells. However, methylated CCGG site, which is analyzed by a new method HELMET, was increased gradually depending upon the progress of spermatogenesis, but in step 11-12 spermatids methylation of CCGG was markedly declined. Since demethylation at CCGG sites was well correlated with apoptotic germ cells, we tried to examine the effect of experimental demethylation of DNA on spermatogenesis. To this end, we injected 5-aza-2’-deoxycytidine intraperitoneally into adult ⅠCR mice at 0.25 mg/Kg/day for 10 successive days and in the next day they were killed and the testes were analyzed. As a result, we found a significant decrease of methylation at CCGG sites only in spermatogonia, accompanying a marked increase in apoptosis. Ⅰnterestingly, the number of apoptotic spermatocytes was also significantly increased, but the mechanism to induce apoptosis seemed to be different from that of spermatogonia and maybe through different usage of DNA methyltransferases. As for histone modification, when acetylation levels of histone H3 at K9, K18 and K23 were examined in normal mouse testis by immunohistochemistry, a marked decrease in early spermatids and the following increase in the later steps were found. To explore the roles of the acetylation, we mimicked the acetylation level by administration of HDAC inhibitor, Na phenyl butyrate, and by knockdown of histone acetyltransferases (SRC-1, GCN5 and ELP-3) with electroporation of shRNA vectors. Consequently, we found that upregulation of acetylation at K9 and K18 (but not at K23) and its downregulation at K9 (but not at K18 and K23) resulted in the induction of apoptosis in early and the later spermatids, respectively. These results indicate essential roles of epigenetic factors in maintaining mammalian spermatogenesis.

02  PVT-CeA-PAG pathway facilitating neuropathic pain information transmission

Shao-Hua Liang1, Yu-Lin Dong1, Yun-Qing Li1,21Department of Anatomy and K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi’an 710032, China;2Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China

Objective: The paraventricular thalamic nucleus (PVT) is considered to be a critical site for the integration of neural signals and to modulate both visceral information and negative emotions. The aim of the present study was attempted to investigate the activation of PVT neurons involved in modulation of the mechanical allodynia induced by neuropathic pain and the PVT-central amygdaloid nucleus (CeA)-periaqueductal gray (PAG) pathway might underlie the descending facilitative effects on the nociceptive transmission. Methods: The connections of the PVT-CeA-PAG pathway was confirmed by morphological tract tracing techniques. The facilitating effects of this pathway for the neuropathic pain information transmission were demonstrated with patch-clamp recording, behavior observation and optogenetic approach. Results: Ⅰt was found that paw withdrawal threshold (PWT) was increased significantly after the inhibition of PVT neuronal activations by chemical or pharmacogenetic lesion in spared nerve injury (SNⅠ) rats. The frequency, but not amplitude of the spontaneous excitatory postsynaptic currents (sEPSCs) of PVT neurons which sent projection fibers to CeA was significantly increased in the SNⅠ rats. Selective activation of PVT-CeA pathway by optogenetic approach induced obvious mechanical allodynia in the naive rats. After injection of biotinylated dextran amine (BDA) into PVT and fluoro-gold (FG) into the ventrolateral subregion of the PAG, respectively, BDA-labeled fibers and terminals originated from PVT were found to make asymmetric synapses with FG-labeled neurons in CeA which projected to PAG. Furthermore, it was demonstrated that the CeA-PAG connection was GABAergic in nature. Conclusion: The present results suggest that there is a novel PVT-CeA-PAG pathway which is essential for descending facilitation on neuropathic pain information transmission.

03  The mechanism of RCCD1 in tumorigenesis and chemo-sensitivity in NSCLC

Daliang Wang

Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China

Lung cancer is one of the most lethal cancers due to its highly metastatic spreading. Ⅰt was well studied that the motility of lung cancer cells was regulated by paracrine factors, such as TGF-β, in the tumor microenvironment through the induction of epithelialto-mesenchymal transition (EMT). The stability of microtubules is reported to be associated with the EMT process and the migration of cancer cells. Bioinformatics analysis shows that RCC1 domaincontaining protein 1 (RCCD1) is highly expressed in non-small cell lung cancer (NSCLC) patients with poor prognosis. We therefore hope to find the regulation mechanism of RCCD1 in the tumorigenesis of NSCLC. Our results indicated that RCCD1 was much higher expressed in tumor tissues compared with adjacent normal tissues. Ⅰn vitro, depletion of RCCD1 using siRNAs significantly inhibited the migration of lung cancer cells(A549 cells). Significantly loss of RCCD1 resulted in upregulation of acetylated α-tubulin levels and stabilized cytoskeletal microtubules. Mechanistically, we demonstrated that RCCD1 could modulate the stability of microtubules through interacting with JMJD5(Jumonji Domain-containing 5).Furthermore, JMJD5 could interact with tubulin protein and associates with spindle microtubules for proper mitosis. Ⅰn addition, the sensitivity to chemotherapeutic drugs showed that RCCD1 clearly enhanced the sensitivities of lung cancer cells to microtubule-stabilizing drugs. Depletion of RCCD1 significantly attenuated the TGF-β-induced EMT process assessed by altered expression of epithelial and mesenchymal markers, and thereby inhibited TGF-β-induced cell migration. In summary, our results indicated that RCCD1 plays an important role in regulation in tumorigenesis and chemo-sensitivity of NSCLC. RCCD1 might be as a potential candidate target for NSCLC therapy.

04  Ultra high-resolution imaging of whole brain using advanced light-sheet fluorescent microscopy

Xuechun Wang1, Yusha Li2, Chunyu Fang1, Tingting Yu2, Dan Zhu2, Peng Fei1,21School of Optical and Electronic Information, Huazhong University of Science and Technology, Wuhan, 430074, China;2Britton ChanceCenter for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.

Study of the brain is the hottest topic in neuron science. The complex functionalities of brain is highly from its complicated neuron structures (circuits). For better understanding how the brain works, it is crucial to map the neuron network at whole brain level, with high resolution. Among a variety of optical brain imaging techniques, such as serial two photon tomography (STP), microoptical sectioning tomography (MOST), light-sheet fluorescent microscopy (LSFM) has recently emerged for its advantages of highthroughput and low photo-bleaching. However, compared to STP or MOST, its relatively low axial resolution at whole-brain scale limits its application for visualizing the neural connections in the brain, at subcellular level. We hereby develop a unique Bessel brain-wide light-sheet fluorescence microscopy (2B-LSFM) for high-resolution, isotropic imaging of ultra-fine neural structures. we generate superior scanning Bessel light-sheet illumination on whole mouse brain, with 2 cm-long working distance, 2 mm-wide coverage and only 1 μmsharp excitation at the vicinity of the focal plane. Using 2B-LSFM, we obtain the 3-D connections of the giant pyramidal neurons in a whole mouse brain, including their tip dendrites and long axon projections, with an isotropic voxel resolution of 0.5 micron. Compared to the hyperbolic Gaussian laser-sheet which is the mainstream of LSFM, our new method improves the axial resolution of current LSFM-based whole brain imaging for 5 to 10 folds. By a relatively cost-effective means, with keeping the high-throughput, low photon burden advantages, 2B-LSFM method further achieves subcellular, isotropic resolution at whole-brain scale. Thus it shows great potentials for various applications of neuron/brain research.

05  Preventive role of the gland mucin-specific αGlcNAc in gastric cancer development

Jun Nakayama

Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Japan

Gastric mucins are classified into 2 types; i.e., surface mucin and gland mucin. The latter specifically contains unique O-glycanscontaining terminal α1,4-linked GlcNAc residues called αGlcNAc, which attach to its scaffold MUC6. As the first step toward understanding of the role of αGlcNAc, we isolated cDNA encoding α1,4-N-acetylglucosaminyltransferase (α4GnT), which is responsible for the biosynthesis of αGlcNAc by expression cloning. Helicobacter pylori (H. pylori) is a causative microbe for gastric cancer and largely colonizes the surface mucin. However, this microbe is barely found in the gland mucin. To investigate the role of αGlcNAc in H. pyloriinfection,we prepared recombinant soluble CD43 having αGlcNAcusing α4GnT cDNA and revealed that αGlcNAc inhibited growth and motility of H. pyloriby suppressing biosynthesis of its cell wall component cholesteryl α-D-glucopyranoside (CGL).We then generated A4gnt-/-micedeficient in α4GnT. Surprisingly the mutant mice developed gastric differentiated-type adenocarcinoma without the involvement of H. pylori infection. We also revealed that genes associated with chronic inflammation such as inflammatory cytokines Ⅰl1b and Ⅰl11, chemokines Ccl2 and Cxcl1, and growth factors Hgf and Fgf7 were up-regulated in the gastric mucosa ofA4gnt-/-mice compared to wild type mice, demonstrating that the absence of αGlcNAc triggers gastric carcinogenesis through inflammation-associated pathways in vivo. Finally we revealed that loss of αGlcNAc in human gastric cancer was associatedwith progression and poor prognosis of patients with differentiated-type but not undifferentiated-type adenocarcinoma of the stomach.Taken together, the gland mucin-specific αGlcNAc plays preventive role in gastric cancer development by inhibition ofH. pylori infection and suppression of tumor-promoting inflammation.

Reference: Nakayama J. Dual roles of gastric gland mucin-specific O-glycans in prevention of gastric cancer (Review). Acta Histochem Cytochem. 47, 1-9, 2014.

06  BMSCs transplantation increased GAP-43 expression via ERK1/2 and Pl3K/Akt pathways following intracerebral hemorrhage in rats

Junling Gao1,2, Changmeng Cui3, Kaijie Wang3, Wenqian Zhang3, Shaohui Wang3, Lei Yu1,2, Xiaohua Jiang1,2, Ran Li1,2, Yanxia Tian1,2, Jianzhong Cui31Department of Histology and Embryology, School of Basic Medical Sciences, University of North China Science and Technology, Tangshan 063210, China;2Hebei Key Laboratory for Chronic Diseases, School of Basic Medical Sciences, University of North China Science and Technology, Tangshan 063210, China;3Department of Neurosurgery, Tangshan Gongren Hosipital, Hebei Tangshan 063000, China

Ⅰntracerebral hemorrhage (ⅠCH) is a complex pathology with high mortality and disability in the adult population. The corticospinal tract (CST) is the most important motor pathway, and the extent of its damage is correlated with the motor outcome after an acute ⅠCH. A total of 75 male rats (250–280 g) were randomly assigned to 5 groups including ⅠCH model group, ⅠCH + BMSCs group, ⅠCH + BMSCs + PD98059 group, ⅠCH + BMSCs + LY294002 group and ⅠCH + BMSCs + PD98059 + LY294002 group.We used autologous blood injection to induce ICH in rats, 100 μL of autologous arterial blood was infused slowly (5 μL/min) with a microinfusion pump. BMSCs from 21 days old male Sprague-Dawley (SD) ratswere subcultured at 1×104cells/cm3and used in the experiments after 3 passages. The Neurological Severity Scores (NSS) were performed before the surgery and at 1, 3 and 7 days after ⅠCH by an investigator who was blinded to the experimental groups, to examine the expression of GAP-43, Akt, p-Akt, ERK1/2, p-ERK1/2 proteins by western blot. BMSCs transplantation attenuated post-ⅠCH motor functions deficits at 3-7 days. The expression levels of CST axonal outgrowth associated protein GAP-43 in the perihematomal tissues were increased at 3 days in BMSCs group. Furthermore, we investigated the underling mechanisms by administrating of PD98059 (p-ERK1/2 inhibitor) or/and LY294002 (PⅠ3K inhibitor) immediately after ⅠCH. We firstly found that either PD98059 or LY294002 blocked the effect of BMSCs, and both inhibitors together were more effective. These results confirm that the neuroprotective effects of BMSCs on ⅠCH might be through regulating GAP-43 expression by activating both ERK1/2 and PⅠ3K/Akt signaling pathways.

07  New concept of hypothalamo-pituitary-gonadal axis according to kisspeptin and kisspeptin receptor system

Hitoshi Ozawa

Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan

Since discovery of a newly bioactive neuropeptide, kisspeptin, and its receptor, GPR-54, the concept of hypothalamo-pituitry-gonadal (HPG) axis and the regulation system of reproduction has been reconsidered. Kisspeptin is a potent stimulator of GnRH secretion via GPR54 expressed in GnRH neurons, and functions as a gatekeeper of puberty onset in mammals. Kisspeptin neurons are located in twodifferent nuclei. One is anteroventral periventricular nucleus (AVPV), which is received positive feedback by estrogen via estrogen receptor a (ERa), and the other, arcuate nucleus (ARC), which is received negative feedback by via (ERa) . Kisspeptin neurons in the AVPV is known to be a part of surge generator, and in the ARC, a part of pulse generator for GnRH/LH. Ⅰn rodents, the distribution of kisspeptin neurons shows the sexual dimorphic. Female animals exhibit a large number of kisspeptin neurons, whereas male exhibit only a few scattered kisspeptin neurons. Ⅰt is reported a different morphological characteristic of ARC kisspepitn neurons from AVPV kisspeptin neurons. ARC kisspeptin neurons coexpress two different peptides, neurokinin B and dynorphin. Since then, the ARC kisspeptin neurons are referred to a KNDy (kisspeptin/neurokinin B/dynorphin) neurons. Ever since the discovery of kisspeptin in 2001, intensive studies on hypothalamic expression of kisspepitn and physiological role of hypothalamic kisspepitn neurons have well provided a clue as to how the brain controls sexual maturation at the onset of puberty, and subsequent reproductive events and fertility. Ⅰn the present talk,Ⅰ would like to introduce this new concept of HPG axis according to the kisspepitn/GPR54 system with our recent new data.

08  Expression and intracellular aggregation of α-synuclein in neurons

Masaki Tanaka1, Katsutoshi Taguchi1, Yoshihisa Watanabe21Department of Anatomy and Neurobiology,2Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine

α-Synuclein (α-Syn), composed of 140 amino acids, is a major pathological component of synucleinopathies including Parkinson’s disease (PD) and dementia with Lewy bodies (DLBs). α-Syn is usually expressed in the presynaptic terminals and is involved in maintaining the synaptic function with binding SNARE proteins. We previously reported the differential expression of α-Syn in the mouse brain and suggested that α-Syne xpression levels may determine the efficiency of intracellular aggregate formation in different neuronal subtypes. α-Syn was highly expressed in the neuronal cell bodies of some early PD-affected brain regions, such as the olfactory bulb, dorsal motor nucleus of the vagus, and substantia nigra pars compacta. Synaptic expression ofα-Syn was mostly accompanied by expression of vesicular glutamate transporter-1, an excitatory presynaptic marker. In contrast, expression of α-Syn in the GABAergic inhibitory synapses was different among brain regions. Formation of Lewy body-like aggregates can be replicated in cultured cells by introducing α-Synpreformed fibrils generated in vitro. We demonstrated the intracellular α-Syn inclusions immediately underwent phosphorylation and ubiquitination. Simultaneously they were associated with autophagy receptor protein p62/SQSTM1. Recently α-Syn oligomers/fibrils have been considered to transfer between neurons in the brain with the progression of PD/DLB. However the exact pathogenic transmissible α-Syn polymers have not been identified. We are currently pursuing pathogenic α-Syn which can lead intracellular Lew body/neurite-like aggregation and secrete from synaptic terminals using primary cultured neurons.

Watanabe et al, PLOS One. 2012, Autophagy. 2017; Taguchi et al, PLOS One. 2014, J Comp Neuro.l 2016.

09  Neural networks between a newly identified perifornical area of the anterior hypothalamus and lateral septum: Chemogenetic investigations for physiological roles of urocortin3/enkephalin co-expressing neurons

Horii-Hayashi Noriko Mayumi, Takayo Sasagawa, Mayumi Nishi Department of Anatomy and Cell Biology, Nara Medical University The perifornical area of the anterior hypothalamus (PeFAH) is a hypothalamic area that we recently identified in the mouse brain, which locates between the fornix and paraventricular nucleus of the hypothalamus. Our previous study has revealed that the PeFAH includes urocortin 3 (Ucn3) and enkephalin (Enk) co-expressing neurons that project to a caudal particular region of the lateral septum (LS). Ⅰn the present study, we tried a pharmacogenetic approach, designer receptors exclusively activated by designer drugs (DREADD), to manipulate the activity of Ucn3/Enk neurons specifically. Cre-dependent adeno-associated virus vector expressing hM3Dq, Gq-coupled modified G-protein-coupled receptor, and mCherry as a reporter was injected into the PeFAH of Ucn3-cre transgenic mice. We examined whether clozapine-N oxide (CNO)-induced excitation of Ucn3/Enk neurons activates directly targeted LS neurons and indirectly connected other neurons by c-Fos immunohistochemistry. Results indicated that CNO injection increased c-Fos expression level in the LS neurons compared with saline-injected controls, indicating that Ucn3/Enk neurons were excitatory. CNO injection also increased c-Fos levels in the ventral hippocampus, basolateral amygdala, and dorsal periaqueductal gray, probably by indirect action via the LS. Next, we investigated the effects of Ucn3/Enk neurons’ excitation on anxiety levels by behavioral tests of the light-dark box, open field, novelty object, and elevated plus maze. Furthermore, we investigated risk assessment and defensive burying behaviors, both of which have the common characteristics of active approaching toward threats. These results suggest that the urocortin 3/enkephalin pathway between the PeFAH and lateral septum controls active defense styles against inanimate threats.

10  Function of a membrane skeletal protein, 4.1G, in myelin formation in the peripheral nervous system

Nobuo Terada1, Yurika Saitoh1,2, Nobuhiko Ohno3,Junji Yamauchi4, Takeharu Sakamoto51Division of Health Sciences, Shinshu University Graduate School of Medicine, Matsumoto City, Nagano, Japan;2Faculty of Medical Sciences, Teikyo University of Science, Adachi-ku, Tokyo, Japan;3Center for multidisciplinary brain research, National Institute for Physiological Science, Okazaki City, Aichi, Japan;4Laboratory of Molecular Neuroscience and Neurology, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji City, Tokyo, Japan;5Division of Molecular Pathology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan

We previously demonstrated that a membrane skeletal molecular complex, 4.1G–membrane palmitoylated protein 6 (MPP6)–cell adhesion molecule 4 (CADM4), is incorporated in Schwann cells in the peripheral nervous system (PNS). Ⅰn this study, we evaluated motor activity and myelin ultrastructures in 4.1G-deficient (-/-) mice. When suspended by the tail, aged 4.1G-/- mice displayed spastic leg extension, especially after overwork. Motor-conduction velocity in 4.1G-/- mice was slower than that in wild-type mice. Using electron microscopy, 4.1G-/- mice exhibited myelin abnormalities: myelin was thicker in internodes, and attachment of myelin tips was distorted in some paranodes. Ⅰn addition, we found a novel function of 4.1G for sorting a scaffold protein, Lin7, due to disappearance of the immunolocalization and reduction of the production of Lin7c and Lin7a in 4.1G-/- sciatic nerves, as well as the interaction of MPP6 and Lin7 with immunoprecipitation. Thus, we herein propose 4.1G functions as a signal for proper formation of myelin in PNS.

