Rebamipide Reduces Gastric Epithelial Cell Apoptosis Induced By Ethanol Overstimulation Through Inhibiting Endoplasmic Reticulum Stress And Activating Autophagy

Aim:Alcohol-induced gastric mucosal epithelial cell apoptosis is an important cause of gastric mucosal injury,acute and chronic gastritis.In recent years,it has been found that endoplasmic reticulum stress also plays an important role in mediating apoptosis,and autophagy related pathways are important mechanisms for protecting cells.Some studies have also found that rebamipide can effectively protect the gastric mucosa,mainly by inhibiting leukocyte activation and inflammatory cytokine production,scavenging oxygen free radicals,increasing the amount of gastric mucus and prostaglandins in the gastric mucosa.This study intends to explore whether ethanol overstimulation of gastric epithelial cells activates endoplasmic reticulum stress and induces apoptosis.And further in-depth studies on whether rebamipide can reduce gastric epithelial cell apoptosis induced by ethanol overstimulation,through inhibiting endoplasmic reticulum stress and activating autophagy pathway,in order to find a new role of rebamipide and reveal new effects.The mechanism provides a more powerful theoretical basis for clinical application of rebamipide.Methods:The gastric mucosal epithelial cell line GES-1 was the main research object,ethanol builded model,and rebamipide intervention.Apoptotic rate was detected by flow Annexin V-FITC/PI double staining;mitochondrial membrane potential(Δψ)was detected by flow cytometry equipped with JC-1 probe;the expression level of apoptosis,endoplasmic reticulum stress and autophagy related protein was detected by Western blot.Changes in autophagy-related proteins were observed by fluorescence microscopy.Results:1.Ethanol induced apoptosis of GES-1 cells in a concentration-dependent manner.After ethanol treatment,the expression levels of apoptosis-related proteins caspase3,CHOP and Bax were significantly increased,and Bcl-2 was significantly decreased,but caspase8 was not statistically different;Δψ was significantly decreased.Rebamipide effectively reduced the expression levels of cleaved caspase3,CHOP,and Bax proteins,restored the expression level of Bcl-2 protein,and partially restored the Δψ level.2.Ethanol induced GES-1 production of endoplasmic reticulum stress,significantly increased the expression level of ATF6 protein,increased thephosphorylation level of p-IRE α,and had no significant effect on the phosphorylation level of p-eIF2α.Rebamipide reduced the expression level of ATF6 protein and decreased the phosphorylation level of p-IREα.At the same time,the expression levels of NF-κB and IκBα protein in the downstream pathway of endoplasmic reticulum stress were significantly decreased after ethanol treatment,and the expression levels of NF-κB and IκBα protein were restored after treatment with rebamipide.3.Further studies on autophagy pathway-related proteins revealed that compared with ethanol alone,after intervention with rebamipide the expression level of P62 protein was significantly decreased,and the autophagic flow of LC3-II/LC3-I was significantly increased,the expression levels of Autophagy related protein Atg5,Atg7 was significantly increased,but had no effect on the expression level of Beclin1 protein.By transfecting the GFP-LC3 plasmid,it was found that the number of GFP-LC3 spots after ethanol treatment had no significant changes;the amount of GFP-LC3 spots was significantly increased after the intervention with rebamipide.Autophagy inhibitor 3-MA inhibited the autophagic activation by rebamipide.4.A further study on the upstream signaling pathway of autophagy revealed that compared with ethanol alone,rebamipide significantly increased the phosphorylation levels of p-ERK and p-p38,but had no effect on the phosphorylation levels of p-JNK and p-mTOR..Conclusion:Ethanol overstimulation of gastric mucosal epithelial cells can activate endoplasmic reticulum stress and induce apoptosis,and can not activate its autophagy pathway.Rebamipide effectively inhibited cell apoptosis by decreasing cleaved caspase3,CHOP,Bax and restoring Bcl-2 protein expression levels;it effectively inhibited the activation of endoplasmic reticulum stress by decreasing the expression level of ATF6 protein and phosphorylation of p-IRE α;it effectively activated autophagy-related pathways by decreasing expression of P62 protein,increasing autophagic flow of LC3-II/LC3-I,increasing expression levels of autophagy associated proteins Atg5 and Atg7,increasing number of GFP-LC3 spots;by increasing Phosphorylation levels of p-ERK and p-p38 activates the autophagy upstream signaling pathway.Therefore,rebamipide can effectively reduce gastric mucosal damage induced by ethanol overstimulation.Further speculated that thecombination of rebamipide and damaged gastric mucosal drugs can effectively protect the gastric mucosa.