Pectin rhamnoside inhibits alcohol-induced gastric epithelial cell pyroptosis through EGFR
Objective To clarify the improvement effect of Vitexin-2"-o-rhamnoside(RHV)on alcohol-induced gastric epithelial cell pyroptosis and its mechanism.Methods GES-1 cells were intervened with different concentrations of alcohol(0,200,400,600,800,1 000 mmol/L),and then the cell viability,the expressions of pyroptosis-related proteins and the lactate dehydrogenase(LDH)release were detected to screen the optimal intervention concentration for inducing the cell apoptosis.GES-1 cells were intervened with 800 mmol/L alcohol for 0,2,4,6,8 h respectively,and the cell viability was detected using CCK-8 to screen the optimal intervention time.GES-1 cells were pretreated with different concentrations of RHV(5,15,30,60 μmol/L)and then treated with 800 mmol/L alcohol,and the cell viability and the expressions of pyroptosis-related proteins were detected to screen the optimal intervention concen-tration of RHV.Network pharmacology analysis and molecular docking were employed to predict the possible action targets of RHV.GES-1 cells were intervened with alcohol or combined with RHV pretreatment,and the expression of phosphorylated epidermal growth factor receptor(p-EGFR)was detected.GES-1 cells were treated with different concentrations of EGFR inhibitor(0,1,2,4,8,10 nmol/L)and activator(0,5,10,20 μmol/L),and the expression of p-EGFR protein was detected to observe whether the changes of core targets affect the cell pyroptosis.GES-1 cells were pretreated with different concentrations of RHV(0,15,30,60 p.mol/L)and then intervented with EGFR activator,and the expression of p-EGFR was detected.Western blot and immunofluorescence were used to detect the expressions of pyroptosis-related proteins,and the lactate dehydrogenase(LDH)assay kit was used to measure LDH release.Results After alcohol treatment,the survival rate of GES-1 cells was decreased in a dose-and time-dependent manner(P＜0.05),while LDH release,and the protein expressions of cleaved caspase-1,GSDMD-N,NLRP3,and IL-1β were dose-dependently increased(P＜0.05).In view of the stability of the model,800 mmol/L alcohol intervention for 4 h was chsoen for the subsequent experiments.After RHV pretreatment,the alcohol-induced GES-1 cell survival rate was dose-dependently increased(P＜0.01),while the protein expressions of NLRP3,COX-2,cleaved caspase-1,and IL-18 were decreased dose-dependently(P＜0.05).The optimal intervention concentration of RHV was 60 μmol/L.The core targets of the combined action of alcoholic gastritis and resveratrol may be IL-6,EGFR,IL-1 β,etc.,the related pathways focused on oxidation-reduction,metabolism,receptor activation,and EGFR-related pathways,and RHV may have good binding affinity with EGFR.Compared with alcohol treatment alone,the level of p-EGFR was increased after combined with RHV pretreatment(P＜0.05).EGFR inhibitor dose-dependently reduced the levels of LDH release and p-EGFR,GSDMD-N,and IL-1 β protein expression induced by alcohol(P＜0.05),while EGFR activator increased the expressions of p-EGFR and GSDMD-N(P＜0.05).Pretreatment with 30,60 μmol/L RHV reduced the protein level of p-EGFR induced by EGFR activator(P＜0.05).Conclusion RHV can improve the alcohol-induced pyroptosis of GES-1 cells by inhibiting EGFR phosphorylation.
国家科技期刊平台
NSTL
万方数据
