Autophagy Protects Gastric Mucosal Epithelial Cells From Ethanol-induced Oxidative Damage Via MTOR Signaling Pathway

Part ? Ethanol Induced Autophagy via down regulation of the mTOR Signaling Pathway in gastric mucosa epithelial cellsObjective:To reconstruct the damage model of gastric mucosa epithelial cell and tissue from ethanol, and to explore the expression of autophagy and regulation of the signaling pathways.Methods:The MTT assay, flow cytometry, histopathological examination were used to estimate the injury of gastric mucosal epithelial cells (GES-1 cells) and Wistar rats gastric mucosa which were treated beforehand with different concentrations of ethanol. The western blot, immunofluorescence staining and immunohistochemical staining were applied to detect the expression of autophagy-related proteins, as well as to evaluate the regulation of the mTOR signaling pathway, so as to explore the molecular mechanism of autophagy in gastric mucosal epithelial cells induced by ethanol.Results:Cell line experiments demonstrated that ethanol treatment could inhibit GES-1 cell viability and induce apoptosis. Animal experiments showed that gastric pit damage and mucosal hemorrhage were visible in the gastric mucosa sections of the rats gavaged with ethanol. In addition, the western blot results reflected that ethanol treatment could increase the expression levels of Beclin-1, Atg12-Atg5, and promote LC3-I converting to LC3-?. Furthermore, this process was also associated with the dephosphorylation of mTOR and its downstream p70S6 kinase.Conclusion:Ethanol exposure could induce gastric mucosal epithelial cell damage, which was accompanied by the downregulation of mTOR signaling pathway and activation of autophagy.Part ? The Influence and Related Mechanism of Autophagy in Ethanol-Induced Gastric Epithelial CellsObjective:To explore the relationship and related mechanisms between autophagy and ethanol-induced gastric epithelial cell injury.Methods:Autophagy inhibitor 3-MA or silencing autophagy-related gene via Atg7-siRNA was used to suppress autophagy in GES-1 cells. And the cells were incubated in the presence of ethanol before processing for PCR or Western blotting which was applied to detect the expression levels of autophagy-related proteins. Cell viability and apoptosis level were examined by the MTT assay and flow cytometry. Autophagy inhibitor CQ was pretreated to rat gastric mucosa by gavage, and following ethanol exposure, gross gastric mucosa was excised for pathological scoring and hematoxylin-eosin (H&E) staining to evaluate the injury degree, thus to investigate the role of autophagy plays in gastric mucosa.Results:In the cells experiment, results of western blot revealed that the autophagy inhibitor 3-MA could decrease the level of LC3-II in GES-1 cells, and Atg7-siRNA could inhibit autophagy through silencing ATG7. Cell viability was significantly decreased after treatment with ethanol and 3-MA/Atg7-siRNA compared with ethanol alone, while the rate of apoptosis was significantly increased. Animal experimental results showed that compared with ethanol alone, CQ pretreatment significantly increased gastric mucosal lesion index, hemorrhage and disruption of gastric pits following ethanol treatmentConclusion:Inhibition of autophagy enhancing ethanol-induced damage to gastric mucosal epithelial cells, which indicates an important role for autophagy in limiting ethanol-induced damage of the gastric mucosal epithelial cells in vitro and in vivoPart ? Cell Autophagy Involved in the Regulation of Ethanol-Induced Oxidative Stress of Gastric Mucosal Epithelial CellsObjective:To investigate the relationship between autophagy and ethanol-induced oxidative stress of gastric mucosal epithelial cells.Methods:GES-1 cells were treated with ethanol in the presence or absence of the autophagy inhibitor respectively or simultaneously, and then were incubated with DCFH-DA. Spectrophotometer was used to detect the intracellular ROS. In the animal modal, following the treatment of ethanol and/or CQ to rats through administration by gavage, the tissue homogenate of gastric mucosa was collected to measure the expression of SOD activity and MDA contents.Results:Treatment with ethanol and autophagy inhibitor 3-MA led to a significant higher DCF fluorescence than ethanol alone. The MDA concentration was dramatically increased, and SOD activity was significantly reduced in the homogenate of gastric mucosal tissue of rats feeding with ethanol and CQ compared with ethanol alone.Conclusion:Autophagy plays an important role in ethanol-induced damage to gastric mucosal epithelial cells, which may inhibit the generation of ROS, the degradation of antioxidase and lipid peroxidation.