化浊解毒活血通络方对脑缺血再灌注损伤大鼠线粒体自噬相关蛋白PINK1和Parkin的影响

Effects of Huazhuo Jiedu Huoxue Tongluo Formula on mitochondrial autophagy related protein PINK1 and Parkin in rats with cerebral ischemia-reperfusion injury

目的:研究化浊解毒活血通络方对脑缺血再灌注损伤大鼠的脑保护作用及对线粒体自噬相关蛋白PTEN诱导激酶1(PINK1)、Parkin的影响。方法:将48只雄性SPF级SD大鼠随机分为假手术组、模型组、中药组(化浊解毒活血通络方)、抑制剂组[3-甲基腺嘌呤(3-MA)抑制剂],每组12只。按照Longa法制备大脑中动脉栓塞(MCAO)大鼠模型,造模成功给药3 d后,行大鼠神经功能缺损评分,HE染色观察脑组织病理改变,TTC染色观察脑梗死体积,Western Blot检测脑组织PINK1、Parkin蛋白的表达,实时荧光定量PCR检测脑组织中PINK1、Parkin mRNA的表达。结果:与假手术组比较,模型组神经功能缺损评分和脑梗死体积显著增加(P<0.05),病理损伤加重,PINK1、Parkin蛋白及mRNA表达水平显著升高(P<0.05);与模型组比较,中药组大鼠神经功能缺损评分和脑梗死体积显著减少(P<0.05),病理损伤减轻,PINK1、Parkin蛋白及mRNA表达水平显著升高(P<0.05);与模型组及中药组比较,抑制剂组大鼠神经功能缺损评分和脑梗死体积显著增加(P<0.05),病理损伤加重,PINK1、Parkin蛋白及mRNA表达水平显著降低(P<0.05)。结论:化浊解毒活血通络方对脑缺血再灌注损伤大鼠具有脑保护作用,其机制可能与上调线粒体自噬相关蛋白PINK1、Parkin的表达相关。

Objective:To study the protective effect of Huazhuo Jiedu Huoxue Tongluo Formula on the brain of rats with cerebral ischemia-reperfusion injury and its influence on mitochondrial autophagy related proteins PINK1 and Parkin.Methods:Forty-eight male SPF SD rats were randomly divided into sham operation group,model group,traditional Chinese medicine group(Huazhuo Jiedu Huoxue Tongluo Formula)and inhibitor group[3-methyl adenine(3-MA)inhibitor],with 12 rats in each group.MCAO rat model was established according to Longa method.After 3 days of successful administration,the neurological deficit of rats was scored,the pathological changes of brain tissue were observed by HE staining,the volume of cerebral infarction was observed by TTC staining,the protein expressions of PINK1 and Parkin in brain tissue were detected by Western Blot,and the mRNA expressions of PINK1 and Parkin in brain tissue were detected by real-time fluorescence quantitative PCR.Results:Compared with the sham operation group,the neurological deficit score and cerebral infarction volume in the model group were significantly increased(P<0.05),the pathological injury was aggravated,and the expression levels of PINK1,Parkin protein and mRNA were significantly increased(P<0.05).Compared with the model group,the neurological deficit score and cerebral infarction volume of rats in the traditional Chinese medicine group were significantly reduced(P<0.05),the pathological damage was alleviated,and the expression levels of PINK1,Parkin protein and mRNA were significantly increased(P<0.05).Compared with the model group and the traditional Chinese medicine group,the neurological deficit score and cerebral infarction volume in the inhibitor group were significantly increased(P<0.05),the pathological damage was aggravated,and the expression levels of PINK1,Parkin protein and mRNA were significantly decreased(P<0.05).Conclusion:Huazhuo Jiedu Huoxue Tongluo Formula has brain protection effect on rats with cerebral ischemia-reperfusion injury,and its mechanism may be related to upregulating the expression of mitochondrial autophagy-related proteins PINK1 and Parkin.