【作者】 王桂斌；

【导师】 刘运海； 张宁；

【作者基本信息】 中南大学 ， 临床医学， 2014， 博士

【摘要】 第一章依达拉奉减轻缺氧复氧酸中毒诱导的神经元毒性损伤的作用及分子机制研究摘要目的：1研究依达拉奉对缺氧复氧酸中毒诱导的神经元毒性损伤的作用；2探讨依达拉奉对缺氧复酸中毒损伤的神经元保护的分子作用机制。方法：1建立缺氧缺糖复氧酸中毒(OGD-A/R)神经元损伤模型,为消除谷氨酸盐活性及电压依赖的Ca2+通道作用,所有操作中均加入了NMDA和AMPA受体阻滞剂和电压门控Ca2+通道拮抗剂；2分空白对照组、OGD-A/R组、OGD-A/R加不同浓度依达拉奉组、OGD-A/R加ASICs阻断剂组,检测不同时段LDH释放量、Caspase-3活性、Hoechst33258染色,并进行比较来评价依达拉奉对OGD-A/R损伤神经元的保护作用；3选择适当浓度的依达拉奉、ASIC1a阻滞剂及MEK/ERK阻滞剂分别对不同组别OGD-A/R模型培养中的细胞进行预处理,用Western-blot检测ERK、AKT及Bcl-2蛋白表达,RT-PCR检测BDNF、 Bcl-2mRNA表达,ELISIA检测BDNF蛋白表达,相应试剂盒或试剂检测LDH释放量(检测细胞损伤)及]Hoechst33258染色(观察细胞凋亡)。分析比较相应数据探讨依达拉奉在OGD-A/R模型中可能的保护机制。结果：1成功建立了OGD-A/R模型,并发现4h和8h即可出现LDH释放量爆发性增加。2ASICs阻滞剂PcTX和amiloride预处理后予OGD-A/R操作4h和8h LDH释放量显著减少。与ASICs阻滞剂相对应,依达拉奉预处理继以OGD-A/R操作可剂量依赖地减少损伤神经元LDH释放而产生与前者类似但更显著的保护作用。3与OGD-A/R组相比,依达拉奉预处理在各时点提高了BDNF和Bcl-2mRNA表达,且在OGD-A/R后8h作用最强；ELISIA检测提示依达拉奉在同一时间点使BDNF蛋白表达增多；Western-blot则显示在同一时间点依达拉奉在OGD-A/R后8h上调了Bcl-2蛋白表达；Caspase-3活性检测结果显示OGD-A/R极大地提高了Caspase-3活性,依达拉奉预处理可明显降低这种Caspase-3活性的升高,且具有剂量依赖性。这种保护作用与ASICs阻断剂使用后相当。4与正常对照组比较,OGD-A/R神经元p-AKT水平显著提高,而依达拉奉或PcTX预处理后OGD-A/R则显著抑制了AKT磷酸化。5与正常对照组相比,OGD-A/R组或OGD-A/R并PcTX预处理组可引起p-ERK1/2活性轻度升高,而依达拉奉干预OGD-A/R后则可引起p-ERK1/2活性明显升高。这种作用可被MEK/ERK阻断剂PD98059(10μM) and U0126(5μM)阻断；6OGD-A/R诱导细胞损伤引起核染色质断裂和LDH释放增多。而依达拉奉预处理可在很大程度上防止细胞核形态变化和减少LDH释放。然而MEK/ERK阻滞剂PD98059和U0126预处理几乎完全抵消了依达拉奉对OGD-A/R损伤神经元抗凋亡能力。结论：1本研究首次发现依达拉奉能减轻缺氧复氧酸中毒诱导的神经毒性损伤,且这一作用至少部分地依赖于ERK1/2的活性增强。2缺氧复氧酸中毒条件下,依达拉奉可通过上调BDNF及Bcl-2的表达、抑制caspase-3活性达到神经保护作用。3ASICs通道激活是缺氧复氧酸中毒诱导的细胞毒性损伤重要机制之一,依达拉奉对缺氧复氧酸中毒损伤神经元的保护作用可能与阻断ASICs有关。第二章依达拉奉减轻缺氧复氧诱导的神经元毒性损伤的分子机制研究摘要目的：1研究依达拉奉对缺氧复氧损伤神经元的保护作用；2探讨依达拉奉减轻缺氧复氧引起的细胞毒性损伤、保护神经元细胞的分子作用机制。方法：1建立缺氧缺糖复氧(OGD-R)神经元损伤模型；2分空白对照组、OGD-R组、OGD-R加不同浓度依达拉奉组,检测不同时段LDH释放量、Caspase-3活性、Hoechst33258染色(计算核固缩比例),并进行比较来评估依达拉奉对缺氧复氧损伤神经元的保护作用；3选择适当浓度的依达拉奉及依达拉奉并特异性AKT阻断剂LY294002和MK2206对细胞分别进行预处理后再行OGD-R处理,用Western-blot检测ERK、AKT及Bcl-2蛋白表达,RT-PCR检测BDNF、Bcl-2mRNA表达,ELISIA检测BDNF蛋白表达,相应试剂盒或试剂检测LDH释放量及Hoechst33258染色。分析比较相应数据探讨依达拉奉在OGD-R模型中可能的保护机制。结果：1成功建立了OGD-R模型,并发现在复氧后4h和8h即可出现明显的病理学改变。2依达拉奉预处理培养细胞减轻了缺氧复氧诱导的神经元损伤程度(LDH释放减少、Caspase-3活性和核固缩比例下降),且具有剂量依赖性。3确定LDH释放量检测可以作为一个简易可靠的衡量神经元损伤的定量指标,该结论经Caspase-3活性检测和Hoechst33258染色检测验证。4依达拉奉预处理后OGD-R, p-ERK的表达量没有明显变化,而p-AKT的表达量明显增加,差异有统计学意义,p<0.01。5依达拉奉预处理可明显降低OGD-R处理细胞中LDH释放和核固缩比例,p<0.01,但该过程可被MK2206抑制。6与OGD-R组比较,依达拉奉预处理显著上调了BDNF和Bcl-2mRNA的表达,且在OGD-R8h时尤其明显；ELISIA检测提示依达拉奉在同一时间点使BDNF蛋白表达增多；Western-blot则显示依达拉奉上调了Bcl-2蛋白表达,这种作用可被MK2206抑制。结论：1依达拉奉通过上调BDNF和Bcl-2的表达、抑制Caspase-3活性减轻缺氧复氧引起的神经元毒性损伤,即在发生缺氧复氧不合并酸中毒损伤条件下,依达拉奉已经具有神经保护作用。2PI3K/KT通路的激活可能是上述作用实现的重要途径之一。更多还原

【Abstract】 Part Ⅰ To investigate the molecular mechanism of edaravone of mitigating the neurotoxicity induced by hypoxia-acidosis/reoxygenationAbstractObjective:1To evaluate the effects of edaravone on cytotoxicity induced by hypoxia/reoxygenation combined with acidosis injury;2To investigate the molecular mechanism how edaravone reduces cytotoxicity caused by hypoxia with acidosis and protects the neurons.Methods:1Establish ischemia model--oxygen and glucose deprivation combined with acidosis/reoxygenation model(OGD-A/R).To eliminate the activation of glutamate and voltage-dependent Ca2+channels,all experiments were conducted in the presence of blockers for NMDA and AMPA receptors and voltage-gated Ca2+channels.2Cells were divided into several groups:control group,OGD-A/R group,OGD-A/R and edaravone of diferent