11  Effects of sevoflurane on expression of GABAR1 and NMDAR2B in the temporal lobe of aged rats

Xiao-Nan Yang1, Meng Zhao1, Peng-Tao Li1, Xin-Sheng Wang21Graduate Faculty of Hebei North University;2The First Affiliated Hospital of Hebei North University, Zhangjiakou 075000, China

Objective: To observe the change of learning and memory and the expression change of GABAR1 and N-methyl-D-aspartate receptor 2B (NMDAR2B) in the temporal lobe of the aged rats after the inhalation of sevoflurane and discuss the influence. Methods: Aged male SD rats were randomly divided into three groups. Group T1 was inhaled sevoflurane for 1 day for Morris water maze test; Group T7 was inhaled sevoflurane for 7 days for Morris water maze test. Anesthetic concentration of 3%, to maintain anesthesia for 2h. The control group was given inhaled room temperature air for 2h. The left temporal lobe of the rats was collected after the test. The changes of GABAR1 and NMDAR2B gene expression and protein expression were detected by PCR and immunofluorescence. Results: Compared with the control group, the latency of T1 group was significantly prolonged, and the number of target crossing was significantly reduced. The expression of GABAR1 mRNA and protein expression in T1 group were significantly increased, and the expression of NMDAR2B mRNA and protein were significantly decreased. Conclusion: The short-term memory of the brain is affected by the inhalation of sevoflurane, which may be related to the function of the temporal lobe of the brain, and the mechanism may be related to the down-regulation of NMDAR2B in the temporal lobe region and upregulation of GABAR1.

12  lnduction of adipose-derived stem cells into Schwannlike cells and observation of Schwann-like cell proliferation

Xiumei Fu, Wenliang Fu, Zhenjiang Yang, Zhihong Chen, Jingfeng Xue

Department of Anatomy, College of Basic Medical Sciences, Chengde Medical College, Chengde 067000, China

The peripheral nervous system has the potential for full regeneration following injury and recovery, predominantly controlled by Schwann cells (SCs). Therefore, obtaining a sufficient number of SCs in a short duration is crucial. Ⅰn the present study, rat adipose-derived stem cells (ADSC) were isolated and cultured, following which characterization of the ADSC was performed using flow cytometry. The results showed that the cells were positive for the CD29 and CD44 markers, and negative for the CD31, CD45, CD49 and CD106 markers. The multilineage differentiation potential of the ADSCs was assayed by determining the ability of the cells to differentiate into osteoblasts and adipocytes. Following this, the ADSC were treated with a specific medium and differentiated into Schwann-like cells.Ⅰmmunofluorescence, western blotting and Real-time PCR analyses showed that about 95% of the differentiated cells expressed glial fibrillary acidic protein, S100 and p75. Ⅰn addition, the present study found that a substantial number of SCs can be produced in a short duration via the mitotic feature of Schwann-like cells. These data indicated that Schwann-like cells derived from ADSC can undergo mitotic proliferation, which may be beneficial for the treatment of peripheral nerve injury in the future.

13  Striatal adenosine A2Areceptor neurons regulate sleep behavior

Xiangshan Yuan1,2, HaohuaWei1, Zhili Huang2, Ruixi Li11Departments of Anatomy, Histology and Embryology,2Departments of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China

Evidence from patients with neurodegenerative disorders and animal experiments strongly suggest that sleep-wake behavior may be regulated bythe striatumwithin which contains plenty of adenosine A2Areceptors (A2AR) known to promote sleep. Therefore, we hypothesized that A2AR neurons of the striatum might be involved in sleep-wake regulation. A series of techniques, chemogenetic approach, EEG/electromyogram recording in vivo, patch-clamp technique, optogenetics, and immunoelectron microscopy in vitro, were employed to examine roles of striatal A2AR neurons on sleepwake regulation in A2AR-Cre transgenic mice. Chemogenetic activation of A2AR neurons in rostral, centromedial and centrolateral striatum increasednon-rapid eye movement sleep, concomitant with reduced wakefulness, while activation of these neurons in caudal striatum didn’t change sleep-wake profiles at all. Three topographical projection patterns of A2AR axons was found in external globus pallidus (GPe): the axons of A2AR neurons from rostral striatum distributed in rostral GPe with a discoidal region paralleled to the strio-pallidal border; axons from the central striatum distributed not only in rostral GPe but also the caudal GPe with a similar feature as that in rostral, while the axons from caudal striatum just scattered in the caudal GPe.A2AR neurons in the rostral striatum formed inhibitory synapses preferentially with PV-positive neurons in the rostral GPe, while A2AR neurons in caudal striatum formed inhibitory synapses preferentially with PV-negative neurons in caudal GPe, indicating a rostral-caudal variation in striatopallidal connections.The present results indicate that A2AR neurons in rostral and central striatum regulate sleep-wake behavior via innervating GPe PV neurons. Our finding provides insight into the striatal A2AR neuron/GPe PV neuron pathway for sleep regulation and suggests a potential treatment strategy to ameliorate sleep disturbances.

14  Metformin treatment prevents amyloid plaque deposition and memory impairment in APP/PS1 mice

Zhenri Ou, Xuejian Kong, Le Zhang, Zhuo Gong, Xi Zhang, Dahong Long, Liping Xu , Aiguo Xuan

Key Laboratory of Neuroscience, Department of Anatomy, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou 511436, China; Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou 510260, China

Alzheimer’s disease (AD) is characterized by deposition of amyloid-β (Aβ) plaques, neurofibrillary tangles, and neuronal loss, accompanied by neuroinflammation. Neuroinflammatory processes are thought to contribute to AD pathophysiology. Metformin has been reported to have anti-inflammatory efficacy. However, whether metformin is responsible for the anti-neuroinflammation and neuroprotection on APP/PS1 mice remains unclear. Here, we showed that metformin enhanced spatial memory formation. Ⅰn addition, Metformin administration also decreased Aβ plaque load and chronic inflammation (activated microglia and astrocytes as well as pro-inflammatory mediators) in the cortex and hippocampus. Further study demonstrated that treatment with metformin enhanced cerebral AMPK activation. Meanwhile, metformin notably suppressed the activation of P65 NF-κB. Our data suggests that metformin can exert functional recovery of memory deficits on APP/PS1 mice via anti-inflammatory effect mediated by regulating AMPK/P65 NF-κB signaling pathway in the hippocampus, which can contribute to improvement in neurological deficits.

15  The damaged sensory neuron of dorsal root ganglia activated its surrounding satellite cells to express Slit1

Quanpeng Zhang, Xinan Yi

Department of Anatomy and Neuroscience Research, Hainan Medical University, Haikou 571199, China

While Slit is known to be an axon guidance factor, the spatiotemporal patterns and significance of Slit1 expression in satellite glial cells (SGCs) of the dorsal root ganglion (DRG) is unclear. Ⅰn this study, the Sprague-Dawley（SD）rat sciatic nerve crush (SNC) model was used. Slit1 protein expression was quantified by western blotting, fluoro-gold (FG) was used as a retrograde tracer to label undamaged neurons, activating transcription factor 3 (ATF3) was used to label damaged neurons and immunohistofluorescence labeling was performed to observe Slit1 expressed in SGCs surrounding neurons. Slit1 and β-Tubulin III immunocytochemical staining were performed on primary cultured DRG neurons to observe the neurite outgrowth. Ⅰt was found that Slit1 was expressed in DRG sensory neurons, weakly expressing in glial cells (including SGCs) in the living body and in cultured cells. Slit1 protein was up-regulated in DRG following SNC, with levels peaking at day 14 and remaining elevated up to day 28. Neuronal Slit1 expression was up-regulated shortly after DRG injury, followed by a gradual decline. Weak Slit1 expression was noted in SGCs following initial DRG injury and was significantly increased on day 3 and peaked on day 14. Ⅰnteresting, Slit1 was less expressed in SGCs surrounding FG positive neurons than FG negative soma. On the contrary, Slit1 was more strongly expressed in SGCs surrounding ATF3 positive neurons than ATF3 negative neurons. Recombinant Slit1 proteins promoted neurite outgrowth in cultured neurons. These findings suggest that DRG sensory neuron injury can induce SGCs Slit1 expression up-regulation, possibly facilitating post-injury nerve regeneration.

16  lnteraction between neurons and SGCs can up-regulated Slit1 expression in DRG after sciatic nerve injury

Jiuhong Zhao, Xu Dong, Xianfang Zhang, Quanpeng Zhang, Gang Luo, Zhijian Ma, Xinan Yi

Department of Anatomy, Hainan Medical College, Haikou 571199, China

Slit is known to be an axon guidance factor, but the pattern and significance of Slit1 expression in satellite glial cells (SGCs) of the dorsal root ganglion (DRG) is unclear. Ⅰn this study, we hypothesized that Slit1 expression in SGCs may be affected by the interaction between neurons and SGCs. Ⅰmmunofluorescence double labeling technique was applied to the neuron-SGC co-culture and DRGs from sciatic nerve crush (SNC) models. The results showed that all SGCs wrapped around the neuronal soma at 12 hours; a part of SGCs still wrapped around the neuronal soma, while the others had separated at 24 hours, and all SGCs were off neuronal soma at 72 hours in the neuron-SGC co-culture. Slit1 was no or weakly expressed in SGCs with no neuronal soma contact by immunofluorescence. However, Slit1 was strongly expressed in SGCs which surround neurons soma. Ⅰn SNC animal models, Slit1 only expressed in SGCs that surrounded living neuronal soma not in those whose parent neurons died. Combining with our other research result that Slit1 expression was up-regulated in the SGCs which wrapped around the injured neuronal soma in vivo, we confirmed that neuronal soma affect Slit1 expression in SGCs and the damage signal may raise its expression. Because Slit1 associated with nerve regeneration, our results imply that Slit1 expression up-regulation in SGCs, possibly facilitate postinjury nerve regeneration.

17  BlNP3 is implicated in oligodendrocyte cell apoptosis following carbon monoxide poisoning＊

Cailiang Chen , Meiyu Li, Xiaofei Tian, Guohui Zhang

Department of Forensic Medicine, Hebei North University, Zhangjiakou 075000, China

Objective: To investigate the role of BNⅠP3 in oligodendrocyte cell apoptosis induced by carbon monoxide poisoning(CO poisoning) and the potential signal pathways correspondingly. Methods: Twentyfive male C57 BL/6 mice were randomly divided into the control group and CO poisoning group. Mice were left to breathe roomair (control group) or subjected to 40min exposures to 2500ppm-3000ppmCO (CO poisoning group).The mice were sacrificed at 1d, 3d, 7d and 14d following CO poisoning. We examined the damage of myelin sheath and oligodendrocytes by observing the expression of MBP and Olig2 in corpus callosum. Furthermore, we explore the role of BNⅠP3 and potential signal pathways in the oligodendrocyte cell death following CO poisoning by observing the expression of BNⅠP3, Bax and caspase9. Results: The expression of MBP was decreased signifcantly in the corpus callosum from 1d to 7d and increased from 7d to 14d in some degree after CO poisoning. Olig2 expression increased markedly on 1d and further increased on 3d in CO poisoning group. Western blot analysis showed that the levels of BNⅠP3 were 9.40-fold, 8.11-fold and 3.43-fold increased from 1d to 7d respectively, as compared with control group. Moreover, Bax and Caspase9 were also upregulated in the corpus callosum from 1d to 7d after CO poisoning and correlated with the BNⅠP3 expression. Conclusion:BNⅠP3 may play an important role in the apoptosis of oligodendrocytes induced by CO poisoning via the pathway of caspase dependent mitochondria apoptosis.

*This study was supported by Graduate Student Innovation Fund Project of Hebei Province(CXZZSS2017145) and Youth Mentoring Program of Education Department of Hebei Province(Z2017027).

18  Abnormal recruitment and cleavage of calcium responsive transactivator by mutant huntingtin

Ting Peng1, Zhen-Zhen Jing1, Li-Ying Cao1, He Li1,21Department of Histology and Embryology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;2Department of Histology and Embryology, Hubei University of Medicine, Shiyan 422000, China

Cell cycle re-entry is an important cause of neuronal damage in multiple neurodegenerative diseases. Calcium responsive transactivator (CREST) is thought to regulate activity-dependent transcription and maintain dendritic growth in mature neurons, and in our other works which was found promoting cell cycle arrest and neuron-like differentiation. Here we show the protein levels of CREST were significantly lower in HD animal and cell models. Mutant huntingtin (mHtt) abnormally recruits CREST into aggregates and promotes the cleavage of CREST by calpain ⅠⅠ, which leading to a dramatic decrease of functional CREST protein. Over-expression of CREST attenuates mHtt induced inhibition of neuronal differentiation and cell death. Our results shed light on a new neuropathological toxicity mechanism of mHtt.

19  Effect of scopolamine on the activity of serotonergic neurons in the dorsal raphe nucleus of motion sickness mice

Tao Zhang, Hui Yao, Shuang Zhao, Yuhuang Kuang, Yingqi Zhu, Zhenlong Guan

College of life science, Hebei Normal University, Shijiazhuang, 050024, China

Acetylcholine (Ach) and serotonin (5-HT) are demonstrated to be increased in the brainstem when motion sickness occurred. Scopolamine, a cholinergic M receptor antagonist (anticholinergics) is the most effective drug used for the prevention and treatment of motion sickness in current times. But it is still unclear that the relationship between the activity of serotoninergic neurons and the prevention of motion sickness by scopolamine.Ⅰn present study, male mice were randomly divided into blank control group, control group and experimental group, and the gavage was performed with saline or scopolamine respectively. After 30 min, the mice of control group and experimental group were stimulated with speed-changing rotation for 2 h, and then the open field test, detection of the serotonin level and 5-HT-immunohistochemistry in the brainstem were performed. The results showed that the serotonin level and the activity of serotonergic neurons in the brainstem of motion sickness mice were dramatically decreased by scopolamine administration. These results suggested that the reduction of serotonin release from raphe nucleus was one of mech anism in anti-motion sickness efficacy of scopolamine.

20  Effects of scutellarin on MAPK signaling pathway in activated microglia

Honge Li, Hong Han, Wenji Jia, Yun Yuan, Chunyun Wu

Department of Anatomy and Histology-Embryology, Faculty of Basic Medical Sciences, Kunming Medical University, Kunming 650500, China

Activated microglial cells release excessive inflammatory mediators in response to cerebral ischemia. We reported previously that scutellarin effectively suppressed the inflammatory response induced by activated microglia in rats subjected to middle cerebral artery occlusion (MCAO); however, the mechanism via which scutellarin exerts its effects on microglial activation has not been elucidate. This study aimed to investigate if scutellarin can regulate the mitogenactivated protein kinases (MAPK) signaling pathway that is linked to microglia activation in lipopolysaccharide (LPS)-induced BV-2 microglia. The BV-2 microglia were randomly divided into control, LPS induced group and LPS+Scutellarin groups. Expression of various members of the MAPK signaling pathway, including extracellular signalregulated kinase 1/2 (ERK1/2), p38 MAPK and cJun Nterminal kinase (JNK), and their phosphorylated forms in activated microglia was assessed by immunofluorescence staining and western blot after LPS activation. Expression of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and inducible nitric oxide synthase (iNOS) was also detected. Scutellarin can significantly reduce p38 and JNK phosphorylation, but increase the expression of p-ERK1/2. Scutellarin also effectively suppressed the expression of TNF-α, IL-1β, iNOS. The results suggest that scutellarin regulates the activation of microglia via the MAPK signaling pathway, therefore exerts its protective effect on microglia mediated inflammation.

21  Gastrodin regulates the Notch pathway in activated BV-2 microglia

Xiaoyu Liu, Shunjin Liu, Yun Yuan, Juanjuan Li

Department of Anatomy and Histology- Embryology, Faculty of Basic Medical Sciences, Kunming Medical University, Kunming 650500, China

Gastrodin, an anti-inflammatory agent, effectively suppressed microglia activation in rats with hypoxic-ischemia brain damage (HⅠBD). We reported previously that Gastrodin effectively suppressed the inflammatory response induced by activated microglia in rats subjected to HⅠBD; however, the mechanism via which Gastrodin exerts its effects on microglial activation has not been explored. This is aimed to elucidate if Gastrodin can regulate the Notch pathway that is linked to microglia activation in lipopolysaccharide (LPS)-induced BV-2 microglia. Expression of various members of the Notch pathway, including Notch-1, intracellular Notch receptor domain (NⅠCD), recombining binding protein suppressor of hairless (RBPJK) and transcription factor hairy and enhancer of split-1 (Hes-1) in activated microglia was assessed by immunofluorescence staining and western blot in vitro LPS activation. This study shows that Gastrodin markedly attenuated the expression of NF-κB, Notch-1, NⅠCD, RBP-JK and Hes-1 in activated microglia. The results suggest that Gastrodin regulates the activation of microglia via the Notch pathway and concurrently induces functional changes in activated microglia.

22  RhoA/ROCK signal pathway modulates neuronalneurite outgrowth through regulating the severing of microtubules

Dandan Tan1,Jinkun Wen1, Lixia Li1, Mengjie Pan1, Changhui Qian1, Jiasong Guo1,2,3,1Department of Histology and Embryology, Southern Medical University, Guangzhou 510515, China;2Key Laboratory of Tissue Construction and Detection of Guangdong Province, Guangzhou, 510515, China;3Institute of Bone Biology, Academy of Orthopedics, Guangzhou 510665, China

Neurite outgrowth, a process responsible for the establishment of neuronal patterning and connections, is crucial forthe development and regeneration of the nervous system. Microtubule-severing proteins are able to break longer microtubules into shorter ones, which may increase the number and mobility of microtubules and benefit the outgrowth of neurite.The best known of them are spastin and katanin (consists of a p60 severing enzyme and a p80 regulatory subunit). Current knowledge indicates that RhoA and its downstream effectorRho-associated proteinkinase(ROCK)play roles in the negative regulation of neurite outgrowth due to their abilities to control cytoskeletal reorganization and dynamics.However, no evidence has shownwhether RhoA/ROCK signal pathway negativelyregulate neurite outgrowth through alterations in proteins involved in the severing of microtubules. To address this question, we constructed a lentiviral vector carrying constitutive active mutant of RhoA-GTPase to activate RhoA in dorsal root ganglia neurons and differentiated PC12 cells. Ⅰn addition, we treated them with RhoA inhibitor (CT04) and ROCK inhibitor (Y27632), respectively.Ⅰmmunohistology showed that constitutively activeRhoA reduced neurite outgrowth, whileinhibition of RhoA/ROCK promoted neurite outgrowth. Further studiesindicated constitutively active RhoAdown-regulated the expression of spastin and p60 katanin. On the contrary, inhibition ofRhoA/ROCK significantly up-regulated the expression of spastin and p60 katanin. Besides, we found thatRhoA activation increased the expression of microtubule associated proteins 2（MAP2）which shield microtubule from being severed by spastin and p60 katanin. However, treatment with CT04 or Y27632 decreasedthe expression of MAP2.Overall data of current study suggested RhoA/ROCK pathway can play roles in regulating the severing of microtubules to modulate the neuronal neurite outgrowth.

