| Objective:Enterovirus 71(EV71)is one of the major pathogens causing childhood hand-foot-mouth disease(HFMD).At present,the pathogenic mechanism of EV71 virus is not fully understood,and no specific drug can be used to anti-EV71 therapy.Our previous study found that EV71 can induce apoptosis and pyroptosis.The pannexin1(Panx1)is a macroporous membrane channel associated with gap junctions,with high permeability to Adenosine triphosphate(ATP).While whether Panx1 is involved in the regulation of EV71 induced apoptosis and pyroptosis remains unclear.If the answer proves to be yes,we want to know whether Panx1 can be used as a regulatory target to inhibit EV71 induced pyroptosis for the screening and design of anti-EV71 drugs? Next,we will try to find the anti-EV71 active substances in the plants which have the same medicine and food origin.In this study,we conducted the research from two aspects: first,whether Panx1 participates in the regulation of viral replication and EV71 induced apoptosis and pyroptosis;second,whether seabuckthorn flavonoids and its main components inhibit EV71 virus and its induced apoptosis and pyroptosis function through Panx1.Methods:1.Human gastric mucosal epithelial cells(GES-1)were infected with EV71 with certain multiplicity of infection(MOI)values to model EV71 infection.The viability of the infected cells was determined by CCK-8 assay,the damage of the infected cells was determined by lactate dehydrogenase(LDH)assay,the morphology of apoptosis in infected cells were detected by propidium lodide(PI)staining,the ATP kit detected ATP changes intracellular and extracellular the infected cells,the protein levels of PARP,cleaved-PARP,caspase-3,NLRP3,GSDMD,caspase-1,mature IL 1β,pro-IL 1β,Panx1 and VP1 in GES-1 cells were detected by Western Blotting.To explore the apoptosis,pyroptosis and Panx1 activation induced by EV71.2.The EV71-infected cells were intervened with Z-VAD-FMK or Z-DEVD-FMK,the effect of Z-VAD-FMK or Z-DEVD-FMK on the damage condition of infected cells was tested by LDH,PI staining was used to detect the morphological effects of EV71 induced apoptosis by Z-VAD-FMK or Z-DEVD-FMK,the ATP kit to detect the effect of Z-VAD-FMK or Z-DEVD-FMK on ATP intracellular and extracellular of infected cells,the effect of Z-VAD-FMK or Z-DEVD-FMK on NLRP3 inflammasome was detected by immunofluorescence(IF),the protein levels of Poly ADP-ribose polymerase(PARP),cleaved-PARP,Cysteinyl aspartate specific proteinase-3(caspase-3),NOD-like receptor protein 3(NLRP3),Gasdermin-D protein(GSDMD),caspase-1,mature Interleukin 1β(mature IL 1β),pro-Interleukin 1β(pro-IL 1β),Panx1 and VP1 in GES-1 cells were detected by Western Blotting after treatment with Z-VAD-FMK or Z-DEVD-FMK.To explore the effect of caspase-3 inhibition on apoptosis,pyroptosis and Panx1 activation induced by EV71.3.The expression of caspase-3 was interfered by si RNA,the protein levels of PARP,cleaved-PARP,caspase-3,NLRP3,GSDMD,caspase-1,mature IL 1β,pro-IL 1β,Panx1 and VP1 were detected by Western Blotting.To observe the effect of si Caspase-3on EV71 induced apoptosis,pyroptosis and Panx1 activation.4.The EV71-infected cells were intervened with probenecid(PBD),the effect of PBD on the damage condition of infected cells was tested by LDH,PI staining was used to detect the morphological effects of EV71 induced apoptosis by PBD,the ATP kit to detect the changes of PBD on ATP intracellular and extracellular of infected cells,the effect of PBD on NLRP3 inflammasome was detected by IF,the protein levels of PARP,cleaved-PARP,caspase-3,NLRP3,GSDMD,caspase-1,mature IL 1β,pro-IL 1β,Panx1 and VP1 in GES-1 cells were detected by Western Blotting after treatment with PBD.To explore the effect of Panx1 inhibition on apoptosis,pyroptosis and Panx1 activation induced by EV71.5.The expression of Panx1 was interfered by si RNA,the protein levels of PARP,cleaved-PARP,caspase-3,NLRP3,GSDMD,caspase-1,mature IL 1β,pro-IL 1β,Panx1 and VP1 were detected by Western Blotting.To observe the effect of si Panx1 on EV71 induced apoptosis,pyroptosis and Panx1 activation.6.The EV71-infected cells were intervened with seabuckthorn flavone(SF),isorhamnetin(Iso),quercetin(Que),and kaempferol(Kae),the viability of infected cells after intervention with SF,Iso,Que,and Kae was measured by CCK-8,the effect of SF,Iso,Que,and Kae on the damage condition of infected cells was tested by LDH,PI staining was used to detect the morphological effects of EV71 induced apoptosis by SF,Iso,Que,and Kae,the effects of SF,Iso,Que and Kae on NLRP3 inflammasome and caspase-1 P20 protein were detected by IF,the ATP kit to detect the effects of Iso on ATP intracellular and extracellular of infected cells,the protein levels of PARP,cleaved-PARP,caspase-3,NLRP3,GSDMD,caspase-1,mature IL 1β,pro-IL 