concentration groups, OGD-A/R and blocker of ASICs channels groups. The levels of LDH releasing、Caspase-3activity and Hoechst33258staining are detected at different time point of reoxygenation after OGD-A/R to evaluate the neuroprotective effect of edaravone against hypoxia/reoxygenation combined with acidosis induced injury; 3The appropriate concentration of edaravone and ASIC1a blockers and MEK/ERK antagonists were used on cells of OGD-A/R model in different groups individully; The expression levels of ERK、 AKT and Bcl-2were detected with Western-blot, while the expression levels of BDNF and Bcl-2mRNA detected with RT-PCR, the expression levels of BDNF protein detected with ELISIA,the levels of LDH releasing and Hoechst33258staining detected with homologue assay kit or regents. The data above were analyzing to probe the possible protective mechanism of edaravone in OGD-A/R model.Results:1OGD-A/R model is established successfully, and OGD-A/R for4h and8h induced a robust increase in LDH release;2Pretreatment with PcTX or amiloride remarkably reduced the LDH release after4h and8h of OGD-A/R.Relative to ASICs blockade, the pretreatment of edaravone followed by OGD-A/R elicited a similar but greater protection by a dose-dependent suppression of LDH release after reperfusion;3Compared to OGD-A/R,edaravone pretreatment markedly promoted BDNF and Bcl-2mRNA expression at each time point,and the most pronounced effects occured at8h after OGD-A/R.ELISIA analysis showed that BDNF protein levels were significantly elevated after edaravone pretreatment at the same point. Western-blot analysis indicted that edaravone pretreatment significantly enhanced Bcl-2expression at8h after OGD-R. Results from caspase-3activity assay indicated that OGD-A/R insults greatly elevated caspase-3activity which could be relieved by edaravone in a dose-dependent manner. This protective effects were comparable to those from blockade of ASICs similarly.4Neurons exposed to OGD-A/R had a marked increase in the p-AKT level compared to the normal control,p<0.01. However, the high activity of p-AKT was significantly inhibited by edaravone or PcTX pretreatment during OGD-A/R,p<0.01;5Compared to the normal control, OGD-A/R or OGD-A/R combined with PcTX treatment induced a mild increase in p-ERK1/2, whereas edaravone administration resulted in a dramatic increase in p-ERK1/2during OGD-A/R.The effect was blocked by the MEK (MAPK/ERK kinase) antagonists PD98059(10μM) and U0126(5μM).6The data from Hoechst33258staining and LDH release assay showed that OGD-A/R-induced cell death triggered fragmentation of nuclear chromatin and augmentation of LDH release. Edaravone pretreatment largely prevented nuclear morphological changes and LDH release in injured cells. However, the MEK inhibitors PD98059and U0126administration30min prior to OGD-A/R, almost completely abrogated the antiapoptotic ability of edaravone on neurons exposed to OGD-A/R.Conclusions: 1Edaravone is capable of attenuating hypoxia-acidosis/reoxygenation-mediated neurotoxicity and the effect,at least partially,depends on the enhanced ERK1/2activation.2Edaravone confers neuroprotective effects through enhancing BDNF and Bcl-2expression and supressing caspase-3activity in the presence of hypoxia-acidosis/reoxygenation.3Acidosis and ASICs play a critical role in the