基于Nix介导的线粒体自噬通路探讨清热化瘀方对脑缺血再灌注损伤大鼠的神经保护机制

Mitochondrial autophagy and apoptosis is the key pathological injury of neuronal death in cerebral ischemia reperfusion.Studies have shown that Nix signaling pathway is one of the most important pathways to regulate both of them, and it was activated in cerebral ischemia reperfusion injury, produces neuroprotective effects, but the mechanism is not clear.Our Previous studies have confirmed that Qingre Huayu decoction has significant effect in treating cerebral infarction. Then suggest a hypothesis that Qingre Huayu decoction can protect the neurons by interfering the Nix signaling pathway, which through the interaction of mitochondrial autophagy and apoptosis. Thus,we will make rat MCAO models,obverse its neurologic impairment by the mNSS and infarction by TTC,then determine neuronal apoptosis with TUNEL, test Nix signaling pathway and its downstream protein Nix, LC3 I, LC3 II and Beclin-1, Bcl-2 through real-time quantitative PCR and westernblot and test the co -localization of Nix/ LC3Ⅱ、Nix /Bcl-2 in laser confocal microscopy.Our purpose is to and make further efforts to provide more evidence for theory of Toxin damaging brain collaterals，through discussing the mechanism of Qingre Huayu decoction on mitochondrial autophagy and apoptosis in rat cerebral ischemia reperfusion injury，clarifing essence of TCM pathogenesis of cerebral infarction.

线粒体自噬和细胞凋亡是脑缺血再灌注损伤中神经元死亡的病理关键，研究表明Nix信号通路是调控两者的最重要一条通路，其在脑缺血再灌注损伤后激活，具有保护作用，但机制尚不清楚。前期研究证实清热化瘀方治疗脑梗死的效果肯定，我们假设清热化瘀方可干预Nix信号通路，通过线粒体自噬与细胞凋亡交互调控，保护神经元。采用Longa’s的线栓法复制大鼠大脑中动脉缺血再灌注模型，观察神经功能缺损，TTC染色法观察梗死面积，TUNEL法观察细胞凋亡，实时荧光定量PCR、及westerblot检测Nix信号通路及下游特异蛋白Nix、LC3Ⅰ、LC3Ⅱ及Beclin-1 、Bcl-2、 Bax基因及蛋白表达，激光共聚焦检测Nix/ LC3Ⅱ、Nix /Bcl-2共定位。探讨脑缺血再灌注损伤中线粒体自噬及凋亡交互调控机制及清热化瘀方干预的效应机制，阐明脑梗死中医病机的本质，为揭示中医“毒损脑络”理论提供科学依据。

粒体自噬和细胞凋亡是脑缺血再灌注损伤中神经元死亡的病理关键，研究表明Nix信号通路是调控两者的最重要一条通路，其在脑缺血再灌注损伤后激活，具有保护作用，但机制尚不清楚。前期研究证实清热化瘀方治疗脑梗死的效果肯定，我们假设清热化瘀方可干预Nix信号通路，通过线粒体自噬与细胞凋亡交互调控，保护神经元。采用Longa’s的线栓法复制大鼠大脑中动脉缺血再灌注模型，观察神经功能缺损，TTC染色法观察梗死面积，TUNEL法观察细胞凋亡，实时荧光定量PCR、及westerblot、免疫组化检测Nix信号通路及下游特异蛋白Nix、LC3Ⅰ、LC3Ⅱ及Beclin-1 、Bcl-2、 Bax基因及蛋白表达，激光共聚焦检测Nix/ LC3Ⅱ、Nix /Bcl-2共定位。结果：①神经功能缺损评分比较： mdivi-1组各时间点神经功能缺损评分较MCAO/R组无明显改善（P>0.05）；QRHYF组3d、7d、14d亚组神经功能缺损评分明显降低（P＜0.01）。②TTC染色提示各时间点QRHYF组较MCAO/R组脑梗死面积显著减少。③各组Nix、LC3 II、Beclin-1、Bcl-2、Bax mRNA及蛋白的表达表达水平比较： 各时间点QRHYF组Nix、LC3 II、Beclin-1、Bcl-2的mRNA及蛋白表达较MCAO/R组明显上调（P＜0.05或P＜0.01），而Bax mRNA表达明显降低（P＜0.01）。④神经元凋亡TUNEL检测显示：各时间点MCAO/R组、mdivi-1组较假手术组细胞凋亡数显著增加，而QRHYF组较MCAO/R组细胞凋亡数显著减少。⑤激光免疫共聚焦提示：Nix/LC3Ⅱ、Nix /Bcl-2 存在同时表达。.结论：清热化瘀方可激活脑缺血再灌注损伤大鼠的Nix表达上调，并调控下游因子，上调LC3、Beclin-1、Bcl-2的表达，下调bax的表达，减轻缺血再灌注损伤，发挥神经保护作用，可能与通过调控Nix介导的线粒体自噬通路发生线粒体自噬以及凋亡相关。