【作者】 黄志华；

【导师】 韩立民；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2014， 博士

【摘要】 目的：在中医学理论的指导下,应用现代生物医学技术,从氧化应激、能量代谢、兴奋性毒性损伤、炎症反应、细胞凋亡等多角度,研究葛根有效成分结构改造物——染料木素磺酸钠(genistein sodium sulfonate, GSS)对脑缺血再灌注损伤的保护作用及机制。为进一步拓展葛根的药理作用提供理论和实践依据。方法：采用大鼠在脑中动脉栓塞动物模型、谷氨酸损伤PC12细胞及原代培养的皮层神经元细胞模型,运用神经功能学评分、TTC染色、酶活性分析检测技术、放射免疫学方法、ELISA、MTT:方法、流式细胞术、Western blot等多种研究方法,分别从离体和在体水平研究GSS对脑缺血再灌注损伤的保护作用及机制。结果：(1)MCAO再灌注模型组大鼠均出现不同程度的神经功能损伤和脑梗死,血清LDH及CK活性升高,缺血侧脑组织LDH活性降低;GSS治疗后,神经功能评分降低,脑梗死体积缩小,血清LDH及CK活性降低,缺血侧脑组织LDH活性升高。(2)MCAO模型大鼠缺血侧脑组织SOD、GSH-Px、CAT、tNOS、cNOS活性降低,T-AOCP争低,iNOS活性升高,NO含量降低,MDA含量升高; GSS治疗后,大鼠缺血侧脑组织SOD、GSH-Px、CAT、tNOS、cNOS活性升高,T-AOC升高,iNOS活性降低,NO含量升高,MDA含量降低。(3)MCAO模型大鼠缺血侧脑组织钠钾ATPase及钙镁ATPase活性降低;GSS治疗后,大鼠缺血侧脑组织钠钾ATPase及钙镁ATPase活性升高。(4)谷氨酸损伤组PC12细胞活性降低,GSS处理后,PC12细胞活性升高。(5)正常组原代皮层神经元细胞折光性好,胞体饱满,有较多树突生成;GSS对正常皮层神经元形态及LDH释放率无明显影响;谷氨酸损伤模型组细胞折光性差,胞体饱满性差,树突减少,LDH释放率升高；GSS处理谷氨酸损伤细胞较单纯模型组细胞状态明显好转,表现为细胞折光性较好,胞体较饱满,树突较多,LDH释放率降低。(6)MCAO大鼠海马组织NMDA受体GluN2B蛋白表达升高,GSS治疗后,海马组织GluN2B蛋白表达降低。(7)MCAO模型组大鼠血清MPO、IL-6、TNF-α水平升高,IL-10水平降低,缺血侧脑组织IL-10含量降低,脑组织皮层半损伤区及海马IL-10和IL-10R蛋白(8)MCAO模型组大鼠脑组织皮层半损伤区Bax、Caspase-3及Caspase-.9蛋白表达升高,Bcl-2蛋白表达降低;GSS治疗后,皮层半损伤区Bax Caspase-3及Caspase-9蛋白表达降低,Bcl-2蛋白表达升高。(9)谷氨酸损伤模型组细胞出现较多的凋亡细胞,Bax及Caspase-3表达升高,Bcl-2表达降低；GSS处理后,凋亡细胞减少,BaX及Caspase-3表达降低,Bcl-2表达升高。(10)MCAO模型组大鼠脑组织皮层半损伤区及海马DREAM蛋白表达降低;GSS治疗后,DREAM蛋白表达升高。结论：(1)GSS对脑缺血再灌注损伤具有保护作用；(2)GSS可通过提高机体的抗氧化能力,减轻氧化应激损伤,升高ATP酶的活性,改善脑细胞能量代谢障碍,抑制炎症反应,调控NMDA受体的表达及活化抑制兴奋性毒性损伤,减少细胞凋亡对脑缺血再灌注损伤起保护·作用;(3)DREAM可能在GSS的脑保护效应中起关键作用。更多还原

【Abstract】 Objective:On the basis of traditional chinese medicine, with the modern biotechnology to investigate the protective effect and its mechanisms of genistein sodium sulfonate (GSS), a derivatives of the effective component in pueraria, on brain ischemia/reperfusion injury in rats from different angles of oxidative stress, energy metabolism, excitatory toxic injury, inflammatory response and so on. It can provide the theroretical and practical foundation for expanding pharmacological effects of pueranae.Methods:A rat ischemia/reperfusion injury animal model was establish through embolization of the middle cerebral artery, and a cell injured models were induced by glutamate. Through different research methods like nerve function scores, TTC staining, enzymatic assay and test technology, radioooimmunoassay, enzyme-linked immuno sorbent assay, MTT method, flow cytometry, Western blot and so on, to study the protective effect and mechanisms of GSS on brain ischemia/reperfusion injury in vitro and in vivo.Result:(1) MCAO model rats showed significant injury in neurological function and infarction volume, increases in LDH and CK activities in serum while decreases in brain tissue. After treated with GSS, the neurological score, infarction volume and LDH and CK activities in serum were reducted, and the LDH activitiy in brain tissue were increased.(2) Compared with control rats, the rats suffered ischemia showed decreases in the activities of SOD, GSH-Px, CAT, tNOS and cNOS in the injuried brain tissue and similarly in T-AOC. But increases in activity of iNOS. Moreover, its content of NO is lowered while increased of MDA. After treated with GSS, these changes were transformed.(3) GSS can enhance the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase on the affected brain.(4) GSS treatment significantly increased the viability of PC12cells injuried by glutamate.(5) The control primary culture cortical neurons have good refraction, body full and more dendrite meanwhile. The morphology and ratio of LDH release do not change in neurons by simple treated with GSS. The neurons injuried by glutamate showed worse refraction, body full, less dendrite and more LDH release, compared with the normal neurons. However, the resulted is opposite by GSS treatment. These dataes show that GSS can protect the cortical neurons against glutamate induced neurotoxicity in vitro.(6) GSS can derease the expression of GluN2B, a NMDAR subunit, in MCAO rats’ hippocampus.(7) Treated with GSS, the rats suffered cerebral ischemia/reperfusion injury showed an increase in IL-10content in serum and expression of IL-10and IL-10R in half injury area of cortex and hippocampus, while decreases in IL-6and TNF-α levels, and the activity of MPO in serum.(8) The protein of Bax, Caspase-3and Caspase-9are increased in the ischemic penumbra area of cortex, while Bcl-2is decreased. After the treatment by GSS, all that is reversed.(9) It’s showed that the protein expression of Bax and Caspase-3is increased in the injuried neurons but Bcl-2is down. These changes were transformed through treatment by GSS.(10) The protein expression of downstream regulatory element antagonist modulator(DREAM) is decreased in the ischemic penumbra area of cortex and hippocampus. After treated with GSS, the expression of DREAM in the same tissues was increased.Conclusion:(1) GSS had protective effects on brain ischemia/reperfusion injury in rats.(2) GSS can increase antioxidant capacity and then alleviat oxidative stress injury, improve the activity of ATP and then modulate the disorder of energy metabolism, antagonize the excitatory by regulating the expression and activation of NMDA receptor, inhibit inflammatory response and cell apoptosis to provide protective effects on brain ischemia/reperfusion injury in rats.(3) DREMAM may play an important role in protective effects on brain ischemia/reperfusion injury in rats.更多还原