【作者】 薛晶；

【导师】 冯加纯；

【作者基本信息】 吉林大学 ， 神经病学， 2009， 博士

【摘要】 本研究旨在动态观察反映脑缺血再灌注损伤的各项指标,寻找其高峰时间点,并在此时单独及联合应用依达拉奉和万珂,通过HE、TTC染色;化学比色法、免疫组化法、Western-blot法和TUNEL法测MDA、SOD、Cyt C、Caspase-3、NF-κBp65、IL-1β和细胞凋亡,观察二者对脑缺血再灌注损伤的作用,探讨其脑保护机制。结果发现:1.脑缺血再灌注后炎症反应、自由基增加、线粒体损伤和细胞凋亡等呈动态改变;2.依达拉奉和万珂均可减轻线粒体损伤,减少细胞凋亡,缩小脑梗死体积,有脑保护作用;3.两药联合应用疗效优于单独用药。本实验创新点:1.国内首次将万珂用于治疗脑缺血再灌注损伤,国内外首次证实蛋白酶体抑制剂的脑保护作用机制可能主要通过抑制线粒体凋亡途径而减轻脑损伤;2.依达拉奉除抗脂质过氧化外还有抑制后续炎症反应的作用,可能主要通过抑制线粒体凋亡途径发挥脑保护作用,尚未有学者提出此观点;3.国内外首次证实早期联合应用蛋白酶体抑制剂和自由基清除剂的“鸡尾酒疗法”治疗脑缺血再灌注损伤效果明显,二者可能共同抑制线粒体凋亡途径实现脑保护作用。更多还原

【Abstract】 Background and purpose Many factors are involved in cerebral ischemia-reperfusion injury, among them inflammatory reaction and free radicals play an important role in early stage. Edaravone, a kind of free radical scavenger, has been used to treat cerebral infarction and verified effective, while the precise mechanisms are unclear. At present, there is no an effective medicine on restrain inflammatory reaction. The proteasome inhibitors can induce apoptosis on proliferating tumor cells and are permitted to use for the multiple myeloma. Recent research shows that the proteasome inhibitors can control inflammatory reaction on transcriptional level and are used in treating cerebral infarction. Now the mechanism is unclear.The study plans to apply the animal model of cerebral ischemia-reperfusion to observe the changes of inflammatory reaction, free radicals, mitochondria and cell apoptosis, and to treat with Edaravone and Velcade on the proper time. Comparing the changes pre- and post–treatment and observing the effects on the cerebral ischemia-reperfusion injury, the aims is to explore the protective mechanisms of Edaravone and Velcade on cerebral reperfusion inury.Methods1. Experimental animal and group 175 Wistar rats were randomly divided into the normal group(5), the sham operation group and cerebral ischemical reperfusion group (55 in each group), the physiological saline control group, Edaravone treatment group, Velcade treatment group and combined treatment group (15 in each group). The transient middle cerebral artery occlusion models were applied and reperfused after 2 hours. All the groups were divided into 5 subgroups according to different reperfusion time points, 0h, 6h, 12h, 24h, 48h (10 in each group). The others were used to measure infarct volume with TTC stain.2. Decapitation and detection methods Edaravone was given instantly and on 12h (3mg/kg), and Velcade was given instantly too (0.2mg/kg) after reperfusion via vena caudalis injection. Combined treatment method was given according to the above all. The physiological saline was used in control group. After 24h 5 rats were infused , decapitated and fixed with 4% paraformaldehyde after reperfusion 24h, alcoholic dehydration, dimethyl benzene transparence, paraffin imbedding and sect serial sections. Immunohistochemistry was used to detect the expression of Cyt C, Caspase-3, NF-κBp65 and IL-1βprotein, while cell apoptosis was detected by TUNEL. At the same time, the expression of Cyt C and Caspase-3 protein were detected by Western-blot. Another 5 rats were decapitated on ice plate immediately. The 2/3 brain tissue was used to make brain homogenate for detecting MDA and SOD, the others were preserved in -70℃refrigerator for Western-blot. After TTC stain, the images were measured and the cerebral infarct volume were calculated with Motic Images Advanced 3.2 image analysis system.Results1. Histological changes: The chromatospherites was clear, endochylema was profuse in normal group and sham operation group, while in reperfusion group karyopyknosis,工团chromoplasm condense. The interspace between cells became wide. brain tissue became loose. These changes were gradually obvious with time .2. There were seldom immune reaction positive cells and apoptosis cells in normal group and sham operation group. The difference between MDA and SOD was not significant (p>0.05) in them. MDA increased gradually after reperfusion and peaked at 12h, while SOD decreased obviously at that time. Compared with the physiological saline control group , the combined treatment group and single treatment groups MDA decreased and SOD activity restored, the differences were significant (p<0.05). There were lots of NF-κBp65, IL-1β, CytC and Caspase-3 positive cells in all the intervention groups. All kinds of positive cells increased gradually until 24h, compared with all the subgroups of the sham operation group, the differences were significant (p<0.05). Compared the single treatment group with the physiological saline control group and combined treatment group with all the other groups, NF-κBp65, IL-1β, CytC and Caspase-3 positive cells and apoptosis cells decreased obviously, the differences were significant (p<0.05). The expression of Cyt C, Caspase-3 by Western-blot in different time points and pre-and post-treatment were consistent with the results of immunohistochemistry methods.3. Cerebral infarct was found in all the operation groups except the sham operated group, and the infarct volumes with TTC stain in treatment group became smaller than the physiological saline group (p<0.05).Conclusion1. The change of inflammatory reaction, free radicals and mitochondria damage involved in cerebral ischemical reperfusion injury were dynamic . Cell apoptosis was coincident to the above changes.2. Edaravone can inhibit lipid peroxidation, scavenge free radicals, restrain inflammatory reaction, protect mitochondria and reduce cells apoptosis. So Edaravone maybe mitigate cerebral ischemical reperfusion injury through blocking mitochondria apoptosis pathway. On the other hand, Velcade can restrain inflammatory reaction and free radicals following that which can protect mitochondria and reduce cells apoptosis. So we assumed that Velcade mitigate cerebral ischemical reperfusion injury by blocking mitochondria apoptosis pathway too.3. Edaravone can scavenge free radicals and Velcade can inhibit inflammatory reaction. Combined treatment of them may deduce synergistic inhibition to mitochondria apoptosis pathway. The protection will be more effectively than single treatment.更多还原