从调控内质网稳态探讨化痰通络法对急性脑梗死溶栓后神经细胞适应性保护效应机制

Thrombolysis therapy is one of the most direct and effective methods for the treatment of acute cerebral infarction, however, the ischemia reperfusion injury after thrombolysis would tend to increase the brain damage, the mechanism is closely related to the destruction of endoplasmic reticulum steady-state inducing the endoplasmic reticulum stress, and then activate the nerve cell apoptosis way and autophagy reactions. This topic found that phlegm-resolving and collateral-unblocking method has the trend to relieve the ischemia reperfusion injury after thrombolysis in our earlier clinical and animal experiments studies.Based on this, we are ready to use MCAO rats to be the observation object, using eliminating phlegm traditional Chinese medicine for intervention validates method, through the animal behavior, microcosmic morphology and molecular biological detection method to observe the neural function defect recovery degree change of model rats; observe the change of half dark area with nerve cells microcosmic morphology; observe the ischemia half dark area with brain nerve cells endoplasmic reticulum stress response after the different development phase (protection stage and apoptosis stage) signal transduction pathways in the key target protein and gene expression changes,and combined with observing the neural cell apoptosis and pathological autophagy reaction conditions,in order to clarify part of the mechanism of phlegm-resolving and collateral-unblocking method regulating the endoplasmic reticulum steady-state after thrombolysis of acute cerebral infarction, to explore the mechanism of adaptive cytoprotection to nerve cells in the area of ischemic half dark with the traditional Chinese medicine, so as to provide preliminary experimental basis of the combined application of traditional Chinese medicine and thrombolytics in the treatment of acute cerebral infarction.

溶栓疗法虽为治疗急性脑梗死最直接、最有效方法之一，但溶栓后缺血再灌注损伤则易加重脑损害，其机制与内质网稳态破坏，诱发内质网应激，进而激活神经细胞凋亡途径及自噬反应密切相关。本课题在前期临床与动物实验研究中发现，化痰通络法具有减轻溶栓后缺血再灌注损伤的趋势。基于此，拟采用MCAO大鼠为观察对象，以化痰通络法中药为干预手段，通过动物行为学、微观形态学与分子生物学检测手段，观察模型大鼠神经功能缺损恢复情况；观察半暗带区神经细胞微观形态结构变化；观察缺血半暗带区脑组织神经细胞内质网应激反应后不同发展阶段（保护阶段与凋亡阶段）信号转导途径中关键靶点的蛋白与基因表达变化；结合观察神经细胞病理性凋亡及自噬反应情况，以期阐明化痰通络法调控急性脑梗死溶栓后内质网稳态的部分作用机制，探索中医药对缺血半暗带区神经细胞适应性保护效应机制，为中医药在急性脑梗死治疗中参与溶栓剂联合应用提供初步实验依据。

急性脑梗死是以致死、致残率高为特点的疾病。其起病迅速，早期确诊及治疗对患者预后影响较大。美国卒中协会2014指南推荐，脑梗死急性期（3h内）静脉rt-pa溶栓治疗可以改善脑梗死患者的预后，研究证实rt-pa溶栓是唯一推荐的急性脑梗死治疗方法。但临床运用rt-pa治疗的比率较低，溶栓治疗所带来的并发症是限制其临床运用的主要原因，其中溶栓后的缺血再灌注损伤受到最广泛的关注。脑梗死缺血再灌注后，氧自由基蓄积、钙离子超载等一系列反应，导致了神经元的死亡。.本研究以SD大鼠为实验对象，建立大鼠自体血栓经大脑颈内动脉注射的大脑中动脉栓塞的动物模型，以5.67mg/Kg的rt-PA静脉溶栓与7.2g/Kg化痰通络法中药灌胃，分为假手术组、模型组、rt-PA组、化痰通络法联合rt-PA组，分别采用Western Blot与RT-PCR法从分子生物学水平上观察化痰通络法中药对急性脑梗死大鼠溶栓再灌注损伤后内质网应激途径中的保护性途径与凋亡性途径中关键靶点蛋白与基因的表达。相关基因表达检测结果显示，与rt-PA相比，化痰通络法联合rt-PA组IRE-1、ATF6基因表达显著增加（P＜0.05），Caspase-12基因表达显著下降（P＜0.05），其余基因表达下降不明显（P＞0.05）。相关蛋白检测结果显示，与rt-PA组比较，化痰通络法联合rt-PA组IRE-1、Ask1蛋白表达显著增加（P＜0.05），TRB3、Caspase-9、Caspase-3蛋白表达量下降明显（P＜0.05）。.根据实验结果，发现化痰通络法具有改善急性脑梗死大鼠溶栓后神经功能缺损及神经元形态学的作用，同时可以减少神经元的凋亡；化痰通络法抑制急性脑梗死大鼠溶栓后神经元损伤的作用，其机制可能与调控内质网稳态中保护性通路及凋亡性途径相关。研究结果已发表期刊论文6篇，培养硕士研究生2名（已毕业）。.本实验为化痰通络法防治急性脑梗死大鼠溶栓后缺血再灌注损伤的提供实验依据，促进临床上中医药联合溶栓剂对治疗急性脑梗死等危重疾病的应用和推广，进一步推动中医药对脑神经细胞保护作用的研究。