加味五虎追风散灌胃对帕金森病大鼠脑组织多巴胺能神经元自噬活性的影响及机制

Effects of Jiawei Wuhu Zhuifeng powder on autophagy activity of dopaminergic neurons in brain tissues of Parkinson's disease rats

目的观察加味五虎追风散灌胃对帕金森病(PD)大鼠脑组织多巴胺能神经元自噬活性的影响,并探讨其机制。方法将100只大鼠随机分为4组各25只,中药组、西药组、模型组大鼠通过微量注射泵予以6-羟基多巴胺溶液以制备PD模型,对照组给予4μL生理盐水。注射后休养1 d,第2 d进行模型评价(剔除制模不成功者和死亡者)。制模成功后次日,中药组灌胃1. 2 g/kg加味五虎追风散,西药组灌胃0. 27 mg/kg西罗莫司,对照组和模型组均灌胃等体积无菌蒸馏水,1次/d,连续1周。1周后处死大鼠,并取脑组织。免疫蛋白印记实验检测Beclin 1、LC3B蛋白及磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR);线粒体呼吸检测仪检测state3的呼吸速率、state4的呼吸速率,并计算线粒体呼吸控制比(RCR);荧光素-荧光素酶发光法检测线粒体ATP合成酶活力。结果与对照组比较,模型组Beclin 1、LC3B蛋白表达升高,线粒体RCR、ATP合成酶活力、p-mTOR表达降低(P均<0. 05);与模型组比较,中药组和西药组Beclin 1、LC3B蛋白和线粒体RCR、ATP合成酶活力升高,p-mTOR表达降低(P均<0. 05);与西药组比较,中药组Beclin 1、LC3B蛋白表达降低,p-mTOR表达升高(P均<0. 05)。结论加味五虎追风散灌胃能够通过线粒体-ATP-mTOR途径促进脑组织多巴胺能神经元自噬。

Objective To investigate the effects of Jiawei Wuhu Zhuifeng powder into the stamoch on the autophagy activity of dopaminergic neurons in the brain tissues of Parkinson′s disease(PD)rats,and to explore its mechanism.Methods A total of 100 rats were randomly divided into 4 groups,25 in each group.The rats in the Chinese medicine group,Western medicine group and model group were subjected with a 6-hydroxydopamine solution by a microinjection pump for preparing the PD models,and the rats in the control group were administered 4μL physiological saline.The rats rested for 1 d after the injection,and the model was evaluated on the 2nd day(excluding the unsuccessful model and the death one).On the following day after the successful model,the rats in the Chinese medicine group were given Jiawei Wuhu Zhuifeng powder 1.2 g/kg,the Western medicine group was intragastrically administered 0.27 mg/kg sirolimus,the control group and the model group were given equal volume of sterile distilled water,once a day,for a week.The rats were sacrificed and the brain tissues were taken.We detected the tyrosine hydroxylase(TH)andα-synuclein(α-syn)level in brain tissues by immunohistochemistry,detected Beclin 1,LC3B protein and phosphorylated mammalian target of rapamycin by Western blotting;the mitochondrial respiration detector was used to detect the state3 respiration rate,state4 respiration rate,and we calculated mitochondrial respiratory control ratio(RCR);fluorescein-luciferase luminescence assay was used for mitochondrial ATP synthase activity.Results Compared with the control group,the expression ofα-syn,Beclin 1 and LC3B protein increased,TH expression,mitochondrial RCR,ATP synthase activity and p-mTOR expression decreased in the model group(all P<0.05);compared with the model group,the expression of TH,Beclin 1,LC3B protein and mitochondrial RCR and ATP synthase increased,and the expression of p-mTOR andα-syn decreased in the Chinese medicine group and Western medicine group(all P<0.05);compared with the Western medicine group,Beclin 1 and LC3B protein decreased and p-mTOR expression increased in the Chinese medicine group(all P<0.05).Conclusion Jiawei Wuhu Zhuifeng powder can promote the autophagy of DA dopaminergic neurons in the brain tissues through the mitochondria-ATP-mTOR pathway.