Research On Astragaloside Inhibits Apoptosis And Pyroptosis Of Enterovirus 71 Induction By TXNIP

Objective:Enterovirus 71(EV71)is one of the most main pathogens of children hand-footmouth disease.EV71 infection causes a lot of programmed cell deaths in most cases.However,there is no antiviral drug available for the prevention and treatment of EV71 infection currently.Astragalus membranaceus(AM),a traditional Chinese medicine(TCM),has exhibited encouraging therapeutic effects in several viral infectious diseases.We aim to explore the protective effect of AM on EV71 infected cells and find a component of AM directly against EV71 virus to further reveal its antiviral mechanism.Methods:1.GES-1 cells were infected with EV71 of different MOI value to establish virus infected cell model.Morphological changes of EV71 infected cells were observed under an inverted microscope.CCK8 was used to detected the EV71 infected cell vitality.The damage of EV71 infected cells was measured by the LDH release.DAPI was used to dye nucleus to observe the apoptosis morphologic alteration of EV71 infected cells.The protein lever changes of Caspase-3,PARP,NLRP3,GSDMD,Pro caspase-1,Pro IL-1β,P62,LC3,TXNIP were investigated by Western blot to evaluate EV71 induced apoptosis,pyroptosis and autophagy.2.To explore the effect of AM on EV71 induced apoptosis,GES-1 cells were treated with different concentrations AM.The cell vitality was measured by CCK8 and the protein levels of Caspase-3,PARP in GES-1 cells were defected by Western blot.3.To defect the effect of APS,TFA,ASTs,main effective component of AM,on EV71 induced apoptosis and pyroptosis,the protein lever changes of Caspase-3,PARP,NLRP3,Pro IL-1β were measured by Western blot.4.For the purpose of evaluating the effect of ASTs,AST-IV on EV71 induced programmed cell deaths and replication of EV71,CCK8 was utilized to detect cell vitality.LDH was applied to evaluate cell damage.The apoptosis morphologic alterations were observed by DAPI fluorescent staining.Western blot was applied to measure the expressions of Caspase-3,PARP,NLRP3,GSDMD,Pro caspase-1,Pro IL-1β,P62,LC3,VP1,TXNIP in the cells.TCID50 was used to detect virus titer of supernatant.5.To defect the effect of TXNIP on EV71 induced programmed cell deaths and replication of EV71,the expression of TXNIP was interfered by si RNA transfection,and the protein changes of Caspase-3,PARP,NLRP3,GSDMD,Pro caspase-1,Pro IL-1β,P62,LC3,TXNIP and viral structural protein VP1 were observed by Western blot.Virus titer of supernatant was detected by TCID50.Results:1.EV71 infection caused demage in GES-1 cells,which was characterized by cytoplasm shrinkage,cell density reduced,cell viability decreased,LDH release increased,nuclear uneven,and changes in marker proteins of apoptosis,pyrotosis and autophagy.2.AM improved cell viability of EV71 infected GES-1 cells and inhibited degradation of Caspase-3,PARP to inhibit EV71 induced apoptosis in a dose-dependent manner.3.APS or TFA inhibited degradation of Caspase-3,PARP to inhibit EV71 induced apoptosis.ASTs not only inhibited degradation of Caspase-3,PARP,but also inhibited degradation of Pro IL-1β,increase of NLRP3 to restrain EV71 induced apoptosis and pyroptosis.4.ASTs or AST-IV inhibited degradation of Caspase 3,PARP,Caspase 1,GSDMD,Pro IL-1β,P62 protein,protein levers increase of NLRP3 and EV71 structure protein VP1,conversion of LC3 to inhibit EV71 induced apoptosis,pyroptosis,autophagy and replication of EV71.5.EV71 infection increased the expression of TXNIP,while ASTs or AST-IV could restrain this process.TXNIP si RNA inhibited the degradation of Caspase 3,PARP,Caspase 1 GSDMD,Pro,Pro IL-1β,transform of LC3 and increased of NLRP3,VP1 and virus titer of supernatant.These results indicated that TXNIP si RNA inhibits EV71 induced apoptosis,pyroptosis,autophagy and replication of EV71.Conclusion:1.EV71 induces apoptosis,pyroptosis and autophagy of GES-1 cells.2.AM attenuates the cytopathic of GES-1 cells by inhibiting EV71 induced apoptosis.3.APS or TFA inhibits EV71 induced apoptosis and ASTs inhibits EV71 induced apoptosis and pyroptosis of GES-1 cells.4.ASTs or AST-IV inhibits replication of EV71 to inhibit EV71 induced apoptosis and pyroptosis.5.ASTs or AST-IV inhibits EV71 induced autophagy.6.The inhibitory effect of ASTs or AST-IV on EV71 replication through downregulating TXNIP.