Investigation On The Mechanism Of GSDME-mediated Pyroptosis Induced By Coxsackievirus B3

Coxsackievirus B3(CVB3)belongs to Enterovirus of Picornaviridae family.It is one of the pathogens causing many diseases,such as viral myocarditis,aseptic meningitis and pancreatitis.The necrotizing rupture of cells in CVB3-infected tissues is also the main cause of pathological inflammation.Early virological studies suggested that CVB3 infection induces apoptosis by activating caspase 8/ 9 in host cells and its downstream caspase 3,while apoptosis does not induce inflammation.This contradiction has not been fully explained,which is not conducive to the study on the pathogenesis of CVB3 infection.In recent years,cell biology studies have revealed that pyroptosis is a form of cell death which can cause cell breakdown and inflammation,and activated caspase 3 can cause pyroptosis by cleavage of gasdermin E(GSDME).Therefore,we hypothesized that CVB3 infection can induce pyroptosis by activating caspase 3 and GSDME,and this hypothesis can reasonably explain the molecular mechanism of CVB3-induced inflammation.In order to verify this hypothesis,we selected He La cell lines with high GSDME background expression as the virus infection model.After He La cells were infected with CVB3,morphological observation,propidium iodide,PI staining and lactate dehydrogenase(LDH)assay confirmed the occurrence of pyroptosis,and activation of caspase 3 and GSDME was observed by Western blot.Then,specific si RNA was used to knock down the expression level of GSDME in cells and CVB 3 was used to infect them,and it was found that pyroptosis was significantly inhibited,which proved that GSDME was indeed necessary for CVB3-induced pyroptosis.In order to confirm the activation of the upstream signaling pathway of GSDME,the broad-spectrum caspase inhibitor z-VAD-FMK was used to inhibit the activity of caspase family proteins,and it was found that CVB3-induced GSDME activation and pyroptosis were significantly inhibited.The results showed that the caspase family proteins were involved in the upstream process of CVB3 activation of GSDME.To further identify the members of the involved caspase protein family,specific inhibitors of caspase 3,8,and 9 were used to treat cells,and it was found that only inhibition of caspase 3 or both inhibition of caspase 8/9 attenuated CVB3-induced GSDME activation and pyroptosis.Because caspase 8 and 9 are upstream signals of caspase 3,the above results proved that CVB3 infection can activate both caspase 8 and 9 simultaneously,and both of them can activate caspase 3 together,thus causing pyroptosis.In summary,this study reveals the molecular mechanism of CVB3 induced pyroptosis through the caspase 3/GSDME pathway.The results of this study provide a possible reasonable explanation for the mechanism of CVB3-induced inflammation,and provide clues for the treatment of CVB3-related inflammation.