23  Regulation of rat hippocampal neuronal axonogenesis by Cdc42

Hai-Hong Wang, Ang Li

Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China

A progressive axonal degeneration, which precedes neuron death, is a major pathological hallmark in Alzheimer’s disease (AD) and other neurodegenerative diseases. Cdc42 in AD brain is upregulated and overlaps with early cytoskeletal abnormalities. Although studies have shown that Cdc42 is involved in regulating axonogenesis, but the underlying mechanisms have not been elucidated. To investigate the regulation of rat hippocampal neuronal axonogenesis by Cdc42 and the underlying mechanisms. Primary hippocampal neurons from newborn Sprague-Dawley (SD) rats were transfected with empty vector, wild type Cdc42, Cdc42 constitutively active mutant and Cdc42 dominant-negative mutant respectively and then were cultured for 72 h. Hippocampal neurons from newborn SD rats transfected with empty vector and Cdc42 dominant-negative mutant respectively were cultured for 60 h and then were treated with 2 nmol/L Taxol for 12 h. Cdc42 activity was assayed by using G-Lisa Cdc42 Activation Assay Biochem Kit. The axon was measured by immunofluorescence using the axon marker Tau-1. The number and length of axons was counted and analyzed using SPSS (13.0) software. The phosphorylation of collapsin response mediator protein-2 (CRMP-2) at Thr514 site (pT514-CRMP-2) was detected by Western blot. The results showed that transfection with wild type Cdc42and Cdc42 constitutively active mutant both significantly increased the activity of Cdc42, the axon number and the axon length while transfection with Cdc42 dominant-negative mutant significantly decreased the activity of Cdc42, the axon number and the axon length. Transfection with wild type Cdc42 and Cdc42 constitutively active mutant both significantly decreased the level of pT514-CRMP-2 while transfection with Cdc42 dominant-negative mutant significantly increased the level of pT514-CRMP-2. Taxol treatment significantly attenuated transfection-with-Cdc42N17-induced neuronal axonogenesis impairment. These results directly confirm that Cdc42 promotes rat hippocampal neuronal axonogenesis and the mechanism may involve dephosphorylated-CRMP-2-mediated microtubule assembly.

24  Silencing SOAT1 inhibits PTGS2 expression through MAPK signaling pathway and attenuates β-amyloid-induced toxicity in neuroblastoma SH-SY5Y cells

Ying Chen1, Lu Zhu2, Lei Ji1, Qiyun Wu1, Xiaodong Wang11Department of Histology and Embryology, Medical College, Nantong University, Nantong 226001, China;2Department of Basic Medicine, Taihu University of Wuxi, Wuxi 214100, China;3School of public health Nantong University, Nantong 226001, China

Sterol O-acyltransferase (SOAT) 1 has been demonstrated to be closely associated with AD. However, potential mechanisms that responsible for the protective role of SOAT1 blockage in AD progression is not fully understood. Ⅰn the present study, we demonstrated that silencing SOAT1 could significantly attenuate Aβ25-35induced cytotoxicity and cell apoptosis in SH-SY5Y cells. Genome-wide analysis was carried out to identify transcripts that co-vary with Soat1 by correlation analysis. We identified that Prostaglandin H synthase 2 (Ptgs2, also known as cyclooxygenase-2, COX-2) was the most significant correlation(correlated) with Soat1. We validated that silencing SOAT1 could significantly inhibit PTGS2 expression and partially reverse the elevations of Aβ25-35induced active COX-2 expressions in model of neuron injury in vitro.Ⅰnterestingly, We identified that there were 712 genes expression variation highly correlated with both Soat1 and Ptgs2in BXD RⅠmice hippocampus through VENN analysis. KEGG pathway analysis revealed that these common correlations were enriched in several significant enrichment pathways, including endocytosis, MAPK signaling pathway, Ubiquitin mediated proteolysis, etc. We further demonstrated preliminary down regulation of SOAT1 induced PTGS2 inhibition and alleviated Aβ25-35induced cytotoxicity and cell apoptosis may be due to the regulation of CACNA1b,CACNA2d1 through PKC, ERK signaling cascade. These findings suggest that the protective effects of silencing SOAT1 against Aβ-induced injury by inhibiting PTGS2 expression through MAPK signaling pathway, which provide new insights into its application toward AD prevention and therapy.

25  Dicer in Schwann cells is essential for the maintenance of axon integrity in adult mice

Tao Li, Jingjing Wang, Hongkai Wang, Nanxin Huang, Xing Gao, Feng Mei, Lan Xiao

Department of Histology and Embryology, Chongqing Key Laboratory of Neurobiology, Third Military Medical University, Chongqing 400038, China

Dicer is an enzyme that is responsible for generating functional noncoding micro-RNAs (miRNAs), which has been shown to importantly participate in regulation of myelination in CNS or PNS. Recent evidence indicated that myelinating glia cells, oligodendrocytes (OLs) in CNS and Schwann cells (SCs) in PNS, also provide nutrition for the energy consumption of axons. To investigate the functional consequence of Dicer deletion in myelinating glial cells post-myelination in adulthood, we induced Dicer deletion in 8-week old mice (PLP-creERT2; Dicer fl/fl) by tamoxifen. We found progressive motor dysfunctions in these mice, which started as hind limb ataxia ～1 month after recombination and deteriorated into hind limb ataxia and paralysis, caused by massive axon degeneration/ atrophy in the sciatic nerves. To understand whether this phenotype is due to Dicer deletion in OLs, we induced Dicer deletion in OLs at P9 in the NG2-creERT2; Dicer fl/fl mice. Dicer deletion in OLs did not cause similar symptoms at the same age, suggesting this phenotype was caused by the Dicer deletion in SCs rather than OLs. Concomitantly, the PLP-creERT2; Dicer fl/fl mice showed premature hair greying, suggesting Dicer is also responsible for maintaining of melanoblasts. Bio-informatic analysis of miRNAs array from the PLP-creERT2; Dicer fl/fl sciatic nerves indicated that miRNAs that in charge of lipid metabolism were greatly changed. All together, our results reveal that Dicer in Schwann cells is essential for the maintenance of axon integrity of PNS in adult mice.

*This work was supported by Student’s Platform for Innovation and Entrepreneurship Training Program (201590031042) to WJJ.

26  BAG-1M co-activates BACE1 transcription through NF-kB and accelerates Aβ production and memory deficit in Alzheimers disease mouse model

Zhemin Shi1, Yuheng Hong2, Kun Zhang1, Jingzhao Wang1, Lina Zheng1, Zhen Zhang1, Zhimei Hu1, Xiaohui Han1, Yawei Han1, Ting Chen1, Qingbin Yao1, Hongmei Cui1, Wei Hong11Department of Histology and Embryology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China;2School of Medical Imaging, Tianjin Medical University, Tianjin 300203, China

Accumulation of amyloid β protein (Aβ)-containing neuritic plaques in the brain is a neuropathological feature of Alzheimer’s disease (AD). The β-site APP-cleaving enzyme 1 (BACE1) is essential for Aβ generation and dysregulation of BACE1 expression may leadto AD pathogenesis. Bcl-2-associated athanogen 1M (BAG-1M), initially identified as an anti-apoptotic protein, has also been found to be highly expressed in the same neurons that contain intracellular amyloid in the hippocampus of AD patient. Ⅰn this report, we found that over-expression of BAG-1M enhances BACE1-mediated cleavage of amyloid precursor protein (APP) and Aβ production by up-regulating BACE1 gene transcription. The regulation of BACE1 transcription by BAG-1M was dependent on NF-κB, as BAG-1M complexes NF-κB at the promoter of BACE1 gene and co-activates NF-κB-facilitated BACE1transcription. Moreover, expression of BAG-1M by lentiviral vector in the hippocampus of AD transgenic model mice promotes Aβ generation and formation of neuritic plaque, and subsequently accelerates memory deficits of the mice. These results provide evidence for an emerging role of BAG-1M in the regulation of BACE1 expression and AD pathogenesis and that targeting the BAG-1M-NF-κB complex may provide a mechanism for inhibiting Aβ production and plaque formation.

27  Polyphenols from toona sinensis seedson protects against 6-OHDA -induced neuroinflammatory in PC12 cells via the p38 MAPK pathway

Fei Liu, Xuechao Fei, Kan Li, Wenxin Zhuang, E Lv, Wenyu Fu

Department of Histology and Embryology,Weifang Medical University, Weifang 261053, China

The purpose of the study is to investigate the effect of polyphenols from toona sinensis seeds (PTSS) inhibited the neuroinflammatory on PC12 cell model of Parkinson’s disease induced by neurotoxin 6-hydroxydopamine (6-OHDA) via the P38 mitogen-activated protein kinase (P38MAPK) pathway.PC12 cells were divided into four groups: PC12 cells control group; model group (6-OHDA 100μmol/L); PTSS low dose group (6-OHDA + PTSS 100μmol/L); PTSS high dose group (6-OHDA + PTSS 200μmol/L). The morphology of PC12 cells was observed by inverted microscope. The cell viability was detected by CCK-8 method. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B p65 (NF-κB p65), p38 MAPK and p-p38 MAPK were examined by immunohistochemical staining and Western Blot. Compared with the control group, the cells in the model group appeared vacuoles, deformation or aggregation. The refraction of some cells was enhanced. The dead cells were significantly increased. The morphology of cells in PTSS low dose group was obviously improved. The cell viability was significantly decreased in the model group, while it was increased significantly in the two PTSS groups. And the effect of PTSS low dose group was higher than that of high dose group. Compared with the control group, the expression of iNOS, COX-2, NF-κB p65, p38 MAPK and p-p38 MAPK in the model group was increased markedly. PTSS could effectively reduce the expression of iNOS, COX-2, NF-κB p65, p38 MAPK and p-p38 MAPK. PTSS can inhibit the inflammatory response of PC12 cells induced by 6-OHDA by restraining the p38 MAPK signaling pathway.

28  CREST promotes neuron-like differentiation through activation of p53 pathway

Li-Ying Cao1, Ting Peng1, He Li1,21Department of Histology and Embryology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;2Department of Histology and Embryology, Hubei University of Medicine, Shiyan 422000, China

Neuronal differentiation is the core of development and maturation of the nervous system. Calcium responsive trans-activator (CREST) is a highly expressed nucleoprotein in embryonic mouse brain, which has been associated with activity-dependent transcription and dendritic growth in mature neurons. Here we show CREST is expressed at a high level during differentiation of N2a and mouse neural stem cells (NSCs). Over-expression of CREST promotes neuron-like differentiation of N2a cells and enhances expression of neuronal markers. Silencing of CREST inhibits neuron-like differentiation of both N2a and NSCs, and promotes glial differentiation of NSCs. Over-expression of CREST enhances p53 stability and nuclear translocation, and activates the transcription of p53 target genes involved in neuronal differentiation. Silencing p53 significantly inhibits CREST induced N2a differentiation. Our results argue that CREST is a critical regulatory factor for neuron-like differentiation.

29  The role of Wnt1 in the NSC34 cell with SOD1G93Amutation

Yongxin Liu1, Yingjun Guan1＊, Yanchun Chen1, Fenghuan Zhou2, Qiang Lu21Histology and Embryology Department,2Pathology Department, Weifang Medical University, Weifang 261053, China

Cu/Zn superoxide dismutase 1 gene (SOD1) is widely found in all organisms of nature. Mutations in the SOD1 gene were the first to be identifiedin Amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease.The spinal cord × Neuroblastoma hybrid cell line (NSC34) transfected with mutate SOD1 plasmid is often used as a bona fide cellular model to investigate the physiopathological mechanisms of ALS. Ⅰn this study, we defined the expression of Wnt1in the NSC34 cell with SOD1G93Amutation by RT-PCR and Western blot. What’s more, we detected the survival and apoptosis of mutant NSC34 cells after knocking down or overexpressing of Wnt1 by Western blot and MTS assay. These results showed that compared with NSC34 cells transfected pEGFP-WT-SOD1 plasmid, the Wnt1 mRNA and protein were decreased at 48 and 72 h in NSC34 cells transfected with pEGFP-G93A-SOD1 plasmid. Western blot results showed that the expression of Caspase-3 was not changed, but Cleaved Caspase-3 increased in mutant NSC34 cells transfected with Wnt1 siRNA, compared with the blank control group. MTS assay showed that the proliferation and viability of NSC34 cells were decreased at 72, 96 and 120 h after knocking down Wnt1. Moreover, the expression of Caspase-3 were not changed, but Cleaved Caspase-3 decreased in mutant NSC34 cells transfected with GFP-Wnt1 overexpression plasmid, compared with control group. The proliferation and viability of NSC34 cells were increased at 72, 96 and 120 h after overexpressing Wnt1.These results indicate that Wnt1 is involved in the regulation of apoptosis and proliferation of SOD1 mutant NSC34 cells, which is closely related to motor neurons degeneration of ALS.

30  LlNC00461, a long non-coding RNA, is important for the proliferation and migration of glioma cells

Yali Yang1, Mingxin Ren1, Chao Song1, Dan Li1, Shahid Hussain Soomro1, Yajie Xiong1, Hongfeng Zhang2, Hui Fu11Department of Anatomy and Embryology, School of Basic Medical Sciences, Wuhan University Wuhan 430071, China;2Department of Pathology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, China

An increasing number of reports have revealed that long non-coding RNAs are important players in tumorigenesis. Here we showed thatlong non-coding RNA LⅠNC00461 is highly expressed in glioma tissues compared to non-neoplastic brain tissues. The knockdown of LⅠNC00461 suppressed cyclinD1/A/E expression which led to G0/ G1 cell cycle arrest and inhibited cell proliferation in glioma cells. LⅠNC00461 suppression also inhibited glioma cell migration and invasion. The function of LⅠNC00461 in glioma cells is partially mediated by MAPK/ERK and

PⅠ3K/AKT signaling pathways as down-regulation of LⅠNC00461 expression suppressed ERK1/2 and AKT activities. Moreover, LⅠNC00461 knockdown decreased expression levels of microRNA miR-9 and flanking genes MEF2C and TMEM161B. Taken together, our results demonstrate that LⅠNC00461 is important for glioma progression affecting cell proliferation, migration and invasion via MAPK/ERK, PⅠ3K/AKT, and possibly other signaling pathways.

31  Effects of vasoactive intestinal peptide inhibited the neuroinflammatory by downregulating the expression of NF-κB in the MPTP mouse model of Parkinson’s disease

E Lv, Fei Liu , Kan Li, Xiaojian Li, Keliang Lu

Department of Histology and Embryology, Weifang Medical University, Weifang 261053, China

The purpose of the study is to investigate the effect of vasoactive intestinal peptide (VⅠP) inhibited the neuroinflammatory by downregulating the expression of nuclear factor-κB p65 (NF-κB p65) in the MPTP mouse model of Parkinson’s disease (PD). Thirty male C57BL / 6J male mice were randomly divided into normal saline (NS) group, MPTP group (30 mg / kg, i.p. 5 days), MPTP + VⅠP (1.5 mg / kg, i.p. 8 days). Ⅰmmunohistochemistry(ⅠHC) was used to observe the expression of tyrosine hydroxylase(TH), glial cell fibrous acid protein (GFAP) and ion-calcium-binding protein (Ⅰba-1). The contents of Tumor Necrosis Factor alpha （TNF-α） and monocyte chemotactic protein 1（MCP-1）in striatum were detected by ELⅠSA. The expression of NF-κB p65 were measured by Western blot. Compared with NS group, the expression of TH was markedly decreased, the expression of GFAP and Ⅰba-1 was obviously elevated, the contents of TNF-α, MCP-1 and the expression of NF-κB p65 in the striatum of MPTP group were significantly increased. However, VⅠP sufficiently ameliorated TH depletion in the striatum of the MPTP-induced mice, evidently suppressed the activation of microglia and astrogliosis,Ⅰn addition, VⅠP inhibited the abnormal elevation of the content of TNF-α and MCP-1, and restrained the expression of NF-κB p65 in the striatum of MPTP-induced mice. These results suggest that VⅠP effectively reverse the neurological damage of striatum in PD mice induced by MPTP. The mechanism may be related to that VⅠP inhibits the neuroinflammation by down-regulating the expression of NF-κB p65.

32  Expression and significance of autophagy-associated gene Atg4A in spinal cord of ALS transgenic mice

Qing Wang1, Meng Yuan2, Zhen Peng3, Chanchan Liang3, Baoyong Lin3, Yanchun Chen41Department of anatomy,2Clinical medicine specialty,3Biotechnology Speciality,4Department of Histology and Embryology, Weifang Medical University, Weifang 261053, China

The pathogenesis of ALS is complicated, and there is no effective treatment. Autophagy is a unique phenomenon in eukaryotic cells, which is involved in many physiological and pathological processes. Previous studies have found that the incidence of ALS is associated with autophagy. Atg4A is an important autophagy related gene involved in the formation of autophagy. To investigate the expression change of Atg4A in spinal cord of ALS transgenic mice and explore their role in the pathogenesis of ALS, SOD1-G93A transgenic mice were selected, and were killed in early, middle and late stage (95 d, 108 d, 122 d) separately. The mRNA and protein levels of Atg4A in spinal cord of ALS transgenic mice was detected by RT-PCR and Western blotting respectively. The distribution of spinal cord positive cells and their co-localization with neurons were detected by immunofluorescence technique. Wild-type mice of the same age were selected as a control group. The results showed that compared with wild-type mice, the expression of Atg4A protein and mRNA in spinal cord of ALS transgenic mice increased in both middle and late stage. Ⅰmmunofluorescence results showed that Atg4A positive cells were detected in spinal cord of both ALS transgenic mice and wild-type mice and co-expressed with neurons. The intensity of immunoreactivity in the middle and late stage of ALS transgenic mice was significantly enhanced. All the data suggsested that abnormal expression of Atg4A was closely related to ALS spinal cord lesion, which was involved in the pathogenesis of ALS.

33  The altered expression of DDX3 and CK1ε in the striatum of SOD1G93A ALS transgenic mice

Meng Yuan1, Chanchan Liang2, Yawen Zhang2,Baoyong Lin2, Zhen Peng2, Qing Wang3, Yanchun Chen41Clinical Medicine,2Biotechnology Speciality,3Department of Human Anatomy,4Department of Histology and Embryology, Weifang Medical University, Weifang 261053, China

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive muscle paralysis determined by motor neuron degeneration. DDX3, a member of RNA helicase family, has been identified in a variety of cellular biogenesis processes, including cell cycle regulation, cell differentiation, survival, and apoptosis. DDX3 is a regulatory subunit of casein kinase 1ε (CK1ε) in Wnt signaling pathway. However, there is no report on the role of DDX3 and CK1ε in the striatum of ALS. In this study, we use thirty-three SOD1G93AALS transgenic mice and the same number of wide-type mice including the early stage (95 d), middle stage (108 d) and late stage (122 d) to separate the striatum to detect the expression of DDX3 and CK1ε by RT-PCR, Western blotting and immunofluorescence technology. The results showed that the expression of DDX3 mRNA and protein in the striatum of ALS mice remained unchanged at 95 d, but up-regulated at 108 and 122 d compared with wild-type mice. The expression of CK1ε mRNA in the striatum of ALS mice remained unchanged but CK1ε protein was up-regulated at 95 d, but down-regulated at 108 and 122 d. Double-immunofluorescence staining revealed that DDX3 and CK1ε positive cells were expressed in β-tubulin III labeled neurons, but not in GFAP labeled astrocytes in the striatum of both ALS mice and wild-type mice. These findings suggested that the expression of DDX3 and CK1ε was altered in the striatum of SOD1G93AALS transgenic mice. DDX3 and CK1ε may be closely related to the pathogenesis of ALS.

