| Purpose:1 To verify the regulating effect of He Wei Li Qi Formula on gastrointestinal motility disorder in functional dyspepsia rats model with discordant liver and stomach.2 To observe the effect of He Wei Li Qi Formula on Cellular Autophagy,Mitochondrial autophagy and mitochondrial function in functional dyspepsia rats model.3 To observe the expression of mitochondrial autophagy-related protein Pink1,Parkin,P62,LC3 and mitochondrial autophagy-related pathway protein AMPK,ACC,S6 K in gastric tissue of rats with liver-stomach disharmony,to explore the mechanism of regulating mitochondrial autophagy by He Wei Li Qi Formulathrough AMPK pathway to improve gastrointestinal motility in functional dyspepsia rats model.Material and method:1 The rat models of functional dyspepsia with liver and stomach disturbance were established by trapping tail stimulation method.Fifty healthy Wistar(SPF grade)rats were randomly divided into 5 groups: blank control group,model control group,mosapride group,He Wei Li Qi formula group,and He Wei Li Qi formula + mitochondrial autophagy inhibitor group,with 10 rats in each group.Except for the blank control group,the other groups were used to make FD liver depression rat model by tail-clip irritation method.Each daythe rat trap was used to clamp the distal 1/3 of the rat tail for 30 minutes each time,and continued to stimulate within half an hour,once every 3 hours,four times a day with consecutive 7 days.After the end of the modeling period,the rats were given intragastric administration,and He Wei Li Qi formula were given intragastric treatment with a concentration of 0.9702g/m L,twice a day for consecutive 7 days.mosapride group was given Mosapride suspension with a concentration of 0.297mg/ m L twice a day for consecutive 7 days.In addition to traditional Chinese medicine intervention,He Wei Li Qi formula + mitochondrial autophagy inhibitor group was given intraperitoneal injection of U026-ETOH 20 mg /kg for consecutive 7 days.The general state,water intake and food intake of rats in each group were observed.The gastric emptiness rate and small intestinal proptosis rate were detected.The anatomical structure of gastric mucosa and the pathological examination of gastric tissue were observed.2 According to the method of the first part,after the model was made and the corresponding intervention,the changes of autophagy of gastric tissue were detected by immunofluorescence staining,to explore the regulating effect of He Wei Li Qi formula on gastrointestinal motility disorder in functional dyspepsia rats model on the autophagy of gastric tissue in rats with functional dyspepsia.3 On the basis of the first part of the experiment,the changes of mitochondrial membrane potential in the blank control group,model control group,mosapride group and He Wei Li Qi formula group were detected by JC-1 flow cytometry,enzyme-linked immunosorbent assay(ELISA)was used to determine the contents of mitochondrial ATP and Reactive oxygen species(Ros),and to investigate the regulation of HEWei Li Qi formula on mitochondrial membrane potential,mitochondrial ATP and ROS.4 On the basis of the first part of the experiment,western-blot was used to detect the expression of Mitochondrial autophagic proteins PINK1,LC3,Parkin,P62,AMPK pathway proteins AMPK,ACC,S6 K in rat gastric tissues,to investigate the regulation of He Wei Li Qi formula on mitochondrial autophagy by detecting the gastric emptying in rats treated with mitochondrial autophagy inhibitor.Results:1 After modeling,the rats in the model control group were listless,less moving,dark and dull fur,loose stool or sometimes dry,water consumption,food intake and body weight were significantly reduced compared with the rats in the blank control group.The abdominal anatomy showed that there was no organic change in each organ,no ulcer in the stomach and intestine,and the color was normal.Mild lymphocyte infiltration could be seen in the gastric mucosa,and the gastric emptiness rate and small intestinal propulsive rate were slowed down,indicating the success of modeling.Compared with model control group,the water intake and food intake of rats in He Wei Li Qi formula group were significantly increased(P<0.05),and the gastric empty-rate and small intestinal propelling rate were significantly increased(P<0.05).2 The expression of Cyt-c was almost not expressed in the blank group.After the establishment of the functional dyspepsia model,the expression of Cyt-c rapidly accumulated.The expression of Cyt-C was inhibited in He Wei Li Qi formula group and mosapride group.LC3 b,the marker protein of autophagy,was significantly accumulated in the model group,and was significantly decreased after treatment with He Wei Li Qi Formula group and mosapride group.3 After JC-1 staining,the changes of mitochondrial membrane potential in blank control group,model control group,mosapride group and He Wei Li Qi formula group were detected by flow cytometry.In the control group,the mitochondrial membrane potential remained normal,and the mitochondrial membrane potential decreased after the establishment of functional dyspepsia model(P<0.05).Mitochondrial membrane potential was significantly increased in He Wei Li Qi formula group and mosapride group(P<0.05),and the normal mitochondrial membrane potential was maintained.Compared with blank control group,the percentage of mitochondrial membrane potential in the model control group was significantly increased(P<0.05).The percentage of mitochondrial membrane potential decreased after Heweiliqifang and mosapride