基于PINK1/Parkin通路探讨和胃理气方调控线粒体自噬对功能性消化不良胃肠动力障碍的作用机制

Study on mechanism of the regulation of mitochondrial autophagy by Hewei Liqi Decoction on gastrointestinal motility disorders in functional dyspepsia based on PINK1/Parkin pathway

目的 观察和胃理气方对功能性消化不良(FD)肝胃不和模型大鼠胃肠动力及PINK1/Parkin信号通路的影响,探讨其对线粒体自噬的调节机制。方法 健康Wistar大鼠40只,随机分为空白对照组、模型对照组、莫沙必利组、和胃理气方组,每组10只。除空白对照组外,其余各组用夹尾激怒法制作FD肝胃不和大鼠模型。造模成功后,对和胃理气方组和莫沙必利组进行药物治疗。观察各组大鼠一般状态、饮水量、进食量、胃黏膜表面有无器质性改变,检测胃排空率及小肠推进率以评价胃肠运动功能。通过HE染色观察胃窦组织有无糜烂、溃疡、化生、萎缩、不典型增生等病理改变。通过免疫荧光染色检测胃组织线粒体自噬的变化,JC-1流式细胞技术检测线粒体膜电位的变化,酶联免疫吸附测定法分析线粒体腺嘌呤核苷三磷酸(ATP)的含量、活性氧(ROS)的水平,Western blotting检测线粒体自噬蛋白PINK1、LC3、Parkin、P62的表达情况。结果 与空白对照组比较,模型对照组大鼠饮水量、进食量明显降低(均P<0.05),胃排空及小肠推进功能减缓(均P<0.05),胃组织中CYT-C迅速表达、LC3B表达明显积累,线粒体膜电位下调(P<0.05),线粒体膜电位变化比例明显上调(P<0.05),线粒体产生ATP的能力显著下调(P<0.05),线粒体ROS水平显著上调(P<0.05),线粒体自噬蛋白PINK1、Parkin、P62和LC3表达显著增加(均P<0.05)。与模型对照组比较,莫沙必利组与和胃理气方组大鼠饮水量、进食量明显升高(均P<0.05),胃排空及小肠推进功能显著升高(均P<0.05),CYT-C的表达被抑制、LC3B积累明显减少,线粒体膜电位显著上调(P<0.05),线粒体膜电位变化比例明显下调(P<0.05),线粒体产生ATP的能力显著上调(P<0.05),线粒体ROS水平显著下调(P<0.05),和胃理气方组ROS水平下调更显著(P<0.01),线粒体自噬蛋白PINK1、Parkin、P62和LC3的表达显著下调(均P<0.05)。结论 FD存在胃肠动力障碍,和胃理气方通过调控PINK1/Parkin信号通路介导的线粒体自噬,增加胃肠动力,改善FD肝胃不和大鼠症状。

Objective To observe the effects of Hewei Liqi Decoction on gastrointestinal motility and PINK1/Parkin signaling pathway in functional dyspepsia(FD)rat model with liver-stomach disharmony,and to explore its regulatory mechanism on mitochondrial autophagy.Methods Forty healthy Wistar rats were randomly divided into blank control group,model control group,mosapride group,and Hewei Liqi Decoction group,with 10 rats in each group.Except for the blank control group,the other groups were made into FD rat model with liver-stomach disharmony by clipping tails.After the model was established,the Hewei Liqi Decoction group and the mosapride group were treated with medication.The general state,water intake,food intake and organic changes on the surface of gastric mucosa were observed.The gastric emptying rate and small intestinal propulsion rate were detected to evaluate the gastrointestinal motor function.HE staining was used to observe the pathological changes of gastric antrum such as erosion,ulceration,metaplasia,atrophy and atypical hyperplasia.Immunofluorescence staining was used to detect the changes of mitochondrial autophagy in gastric tissues,JC-1 flow cytometry technology to detect the changes of mitochondrial membrane potential,enzyme linked immunosorbent assay to analyze the content of mitochondrial adenine nucleoside triphosphate(ATP)and the level of reactive oxygen species(ROS),and Western blotting to detect the expressions of mitochondrial autophagy protein PINK1,LC3,Parkin,and P62.Results Compared with the blank control group,the water intake and food intake of the model control group significantly decreased(all P<0.05),the gastric emptying rate and small intestinal propulsion rate slowed down(all P<0.05),CYT-C quickly expressed in gastric tissues,LC3B expression significantly accumulated,mitochondrial membrane potential down-regulated(P<0.05),the proportion of mitochondrial membrane potential changes significantly up-regulated(P<0.05),the ability of mitochondria to generate ATP significantly down-regulated(P<0.05),mitochondrial ROS levels significantly increased(P<0.05),and the expression of mitochondrial autophagy protein PINK1,Parkin,P62 and LC3 significantly increased(all P<0.05).Compared with the model control group,the water intake and food intake of rats in the mosapide group and Hewei Liqi Decoction group increased significantly(all P<0.05),the gastric emptying rate and small intestinal propulsion rate increased significantly(all P<0.05),the expression of CYT-C was inhibited,LC3B accumulation decreased significantly,mitochondrial membrane potential up-regulated significantly(P<0.05),the proportion of changes of mitochondrial membrane potential down-regulated significantly(P<0.05),the ability of mitochondria to generate ATP up-regulated significantly(P<0.05),mitochondrial ROS levels significantly down-regulated(P<0.05),the ROS level of Hewei Liqi Decoction group was down-regulated more significantly(P<0.01),and the expression of mitochondrial autophagy protein PINK1,Parkin,P62 and LC3 significantly down-regulated(all P<0.05).Conclusion There are gastrointestinal motility disorders in FD,and Hewei Liqi Decoction increases gastrointestinal motility and improves the symptoms of FD rats with liver-stomach disharmony by regulating mitochondrial autophagy mediated by PINK1/Parkin signaling pathway.