| Objective:Metformin,as a first-line hypoglycemic agent,has various pharmacological effects such as anti-tumor,anti-oxidant,and aging retardation.Recently research have found that metformin has effective anti-inflammatory and anti-pyroptosis effects.EV71 is one of main causes of severe hand-foot-and-mouth.EV71 infection cause excessive inflammation in the body,so inhibiting pyroptosis could be new strategy to treat EV71 infection.In general,antiinflammatory drugs have side-effects as induce gastric ulcers.Metformin has the characteristics including regulating metabolism,inhibiting oxidation and low side-effects.Metformin has been used in a large mount of patients in the clincal treatment process as a first-line hypoglycemic agent.We observe anti-pyroptosis effect of metformin and explored the molecular mechanism on anti-pyroptosis of metformin in EV71-infected gastric epithelial cells(GES-1)model.The study provided a new reference for clinical treatment of EV71 infection.Methods:1.GES-1 cells were infected with EV71 virus with different MOI for 24 hours.After different dose of virus incubate with GES-1 cell,the effect of cell damage was measured by LDH release kit to evaluate cell damage condition,and the effect of cell viability was measured by CCK8 to evaluate cell viability.The levels of NLRP3,GSDMD,Caspase-1 and Pro-IL-1β were measured by Western blot to evalute pyroptosis effect of EV71 infection in vitro.GES-1 cells were infected MOI for different times.The levels of NLRP3,Caspase-1,Pro-IL-1β protein were detected by Western blot,and inflammasome protein were labled by immunofluorescence staining.The puncta of NLRP3 and the level of NLRP3 was observed by high intension imaging system.2.GES-1 cells were treated with EV71 virus and metformin for 24 hours.After different dose of virus incubated with GES-1 cell,the effect of cell damage was measured by LDH release kit to evaluate cell damage condition,and the effect of cell viability was measured by CCK8 to evaluate cell viability.cytomembrane were labled by Dil staining.The integrity of cytomembrane was observed by Fluorescence microscope to evaluate cell damage condition in vitro.3.GES-1 cells were treated with EV71 virus and 2mM concentration of metformin for24 hours.The levels of NLRP3,GSDMD,Caspase-1 and pro-il-1β were measured by Western blot to study the effect of metformin on the level of pyroptosis induced by EV71 infection.4.GES-1 cells were infected with EV71 virus with different MOI for 24 hours,and GES-1 cells were treated with EV71 virus for different times.The level of DEPTOR protein in GES-1 cells was detected by Western blot to study the changes of DEPTOR protein level under EV71 infection.GES-1 cells were treated with concentration of metformin for 24 hours.The level of DEPTOR protein in GES-1 cells was detected by Western blot.GES-1cells were treated with EV71 virus and different concentration of metformin for 24 hours.The level of DEPTOR in EV71 infected cells was detected by Western blot.5.After overexpression of DEPTOR in GES-1 cells,GES-1 cells were infected with EV71 virus.The levels of DEPTOR,NLRP3,GSDMD,Caspase-1,Caspase1,P20 and Proil-1β in GES-1 cells were detected by Western blot to study the effect of overexpression of DEPTOR in GES-1 cells on pyroptosis.Results:1.EV71 decrease cell viability and cause cell damage in dose-dependent manner.EV71 increase intracellular NLRP3 level,decrease GSDMD,Pro-IL-1β level,and inhibited Caspase-1 activation in time-dependent and dose-dependent.EV71 increase NLRP3 level,and aggregate NLRP3.2.Metformin increase cell viability and decrease cell damage in dose-dependent manner.Metformin inhibit cytomembrane damage in dose-dependent manner.3.Metformin increase GSDMD,Pro-IL-1β,Caspase-1 level and decrease Caspase-1P20,NLRP3 level in EV71 infection model in a dose-dependent manner.4.EV71 decrease DEPTOR level in time-dependent and dose-dependent manners.Metformin increase DEPTOR levels in dose-dependent manner.Metformin increase DEPTOR levels in dose-dependent manner in a model of EV71 infection.5.After the expression of DEPTOR 24 h,we found that the activation level of NLRP3 decreased,the activation level of GSDMD increased,the activation level of Caspase-1decreased,the generation level of Caspase-1 P20 decreased,and the level of Pro-IL-1β shear decreased.Conclusion:1.EV71 decrease cell viability,cause cell damage and induces pyroptosis.2.Metformin increase cell viability in EV71 infected model,decrease cell damage and inhibited cytomembrane damage caused by EV71.3.Metformin inhibit EV71 caused pyroptosis.4.Metformin increase DEPTOR protein level in EV71 infection cell model.5.DEPTOR inhibit pyroptosis in EV71 infection cell model. |