treatment(P<0.05).There was no significant difference in regulating mitochondrial membrane potential between He Wei Li Qi formula group and mosapride group(P>0.05).4 The main function of mitochondria is to produce ATP.The experiment monitored the ability of different treatments to produce ATP in mitochondria.Compared with the blank control group,the ability of mitochondria to produce ATP was significantly decreased after the establishment of functional dyspepsia model(P<0.05).The mitochondrial relative ATP level was significantly up-regulated in He Wei Li Qi formula group and mosapride group(P<0.05).Compared with blank control group,there was no significant difference(P>0.05).There was no significant difference in the ability of regulating mitochondrial ATP production between He Wei Li Qi formula group and mosapride group(P>0.05).5 Mitochondria are the main sites where REDOX reactions occur.The effects of different treatments on mitochondrial ROS levels were tested.Compared with the blank control group,the mitochondrial ROS level was significantly increased after the establishment of the functional dyspepsia model(P<0.05),and the mitochondrial relative ROS level was significantly down-regulated in the gastric He Wei Li Qi formula group and the mosapride group(P<0.05),and the lower ROS level was more significant in the He Wei Li Qi formula group(P<0.01).Compared with blank control group,there was no significant difference(P>0.05).There was no significant difference in regulating ROS level between He Wei Li Qi formula group and mosapride group(P>0.05).6 Western blot results showed that compared with blank control group,the protein expressions of Pink1,Parkin,P62 and LC3 in model control group were significantly increased(P<0.05),and the protein expressions of Pink1,Parkin,P62 and LC3 in He Wei Li Qi formula group and mosapshare group were significantly decreased compared with model control group(P<0.05),but had no significant difference compared with blank control group(P>0.05).There were no significant differences in the regulation of Pink1,Parkin,P62 and LC3 between He Wei Li Qi formula group and mosaprides group(P>0.05).7 Changes of mitochondrial autophagy-related AMPK and m TOR pathway proteins in each group.Western blot results showed that the phosphorylated AMPK,ACC and S6 K protein expression in model control group was significantly increased compared with blank control group(P<0.05),and the phosphorylated AMPK,ACC and S6 K protein expression levels in He Wei Li Qi formula group and mosapril group were significantly down-regulated compared with model control group(P<0.05),but there was no significant difference compared with blank control group(P>0.05).There were no significant differences in AMPK,ACC and S6 K between He Wei Li Qi formula group and mosapril group(P>0.05).8 Effects of mitochondrial autophagy inhibitors on the improvement of functional dyspepsia in rats by He Wei Li Qi formula.Compared with the blank control group,the gastric emptying rate of the model control group was significantly decreased(P<0.05),indicating that the model was successfully constructed.He Wei Li Qi formula could significantly increase the gastric emptying rate of rats(P<0.05),indicating that it had a significant improvement effect on dyspepsia in rats.However,in order to verify the effect of mitochondrial autophagy on functional dyspepsia in rats,we gave inhibitors of mitochondrial autophagy to rats,and the results showed that after the inhibition of mitochondrial autophagy,the improvement effect of He Wei Li Qi formula on dyspepsia was partially neutralized.Conclusion:1 The rat model of functional dyspepsia with disharmony of liver and stomach was successfully replicated by tail-clamping stimulation.2 He Wei Li Qi formula has regulatory effect on gastrointestinal motility disorder in rats with functional dyspepsia.3 He Wei Li Qi formula can regulate cellular autophagy,mitochondrial autophagy and mitochondrial dysfunction in rats with functional dyspepsia.The He Wei Li Qi formula group could inhibit the expression of CYT-C and LC3 B and reduce the occurrence of autophagy.The He Wei Li Qi formula group could maintain the normal mitochondrial membrane potential,up-regulate the level of mitochondrial ATP and down-regulate the level of Mitochondrial Ros in order to improve the mitochondrial function of rats with functional dyspepsia and dyspepsia.4 He Wei Li Qi formula can regulate mitochondrial autophagy through AMPK pathway,which may be one of its mechanisms for improving gastrointestinal motility disorders.By down-regulating the expression of Pink1,Parkin,P62,LC3,AMPK,ACC and S6 K proteins in mitochondrial autophagy related pathways,and improving the activation of AMPK pathway,it is speculated that AMPK and downstream target substances may be involved in the Pink1 /Parkin pathway induced mitochondrial autophagy.By regulating the expression of AMPK and its downstream and downstream target substances,and then regulating the PINK1/Parkin pathway,and reducing autophagy of mitochondria,and alleviating functional dyspepsia in rats.In order to verify the effect of mitochondrial autophagy on functional dyspepsia in rats,we gave inhibitors of mitochondrial autophagy to rats.After the inhibition of mitochondrial autophagy,the improving effect of He Wei Li Qi formula on functional dyspepsia was partially neutralized.The results suggest that the improvement effect of He Wei Li Qi formula depends on the process of mitochondrial autophagy in rats. |
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