Effect Of Mieyou Decoction On Autophagy Of Gastric Mucosal Epithelial Cells In Rats Infected With Helicobacter Pylor

Objective: To investigate the effect of drug-containing serum on autophagy of gastric mucosal epithelial cells in rats by detecting the survival rate of RGM-1,the levels of ROS,IL-6 and TNF-α,and the expression of autophagy-related proteins LC3 and p62,and to investigate the effect of the medicinal serum of Mieyou Decoction on autophagy of gastric mucosal epithelial cells in rats infected with Helicobacter pylori(Hp).Methods: The medicinal serum and blank serum of the Mieyou Decoction were prepared by using 30 healthy SD rats,and the maximum non-toxic concentration of serum was determined by CCK-8 method.The experiment was divided into blank control group,Hp-stimulated group,Mieyou Decoction group,Hp+Mieyou Decoction group,3-MA group,3-MA+Hp group,3-MA+Hp+Mieyou Decoction group,a total of7 groups,the RGM-1 cells were cultured in a 96-well plate,and after 24 hours,the medium containing 10% of the medicinal serum of Mieyou Decoction was added to the Mieyou Decoction group,Hp+Mieyou Decoction group,3-MA+Hp+Mieyou Decoction group,3-MA(5mmol/L)medium was added to 3-MA group and 3-MA+Hpstimulated group,and Hp was added to the Hp-stimulated group,Hp+Mieyou Decoction group,3-MA+Hp+Mieyou Decoction group,and after 24 hours of culture,cell survival was detected by CCK-8 method,DCFH-DA staining flow cytometry was used to detect intracellular ROS levels,ELISA method was used to detect levels of ILS-6,TNF-α,and Western-blot detected autophagy-related proteins LC3-II/ LC3-I,p62.Results: The cell survival rate was > 95% when the serum concentration containing the drug was 10%,and the survival rate was the highest.Compared with the blank control group,the cell survival rate of the Hp-stimulated group was significantly reduced,and the levels of IL-6,TNF-α and ROS increased significantly(P<0.01),and the ratio of LC3-II/LC3-I in the 3-MA group decreased significantly(P<0.01),p62 expression was upregulated,and ROS levels were increased(P<0.05).Compared with the Hpstimulated group,the cell survival rate and IL-6 and TNF-α levels in the Hp+Mieyou Decoction group decreased significantly,the LC3-II/LC3-I levels increased significantly,and the p62 protein was significantly downregulated(P<0.01);the ROS level in the 3-MA+Hp group was significantly increased,the p62 protein was significantly upregulated,and the LC3-II/LC3-I level was significantly reduced(P<0.01).Compared with the Hp+Mieyou Decoction,the cell survival rate and TNF-αlevel of the 3-MA+Hp+Mieyou Decoction group were significantly increased,the expression of p62 protein was significantly upregulated(P<0.01),and the ROS content and LC3-II/LC3-I were significantly reduced(P<0.01).Compared with 3-MA+Hp group,the cell survival rate and LC3-II/LC3-I levels increased significantly in the 3-MA+Hp+Mieyou Decoction group,the levels of ROS,IL-6,TNF-α decreased significantly(P<0.01),and the p62 protein expression was down-regulated(P<0.05).Conclusion: 1.The maximum non-toxic concentration of the medicinal serum of the decoction is 10%.2.The stimulation of Hp can lead to an increase in the inflammatory factors IL-6 and TNF-α in RGM-1 cells,causing an inflammatory response and thus damaging the cells.3.3-MA inhibits autophagy in RGM-1 cells,reduces LC3-II/LC3-I levels,upregulates p62 protein,and increases ROS levels.4.The medicinal serum containing the Mieyou Decoction can increase the LC3-II/LC3-I level of RGM-1 cells,downregulate the p62 protein,activate autophagy,and partially antagonize the autophagic inhibition effect of 3-MA,reduce the level of inflammatory factors such as ROS,IL-6,TNF-α,thereby reducing the inflammatory response and protecting RGM-1 cells infected by Helicobacter pylori.