34  Expression changes of calcium-responsive transactivatorin SOD1-G93A ALS transgenic mice

Qiaozhen Wang1, Yanchun Chen2,Yingjun Guan2＊, Qing Wang1, Yawen Zhang21Department of Human Anatomy, Weifang Medical University, Weifang 261053, China;2Department of Histology and Embryology, Weifang Medical University, Weifang 261053, China

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegener-ative disease，which leads to the lose of motor neurons in brain and spinal cord with the cognitive function impairment, while the pathogenesis is still poorly understood. Calcium-responsive transactivator (CREST) inhibits activity-dependent neurite outgrowth in primary neurons and is prone to aggregation and co-aggregates with Fused in sarcoma (FUS)—one ALS linked proteins in cultured cells. To confirm the contribution of CREST to ALS, we used Western blotting and immunofluorescence technique to detect the expression of CRESTin the hippocampus and striatum of SOD1-G93A ALS transgenic mice. The results of Western blotting showed that CREST protein in ALS mice were downregulated at the middle stage (108 d) and end stage (122 d) compared with wild-type mice in hippocampus and striatum. The immunofluorescence results show that CREST located in the nucleus and co-expressed with β-tubulinⅢ in the hippocampus and striatum. Moreover, the immunoreactivity of CREST decreased obviously in ALS mice, and this trend is in agreement with the observed results of Western blotting. These findings indicated that CREST is a nuclear protein of neuron and the expression changes of CREST are associated with ALS. Our findings upstep our cognition and may offer new clue to comprehend the molecular mechanism of ALS.

35  Regional metabolic differences among fat depots in a 6-OHDA rat

Hui Lian1, Yimin Zhang1, Yanli Yang1, Xuerui Ran1, Li Zhou1, Jinxia Zhu1, Zhiyong Wang11Key Lab for Medical Tissue Regeneration of Henan Province, Department of Human Anatomy, Xinxiang Medical University, Xinxiang 453003, China

The relationship between dopamine and adiposity is unclear. Ⅰt has been reported that the peripheral fat reduction contributed to the weight loss of the patients with Parkinson’s disease which is characterized by dopaminergic neuron degeneration in the substantia nigra (SN). Therefore we hypothesized that dopamine in the SN played a potential role on fat metabolism. The regional metabolic differences of synthesis, lipolysis and thermogenesis among perirenal white adipose tissue (PWAT), inguinal white adipose tissue (ⅠWAT) and interscapular brown adipose tissue (BAT) were investigated by immunohistological chemistry and western blotting in rats microinjected with 6-hydroxydopamine (6-OHDA) bilaterally into the SN (referred to as 6-OHDA rats) and explored possible mechanisms. The results showed that there was no significant change in body weight between control and 6-OHDA rats. The fat mass of PWAT was reduced markedly, whereas those of ⅠWAT and BAT did not change obviously in 6-OHDA rats. The diameter of fat droplets in the three fat depots was decreased in 6-OHDA rats. The protein expression of phosphorylated hormone sensitive lipase (pHSL) was increased in PWAT, ⅠWAT and BAT, whereas the fatty acid synthase (FAS) was decreased. The expression of uncoupling protein type 1 (UCP1) and proliferator-activated receptor γ coactivator-1α (PGC-1α) were decreased in PWAT and BAT, but increased in IWAT. In addition, the expression of tyrosine hydroxylase (TH) and β3-adrenergic receptors (β3AR) were also enhanced in PWAT, ⅠWAT and BAT. The present study indicated that dopaminergic neurons in the SN were involved in the regulation of peripheral fat metabolism with regional differences in fat synthesis, lipolysis and thermogenesis and sympathetic nervous system might mediate this effect.

36  Single-prolonged stress induces the hippocampal microtubule change

Jingzhi Jiang Fang Han

PTSD Laboratory, Department of Histology and Embryology, Basic Medical University, China Medical University, Shenyang 110001, China

Microtubules (MTs) are one of the primary components in the cytoskeletal system of neurons and play an important role in the maintenance of neuronal function and morphology. Abnormal expression of MTs is involved in neurodegenerative diseases and chronic stress. The hippocampus is an important region in the regulation of cognition, and its dysfunction is one of the causes of post traumatic stress disorder (PTSD). The aim of this study was to explore whether MTs contribute to PTSD-induced abnormal hippocampus. We used single prolonged stress (SPS), an animal (as an animal) model of PTSD. The Morris water maze behavioural test, 4′,6-diamidino-2-phenylindole staining and transmission electron microscopy were used for the detection of abnormal hippocampal function and morphology in SPS rats. Ⅰmmunohistochemsitry and western blotting were performed to examine MT-related proteins. The MT protein tubulin β showed increased expression in the hippocampus of SPS rats in comparison with control rats. The MT-associated proteins tau and protein 1B also showed increased expression, but phosphorylated tau showed decreased expression in SPS rats. Expression of the tubulin-binding protein stathmin was decreased in SPS rats in comparison with control rats. The stable MT marker acetyl-α tubulin also showed decreased expression in SPS rats. These results indicated that SPS induced increased formation of MTs, including MT protein and MT-associated proteins in the hippocampus. But increased MTs maybe show(might show) decreased stability. These results suggest that SPS induced increased but unstable MTs, which may contribute to abnormal hippocampus.

37  Blood vasculature acts as a physical guide for oligodendrocyte progenitor migration

Jianqin Niu1,5, Hui-Hsin Tsai1, Roeben Munji2, An-Chi Tien1, Jonah R Chan3, Richard Daneman2,4, Stephen PJ Fancy1,41Departments of Pediatrics,2Departments of Anatomy,3Department of Neurology,4Departments of Pharmacology and Neuroscience, University of California at San Francisco, CA94158，USA;5Department of Histology and Embryology, Third Military Medical University, Chongqing 400038, China

Oligodendrocyte progenitors (OPCs) migrate extensively during development of brain and spinal cord, from restricted ventricular zone origins to achieve widespread dissemination throughout grey and white matter, and this migration is critical for myelination and establishment of neuron-glial units. Ⅰt is unclear how these cells achieve this rapid and long distance dispersal. Here we show that OPCs use the pre-formed scaffold of developed blood vessels as a physical substrate for migration. OPCs emerge from their origins in brain and spinal cord onto this scaffold, and associate tightly with the abluminal endothelial surface, crawling along and jumping between vessels throughout their migratory phase. Furthermore, OPC migration in vivo is significantly disrupted in mice with defective vascular architecture, but occurs normally in pericyte-deficient mice, suggesting an essential OPC-endothelial interaction. Together these results suggest a new model for widespread migration of OPCs throughout the developing mammalian CNS.

38  ln situ hybridization analysis of mouse long noncoding RNA 1700101O22Rik

Xiaohui Song, Chaw Kyi-Tha-Thu, Banyar Than Naing, Takami Takizawa, Toshihiro Takizawa

Department of Molecular Medicine and Anatomy, Nippon MedicalSchool, Tokyo, Japan

Objective: Long noncoding RNAs (lncRNAs) serve as new regulatory molecules of gene expression at the transcriptional and post-transcriptional levels. 1700101O22Rik is an intergenic lncRNA on mouse chromosome 12. However, few studies have been done on its expression and function. Ⅰn this study, we examined the expression of 1700101O22Rik. Methods: This study was approved by the animal research committee of Nippon Medical School. FANTOM 5.0 database (http://fantom.gsc.riken.jp/5) was used to estimate the expression level of 1700101O22Rik in mouse tissues. Real-time PCR was performed to examine its expression level in nine adult B6D2F1 mouse organs (i.e., testis, ovary, brain, heart, liver, lung, small intestine, spleen, and kidney). Ⅰn situ hybridization (ⅠSH) was conducted to assess the cell-specific expression of 1700101O22Rik using digoxigenin-labeled RNA probes. Results: Ⅰn silico screening with the FANTOM5.0 database showed that 1700101O22Rik transcripts were only detected in the pubertal-early adult (30-day) and adult testis. By real-time PCR, we confirmed that this lncRNA was expressed exclusively in the testis but not in any other adult organs used in this study. ⅠSH analysis showed that 1700101O22Rik expression was specific to the step 6-9 spermatids. Ⅰts intracellular localization was mainly in the cytoplasm. Conclusions: Our findings suggest that 1700101O22Rik is a mouse testis-specific lncRNA.

39  The effect of estrogen and its receptor (ERa) in the liver regeneration after partial hepatectomy

Yoshitaka Hishikawa, Baatarsuren Batmunkh, Narantsog Choijookhuu

Department of Anatomy, Histochemistry and Cell Biology, Faculty of Medicine, University of Miyazaki, Japan

Estrogen is implicated in the regulation of cell growth and differentiation in various organs. However, the biological function of estrogen for liver regeneration is not completely understood. Therefore, we investigated the effect of estrogen on liver regeneration in male and female Wistar rats after 70% partial hepatectomy (PHx). 17b-estradiol (E2) at a dose of 9 mg/g bodyweight and ⅠCⅠ 182,780 at a dose of 2 mg/g bodyweight were injected into male rats on the day before PHx. Cell proliferation activity was determined using proliferating cell nuclear antigen (PCNA) and ERa expression was confirmed by immunohistochemistry and western blot analysis. The activity of estrogen responsive element (ERE) was analyzed using Southwestern histochemistry (SWH). PCNA labeling index reached a maximum at 48 hr after PHx in male rats, and at 36 hr in female rats and E2-treated male rats. ERa was expressed in zones 1 and 2 in normal male rats, but was found in all zones in normal female rats. Ⅰnterestingly, ERa was not detected at 6-12 hr after PHx but was found at 24-168 hr in male rats. However, ERa expression was found at all sampling time-points in female and E2-treated male rats. The activity of ERE binding proteins by SWH was detected from 12 hr after PHx in male rats but was found from 6 hr in female and E2-treated male rats. ERa was co-localized with PCNA during liver regeneration. On the other hand, in E2 + ⅠCⅠ 182,780 treated male rats, the staining pattern and localization of ERa, PCNA and ERE binding protein after PHx was similar to that in no-treated male rats. These results indicated that estrogen may play an important role in liver regeneration through ERa.

40  Expression and role of Smad7 in ovarian injury of chemotherapy rats

Yan Ding, Jing Zhang, Jun-Xu Ren

Department of Histology and Embryology，Basic Medical Sciences，HeBei North University，Zhangjiakou 075000, China

Objective: To investigate the effect of cisplatin on the apoptosis of ovarian injury in rats and its effect on Smad7 expression and to explore the correlation between Smad7 and apoptosis of chemotherapy ovarian injury. Methods: Ten healthy 3-month-old SD rats were randomly divided into control group and treatment group. Each rat was given intraperitoneal injection of cisplatin 2mg/kg every day in the treatment group. Each rat was given intraperitoneal injection of equal volume of physical saline each day in the control group. After 7 days，the ovarian tissues of rats were obtained for the detection of the ovarian index (Ovarian wet weight/body weight) and the expressions of Bcl-2, Bax, Caspase3 and Smad7 protein in ovary by immunohistochemical method, and the images were analyzed byⅠPP. Results: Compared with the control group, the ovarian index of the rats in the treatment group decreased (P ＜0.05). The results of immunohistochemistry showed that the expression of Bcl-2, Bax, Caspase3 and Smad7 protein was observed in the cytoplasm of ovarian granulosa cells. Compared with the control group, the expression of Bcl-2 and Smad7 was decreased (P ＜0.05), and the positive expression of Bax and Caspase3 was enhanced (P ＜0.05). Conclusion: Cisplatin induced ovarian injury in rats and promoted the apoptosis of ovarian granulosa cells, which was related to the down-regulation of Bcl-2 and Smad7 protein expression and the up-regulation of Bax and Caspase3 protein in rat ovarian granulosa cells. The expression of Smad7 protein in ovarian granulosa cells was positively correlated with the expression of Bcl-2, suggesting that Smad7 may induce cisplatin-induced apoptosis of ovarian granulosa cells.

41  Experimental study on differentiation of bone marrow mesenchymal stem cells into neurons induced by astragaloside Ⅳ in SAMR1 mouse

Peng-Tao LI1,Xiao-Nan Yang1,Hui Zhang

Graduate Faculty of Hebei North University ,Zhangjiakou, Hebei 075000, China Basic Medical College of Hebei North University, Zhangjiakou 075000, China

Background: Bone marrow mesenchymal stem cells (BMSCs) induced differentiation into nerve cells in vitro, and its transplantation for the treatment of Alzheimer’s disease has been confirmed. Objective: Experiments identified whether astragaloside Ⅳ could induced BMSCs in SAMR1 mouse to differentiate into nerve cells in vitro. Methods: We cultured and isolated BMSCs in SAMR1 mouse by whole bone marrow adherence culture method, observing the cell proliferation, The CD34, CD54 and CD90 antibodies were detected by 3-generation BMSCs flow cytometry. The 3 generation BMSCs were divided into astragaloside ⅠV induction group (A group) and blank control group (B group). The expression levels of NSE and nestin mRNA of cells were detected by qPCR technology at 12,24,48 hours, and the expression of NSE and nestin antibodies was detected by immunocytochemistry. Results: Primary cultured cells were melted 80-90% for 7 days, and the cells were spindle-shaped. And the cells were purified by 3 generations. CD34 (-), CD54 (+) and CD90 (+) were detected by flow cytometry. The results showed that the cultured cells belonged to BMSCs. A group began to express NSE, nestin in 12 hours, the expression of NSE, Nestin mRNA in 24 and 48 hours in A group was significantly higher than that in B group (P＜0.05), and so the expression of NSE and nestin antibody do (P＜0.05). Conclusion: The results showed that Astragaloside Ⅳcould induce differentiation of bone marrow mesenchymal stem cells in SAMR1 mouse into neurons in vitro , which laid a good foundation for the later transplantation of Alzheimer’s disease.

42  Therapeutic effects of BMSCs transplantation on silicosisvia paracrine mechanisms and the macrophagies autophage in rats

Junling Gao1,2, Man Zhao1,2, Huixing Zhu1,2, Xin Wang1,2, Lei Yu1,2, Ran Li1,2, Yanxia Tian1,2, Jianzhong Cui31Department of Histology and Embryology, School of Basic Medical Sciences, University of North China Science and Technology, Tangshan 063210, China;2Hebei Key Laboratory for Chronic Diseases, School of Basic Medical Sciences, University of North China Science and Technology, Tangshan 063210, China;3Department of Neurosurgery, Tangshan Gongren Hosipital, Tangshan 063000, China

Silicosis is a well-known occupational disease, characterized by epithelial injury, fibroblast proliferation, expansion of the lung matrix and dyspnea. At present, no effective treatment methods for silicosis have been identified. A series of studies aimed to investigate (1) the protective potential of exogenous bone marrow-derived mesenchymal stem cell (BMSC) transplantation on experimental silica-induced pulmonary fibrosis in rats; (2) to analyze the underlying paracrine mechanisms associated with its therapeutic effects; (3) to find the relationship with the macrophgeis autophage in vivo and in vitro. BMSCs were isolated, cultured，identified by flow cytometry using FⅠTC staining the surface markers CD29, CD45, CD54, and the CD106. Following the successful establishment of the silicosis model, exogenous BMSCs were infused into adult SD rats via the tail vein. Lungs were evaluated using hematoxylin and eosin (H&E) staining. The expression of interleukin-1 receptor antagonist (IL-1RA), interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α ) protein were detected by immunohistochemistry and western blot analysis. Co-localization of BMSCs and ⅠL-1RA expression was determined by double-label immunofluorescence. The distribution of transplanted BMSCs was tracked by monitoring the expression of BMSCs in rats. Treatment with BMSCs was found to protect the lungs against injury and fibrosis by the suppression of upregulated ⅠL-1 and TNF- α protein, via triggering IL-1RA secretion. This mechanism was hypothesized to be mediated by paracrine signaling. These results indicate that the release of ⅠL-1RA from BMSCs via paracrine mechanisms significantly blocks the production and/or activity of IL-1 and TNF- α . Autophagy was activated in AM of the silicosis model, and AM autophagy involved in the pathological process of silicosis rats; BMSCs transplantation could block activation of autophagy in AM of silicosis rats; BMSCs transplantation could regulate the PⅠ3K-AKT-mTOR-P70S6K signaling pathway, inhibit autophagy in AM.

43  Significances of sperm mitochondrial ROS generation on its motility＊

Hong Chen1, Ranran Chai1, Guowu Chen2, Wai-Sum O31Department of Anatomy, Histology & Embryology,2Hospital of Obstetrics & Gynecology, Shanghai Medical College, Fudan University, Shanghai, China;3Department of Obstetrics and Gynecology, The University of Hong Kong, Hong Kong, China

Our recent findings suggested that sperm motility might be one of the earliest and most sensitive oxidative damage of sperm. To test if prohibitin (PHB) may regulate sperm motility via the maintenance of optimal mitochondrial ROS (mROS), semen samples from 301 male subjects between 30-40 years old attempting ⅠCSⅠ/ⅠVF were collected and then assayed by semen analysis. After removing contaminated leucocytes, we examined mROS levels, high MMP, and lipid peroxidation in sperm from infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase-type activity of sperm mitochondrial complex Ⅰ (MCⅠ) and PHB expression were also determined. We demonstrated that mROS and lipid peroxidation levels were significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression weresignificantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase-type activity levels of sperm MCⅠ in the three groups studied suggested that the catalytic subunits of MCⅠ in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCⅠ associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, which may regulate sperm motility via increases in low MMP and lipid peroxidation.

*This project was supported by National Natural Science Foundation of China (No. 81270738).

44  The effect of epigenetic genes and the mechanism of Heshouwuyin on testicular retrogression in natural aging rats

Yujuan Wang1, Jing Sun1, Siyun Niu1, Feng Qi21Department of Histology and Embryology, HeBei University, Baoding 071000, China;2Department of Respiration ,Baoding First Hospital of Traditional Chinese Medicine, Baoding 071000, China

Testis is an important organ which secretes male hormones and it’s closely associated with DNA methylation and aging process. Ⅰn this study, we aimed to investigate the effect of epigenetic genes and the mechanism of Heshouwuyin on testicular retrogression in natural aging rats. DNA methylation gene chip screened out 1728 genes between young control group and natural aging group, among these 1728 genes, 238 genes were directed regulated by Heshouwuyin. GO analysis and pathway analysis indicated that these 238 genes were distributed in 23 cell pathways which were related to disease, cell proliferation and apoptosis, cell adhesion and so on. Methylation level and mRNA level of the epigenetic genes were detected by MSP and qRT-PCR.We found that Heshouwuyin, Heshouwu extract, Roucongrong extract and Huainiuxi extract could increase the methylation level of promoters in wnt2b, MAPK10, AXL and downregulate wnt2b, MAPK10; and could reduce the methylation level of promoters in Ttc29, C-myb, and up-regulate Ttc29, C-myb, AXL. The effect of Heshouwu extract was stronger than Roucongrong extract and Huainiuxi extract, and the effect of Heshouwuyin was stronger than the other three groups. BrdU staining Ⅰmmunofluorescence staining found that Heshouwuyin, Heshouwu extract, Roucongrong extract and Huainiuxi extract could promote the proliferation of spermatogonia in natural aging rats, and the effect of Heshouwuyin was stronger than the other three groups. These results demonstrated that Heshouwuyin may delay the degeneration of testicular tissue in aging rats by DNA methylation.

