雷帕霉素对大鼠脑缺血再灌注损伤的保护效应及其机制研究

Protective Effect and Mechanism of Rapamycin on Cerebral Ischemia-reperfusion Injury in Rats

目的探讨雷帕霉素对大鼠脑缺血再灌注损伤模型的保护效应及其机制。方法将SD大鼠双侧颈动脉结扎10 min后再通,造成大鼠前脑缺血再灌注损伤模型,分别于术前3 d和术后24 h给予雷帕霉素干预。32只大鼠分为假手术组(NC)、模型组(N7d)、雷帕霉素预给药组(Pre-NR7d)和雷帕霉素治疗组(Post-NR7d),术后7 d取大鼠海马组织进行常规病理和Tunel染色观察缺血再灌注后海马神经元坏死和凋亡情况;蛋白印记检测凋亡启动因子细胞色素酶C(cytochrome c,Cyt c)和自噬相关蛋白LC3-Ⅱ和Beclin-1,观察凋亡和自噬情况。结果病理结果显示N7d组大鼠海马CA1区神经元出现坏死性改变,坏死神经元细胞百分比为(89.83±2.91)%,而Pre-NR7d组[(19.48±2.68)%]和Post-NR7d组[(67.80±5.55)%]均降低了大鼠海马坏死神经元细胞数量(P均<0.01),Pre-NR7d组效果优于Post-NR7d组(P<0.01)。Tunel染色结果显示,N7d组大鼠海马CA1区阳性细胞占(54.35±6.18)%,较NC组[(4.62±0.98)%]升高(P<0.01),而Pre-NR7d组[(4.22±0.61)%]和Post-NR7d组[(47.72±6.08)%]均降低了大鼠海马CA1区Tunel阳性细胞数(P均<0.01),Pre-NR7d组较Post-NR7d组凋亡细胞减少(P<0.01)。缺血再灌注引起大脑海马Beclin-1和LC3-Ⅱ的升高及Cyt c由线粒体释放入胞浆,而雷帕霉素抑制Beclin-1和LC3-Ⅱ的表达并减少Cyt c在胞浆的释放(P均<0.01),Pre-NR7d组较Post-NR7d组抑制效果更为显著(P<0.05或<0.01)。结论大鼠脑缺血再灌注后大脑海马组织坏死、凋亡、自噬均存在,雷帕霉素可通过降低自噬和凋亡,减轻脑缺血再灌注损伤,预防性给药效果优于术后24 h给药。

Objective The aim of this study was to clarify the protective effect and mechanism of rapamycin on cerebral ischemia-reperfusion injury in rats.Methods Ten minutes of forebrain ischemia was induced in rats,intraperitoneal injection of rapamycin was carried respectively 3 days before surgery or 24 hours after surgery.Totally 32 rats were divided into 4 groups:sham operation group(NC),cerebral ischemia and reperfution 7 days group(N7d),rapamycin pre-administration before cerebral ischemia and reperfution 3 days group(Pre-NR7d)and rapamycin treated after reperfusion 24 hours group(Post-NR7d),and the brains hippocampal tissue were sampled after 7 days of reperfusion for pathology and Tunel staining to observe the necrosis and apoptosis.Western blot was used to detect cytochrome c(Cyt c),LC3-Ⅱand Beclin-1 to observe apoptosis and autophagy.Results The pathological results showed that neurons in CA1 area of hippocampus in N7d group showed necrotic changes,the percentage of necrotic neurons was(89.83±2.91)%,while that in Pre-NR7d group[(19.48±2.68)%]and Post-NR7d group[(67.80±5.55)%]decreased the number of necrotic neurons in hippocampus(P<0.01),and the effect of Pre-NR7d group was better than that in Post-NR7d group(P<0.01).Tunel staining showed that the number of Tunel positive cells in CA1 area of hippocampus in N7d group was(54.35±6.18)%,which was higher than NC group[(4.62±0.98)%](P<0.01),while the Pre-NR7d group[(4.22±0.61)%]and Post-NR7d group[(47.72±6.08)%]both reduced the number of Tunel-positive cells(both P<0.01).Ischemia-reperfusion induced the increase of Beclin-1 and LC3-Ⅱin hippocampus and the release of Cyt c from mitochondria into the cytoplasm,while rapamycin inhibited the expression of Beclin-1 and LC3-Ⅱand the release of Cyt c in the cytoplasm(P<0.01).The inhibition effect of Pre-NR7d group was more significant than that of Post-NR7d group(P<0.05 or<0.01).Conclusion All together,these data provide evidence that necrosis,apoptosis and autophagy all exist in the hippocampus after cerebral ischemia-reperfusion.Rapamycin can reduce the damage of cerebral ischemia-reperfusion by reducing autophagy and apoptosis.The pre-administration group is better than post-administration group.