电针通过Yes相关蛋白-视神经萎缩蛋白1信号轴激活线粒体融合功能减轻大鼠脑缺血再灌注损伤的机制研究

Electroacupuncture alleviates neurological function via activating the Yap-OPA1 axis and mitochondrial fusion in rats with cerebral ischemia-reperfusion injury

目的:探讨电针通过Yes相关蛋白(Yap)-视神经萎缩蛋白1(OPA1)信号轴激活线粒体融合功能减轻大鼠脑缺血再灌注损伤(CIRI)的机制。方法:SD大鼠随机分为假手术组、模型组、电针组和电针+Ver组,每组12只。采用大脑中动脉闭塞法制备局灶性CIRI模型。电针组和电针+Ver组电针“百会”“神庭”,每次20 min,1次/d,干预7 d;电针+Ver组造模成功后1 h腹腔注射1次Verteporfin (10 mg/kg)。采用Zea Longa法评估神经功能缺损情况,TTC染色法评估各组大鼠脑梗死体积百分比;Western blot法及荧光定量PCR法检测梗死侧大脑皮层Yap、OPA1、线粒体融合素(Mfn)1、Mfn2蛋白和mRNA表达水平;Western blot法检测梗死侧大脑皮层凋亡蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)蛋白表达水平;免疫荧光染色检测梗死侧大脑皮层Yap、OPA1蛋白荧光表达水平。结果:与假手术组比较,模型组大鼠脑梗死体积百分比及神经功能评分升高(P<0.01),梗死侧大脑皮层Yap、OPA1、Mfn2、Mfn1 mRNA及蛋白表达显著降低(P<0.01,P<0.05);Bax蛋白表达升高(P<0.01),Bcl-2蛋白表达降低(P<0.01),Yap、OPA1荧光表达水平下调(P<0.05)。与模型组和电针+Ver组比较,电针组脑梗死体积百分比及神经功能评分降低(P<0.01,P<0.05),梗死侧大脑皮层Yap、OPA1、Mfn2、Mfn1 mRNA及蛋白表达升高(P<0.01,P<0.05),Bax表达降低(P<0.01),Bcl-2蛋白表达升高(P<0.01),Yap及OPA1荧光表达水平上调(P<0.05)。结论:电针“百会”“神庭”可能通过Yap-OPA1信号轴激活线粒体融合功能并减轻CIRI大鼠神经功能损伤。

Objective To investigate the mechanism of electroacupuncture(EA)in alleviating cerebral ischemia injury by activating the Yap-OPA1 signaling axis.Methods A total of 48 male SD rats were used in the present study.The focal CIRI model was established by occlusion of the middle cerebral artery and reperfusion(MCAO/R),fol⁃lowed by dividing the CIRI rats into model group,EA group and EA+Ver(Verteporfin,Yap antagonist)group(n=12 in each group).And another 12 normal rats were used as the sham operation group.For rats of the EA group,EA(4 Hz/20 Hz,0.5 mA)was applied to“Baihui”(GV20)and“Shenting”(GV24)for 20 min,once daily for 7 days.The neuro⁃logical deficit score(0 to 4 points)was given according to Longa's method.The infarct volume of rats in each group was assessed by TTC method,and the expression levels of Yes associated protein(Yap),Optic atrophy protein 1(OPA1),mitofusin 1(Mfn1),mitofusin 2(Mfn2)proteins and mRNAs in cerebral cortex of infarcted side,as well as Bax(proapoptotic factor)and Bcl-1(anti-apoptotic protein)proteins were detected by Westernblot,and real-time PCR,and the immunoactivity of Yap and OPA1 was detected by immunofluorescent staining.Results After modeling,the infarct volume,neurological deficit score and the expression of Bax were significantly increased(P<0.01),while the mRNA and protein expressions of Yap,OPA1,Mfn2,Mfn1,and Bcl-2 were significantly down-regulated in the model group relevant to the sham operation group(P<0.01,P<0.05).Compared with the model group,the neurological deficit score,infarct volume and the expression of Bax were significantly decreased(P<0.01),while the expression levels of Yap,OPA1,Mfn2,Mfn1 proteins and mRNAs and Bcl-2 protein,Yap and OPA1 immunofluorescence intensify were considerably up-regulated in the EA group(P<0.01,P<0.05).Following administration of Ver,the effects of EA in downregulating the neurological score,infarct volume,and Bax expression and up-regulating the expressions of Yap,OPA1,Mfn1,Mfn2 proteins and mRNAs and Yap and OPA1 immunofluorescence intensify were eliminated.Conclu⁃sion EA of GV20 and GV24 can improve the neurological function in rats with CIRI,which may be associated with its functions in activating mitochondrial fusion function and up-regulating Yap-OPA1 signaling axis.