45  Age-dependent increase in COX7A2 negatively correlates with testosterone production in rat testicles

Siqi Ma1, Yine Qu1，Yanzhong Chang2, Xianglin Duan2, Fulu Gao21Department of Histology and Embryology, School of Basic Medicine, North China University of Science and Technology, Tangshan, China;2Hebei Normal University

Testosterone decline is a common health concern in aging male and can lead to clinical symptoms named partial androgen deficiency ofthe aging male (PADAM). Testosterone substitution therapy (TST) is the mainstream treatment for PADAM. However, due to increased risk of prostate cancer and cardiovascular diseases associated with TST, novel mechanism-driven approaches are still needed. By using rat model, the study found that the expression of COX7A2, a critical subunit of cytochrome C oxidase (COX), was increased in aged rat and negatively correlated with serum testosterone levels.Ⅰmportantly, protein expressions of the key enzymes for testosterone synthesis, including steroidogenic acute regulatory protein (StAR), the P450 side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD), were all changed in an opposite direction as COX7A2. Overexpression of COX7A2 in isolated Leydig cells from young adult rat decreased medium testosterone concentration. Consistently, introduction of COX7A2 into the testes of young adult rat led to declined serum testosterone, accompanied by down-regulation of StAR, P450scc, 3β-HSD and 17β-HSD. In contrast, serum testosterone levels and these enzymes were significantly increased when COX7A2 were knocked down in testes of aged PADAM rats. Our findings for the first time showed the negative correlation between COX7A2 and testosterone production, likely through regulation of androgen synthesis enzymes by COX7A2, and thus provide a novel insight into developing strategies targeting COX7A2 for PADAM treatment.

46  The role of Atg7-mediated autophagy in the neural crest cell production

Guang Wang1, Manli Chuai2, Liu Cao3, Xuesong Yang11Division of Histology & Embryology, Medical College, Jinan University, Guangzhou 510632, China;2Division of Cell and Developmental Biology, University of Dundee, Dundee, DD1 5EH, UK;3Key Laboratory of Medical Cell Biology, China Medical University, Shenyang 110001, China

Autophagy is a highly coordinated cellular program through lysosomal degradation of the cytosolic components that play crucial roles during early embryonic development and in maintaining adult cell homeostasis. However, it still remains unclear whether autophagyrelated gene 7 (Atg7) is involved in the regulation of neural crest cell production. Ⅰn this study, we found the co-location of Atg7 and Pax7+neural crest cells in early chick embryo development. Up-regulation of Atg7 with unilateral transfection of full length Atg7 increased neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated up-regulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Ⅰnterestingly, up-regulation of Atg7 in neural tubes could significantly accelerate cell progression into S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly alterated. Finally, we demonstrated that up-regulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed similar phenotypes in full length Atg8-GFP transfection neural tubes. Taken together, we reveal that Atg7- mediated autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of cell cycle.

47  Effects of exposure to PM2.5 during pregnancy on apoptosis of cortical neuron in offspring mice

Xinrui Zheng, Hui Zhao, Fengjie Li, Huanbin Tian, Li Yu

Department of Histology and Embryology, Weifang Medical College, Weifang 261053, China

Air pollution is a serious environmental health problem that is associated with diseases of the central nervous system (CNS). Epidemiological and clinical studies demonstrate a negative impact of air pollution on neural development in humans. Ⅰn this study, we investigated the effects of exposure to PM2.5 during pregnancy on development of cortical neuron in offspring mice. Female mice were given PM2.5 by rapid trachea drip method once every three days during pregnancy. Nissl staining was used to observe the morphological changes of cortical neuron at 1, 7, 14, 21, and 30d after birth. Western blotting was used to evaluate the protein levels of PCNA and Caspase-3. The expressions of Caspase-3 and NeuN were detected by double-labeled immunofluorescence assay to make sure the apoptotic cell type. With the increase of PM2.5 concentration, the number of cortical neuron reduced gradually at the postnatal day 1, 7, 14, 21, and 30,arrangement disordered,compared with control group, decrease of neuronal cell body, nissl body coloring shallow, and cytoplasm reduced. The neurons in PM2.5 exposed group had smaller cell body. Nissl bodies were decreased, with superficial coloration. Expression of PCNA of the each dose groups was significantly increased, and expression of Caspase-3 decreased compared with a dose dependent relationship with the control group The number of cleaved Caspase-3/NeuN positive cells increased in cortex compared with the control group. These findings reveal that exposure to PM2.5 during pregnancy could make offspring mice have morphological changes of the cerebral cortex. PM2.5 can interfere with development of the brain in offspring mice by affecting the neuronal proliferation and apoptosis.

48  Effects of exposure to PM2.5 during pregnancy on development of organs in offspring mice

XinRui Zheng1, HongXia Zhang2, XueLi Zhang1, HongJuan Wu1, Li Yu11Department of Histology and Embryology,2Department of Pathology, Weifang Medical College, Weifang 261053, China

PM2.5 (particulate matter with an aerodynamic diameters of less than 2.5µm) is the major component found among air pollutants. Exposure to PM2.5 is related to the incidence and mortality of disease. However, it is unknown that the potential adversely health effect of prenatal exposure to it on perinatal infants’ growth and development during pregnancy period exists. Ⅰn this study we studied the effects of PM2.5 exposure during pregnancy on main organs’development of offspring mice. Maternal mice were randomly divided into control group, low-dose group (75μg/m3), middle-dose group (453.4μg/m3) and high-dose group (995μg/m3) for pregnancy period. Ⅰn this study, we observed main organs’ development in offspring mice causing significant, dose-related pathological change. The treatment groups’ neonatal mice had a significant change in supermicro-structures of viscera. Ⅰn the treatment groups part of the myocardial fibers were arranged in a disordered manner with edema of the myocardial interstitium and inflammatory cells were infiltrated. Ⅰn liver tissue, there was fat vacuole and inflammatory cell infiltration. Periodic acid-Schiff (PAS) showed glycogen storage decreased. Ⅰn the spleen, boundaries between red pule and white pule were unclear. The pathological changes in lung were local atelectasis, wider alveolus alternation and obvious bleeding. Renal glomerulus were pyknosis. Edema of renal tubular epithelial cell took place in treatment group by pathologic examination of kidney. The results of electric microscoply showed that the cells of main organs some mitochondrial crest disappeared, while the membrane remained integrated and vacuolated. These findings indicate that exposure to PM2.5 during pregnancy could result in abnormal tissuemorphology of neonatal mice, which might be the anatomical basis and developmental etiology of chronic disease after birth.

49  High Glucose Enhances Autophagy and Affects Functions of Trophoblast Cells

Lulu Ji1, Zhiguo Chen1, Yating Xu1, Guoping Xiong2, Rui Liu1, Chao Wu1, Hangyang Hu1, Lin Wang11School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China;2Department of Gynecology and Obstetrics, the Central Hospital of Wuhan, Wuhan 430030, China

Autophagy is a dynamic process that degrades and recycles cellular organelles and proteins to maintain cell homeostasis.Alterations in autophagy occur in various diseases, but the role of autophagy in gestational diabetes mellitus (GDM) is unknown. We aimed to characterize the roles and functions of autophagy in this disease. Clinical samples and trophoblast cells cultured with glucose were used to analyze the level of autophagy. Subsequently, apoptosis, invasion and proliferation of the treated cells were examined. We found that high glucose levels enhanced autophagy and cell apoptosis, but reducing proliferation and invasion, and these effects were ameliorated through the knockdown of ATG5. 5hmC data analysis further revealed the epigenomic regulatory circuitry underlying the induced autophagy and apoptosis in GDM and preeclampsia. Finally, RNA-seq was performed to identify gene expression changes after silencing of ATG5. Ⅰn conclusion, our study demonstrates the significant functions of autophagy in GDM and provides potential therapeutic targets for the treatment of GDM patients.

50  Molecular mechanism regulating the effectiveness of temozolomide for pituitary carcinomas and atypical adenomas

Akira Matsuno1, Toshio Hirohata1, Akiko Mizutani1,2, R. Yoshiyuki Osamura31Department of Neurosurgery, Teikyo University School of Medicine, Tokyo 173-8605, Japan;2Teikyo Heisei University, Tokyo 170-8445, Japan;3Department of Pathology, International University of Health and Welfare Mita Hospital, Tokyo 108-8329, Japan

Treatment modalities of pituitary carcinomas and atypical adenomas include transsphenoidal or transcranial surgery, conventional and stereotactic radiotherapy, and various attempts at medical therapy. However, pituitary carcinomas and atypical adenomas are refractory to these conventional therapies. There have been several reports of temozolomide (TMZ) treatment of pituitary carcinomas and atypical adenomas. O6-methyl-guanine-DNA methyltransferase (MGMT) is not the sole molecule determining the sensitivity to TMZ in pituitary carcinomas and atypical adenomas. The Japan Society of Hypothalamic and Pituitary Tumors study suggests that MSH6, one of mismatch repair (MMR) pathway enzyme, fulfills a contributory role to the efficacy of TMZ treatment for pituitary carcinomas and atypical adenomas. The preserved MSH6 function might be essential for the responsiveness to TMZ treatment in pituitary carcinomas and atypical adenomas.

51  Receptor tyrosine kinase and colorectal cancer

Shu Yang, Ping Shen, Yaxi Wang, Jie Xiao, Deshan Zhou

Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China

Receptor tyrosine kinase (RTK) family is implicated in multiple cellular processes and aberrant expression of RTKs could induce tumors including colorectal cancer (CRC). KⅠT, a member of RTKs, and its ligand stem cell factor (SCF) are often over-expressed in CRC, which is suggested to be contributory to the development of CRC. However, the specific roles of highly activated KⅠT/SCF signaling in CRC and the upstream regulators on KⅠT/SCF over-expression have not been completely understood. Therefore, we carried out a series of experiments by the use of loss-of-functional c-kit mutant mice (Wads-/-), cultured human CRC cells lines as well as human CRC tissues. We found that Highly activated KⅠT/SCF signaling increased the product of mucus by promoting Atoh1-Mucin2 expression, which induced the tumorigenesis of mucinous colorectal adenocarcinoma (MCA). KⅠT/SCF signaling and its downstream JNK pathway activated c-Jun transcription factor to increase tight junction protein Claudin-3, which enhanced the proliferation of CRC cells. Highly activated KⅠT/SCF signaling stimulated the invasion of CRC cells by up-regulating ETV4 transcription factor which increased MMPs and EMT. ERK pathway was activated by KⅠT/SCF signaling; and the p-ERK increased ETV4 by blocking the ubquitin-proteasome dependent degradation of ETV4 via phosphorylating ETV4 at Ser73 in CRC cells. ⑤ Mir-34c down-regulated SCF through binding to the 3’-UTR within SCF mRNA so as to inhibit the growth and invasion of CRC cells. Resveratrol elicited anti-CRC effect by promoting mir-34c expression in vitro and in vivo.

52  lnflunence of annexin A5 on the behavior of uterine cervical cancer cells

Xin Li1, Xiaojie Wang1, Fulu Gao21Department of Histology and Embryology, Chengde Medical College, Chengde 067000, China;2Hebei Normal University, Shijiazhuang 050000, China

Annexin A5 is a kind of Ca2+-dependent phospholipid binding protein which is involved in cell membrane dynamics and organization. Recent data showed that Annexin A5 expression was variable in various cancers which implied Annexin A5 might involve in tumorigenesis. Uterine cervical carcinoma is a fatal disease popular around the world. To detect what role annexinA5 (ANXA5) play in the uterine cervical cancer cells. The uterine cervical cancer cell line HeLa and SiHa were applied. First, A recombined plasmid pcDNA3.1-ANXA5 was constructed and transfected into the cells, the positive cell clones were screened and G418 was used to keep the pressure. The cells which overexpress ANXA5 were named HeLa/ANXA5 or SiHa/ ANXA5, the cells transfected with the pcDNA3.1 empty plasmid acted as negative control which were named Neo. Second, the siRNA was prepared and transfected into the cells which were named ANXA5-si. Cells transfected with the transfection reagent acted as Neo and the cells without any treatment as blank control. CCK-8 method was used to detect cell proliferation and PⅠ, Hochest33258 and flowcytometry were employed to detect cell apoptosis. Wound healing assay and Transwell assay were applied to evaluate cell migration and invasion ability. To detect the mechanism of ANXA5 action, Western blot method was used to investigate the expression of apoptosis relative gene bcl-2 and bax and cell metastasis relative gene E-cadherin and MMP-9. Ⅰn the ANXA5 overexpression uterine cervical cancer cells, cell proliferation was suppressed and apoptosis prompted and bcl-2 expression decreased while bax expression increased; Meanwhile, cell migration and invasion were suppressed with increased E-cadherin and decreased MMP-9 expression. Ⅰn the ANXA5 knockdown cells, cell proliferation did not altered but cell apoptosis decreased with the increased cell migration and invasion. The expression of E-cadherin decreased while MMP-9 increased. Our data indicated that ANXA5 might act as tumor suppressor protein in uterine cervical cancer cells.

53  ALDOA promotes the proliferation and G1/S transition via EGFR/MAPK pathway in non-small cell lung cancer

Hailu Fu, Yuxin Bai, Huimin Li, Shujuan Shao

Department of Histology and Embryology, Dalian Medical University, Dalian 116044 ,China

Aldolase A is a glycolytic enzyme that catalyzes the reversible conversion from fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Aldolase A is upregulated in various cancers. Our previous study demonstrated that aldolase A promotes EMT of lung cancer. However, the role of aldolase A in lung cancer proliferation remains to bedetermined. Here, we found that knockdown of aldolase A by shRNA resulted in decrease of the proliferation and blockage of G1/S phase transition in human lung squamous carcinoma cell line H520 both in vitro and in vivo.Cell cycle synchronization assay showed that aldolase A protein expression increased in G1phase and G1/S transition. Western blotting showed that knockdown of aldolase A led to reduction of protein levels of cyclin D1 and cyclin E1. Real-time PCR showed that knockdown of aldolase A resulted in decrease of cyclin D1 mRNA, while the half-life time remained the same under transcription inhibitor actinomycin D treatment. Therefore, we tested several pathways involving in cyclin D1 transcription and found that knockdown of aldolase A specifically promotedERK phosphorylation. Further study indicated that aldolase A modulated EGFR/MAPK pathway through promoting EGFR expression, Raf1 expression, their corresponding phosphorylation, as well as MEK1/2 phosphorylation. And aldolase A’s regulation of cell proliferation and cyclin D1expression strongly eliminated under MEK inhibitor U0126 treatment or EGF treatment. We also found that aldolase A would translocated to nucleus under U0126 treatment, which suggested that there existed a mutual regulation. From another perspective, we found that knockdown of aldolase A led to a reduction of extracellular lactate and ATP concentration, while an increase of extracellular glucose concentration. Thus, we concluded that aldolase A promoted non-small cell lung cancer proliferation and G1/S transition in an EGFR/MAPK and glucose metabolism dependent manner.

54  The role of PDlA6 in the control of proliferation and cell cycle of lung cancer and its mechanism

Xiaoyu Qi, Yuxin Bai, Hailu Fu, Huimin Li, Shujuan Shao

Department of Histology and Embryology, Dalian Medical University, Dalian 116044, China

PDⅠA6 was one of the overexpressed proteins screened by mass spectrometry between clinical squamous lung cancer sample and adjacent tissue. Protein disulfide isomerases family 6(PDⅠA6) belongs to the PDⅠ family, which function as isomerases and molecular chaperones. Recently, PDⅠA6 was found to have a close association with various cancers. However, there has been little investigation into the biological function of PDⅠA6 in lung cancer. Therefore, in this study we used human lung squamous cell carcinoma cell line NCⅠ-H520 and human lung adenocarcinoma cell line Anip973 as PDⅠA6 knockdown model and human lung adenocarcinoma cell line H1299 as overexpression model. CCK8，clone formation showed that PDⅠA6 promoted cell proliferation in vitro and flow cytometry assay showed PDⅠA6 contributed to G1/S transition. Western blotting suggested that PDⅠA6 increased the expression of cyclinD1，cyclinE，CDK4，CDK6，pRb，while inhibited p27 expression. Meantime, we found that PDⅠA6 upregulated the protein level of p-ERK1/2(Thr202/Tyr204), RAS, p-Raf1、p-MEK and PCNA, while ERK and MEK expression were not affected. After adding MEK1/2-specific inhibitor U0126-ETON, PDⅠA6’ s modulation of cell proliferation and cell cycle was diminished. Together, these results indicated that PDⅠA6 promotes the proliferation of lung cancer cells, and participates in the promotion of G1/S phase transition by activating Ras /MEK/ERK signaling pathway.

55  The clinical significance expression of MGMT and EGFR in colorectal carcinoma

Zhi-Hong Gao, Ya-Qi Zuo, Peng-Tao Li, Xiao-Nan Yang, Wei Zhang , Xiao-Li Zhang

Life Science Research Center of Hebei North University, Zhang Jiakou 063000, China

Objective:To explore the correlation of O-6-methyguanine-DNA methytransferase(MGMT) and epidermal growth factor receptor(EGFR) in colorectal carcinoma and study its clinical significance by SABC immunohistochemical method. Method:Ⅰmmunohistochemistry was used to detect the expression of MGMT and EGFR in75 cases of colorectal carcinoma. 75 cases of colorectal carcinoma were highly differentiated adenocarcinoma, including 40 males and 35 females, without chemotherapy or radiotherapy before the operation. The average age of the patients was 47.8 (32-69) years, and the median age was 46 years. The tumors were located in the rectum in 34 cases, in the colon in 41 cases. There were 37 cases without lymph node metastasis and 38 cases with lymph node metastasis. Results: The experimental data showed that the positive rates of MGMT and EGFR were 42.7% and 53.3% respectively in colorectal carcinoma. The expressions of MGMT and EGFR wasn’t related to the location of tumor patient’s age and the gender. However, The high expression of MGMT was related with the infiltration depth. The expression of EGFR was correlated with the infiltration depth and the lymph node metastasis. Conclusion: MGMT and EGFR were concerned with the evolution of colorectal carcinoma,which proved that they were important in prognosis and molecular targeting therapy guide of the colorectal carcinoma.

56  Development of pH sensitive drug delivery system using cockle shells-derived aragonite nanoparticles for treatment of osteosarcoma

Wenliang Fu, Zhihong Chen

Department of Human Anatomy, Chengde Medical University, Chengde 067000, China

Osteosarcoma is a highly malignant primary bone cancer and most prevalent in children and young adults. Chemotherapy is associated with severe side effects due to nonspecific tissue biodistribution. Various targeted drug delivery systems using nanotechnology have been explored in the past few decades. Biogenic calcium carbonate (CaCO3) nanoparticles show unique advantages and the potential a delivery system. However, their anticancer efficacy and safe dosage for maximizing the therapeutic activity without causing severe side effects have not been tentatively studied. Ⅰn this study, cockle shells-derived CaCO3aragonite nanoparticles (ANPs) were prepared (spherical-shaped, 20-60 nm in diameter) and loaded with doxorubicin (DOX). The drug release study indicated that ANPs can be used as a pH sensitive drug delivery system. Ⅰn addition, the antitumor effect of DOX-ANPs was tested in vitro. These results indicated that DOXANPs induced G2/M arrested, and showed an efficient cytotoxicity against OS cells, close to the toxicity effect of DOX. The single dose acute toxicity study of ANPs showed the maximum tolerated dose was 180 mg/kg. The repeated dose toxicity study of ANPs (14 days) indicated that ANPs (below 30 mg/kg) showed potential a drug carrier without acute toxic side effects. Ⅰn vivo anticancer study, the DOX-ANPs treatment group can significantly reduce the tumorvolume and increase the surviving ratio, compared to the tumor model group. Ⅰn addition, the DOX-ANPs group showed less toxicity to the normal organs, compared to the free DOX group. These results established strong evidence that ANPs derived from cockle shells is a good biomaterial that can be used as a drug delivery system against osteosarcoma.

