| Objective:To explore the role of endoplasmic reticulum stress in hydrogen peroxide(H2O2)-induced gastric epithelial cells(GES-1) apoptosis, as well as the effect of rebamipide in the apoptosis process and its possible mechanisms. Methods:In vitro, cell monolayer model was established by choosing human gastric epithelial cells(GES-1) as the research object. The cells was divided into three groups as control group, H2O2 group and rebamipide joint H2O2 group(400umol/L rebamipide pretreated 2h), and then induced by different concentrations of H2O2 for different times. Changes in cell morphology was observed under an inverted microscope in each group, the growth activity of GES-1 cells was measured by MTT assay, flow cytometey was applied to detect apoptotic rate of cells in each group, Western blot was used to detect the change of expression of endoplasmic reticulum stress-related protein CHOP and GRP78. Experimental data obtained were analyzed statistically using SPSS20.0, and P﹤0.05 was considered significant. Results:1. The morphology of GES-1 cells changed with the increase of H2O2 concentration and treatment time, such as cells gradually shrinkage, the larger gap, cells loss and even death. In rebamipide joint H2O2 group, changes in cell morphology were smaller than H2O2 group. 2. MTT assay showed that the absorbance values of GES-1 cells with increasing concentration of H2O2 and time gradually decreased(P <0.05). Under the same conditions, the absorbance value of rebamipide pretreated group of GES-1 was higher than H2O2 group. 3. Flow cytometry detecting the apoptosis rate of the cells showed that in the 6h group of 100umol/L H2O2, the apoptosis rate of control group was 0.66%±0.09%, while the H2O2 group and rebamipide joint H2O2 group respectively was 41.71%±1.36% and 19.88%±1.16%(P﹤0.05). In the 16 h group, the apoptosis rate of control group was 1.27%±0.25%, while the H2O2 group and rebamipide joint H2O2 group respectively was 47.32%±1.25% and 10.80%±0.96%(P﹤0.05). Meanwhile, comparing with 6h group, apoptosis of GES-1 in 16 h group was mainly late apoptosis and/or necrosis. 4. After GES-1 cells treating by 100umol/L H2O2 for 12 hours, the expression of endoplasmic reticulum stress-related proteins CHOP and GRP78 were detected by Western blot. In the H2O2 group, the expression of CHOP and GRP78 were increased comparing with the control group, while in rebamipide joint H2O2 group the expression of CHOP and GRP78 were decreased comparing with the previous H2O2 group, the difference was significant(P<0.05). Conclusion:1. H2O2 could inhibit the growth vigor of GES-1 cells, and the growth activity of cells was descreasing as the concentration of H2O2 and the treatment time were increasing. Rebamipide played a protective role on GES-1 cells in this process. 2. Apoptosis of GES-1 cell could occur when treated in a short time under low concentrations of H2O2(100umol/L), and the treatment of longer time would lead to cell necrosis, rebamipide could inhibit the percentage of apoptosis and / necrosis in H2O2-induced gastric epithelial cells. 3. The expression of ERS-related proteins CHOP and GRP78 were significantly increased in the H2O2-induced apoptosis of gastric epithelial cells, which indicated that endoplasmic reticulum stress involved in H2O2-induced gastric epithelial cells apoptosis. After treating by H2O2, the expression of endoplasmic reticulum stress-related protein CHOP and GRP78 in GES-1 pretreated by rebamipide were decreased comparing with the unpretreated cells, it suggested that the protective role of rebamipide in H2O2-induced GES-1 apoptosis may related to endoplasmic reticulum stress. |
