补阳还五汤对缺血性脑卒中大鼠神经元自噬的保护作用及机制分析

目的探究补阳还五汤中药对大脑中动脉闭塞(MCAO)大鼠再灌注24 h后抗神经元缺血损伤的治疗效果,并分析其改善神经元自噬相关的可能作用机制。方法采用MCAO法对成年雄性SD大鼠进行大脑中动脉闭塞处理2 h,再灌注24 h后成模,分为假手术组、模型组和治疗组。模型组和治疗组大鼠在MCAO造模手术后进行24 h再灌注,治疗组腹腔注射补阳还五汤方剂,模型组和假手术组大鼠均腹腔注射接受等体积的生理盐水,共计14 d。再灌注损伤后1 d、7 d、14 d进行神经系统损伤严重程度(NSS)评分;第14天进行平衡木行走试验;采用苏木精-伊红染色法测定大脑梗死体积;采用免疫组化分析皮质缺血区存活神经元密度;采用免疫印迹法对自噬相关蛋白表达水平(Beclin1,LC3)和线粒体动力学蛋白水平(Parkin,DRP1,OPA1)。结果与假手术组比较,模型组在MACO术后1 d、7 d、14 d NSS评分显著升高;第7天模型组和治疗组间NSS评分无显著差异(P> 0. 05),第14天时治疗组大鼠NSS评分显著低于同时间点的模型组大鼠评分(P <0. 01)。与假手术组相比,模型组大鼠通过平衡木的时间均显著延长(P <0. 01);而与模型组相比,治疗组大鼠通过平衡木的时间显著缩短(P <0. 01)。第14天后,与假手术组相比,模型组大鼠脑梗死面积百分比显著升高,缺血皮质缺血区神经元密度显著下降(P <0. 01);与模型组比较,治疗组大鼠脑梗死面积百分比显著降低,皮质区神经元丢失程度显著缓解(P <0. 01)。与假手术组相比,模型组大鼠皮质区Beclin1、LC3-II、Parkin、DRP1和OPA1水平显著均下降(P <0. 001),而与模型组相比,治疗组皮质区Beclin1、LC3-II、Parkin、DRP1和OPA1蛋白表达下降程度显著缓解(P <0. 01)。结论补阳还五汤通过调节大鼠脑卒中后皮质缺血区域自噬,稳定线粒体功能,发挥神经保护作用。

Protective effect and the mechanism of buyang huanwu decoction on neuronal autophagy in rats with ischemic stroke

Objective To investigate the therapeutic effect of buyang huanwu decoction on neuronal ischemia injury in rats with middle cerebral artery occlusion( MCAO) after 24 hours of reperfusion,and to analyze the possible mechanism of improving neuronal autophagy. Methods adult male SD rats were treated with middle cerebral artery occlusion for 2 hours by MCAO method. After 24 hours of reperfusion,they were divided into sham operation group,model group and treatment group. Rats in the model group and the treatment group were reperfused for 24 hours after MCAO modeling surgery. The rats in the treatment group were injected with Buyang Huanwu decoction. The rats in the model group and the sham operation group received intraperitoneal injection of normal saline for 14 days. Nervous system Severity scores were conducted 1 d,7 d and14 d after reperfusion injury,and balance beam walking test was conducted on the 14 th day. Cerebral infarction volume was determined by hematoxylin-eosin staining. Immunohistochemistry was used to analyze the density of viable neurons in cortical ischemic areas. Immunoblotting techniques were used to evaluate the expression levels of autophagy associated proteins( Beclin1,LC3) and mitochondrial dynamics proteins( Parkin,DRP1,OPA1). Results Compared with the sham operation group,the NSS score of the model group was significantly increased at 1 d,7 d,and14 d after MACO. There was no significant difference in the NSS score between the model group and the treatment group at 7 d( P > 0. 05). The NSS score of the rats in the group was significantly lower than that of the model group at the same time point( P < 0. 01). Compared with the sham operation group,the rats in the model group passed the balance beam significantly longer( P < 0. 01);compared with the model group,the rats in the treatment group passed the balance beam significantly shorter( P < 0. 01). After 14 d,compared with the sham operation group,the percentage of cerebral infarct area in the model group was significantly increased,and the neuron density in the ischemic area of the ischemic cortex was significantly reduced( P < 0. 01). Compared with the model group,the brain in the treatment group was significantly reduced. The percentage of infarct area was significantly reduced,and the degree of neuronal loss in the cortical area was significantly relieved( P < 0. 01). Compared with the sham operation group,the levels of Beclin1,LC3-II,Parkin,DRP1,and OPA1 in the cortical area of the model group were significantly reduced( P < 0. 001). Compared with the model group,Beclin1,LC3-II,Parkin,DRP1 and OPA1 protein expressions in the cortical area of the treatment group were significantly increased( P < 0. 01). Conclusion Buyang huanwu decoction plays a neuroprotective role by regulating regional autophagy of cortical ischemia after stroke in rats.