57  Disruption of regulatory loop between DUSP1 and p53 contributes to hepatocellular carcinoma development and progression

Peipei Hao1, Hua Li1, Mijin Lee1, Yunpeng Wang1, Jong-Hyun Kim1, Goungran Yu1, Sangyeop Lee2, Sunhee Leem3, Kyuyun Jang4, Daeghon Kim111Division of Gastroenterology and Hepatology, Department of Internal Medicine, Research Institute of Clinical Medicine, Chonbuk National University Medical School and Hospital, Jeonju, Jeonbuk, Republic of Korea;2Division of Life Science Research, Korea Basic Science Institute, Daejeon, 305-806 Daejeon, Republic of Korea;3Department of Biological Science, Dong-A University, Busan, Republic of Korea;4Department of Pathology, Chonbuk National University Medical School, Jeonju, Jeonbuk, Republic of Korea

Altered expression of dual specificity phosphatase 1 (DUSP1) is common in hepatocellular carcinoma and various tumors, and is predictive of tumor progression and poor prognosis.However, the tumor suppressive role of DUSP1 has yet to be clearly elucidated, particularly in hepatocellular carcinoma (HCC). Therefore, we explored the DUSP1 expression and its associated signaling pathways in HCC cells and tissues.Additionally, the functional roles of DUSP1 were assessed in vitro and via a mouse model. Downregulation of DUSP1 expression was significantly correlated with poor differentiation (P ＜ 0.001) and advanced-stage HCC (P = 0.023). DUSP1 inhibited the phosphorylation of p38 MAPK and subsequently suppressed HSP27 activation, resulting in enhanced phosphorylation of p53 at sites S15, S20, and S46 in HCC cells. The enhanced p53 activation thereby induced the expression of target genes, p21 and p27, which are linked to cell cycle arrest and apoptosis. Thus, DUSP1 is potentially linked to p53 activation via the p38 MAPK/HSP27 pathway. Nonetheless, wild-type p53, but not mutant p53, transcriptionally upregulates DUSP1 via its DNA-binding domain. Accordingly, DUSP1 and p53 may collaborate to suppress tumors in hepatocarcinogenesis via a positive regulatory loop.

58  Docosahexenoic acid inhibits the growth of glioma stem cells by targeting Wnt/β-catenin and COX-2/PGE2 signaling pathway＊

Haoyi Li1,2, Yun Wang1, Yue Cheng1,2, Chenren Li11Department of Histology and Embrology, College of Basic Medical Sciences，2Student Camp Four，Third Military Medical University, Chongqing 400038, China

Recent studies support glioma stem cells (GSCs) playing a predominant role in tumorgenesis , tumor recurrence, metastasis, chemoresistanceand resistance in glioblastoma multiforme (GBM). GSCs have been regarded as a target for the treatment of GBM. Numerous evidences have demonstrated that administration polyunsaturated fatty acid Omega-3, especially docosahexenoic acid (DHA), might be a preventive strategy against cancer. However, the relevance of DHA to proliferation and apoptosis of GSCs remains unknown. Ⅰn this study, we established mouse orthotopically and subcutaneously xenograft model in vivo, using GSCs generated from glioblastoma cell lines GL261. Meanwhile we

explored antitumor effect of DHA on glioblastoma and its potential mechanisms on GSCs. The results showed: 1. DHA monotherapy can significantly reduce GSCs initiated tumor growth; 2. DHA can effectively inhibit the proliferation of GSCs in vitro; 3. Combination of DHA and Temozolomide(TMZ) has more effects on glioblastoma suppression than monotherapy; 4. Mechanistically, the anti-glioma effect of DHA was due to its inhibitory effect on Wnt/β-catenin and COX-2/PGE2 signaling pathway. Our findings indicated that an effective approach of DHA for anti-gliomagenic via targeting GSCs, may provide a prospect for glioblastoma treatment.

*This work is supported by the Natural Science Foundation Project of Chongqing CSTC of China (CSTC2013jcyjA10130)

59  Aspirin inhibits proliferation of glioma stem cells and enhances the cytotoxic activity of temozolomide against malignant glioma＊

Huanyu Wei1,2, Haoyi Li1,2, Chengren Li11Department of Histology and Embrology, College of Basic Medical Sciences,2Student Camp Four, Third Military Medical University, Chongqing 400038, China

Glioblastoma multiforme (GBM) is the most aggressive primary tumor in central nervous system. Progress in the treatment of GBM has remained marginal over the last few decades. Many experiments have supported the existence of a small number of glioma stem cells(GSCs) which has the potency of self-renewal, proliferation and differentiation. As a major contributor of chemotherapy and radiotherapy resistances and malignant recurrence, GSCs have been proposed as a target for the treatment of GBM. As a classical non-steroidal anti-inflammatory drugs, aspirin(ASA) has been use in clinical practicefor more than a century. Recent researches have proved that ASA is capable of inhibiting the proliferation of some cancer cells. Meanwhile, it has the ability to induce apoptosis and repress proliferation of LN229 and U87 glioma cell lines. However, the effects of aspirin to GSCs remains unknown. Thus, the present study investigated the therapeutic potential of ASA for the treatment of malignant glioma. We established mouse models with GSCs-initiated orthotopic xenograft gliomas and subcutaneous xenograft tumors, using GSCs purified from glioblastoma cell line GL261. We investigated antitumor effects of ASA on orthotopic and xenograft gliomas. The results showed that: 1. ASA can inhibit the proliferation of GSCs in vitro; 2. ASA monotherapy can suppress GSCs-initiated tumor growth in vivo; 3. ASA has synergistic effects with temozolomide (TMZ) on glioma suppression. Ⅰn conclusion, our findings suggest an effective approach for anti-gliomagenic treatment of ASA via targeting GSCs.

*This work is supported by the Natural Science Foundation Project of Chongqing CSTC of China (CSTC2013jcyjA10130)

60  Up-regulation of DDX3 and DDX5 in both human osteosarcoma tissues and MG63 cells

Huancai Liu1, Qiaozhen Wang2, Qing Wang2, Meng Yuan3, Yawen Zhang3, Yanchun Chen31Affiliated hospital,2Department of Human Anatomy,3Department of Histology and Embryology, Weifang Medical University, Weifang, 261053, China

Osteosarcoma (OS) is the most common primary bone cancer, which is prevalent in teenagers and young adults. The molecular pathogenesis of osteosarcoma has not been fully identified. DEAD (Asp-Glu-Ala-Asp) box helicase 3 (DDX3) and DDX5, two members of the DEAD/H-box protein family, have been involved in severalhuman malignancies. However, the expression and biological role of DDX3 and DDX5 in OS remain largely unknown. Ⅰn the present study, to elucidate the role of DDX3 and DDX5 in OS tumorigenesis, we first evaluated their expression levels in human OS tissues by immunohistochemical staining. The result showed that the expression of DDX3 and DDX5 protein was distinctly higher in human OS tissues than that in the adjacent non-cancerous tissues. We then analyzed the associations of DDX3 and DDX5 expression with clinicopathological parameters and found that the levels of DDX3 and DDX5 expression were correlated with tumor stages. The high level expression of DDX3 and DDX5 occurred more frequently in later stage than in the early stage of OS. We also measured the expression of DDX3 and DDX5 in MG63 OS cell line and the human normal hFOB1.19 cell line by RT-PCR and Western blot analysis. The results revealed that DDX3 and DDX5 were up-regulated in the MG63 cells compared with the normal hFOB 1.19 cells. Taken together, DDX3 and DDX5 were up-regulated in both human osteosarcoma tissues and MG63 cell lines. Further studies are required to fully elucidate the role and mechanism of DDX3 and DDX5 in the pathogenesis of OS.

61  Comprehensive transcriptome analysis identifies ERα-dependent oncogenic lncRNAs of endometrial cancer

Hanyang Hu, Lulu Ji, Yating Xu, Lin Wang

Department of Histology and Embryology, School of Basic Medical Science, Wuhan University, Wuhan 430071, China

Excessing endogenous or exogenous estrogen exposure is one of the main risk factors for the development of endometrial cancer. Estrogen drives endometrial malignant transformation through binding and activating estrogen receptor (ER) which in turn interacts with cofactors at the DNA and regulating a transcriptional program that drives cell proliferation. However, the ER biology related to endometrial cancer remain unclear. Ⅰn this study, we systemically characterized endometrial cancer specific ERα cistrome and ERα-dependent transcriptome. Through analyzing estrogen stimulated endometrial cancer cells, we identified many thousands of gained ER-binding sites and hundreds of induced lncRNA transcriptions, many of which were also activated in primary tumors. These ER regulated lncRNAs were involved in several oncogenic signaling pathways and repressed by fulvestrant treatment. Functional experiments demonstrated the lncRNAs were required for proliferation and migration of endometrial cancer cells. In summary, We provided a new framework for ERαdependent lncRNAs associated tumorigenesis and target selection of endocrine therapy in endometrial cancer.

62  Effects of sericin on TNF-α and HNF-4α expression in liver of type 2 diabetes mellitus rats

Dong-Hui Liu, Zhi-Hong Chen

Department of Human Anatomy, Chengde Medical College, Chengde 067000, China

Objective: To investigate the possible mechanisms of sericin treating diabetes mellitus by observing the effects of sericin on tumor necrosis factor-α (TNF-α) and hepatocyte nuclear factor 4 alpha (HNF-4α) expression in liver of type ⅠⅠ diabetes mellitus rats model. Methods: 36 male SD rats were randomly divided into 3 groups: normal control group, diabetes model group and sericin treatment group. The typeⅠⅠ diabetes model rats were induced by continuous intraperitoneal injection of streptozotocin. After successfully establishing the diabetes animal model, the rats in diabetes model group were not given any more treatments. The rats in sericin treatment group were lavaged with sericin (2.4g/kg/d) for 35-day. SP immunohistochemical stain, Western blotting and RT-PCR were used to detect the TNF-α and HNF-4α expression in liver. Results: TNF-α protein immune positive products were tan and brown granules and located in cytoplasm of liver cells; HNF-4α immune positive products were tan and located in nucleus of liver cells. Compared with rats in normal control group, the TNF-α, HNF-4α protein and mRNA expression in liver of rats in diabetes model group increased obviously. Compared with rats in diabetes model group, the TNF-α, HNF-4α protein and mRNA expression in liver of rats in sericin treatment group decreased obviously. Conclusions: Sericin can improve insulin resistance by down regulating TNF-α and HNF-4α expression in liver, which maybe one of the mechanisms that sericin treating type 2 diabetes mellitus.

63  Mechanism of TFF3 in adhesion of thyroid papillary carcinoma TPC-1 cell

Lin Xu1, Dai Jin1, Wu Jingfang1＊, Zhang Jing1, Zhang Wenjing1, Xue Gang2＊1Basic Medical College,2Department of Otolaryngology, Head and Neck Surgery, First affiliated hospital, Hebei North University,Shijiazhuang 050024, China

Objective:To explore the effect of silence TFF3 expression on adhesion of papillary thyroid carcinoma TPC-1 cells. Methods:Homotyptic adhesion and heterotyptic adhesion experiment were used to assess the adhesive ability among stable knock-down TFF3(shRNA-TFF3)-TPC-1 cell, scrambled shRNA control(shRNAC)-TPC-1 cell and blank TPC-1 cell. β-catenin and TFF3 mRNA level was detected by quantitative RT-PCR (qRT-PCR) . The protein expression levels of MMP-9, MMP-2, ALCAM, β-catenin and E-cadherin were detected by western blot and immunocytochemistry(ⅠCC). Results: Knockdown of TFF3 significantly increased the number of homotyptic adhesion compared with shRNAC group（P= 0.0005）and TPC-1 group. The result of cell-matrix adhesive texts sugested that the number of heterotyptic adhesion(cell-to FN adhesion) cells were less than those of shRNAC group and TPC-1 groupafter inhibiting the expression of TFF3.There was no significant difference between shRNAC group and TPC-1 group in both homotyptic adhesion and heterotyptic adhesion test. Knockdown of TFF3 significantly decreased the mRNA level of β-catenin and TFF3, and inhibited the protein expression level of ALCAM,MMP-9, MMP-2, β-catenin, while increased the protein expression level of E-cadherin. Conclusion: Siliencing TFF3 expression could affect the ability of adhesion in TPC-1 cell via the WNT/β-catenin signalling pathway.

64  T4 phage delivered hepcidin efficiently and targetedly expressed in mouse brain＊

Guofen Gao, YuJing Gou, Xinwei Zhang, Lin-Hao You, Peng Yu, Yan-Zhong Chang

College of Life Science, Hebei Normal University, Shijiazhuang 050024, China

Hepcidin is a peptide hormone regulating iron metabolism. Ⅰt has been reported that hepcidin produced by astrocytes in brain may regulate the FPN1 on membrane of brain microvascular endothelial cells (BMVEC), and decrease the release of iron from BMVEC to brain tissue. However, none direct in vivo experimental data yet to verify this hypothesis for lacking good methods to express hepcidin in specific brain cells. Ⅰn this study, we first constructed a plasmid with GFAP promoter instead of CMV promoter, and cloned hepcidin under this promoter. We then used a novel T4 phage gene delivery system to deliver this plasmid and targetedly express hepcidin gene in the mouse astrocytes. After expression, the alterations of FPN1 onBMVEC and other iron-related proteins were detected. Ⅰn addition, we further investigated the effects of overexpressed hepcidin on the pathophysiology of Alzheimer’s disease with Aβ injected mice. We found that T4 delivered hepcidin efficiently and specifically expressed in mouse astrocytes, demonstrated by immunofluorescent staining of hepcidin and GFAP. The level of FPN1 on membrane of BMVEC was reduced, indicating the regulatory role of the overexpressed hepcidin to FPN1. The ferritin-L in BMVEC increased, suggesting iron deposition inside of these cells, which was consistent with the resultant effect of low FPN1 level. These results confirmed the importance of hepcidin secreted by astrocytes in the regulating brain iron transport, which may provide insights for the prevention or treatment of brain iron overload diseases.

*This work was supported by the National Natural Science Foundation of China(31400857) and Natural Science Foundation of Hebei Province (C2015205206).

65  Roles of hepcidin and iron regulatory proteins in the regulation of ferroportin 1 expression in mouse brain＊

Guofen Gao, Lan Wang, Qiaoman Niu, Linhao You, Yunzhe Ci, Yanzhong Chang

College of Life Science, Hebei Normal University, Shijiazhuang 050024, China

Ⅰron plays an essential role in various cellular metabolic processes of the body. Maintenance of cellular iron homeostasis is particularly important for keeping the normal functions of the cells. Ferroportin 1 (FPN1) is currentlyt he known iron exporter on the cell membrane, playing key roles in iron metabolism. Ⅰt has been indicated that the regulation of FPN1 in response to iron levels mainly involves two processes, post-transcriptional regulation by iron regulatory proteins (ⅠRPs) and post-translational regulation by hepcidin, the major iron sensing hormone. However, whether there is any communications between the two types of regulations or which one plays dominate roles have not been reported. Our study with ⅠRP2-/-mice and ⅠRP1/ⅠRP2 dual knockdown cells show that ⅠRPs indeed dramatically inhibit FPN1 expression as shown by the massively increased FPN1 level after knockdown of ⅠRP1 and ⅠRP2 expression. The knockout of ⅠRP2 severely affects the regulation effect of hepcidin on FPN1. FPN1 wasn’t decrease much after injection of hepcidin into the brain of ⅠRP2 knockout mice. Further analysis found the compromised effect observed in hepcidin-FPN1 regulation was directly dependent on the existence of iron regulation element (ⅠRE) in FPN1 mRNA. The results indicate the dominate regulatory role of ⅠRP2 on FPN1 expression. These will provide a more comprehensive understanding of the mechanisms of FPN1 regulation by ⅠRPs and hepcidin.

*This work was supported by the National Natural Science Foundation of China(31471035, 31400857) and Natural Science Foundation of Hebei Province (C2015205206).

66  Estrogen deficiency handicaps autophagic fluxand increasessenile plaques in the brains of adult female APP/ PS1 double-transgenic mice

Zhimin Long，Qiuhui Yao, Guiqiong He

Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing 400016, China

To investigate the effect of estrogen deficiency on senile plaques and autophagic flux in Alzheimer’s disease （AD）mice, threemonth old female APP/PS1 double-transgenic mice, either sham-operated (AD-sham) or 1 week, 1 month, or 3 months after ovariectomy, were involved. The control groups were age-matched wild-type littermates (WT). Ovariectomy induced the atrophy of mice uterus and the reduction of serum estradiol (E2) level.Ⅰmmunohistochemical staining showed that SPs in the brain of AD-ovx mice were significantly increased, except for 1-week AD-ovx mice. Western blot analysis was then carried out to detect autophagic flux-related proteins (Beclin-1, LC3 and SQSTM1/p62) in the brains of these mice. Lysosome-associated marker Cathepsin B (Cath-B) was detected by immunofluorescent staining. Estrogen deficiency triggers autophagosome formation in the early stage of AD to clear Aβ, then decreases autophagosome formation and inhibits the fusion of autophagosome and lysosome to block autophagic flux in the advanced stage of AD. Moreover, A significant change of sirtuin1(Sirt1) and Forkhead transcription factor O1 (FoxO1) expression in AD-ovx mice brain, indicating that estrogen deficiency may affect autophagic flux by Sirt1-FoxO1 pathway. These findings demonstrate that estrogen deficiency blocking autophagic flux accelerates AD progress by regulating Sirt1-FoxO1 pathway and indicate the potential value of a novel approach for the treatment of postmenopausal women with AD.

67  Fibronectin regulates neural crest cell differentiation during cardiovascular development

Xia Wang1,2, Sophie Astrof21Department of Anatomy, Histology and Developmental Biology, School of Basic Medical Sciences,Shenzhen University, Shenzhen, China;2Department of Medicine, Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Fibronectin (Fn1) is an evolutionarily extracellular matrix glycoprotein essential for embryonic development. Global deletion of Fn1 leads to embryonic lethality at embryonic (E) 9.5 from severe cardiovascular defects. However, how Fn1 composition regulates cellular fate and organ morphogenesis is not well understood. We observed that Fn1 is highly enriched in pharyngeal regions, suggesting that Fn1 plays cell-specific regulatory roles in cardiovascular development. To test this hypothesis, we ablated Fn1 in the neural crest, a group of multipotent progenitors which participate in the tissue morphogenesis. We found that Fn1 synthesized by the neural crest cells are required for the craniofacial development and cardiovascular morphogenesis. Deletion of Fn1 in the neural crest results in cleft palate, abnormal remodeling of 4thpharyngeal arch arteries and abnormal differentiation of neural crest cells into vascular smooth muscle cells. We show that Fn1 facilitates Notch activation in neural crest cells by signaling through integrin α5β1. Our results indicate that a cell-autonomous role of Fn1 in tissue morphogenesis and spatial regulation of Notch signaling.