process of acido-toxicity induced by hypoxia-acidosis/reoxygenation.The neuroprotective effects of Edaravone may be related to ASICs blockade. Part Ⅱ To investigate the molecular mechanism of edaravone of alleviating the neurotoxicity induced by hypoxia/reoxygenationAbstractObjective1To evaluate the neuroprotective effect of edaravone on neurons damaged by hypoxia/reoxygenation-induced injury;2To investigate the molecular mechanism how edaravone reduces cytotoxicity caused by hypoxia/reoxygenation-induced injury and protect the neurons. Methods1Establish ischemia model--hypoxia/reoxygenation model which is also called oxygen and glucose deprivation/reoxygenation model (OGD-R);2Cells were divided into several groups:control group,OGD-R group,OGD-R with edaravone of diferent concentration groups.The levels of LDH releasing、Caspase-3activity and Hoechst33258staining were detected at different time point of reoxygenation after OGD to evaluate the neuroprotective effect of edaravone against hypoxia/reoxygenation induced injury;3Cells of OGD-R model were pretreated with the appropriate concentration of edaravone and edaravone combine with the specific channel blockers LY294002and MK2206individually, the expression levels of ERK NAKT and Bcl-2were detected with Western-blot, while the expression levels of BDNF and Bcl-2mRNA detected with RT-PCR, the expression levels of BDNF protein detected with ELISIA,the levels of LDH releasing and Hoechst33258staining detected with homologue assay kit or regents. The data above were analyzing to probe the possible protective mechanism of edaravone in OGD-R model.Results1OGD-R model is established successfully, significant pathological changes can form in four hours and eight hours;2The pretreatment of cultured neurons with edaravone alleviates hypoxia/reoxygenation induced neuronal injury in a dose-dependent manner(LDH releasing reduced,Caspase-3activity and ratio of nuclear pyknosis declined).3The levels of LDH releasing detection is determined as a simple and reliable quantitative measure for neuronal cell injury, and this conclusion is verified by detecting Caspase-3activity and Hoechst33258staining.4The edaravone pretreatment did not increase the p-ERK expression levels significantly, but the amount of p-AKT expression was significantly increased, p<0.01.5Pretreatment of edaravone(10μM) remarkably reduced the LDH release and declined the ratio of nuclear pyknosis after reoxygenation (p<0.01),which was inhibited by MK2206.6Compared to OGD-R,edaravone pretreatment markedly promoted BDNF and Bcl-2mRNA expression at each time point,and the most pronounced effects occured at8h after OGD-R.ELISIA analysis showed that BDNF protein levels were significantly elevated after edaravone pretreatment at the same point. Western-blot analysis indicted that edaravone pretreatment significantly enhanced Bcl-2protein expression at8h after OGD-R,which was blocked by MK2206.Conclusions 1Edaravone alleviates hypoxia/reoxygenation induced neuronal injury by enhancing BDNF and Bcl-2expression and inhibiting Caspase-3activity,that is to say,Edaravone has already the neuroprotective effects during the period of hypoxia with no acidosis;2Edaravone protects neurons against hypoxia/reoxygenation induced cytotoxicity maybe through activation of the PI3K/Akt pathway.更多还原