1β,Panx1 and VP1 in GES-1 cells were detected by Western Blotting after treatment with SF,Iso,Que,and Kae,To explore the effects of SF,Iso,Que,and Kae on apoptosis,pyroptosis and Panx1 activation induced by EV71.Results:1.EV71 infection induced GES-1 cell pathological changes,which were characterized by decreased cell viability,increased cytotoxicity,increased PI positive rate,decreased intracellular ATP and increased extracellular ATP,the changes of apoptosis,pyroptosis related marker proteins and the activation of membrane channel protein Panx1 were increased.2.The LDH results showed that Z-VAD-FMK or Z-DEVD-FMK attenuated the cytotoxicity caused by EV71 infection,the PI staining results showed that Z-VAD-FMK or Z-DEVD-FMK decreased the increased PI positive rate caused by EV71 infection,the IF results showed that Z-VAD-FMK or Z-DEVD-FMK reduced the activation of the NLRP3 inflammasome caused by EV71 infection,Z-VAD-FMK or Z-DEVD-FMK increased intracellular ATP and decreased extracellular ATP,the Western Blotting results showed that after Z-VAD-FMK or Z-DEVD-FMK intervention,EV71 induced Panx1 activation was inhibited,VP1 protein levels decreased,and the degradation of PARP,caspase-3,GSDMD,caspase-1,pro-IL 1β was inhibited,while the expression of cleaved-PARP,NLRP3 and mature IL 1β was inhibited.The above results indicate that inhibition of caspase-3 activation by Z-VAD-FMK or Z-DEVD-FMK inhibits Panx1 activation and EV71 replication,thus alleviating apoptosis and pyroptosis induced by EV71.3.The Western Blotting results showed that after interfering with caspase-3expression,EV71 induced Panx1 activation was inhibited,VP1 protein levels decreased,and degradation of PARP,caspase-3,GSDMD,caspase-1,pro-IL 1β was inhibited,while expression of cleaved-PARP,NLRP3,and mature IL 1β was inhibited.The above results indicated that Panx1 activation was inhibited by knockdown of caspase-3,EV71 replication was inhibited,thus inhibiting the apoptosis and pyroptosis induced by EV71.4.The LDH results showed that PBD attenuated the cytotoxicity caused by EV71 infection,the PI staining results showed that PBD decreased the increased PI positive rate caused by EV71 infection,the IF results showed that PBD reduced the activation of the NLRP3 inflammasome caused by EV71 infection,PBD increased intracellular ATP and decreased extracellular ATP,the Western Blotting results showed that after PBD intervention,EV71 induced Panx1 activation was inhibited,VP1 protein levels decreased,and the degradation of PARP,caspase-3,GSDMD,caspase-1,pro-IL 1β was inhibited,while the expression of cleaved-PARP,NLRP3 and mature IL 1β was inhibited.The above results indicate that upon inhibition of Panx1 activation by PBD,EV71 replication was inhibited,which subsequently attenuates pyroptosis induced by EV71.5.The Western Blotting results showed that after interfering with Panx1 expression,EV71 induced Panx1 activation was inhibited,VP1 protein levels decreased,and degradation of PARP,caspase-3,GSDMD,caspase-1,pro-IL 1β was inhibited,while expression of cleaved-PARP,NLRP3,and mature IL 1β was inhibited.The above results indicated that EV71 replication was inhibited by Panx1 knockdown,which then attenuated the pyroptosis induced by EV71.6.The CCK-8 results showed that SF,Iso,Que,and Kae increased the viability of EV71-infected GES-1 cells,the LDH results showed that SF,Iso,Que,and Kae attenuated the cytotoxicity caused by EV71 infection,the PI staining results showed that SF,Iso,Que,and Kae decreased the increased PI positive rate caused by EV71 infection,the IF results showed that SF,Iso,Que,and Kae reduced the activation of NLRP3 inflammasome and caspase-1 P20 protein caused by EV71 infection,Iso increased intracellular ATP and reduced extracellular ATP,the Western Blotting results showed that after SF,Iso,Que,and Kae intervention,EV71 induced Panx1 activation was inhibited,VP1 protein levels decreased,and the degradation of PARP,caspase-3,GSDMD,caspase-1,pro-IL 1β was inhibited,while the expression of cleaved-PARP,NLRP3 and mature IL 1β was inhibited.The above results indicated that SF,Iso,Que,and Kae inhibited EV71 induced apoptosis by inhibiting virus replication,then inhibited Panx1 activation,and finally led to the inhibition of EV71 induced pyroptosis.Conclusions:1.EV71 infection induced apoptosis and pyroptosis in GES-1 cells.2.EV71 infection induced Panx1 activation in GES-1 cells.3.Inhibition of caspase-3 activation attenuates EV71 replication and its induced apoptosis,pyroptosis.4.Inhibition of Panx1 activation attenuates EV71 replication and its induced pyroptosis.5.Seabuckthorn flavone and its main components(Isorhamnetin,Quercetin,Kaempferol)inhibite EV71 replication and its induced apoptosis,pyroptosis. |
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