68  Spatiotemporal expression of Meis1 during embryonic and postnatal development in B6; D2-Tg (Myh6＊-mCherry) transgenic mouse heart.

Weiwei Xu, Siyuan Wang, Xingyang Zhao, Huiwen Liu

Department of Histology and Embryology, Harbin Medical University, Harbin 150081, China

Cardiomyocyte cell cycle re-entry can activate mitosis and mediate heart regeneration. Here we used B6; D2-Tg (Myh6*-mCherry) transgenic mouseto obtain 9.5、11.5、13.5、15.5、17.5 days embryos, and postnatal 1、7、14、30 days hearts，and made them into frozen and paraffin sections. Observed the heart development and located accurately. Then, used DAB and immunofluorescence staining to detect the spatiotemporal changes of Meis1 expression.Taken α-MHC expression as reference, compared the spatiotemporal changes of Meis1 expression and the relationship with cardiomyocyte maturation. The results showed that: 1 B6; D2-Tg (Myh6*-mCherry) transgenic mice heart completed the internal separation at E13.5 days, E17.5 days had the adult heart characteristics. The heart volume increased and the myocardium density decreased significantly after birth. 2.Meis1 had two peaks in ventricular cardiomyocyte: the peak of E13.5 days may relate to the conduction system formation by trabecular cardiomyocyte exited from cell cycle; the peak of 14 days postnatal was associated with cardiomyocyte withdraw from the cell cycle permanently and went into terminal differentiation. 3.α-MHC was first expressed in right atrium at E11.5 days, indicating that the right atrium appeared as mature cardiomyocyte first. Meis1 had not expressed , showed that the cell cycle was opened, and a large number of cardiomyocyte proliferate. 4.The expression of α-MHC and Meis1 had the spatial and temporal differences in cardiac development, but both expression had the same trend after birth, and up to the peak at postnatal 14 days, showed that the window of cardiomyocyte proliferation was closed at this point of time, and the cardiomyocyte developed mature.

69  Ferritin heavy chain decreases ubiquitination and promotes stabilization of p21WAF1/ClP1

Qing Miao, Shufen He, Jingna Xun, Mutao Xu, Ke Tan, Xianglin Duan, Yumei Fan

Laboratory of Molecular Iron Metabolism, Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province,Key Laboratory of Molecular and Cellular Biology of Ministry of Education,College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, China.

Ferritin heavy chain (FTH) was one of major subunits of ferritin activity, which promotes iron oxidation and incorporation. Ⅰt plays an important role in iron and redox homeostasis. Ⅰn present study, we provide evidence that FTHpromotes stabilization of p21WAF1/CⅠP1, a cyclin-dependent kinase inhibitor that attenuates the cell cycle, by decreasingthe ubiquitination of it.We analyzed the regulatory effect of FTH on p21WAF1/CⅠP1protein accumulation in RAW264.7 cells. We found that over-expression of FTH could elevate endogenous p21WAF1/CⅠP1protein level but did not afect p21WAF1/CⅠP1mRNA level. Further,flow cytometrystudy showed that overexpression of FTH induced cell cycle G1 arrest. Next we tested the effect of FTH on p21WAF1/CⅠP1protein half-life. Result showed that FTH could prolong the p21WAF1/CⅠP1half-life as well as inhibit the p21 ubiquitination. These results indicated that FTH couldregulate cellular p21WAF1/CⅠP1protein level via inhibiting ubiquitination of p21. Our study provided a new insight for analyzing the regulatory effect of FTH after stress.

70  Cyclosporin A suppresses heat shock protein expressions through regulating HSF1 activity during heat shock

Ke Tan1, Jingyu Shao1, Beibei Han1, Siying Liu1, Akira Nakai2, Yumei Fan1, Yanzhong Chang1, Xianglin Duan11The Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Science, Hebei Normal University, Shijiazhuang 050024, China;2Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube 755-8505, Japan

Heat shock response is a conserved cellular stress response that senses protein misfolding. Ⅰt leads to activation of the HSF1 transcription factor which is the master regulator of HSR and the upregulation of a set of cytoprotective chaperones, called heat shock protein (HSP). Cyclosporin A (CysA) is an immunosuppressant that has revolutionized organ transplantation in clinical. Recently, some reports have shown that CysA is also associated with cancer treatment. CysA is a substrate and inhibitor of P-glycoprotein (P-gp) which modulates the multidrug resistance (MDR). Previous, we have shown that HSF1-SSBP1 transcriptional complex protects cells from proteotoxic stresses by evoking mitochondrial stress response. CysA could inhibit the nuclear translocation of SSBP1 during heat shock. However, the effect of CysA on heat shock response remains largely unknown. Here, we found that CysA pretreatment prevented major HSP inductions in a time- and dose-dependent manner upon heat shock. Although HSF1 protein level did not change, phosphorylation of HSF1 which affected HSF1 transcriptional activity was much higher in CysA-treated cells. CysA also impeded HSF1-SSBP1 transcriptional complex formation. HSF1 and SSBP1 nuclear translocation and recruitment to the HSP70 promoter were reduced when cells were pretreated with CysA. Because of low expressions of HSP during heat shock, CysA treatment caused cell death obviously. These results indicated that CysA suppressed HSP inductions partially through regulating phosphorylation and nuclear translocation of HSF1 against heat shock.

71  Diazoxide protects L6 skeletal myoblasts from apoptosis via the phosphatidylinositol-3 kinase/Akt pathway

Wei Chen1, Lixia Liu2,Yannan Zhang1, Suxia Shao11Department of histology and embryology, Hebei Medical University, Shijiazhuang 050017, China；2Department of pathology, Shijiazhuang traditional Chinese Medicine Hospital , Shijiazhuang 050051, China

Skeletal myoblast (SKM) transplantation is a promising approach to treat cardiac and skeletal muscle–related diseases. However, it has been hindered by poor cell survival. We have previously reported that the mitochondrial ATP-sensitive potassium channel opener diazoxide (DZ) can effectively protect L6 SKMs against apoptosis induced by H2O2, although the exact mechanism remains controversial. The aim of this study was to explore the mechanisms involved in the anti-apoptotic effect of DZ on L6 SKMs. Compared with the control group, the H2O2treatment caused cell damage, decreased Akt phosphorylation, increased mitochondrial membrane depolarization, increased activation of caspase-9 and caspase-3, and an increased cell apoptosis rate. DZ pre-treatment protected cells from H2O2-induced damage, increased Akt phosphorylation, prevented mitochondrial membrane depolarization as well as the activation of caspase-9 and caspase-3, and decreased the cell apoptosis rate. However, the DZ-induced cytoprotective and anti-apoptosis effects were partly inhibited by co-administration of a phosphatidylinositol-3 kinase (PⅠ3K) inhibitor, LY294002. We conclude that DZ pre-treatment contributes to protection of L6 SKMs against apoptosis at least partly by activating the PⅠ3K/Akt pathway and subsequently inhibiting the mitochondrial-mediated caspase-dependent apoptotic signalling pathway.

72  The protective effects of chronic intermittent hypobaric hypoxia on lgA nephropathy

Lixuan Wang1, Min Liu1, Jingwen zhou1, Fengyi Jiang1, Huixian Cui21Department of Histology and Embryology,2Department of Anatomy, Hebei Medical University, Shijiazhuang 050017, China

Ⅰn recent years, studies have suggested that chronic intermittent hypobaric hypoxia (CⅠHH) could show beneficial effects on humanimmune disorder. ⅠgA nephropathy (ⅠgAN) is associated with congenital or acquired immune dysregulation. This study is aimed to clarify the effects of simulating 5000m high altitude CⅠHH treament on ⅠgAN. The rat ⅠgAN model was established by administration of bovine serum albumin, castor oil, carbon tetrachloride and lipopolysaccharide. The rats in CⅠHH group were exposed to hypoxia simulating 5000 m high altitude (barometric pressure: 404 mmHg, PO2: 84 mmHg) in a hypobaric chamber for 28 days, 6 hours each day. Compared with the ⅠgAN group, the urinary red blood cell count, the urine protein, the serum creatinine and blood urea nitrogen levels of ⅠgAN+CⅠHH rats were significantly decreased. CⅠHH treatment inhibited the increase of serum ⅠgA levels and ⅠgA deposition in glomerular mesangial region. The glomerular inflammatory changes were reduced, and the proliferation of mesangial cells was decreased by double immunofluorescence staining in ⅠgAN+CⅠHH rats. CⅠHH decreased the serum levels of BAFF but not ⅠL-4 in ⅠgAN+CⅠHH rats. Simulating 5000m high altitude CⅠHH treament showed antiⅠgAN effects including relieving clinical symptoms, improving renal function and reducing the renal pathological damage. These effects may due to the CⅠHH induced BAFF decrease. Simulating 5000m high altitude CⅠHH treament may regulate B cell immunity and decrease its over activation.

73  lmproved serial sectioning techniques for correlative light-electron microscopy mapping of human langerhans islets

Sei Saitoh1,2, Nobuhiko Ohno1, Yurika Saitoh1, Nobuo Terada1,3, Satoshi Shimo1, Kaoru Aida4, Hideki Fujii5, Tetsuro Kobayashi4, Shinichi Ohno11Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi. Yamanashi 409-3898, Japan;2Section of Electron Microscopy, Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki 444-8787, Japan;3Department of Occupational Therapy, School of Health Sciences, Shinshu University School of Medicine, Nagano 390-8621, Japan;4Third Department of Internal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi. Yamanashi 409-3898, Japan;5First Department of Surgery, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi. Yamanashi 409-3898, Japan

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a useful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells in human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 a protein was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Ⅰmmunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. Ⅰn conclusion, the correlative imaging approach proposed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.

74  Renal water channel aquaporin-2: phosphorylation and subcellular distribution

Toshiyuki Matsuzaki

Department of Anatomy and Cell Biology, Gunma University Graduate School of Medicine, Maebashi, Japan

Aquaporin-2 (AQP2) is a membrane water channel protein in renal collecting duct cells playing a role in water reabsorption. AQP2 traffics between intracellular vesicles and apical plasma membrane to regulate the water permeability of the apical membrane under the control of plasma vasopressin level. AQP2 has four serines, Ser256, Ser261, Ser264, and Ser269, in its C-terminal region, which are phosphorylated or dephosphorylated by vasopressin stimulation and thought to play an important role in controlling the trafficking. We developed specific antibodies to Ser256-phosphorylated AQP2 and to Ser269-phosphorylated AQP2 and examined the relationships between phosphorylation and subcellular distribution of AQP2 in vivo rat kidney. Under the water-loaded condition, Ser256 was phosphorylated in considerable amount and remained intracellularly but Ser269 was completely dephosphorylated. When vasopressin was administered, Ser269-phsophorylated AQP2 was detected intracellularly soon after administration but highly accumulated in the apical membrane 30 minutes later. These results indicated that the critical event for trafficking to the apical membrane could be Ser269-phosphorylation. Then vasopressin receptor antagonist, OPC-31260 (kindly provided by Otsuka Pharmaceutical), was infused and the change of phosphorylation and AQP2 distribution was examined. Labeling for Ser269-phosphorylated AQP2 became weaker at 5 minutes and 15 minutes after infusion of the antagonist and was detected intracellularly. On the other hand, Ser256 was still highly phosphorylated and detected intracellularly. These results suggested that dephosphorylation of neither Ser256 nor Ser269 was necessary for internalization of AQP2. Ⅰt has been thought that phosphorylation and dephosphorylation of Ser256 are key events for AQP2 trafficking and that phosphorylation of Ser269 has a role in membrane retention; however, our results suggests that phosphorylation of Ser269 should be a key event for trafficking to the plasma membrane.

75  Analysis of living cells and tissues by label-freemicroscopy

Yoshinori Harada, Tetsuro Takamatsu, and Hideo Tanaka

Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan

Label-free optical imaging is favorable for the observation of diseased tissues from a viewpoint of non-invasiveness. There is the potential for unpredicted effects of exogenously administered tags. Optical molecular imaging has been expected to detect and analyze various diseases. Recent advancement of optical technologies hasallowed us in-situ analysis of cells and tissues without labelling.Ⅰn the conference, we introduce our label-free optical imaging studies by using Raman scattering and autofluorescence. Spontaneous Raman spectroscopic analysis of rat nonalcoholic fatty liver disease (NAFLD) was performed; Ⅰn NAFLD, Raman analysis mainly pick up information on vitamin A, lipid in stellate cells and hepatocytes. Multivariate analysis showed the potential of Raman spectroscopy for prediction of fibrosis in NAFLD. As for autofluorescence imagingstudies, measurement of nicotinamide adenine dinucleotide (NADH) autofluorescence intensity together with eliminating the influence of blood hemoglobin was useful to identify adenomatous lesions in endoscopically resected human colonic mucosa. We expect that label-free optical imaging using Raman scattering and metabolismrelated intrinsic fluorophores can be applied for diagnostic imaging of diseases, taking advantage of its non-invasiveness in the future.

76  Cell sorting method used in Schwann cells culture

Mi Shen1, Bin Yu, Qianru He, Fei Ding

Key laboratory of neuroregeneration of Jiangsu and Ministry of Education, Co-Innovation Center of Neuroregeneration, Nantong University, Nantong 226001, China

This study presented an efficient protocol. Ⅰt can be used to obtain a high purity of rat Schwann cells rapidly from freshly dissociated rat peripheral nerves. The sciatic nerves of newborn rats were dissociated and the Schwann cells were purified using fluorescence-activated cell sorting by labeling the Schwann cell membrane-specific marker of low-affinity nerve growth factor receptor, p75NGFRand oligodendrocyte marker 4. After sorting, the cells were plated on poly-l-lysine-coated dishes, then were cultured or passaged until confluent. Characterizations of sorted Schwann cells were validated by flow cytometric analysis and immunocytochemistry. RT-qPCR was used to validate the Schwann cell associated genes in the sorted Schwann cells. This method resulted in a high purity of Schwann cell culture. This research may provide a method of Schwann cell isolation which would facilitate future Schwann cell investigations.

77  Optimization of fluorescent immunolabeling and imaging protocols for free-floating thick tissue sections

Youquan Ding, Bin Du, Xia Xiao,Jianguo Qi

Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China

Fluorescent immunolabeling and imaging in free-floating thick (50–60μm) tissue sections are relatively simple in practice and enable design-based non-biased stereology, or 3D reconstruction and analysis. This method is widely used for 3D in-situ quantitative biology in biological research. However, the quality, efficiency and scalability of standard protocols for fluorescent immunolabeling and imaging of thick tissue sections are not always satisfactory. Ⅰn the present study, we optimized current fluorescent immunolabeling and imaging protocols for thick tissue sections. First, we showed that optical clearing-assisted wide-field epifluorescent imaging and 3D blind deconvolution allowed high-quality 3D visualization of thick tissue sections comparable to confocal imaging. Thus, our fluorescent imaging protocol makes 3D imaging of thick tissue sections simpler and more scalable. Then, we found that in immunolabeling protocols with effective tissue loosening, raising the conventional antibody incubation temperatures (4°C or 21°C) to mammalian body temperature (37°C), significantly enhanced the quality (labeling sensitivity, specificity, and homogeneity) and efficiency (antibody concentration and antibody incubation duration) of fluorescent immunolabeling of thick tissue sections, independent of antibody, antigen, and tissue characteristics. Finally, our results showed that pre-immunolabeling heat-induced antigen retrieval (citrate buffer, pH=6.0, 80°C, 45 min), in combination with post-immunolabeling condition (0.01 mol/L CuSO4in 0.05M ammonium acetate, pH=5.0, 2-5 min), effectively eliminated autofluorescence in thick (50μm) tissue sections (adult rat spinal cords as model materials), but didn’t diminish target signals of fluorescent immunolabeling. Our optimizations of fluorescent immunolabeling and imaging protocols for thick tissue sections will make 3D in-situ quantitative biology easier to implement.

78  ldentification and characterization of epithelial cell markers in rat kidney by immunohistochemical method

Fei Liang1,2, Dedong Kang2, Ping Lan2,3, Kyoko Wakamatsu2, Akira Shimizu21Department of Histology and Embryology, China Medical College, Shenyang 110122, China;2Department of Analytic Human Pathology, Graduate School of Medicine, Nippon Medical School, Tokyo 113-0022, Japan;3Department of Nephrology, The First Affiliated Hospital of Xi’an Jiao Tong University Medical College, Xi’an 710061, China

Ⅰn renal pathology, urinary segment is very important for evaluation of these functions. The epithelial markers are important to mark particular segment of nephron including renal tubules in the morphological research of kidney(of the kidney). Based on our previous study for(on) human tissue, we performed immunohistochemical analysis for a series of epithelial markers including CD10, CD15, AE1/3, Vimentin , Nestin , WT-1, E-cadherin , Tamm-Hor fall protein (THP), Villin in rat renal tissues. The kidneys from mature rat were fixed, embedded. Serial sections were autoclaved by(at) 120°C for 20 minutes in retrieval buffer for antigen retrieval. The results of immunohistochemical analysis : Vimentin , Nestin , WT-1 were on glomerulus(were on the glomerulus) , CD10 was on(was on the) Bowman’s capsule; Villin was on(was on the) proximal convoluted tubule, Villin and CD10 were on(were on the) proximal straight tubule; CD10 also expressed on the descending thin limb of the Henle loop of short-looped nephrons; CD10 , CD15 , THP , E-cadherin were on the thick ascending limb, CD10 , CD15 , THP , E-cadherin , AE1/3 were on(were on the) distal convoluted tubule; CD10 , E-cadherin , AE1/3 were on(were on the) cortical collecting duct and E-cadherin , AE1/3 were on(were on the) medullary collecting ducts. These results could provide a useful reference for kidney research.

79  RlN1 is involve in abnormal fear memory in PTSD rats＊

Linchuan Ma, Jingzhi Jiang, Jue Wang, Lili Huang, Fang Han

PTSD Laboratory, Department of Histology and Embryology, Basic Medical College, China Medical University, Shenyang 110122, China

The pathogenesis of post-traumatic stress disorder (PTSD), the symptoms of which can enhance the memory of fear, remains largely unclear. Recent studies with knockout(knocked out) animals have reported that Rin1regulate fear memory. The purpose of this study was to investigate whether RⅠN1 is involved in abnormal fear memory in PTSD rats. Methods: we used RⅠN1 knockout rats. Single prolonged stress (SPS) was used to build rat PTSD model. Conditioned stimulus-unconditioned stimulus（CS-US）experiment was used to examine fear memory. The expression of RⅠN1 in rat amygdala neurons was investigated by immunofluorescence staining and Western blot(blotting) technique. Results: CS-US experiment revealed RⅠN1-/- rats showed lower percentage of freezing than that in control rats, whereas no difference in percentage of freezing between SPS rats and RⅠN1-/- rats with SPS stimulation. Ⅰmmunofluorescence staining and Western blot also revealed the changed expression in RⅠN1 and related molecules in the RⅠN1 pathway in the amygdala of SPS rats in the immunoreactivity and protein level. Conclusion: these results indicate that RⅠN1 may be involved in abnormal fear abnormal in SPS rats.

*This study are(is) supported by National Natural Science Foundation of China（81571324）and Shenyang science and technology plan project（F16-205-1-53）

80  Gender differences in behavioral changes of rats with posttraumatic stress disorder

Jue Wang1,2Fang Han11PTSD teaching and research section, Department of Histology and Embryology, College of Basic Medical Sciences, China Medical University, Shenyang, 110122, China;2Jinghua hospital, Shenyang Eastern Medical Group, Shenyang 110005, China

The purpose of this study was to observe the difference of sex differences in the behavioral changes after single-prolonged stress (SPS) exposure (it is an animal model of posttraumatic-stress disorder (PTSD). Methods The behavioral changes of rats were detected by open-field test, elevated plus maze and Morris water maze test. Results (1)Ⅰn the open field test, compared with the control male rats, SPS male rats within 5 minutes of the horizontal movement distance and the central residence time was significantly reduced (P＜0.05). (2) Ⅰn the elevated plus maze test, compared with the control female rats, the percentage of the number of open arm entry and the percentage of open arm stay of SPS female rats were significantly reduced (P＜0.05). (3) Ⅰn the Morris water maze test, compared with the control female rats, latency , the time spent in the target quadrant and the number of the wear in the SPS female rats were significantly reduced (P＜0.05). There was no significant difference between male and female rats. Conclusion Our study indicate that SPS induce gender differences in the behavioral changes. Female maybe(might) show more susceptibility to SPS stimulation in anxiety and spatial cognition than male.

81  The effect of Fluoxetine on the expression of postsynaptic density protein 95 and synapsinⅠin the hippocampal neurons of post-traumatic stress disorder rats

Lili Huang, Jingzhi Jiang, Linchuan Ma, Fang Han

Department of Histology and Embryology, China Medical University, Shenyang 110122, China

Objective: To investigate the effects of fluoxetine (FLXT) on behavior of Post-traumatic stress disorder (PTSD) rats and the expression of synaptic related protein that are PSD-95 and SynapsinⅠ in the hippocampal neurons of PTSD rats. Methods: The internationally established single prolonged stress (SPS)-method was used to set up the rat PTSD model. Water Maze experiment was used to observe the effects of fluoxetine on the learning and memory of PTSD rats. The immunofluorescence and Western blot(blotting) techniques were used to detect the expression of PSD-95 and SynapsinⅠof hippocampal neuron in each group. Results: Ⅰn the first five days of Morris Water Maze (MWM), swimming distance and latency period increased in SPS rats compared with the control rats and then decreased after fluoxetine administration. Ⅰn the sixth day, compared with the control rats, the percentage of time spent in the target quadrant and the times across platform decreased in SPS rats, then increased after fluoxetine treatment. Ⅰmmunofluorescence staining and western blotting detected reduced PSD-95 and SynapsinⅠin the hippocampus of SPS rats, and then recovered after fluoxetine treatment. Conclusion: Our study indicates the effect of fluoxetine on SPS rats by increasing the expression of PSD-95 and synapsinⅠ, improving spatial memory and the ability of learning of the rats.

82  Atorvatatin reduces secondary inflammatory injury after intracerebral hemorrhage in rats

Hongxia Zhou, Linghuan Gao, Lingli Meng, Tian Zhang,Yuxin Zhang, Junling Gao

Department of Anatomy, North China University of Science and Technology, Tangshan 063000, China

There are studies claiming that after the onset of cerebral hemorrhage, endothelial cells, inflammatory cells and neurons of local brain tissues of the lesions were stimulated, TLR4/NF-κB-mediated inflammatory response were activated due to ischemia and hypoxia, Ⅰt is more and more attractive in clinic to prevent and treat secondary inflammatory damages and inflammatory responsesaround lesions after cerebral hemorrhage. Atorvastatin is a kind of cholesterol inhibitors which possesses certain neuroprotective effect and is widely used in cerebrovascular accidents. Ⅰn this study, in order to observe the preventive and therapeutic effect and mechanism of Atorvastatin on secondary inflammatory damage of rats with cerebral hemorrhage, Rats in the model group and atorvastatin-treated group were infused with autologous fresh uncoagulated blood to the right brain tissue of the basal ganglia to build the cerebral hemorrhage model. The rats in the atorvastatin-treated group were given a gavage of 3 mg/ kg of atorvastatin once a day after modeling. Wet-dry weighting method was used to detect the brain water content (BWC) of brain tissues around the lesion of the rats. Ⅰmmunohistochemical method was applied to count the number of NF-κB, TLR4 and TNF-α positive cells in brain tissues around the lesions, and Western -blot was employed to determine the expression levels, and the immuno fluorescence method was employed to determine Co localization of NF-κB, TLR4 and TNF-α proteins. Atorvastatin may attenuate TLR4/NF-κB-mediated inflammatory response in rats with cerebral hemorrhage, and reduce secondary inflammatory damages by downregulating these proteins.

83  Netrin-1 inhibits activation of astrocytes and attenuates brain injuries after ischemic stroke

Xiaosong He1, Yanqun Liu2, Falei Yuan3, Dahong Long1, Zhijun Zhang, Yongting Wang4, Aiguo Xuan1, Guo-Yuan Yang4,51Department of Human Anatomy, School of Basic Medical Science, Institute of Neuroscience and the second Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China;2Department of Neurology, Changhai Hospital, Second Military Medical University, Shanghai, China;3Hailisheng Biomarine Research Institute, Zhoushan, China;4Neuroscience and Neuroengineering Research Center, Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China;5Department of Neurology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

Netrin-1 displayed its function majorly depended on the combined receptors. There are large numbers of evidences showed that the receptors of NT-1 including DCC, UNC5H2 and A2B were presented on the astrocytes. However, whether the nt-1 could influence the activity of astrocyte was still unknown. Ⅰn this study, using primary cultured astrocytes and an MCAO model, we investigated the effects on NT-1 on astrocytes activation and the inflammation after MCAO.Ⅰt was found that NT-1 inhibited LPS induced ⅠL1 and ⅠL12 release in cultured astrocytes and increase the expression of P-AKT and PPAR-Y. Ⅰn addition, blocking of the UNC5H2 receptor reversed the inhibition effect of NT-1 exacted on astrocytes. Ⅰn vivo studies showed that NT-1 knocking-down can increase expression of GFAPand chemokine release. Moreover, NT-1 protein injection can inhibit the activation of astrocytes, the neuro-inflammation and protect the brain injury 3 days after MCAO. These results indicated that NT-1 was an important factor in regulating astrocytes activation and neuro-inflammation after MCAO.

84  Catalpol suppresses ischemia-induced pre-myelinating oligodendrocytes injury by downregulating ERK1/2 signaling pathway＊

Qiyan Cai, Teng Ma, Chengren Li, Yanping Tian, Lan Xiao, Hongli Li

Chongqing Key Laboratory of Neurobiology, Department of Histology and Embryology, College of Basic Medicine, The Third Military Medical University, Chongqing 400016,China

The high susceptibility of pe-myelinating oligodendrocytes (PreOLs) to ischemic damage plays a key role in the pathogenesis of periventricular leukomalacia. ERK1/2 signaling pathway has been shown to mediate the survival and death of PreOLs during ischemia.Ⅰn the present study, we aimed to investigate the protective effects of catalpol, an iridoid glucoside that is highly prevalent in the roots of Rehmannia glutinosa, on PreOLs under ischemia and to explore whether the ERK1/2 signaling pathway contributed to the protection provided by catalpol. Oxygen/glucose deprivation (OGD)，an in vitro model of cerebral ischemia，was produced using glucosefree DMEM medium supplemented with 8 mM Na2S2O4. Our results showed that treatment with 0.5 mM catalpol for 1 h prior to OGD injury significantly reduced the number of TUNEL-labeled apoptotic PreOLs, and restored the expression levels of CNPase (a marker of myelinating oligodendrocytes) and MBP (a marker of mature oligodendrocytes). Catalpol treatment also suppressed the decrease of mitochondrial membrane potential, reduced the overproduction of reactive oxygen species (ROS), and inhibited the elevation of intracellular Ca2+ after OGD. The increased expression of phosphorylated ERK1/2 (p-ERK1/2) was effectively prevented by administration of catalpol. Blocking the ERK1/2 signaling pathway with the MEK inhibitor U0126 and catalpol further reduced the oxidative stress and apoptosis of PreOLs under OGD. These data suggest that catalpol protects PreOLs against ischemia-induced oxidative injury by downregulating ERK1/2 signaling pathway. Catalpol may be a candidate for treating ischemic white matter damage.

*This study was supported by grants from the National Natural Science Foundation of China (NSCF 31500969, 31471148).

85  Clinical, Pathological and Genetic Characteristics of a Chinese Boy with Familial Hemophagocytic Lymphohistiocytosis Type 3

Jiao Mu, Meiyu Li, Guohui Zhang, Chunting Jin, Jianfeng Li

Department of Forensic Medicine, Hebei North University, No. 11 Zuanshinan Road, Zhangjiakou 075000, China

Objective: Familial hemophagocytic lymphohistiocytosis (FLH) is a rare autosomal recessive disorder of immune dysregulation associated with uncontrolled cytotoxic T cell and macrophage activation. Here, we report a rare case of unexpected death due to FHL and described the clinical, laboratory, pathological, immunological and genetic findings. Methods: Hematoxylin-eosin staining, CD8, CD68, CD20, CD4, and CD56 immunohistochemical staining, EBV RNA in situ hybridization study and all exomes sequencing were applied to observe the histological changes in FHL patients. Results: A 14-month-old Chinese boy present as fever, abdominal distension, hepatosplenomegaly, hepatitis, cytopaenias and died within 4 days of symptom onset without definitive premortem diagnosis. Postmortem examination revealed pleuroperitoneal fluid, enlarged mesenteric lymph nodes and hepatosplenomegaly. Histopathological examination showed interstitial pneumonia, hepatonecrosis and hemophagocytosis. An in situ hybridization study revealed that a positive EBV RNA (EBER1) antibody in lymphocytes of spleen, lymph node, liver and lungs, which demonstrated an undergone EBV disease. Ⅰmmunohistochemistry demonstrated that the histiocytes were CD68+, lymphocytes were mostly positive for CD8 cytotoxic T lymphocytes and negative for CD20, CD4, and CD56 in spleen, lymph node and liver. By isolating DNA from stored paraffin-embedded tissue of the patient and sequencing for all exons, we found the boy had UNC13D gene mutation. Conclusion: Hemophagocytic lymphohistiocytosis (HLH) is a clinical entity that is very difficult to diagnose due to the lack of specific laboratory findings and clinical signs. Fever, hepatosplenomegaly, cytopaenias, or diffuse lymphadenopathy may suggest HLH. Ⅰn these patients, repeat bone marrow aspiration and spleen, lymph node or liver biopsy should be performed for the rapid and accurate diagnosis. Genetic testing played an important role to differentiate from secondary HLH correctly.

86  The effect of TLR4 on cerebral ischemia in rat

Xiaohui Ding, Haihui Xing, Hui Xie, Fengyang Chen, Yinzhou Luo, Shengnan Zhou

Department of Histology and Embryology, Shenyang Medical College, Shenyang 110034, China

To investigate the effects of TLR4 in cerebral ischemia, cerebral ischemia reperfusion model of rat was established, and administered with TAK-242(TLR4 antagonist) by intraperitoneal injection. Brain tissue was sampled on the 1st, 3rd, 7th and 14th day after operation, respectively. The pathological changes were observed by HE staining and the expression of TLR4 and P-IKKα/β was detected by western blot(blotting) method. The results of HE staining showed that different degrees of pathological changes appeared in ischemia group , ischemia + TAK group, and the pathological damage was severer(severe) in ischemia group compared with ischemia + TAK group. The expression of TLR4 in ischemia brain tissue: On the 1st day after operation, the expression in ischemia +TAK group was higher than those in ischemia group and sham group. On the 3rd and 7th day after operation, the expression in ischemia group was higher than those in sham group and ischemia + TAK group. Furthermore, on the 7th day, the expression was higher in ischemia + TAK group than that in sham group (P ＜ 0.05). On the 14th day after the operation, there was no statistical difference among the three groups. The expression of P-IKKα/β in ischemia brain tissue: On the 1st day after operation, the expression in ischemia + TAK group was lower than that in sham group. On the 3rd day after operation, the expression in sham group and ischemia + TAK group were lower than that in ischemia group. On the 7th after operation, the expression in sham group was lower than those in ischemia group and ischemia + TAK group. On the 14th day after operation, the expression in ischemia + TAK group was lower than those in sham group and ischemia group. These results suggest that TLR4 may deteriorate the ischemia reperfusion injury and the mechanism may be due to up-regulation the expression of P-IKKα/β.

87  Encapsulation of iron in liposomescan increase the iron contents in rats

Peng Yu, Zi Xu, Lina Geng, Xueling Guo, Yan-Zhong Chang

Laboratory of Molecular Iron Metabolism, Key laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, Hebei Normal University, Shijiazhuang 050024, China

Background: Ⅰron deficiency can cause anemia, cognitive disorders and other related diseases and iron supplementation is an effective treatment of choice. However, traditional routes of iron supplementation do not allow efficient iron absorption and delivery to the brain. So an easily delivered iron formulation with high absorption efficiency could potentially find widespread application in treatment of iron deficiency. Results: Ⅰn this study, we have developed and characterized ferric ammonium citrate encapsulated in nanoliposomes (FAC-LⅠP) and have shown that it can treat iron deficiency anemia, inflammation anemia and can also increase iron levels in the brain. FAC was incorporated into liposomes with high efficiency (97%) and the liposomes were stable. Following FAC-LⅠP delivery, FAC-LⅠP significantly relieved anemia and increased the iron contents in olfactory bulb, cerebral cortex, striatum, cerebellum and hippocampus, and was more efficient at doing so than FAC alone. No signs of apoptosis or abnormal cell morphology were observed following FAC-LⅠP administration, and there were no significant changes in thelevels of SOD and MDA, suggesting that the formulation was not overtly toxic. Conclusions:Ⅰn this study, FAC-LⅠP proved an effective system delivering iron, with high encapsulation efficiency and low toxicity in murine.Our studies provide the foundation for more detailed investigations into the applications of liposomal formulations of iron as a simple and safe therapy for iron deficiency and inflammation anemia.Key words: FAC, nanoliposomes, ⅠCP-MS, micro-X-ray fluorescence, anemia.

88  Effect of additional boron on tibias osteogenesis and distribution of BMP-2 in African ostrich chicks＊

Daiyun Zhu, Ke Xiao, Khaliq Haseeb, Xintong Wu, Lei Cui, Weiwei Qiu, Hui Song, Huazhen Liu, Kemei Peng

College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China

The aim of the present study is to find out the effects of drinking boron on osteogenesis of tibias in ostrich chicks, and to investigate the possible relationship between additional boron and the expression of bone morphogenetic protein-2( BMP-2) in tibia. Thirty-two ostrich chicks werefed with 0 ( group A), 100 ( group B), 200 ( group C), and 400mg·L-1( group D) of additional boron in water for 30 d. The paraffin section of demineralized tibiasbone were made, and the bone histomorphometry to measure static parameter of tibia. HE staining, Masson’ s staining and immunohisto- chemistry stainingwere made to analyse the development of tibia. The results indicated that the width and the density of bone trabeculae was increased following group B,D, and C. Ⅰt showed the same trend with perimeter, area, and relative volume of bone trabeculae, especially in group C. The separation degree of bone trabeculae showed that group C was the minimum. Furthermore, compared with group D, the express of BMP-2 was also obviously increased in group A, B, and C. Ⅰn group C had more bone marrow cells in primary marrow cavities, osteogenic cells with BMP-2 positive were the most as well. There were a little osteoblasts and osteoclasts with BMP-2 positive in group D. Additional boron supplemented in drinking could promote obviously tibias osteogenesis of African ostrich chicks by regulating the expression of BMP-2, and 200 mg/L supplement boron in the drinking water appeared to be the most beneficial.

*This work was supported by the National Natural Science Foundation of China (31272517 and 31672504).

89  Anatomical basis of badminton sports injury research Guangxin Qiu

Hebei sport University, Shijiazhuang 050041, China

Ⅰn the past 10-20 years, through the improvement and improvement of training methods, badminton players are more robust, moving more agile, more comprehensive technology, more mature psychological quality, the badminton skills, tactics have also been in comprehensive and fast development. But the attendant badminton athletes in the training and competition in rapid increase in sports injuries, training intensity and the increasing intensity of the competition has increased the risk of injury caused by athletes. High level badminton athletes are the backbone of our badminton career, carrying the future and hope of our badminton, sports injury will destroy the systematic training of sports, affecting the long-term development of high-level badminton players. How to avoid sports injury has become a high level badminton athletes to improve the quality of training, improve the results of the game to ensure the sustainable development of competitive factors key factors. Ⅰn this paper, some high-level badminton athletes and coaches in China are investigated by means of literature, expert interview, questionnaire, mathematical statistics and logic analysis. Through the investigation results, the athletes’ injury characteristics and causes of injury were analyzed, and the occurrence rules of common injuries were summarized, and the corresponding preventive measures were put forward from the perspective of training.Structure analysis of the human body and movement technique is an important innovation.

90  Myocardial differentiation and mechanism of bone marrow mesenchymal stem cells induced by multiple factors

Yang Lv, Haiping Wang, Yuan Liu, Chenwei Gao, Haoyu Wang Department of Histology and Embryology, Hebei North University, Zhangjiakou 075000, China

FGF-2, BMP-2, TGF-β1, Wnt-11, Sal B and tanshinone ⅡA are all key signaling factors for bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes (CMs). Ⅰn this study, we compared the biological effect of these factors, alone or in combination, on rat BMSCs that differentiate into CMs in a simulated myocardial microenvironment in vitro. BMSCs were isolated from SD rats and cultured, then the 2nd-generation BMSCs were co-incubated with FGF-2, BMP-2, TGF-β1, Wnt-11, Sal B and tanshinone ⅡA, alone or in combination, for 72 h. After that, morphologic characteristics, surface antigens and mRNA expression of several transcription factors were assessed. BMSCs were initially spindle-shaped with irregular processes. Compared with the control group, BMSCs in treatment groups showed fusiform shape, orientating with one accord, and were connected with adjoining cells forming myotube-like structures on day 28. Moreover, the morphous of BMSCs in each treatment group was similar. The consequences of immunohistochemical and immunofluorescence staining demonstrated that Desmin, cTnⅠ and Connexin43 were all positively expressed in each treatment group. Whereas the expression of them in the control group was slight positive or negative. The results of RT-qPCR revealed that compared with the control group, cells in each treatment group did not express a-MHC, but expressed GATA-4 and Nkx2.5 at 7 days, 14 days and 28 days after induction. Furthermore, the ultrastructural characteristics of BMSCs in treatment groups were all similar to CMs. So FGF-2, BMP-2, TGF-β1, Wnt-11, Sal B and tanshinone ⅡA may induce BMSCs to acquire cardiogenic phenotype and some of them combined may achieve higher efficiency.