The Study Of P21-related Foxo And Ca²⁺ Signaling Pathways Induced By Japanese Encephalitis Virus

Japanese encephalitis?JE?caused by Japanese encephalitis virus?JEV?is an infectious disease severely harming human and livestock in china,and its prominent pathological features are brain inflammation and cell death.The pathogenesis of JEV infection remains unclear,and there are no efficacious drugs against JE.Therefore,elucidating the mechanism of JE virus infection and pathogenicity at the molecular level,which will provide new insights and targets for the development of special anti-JE drugs,is a hot topic and frontier of JE virus research at home and abroad in recent years.Apoptosis is a process of programmed cell death that occurs in multicellular organisms,which is controlled by genes in order to maintain the stability of the internal environment.It involves the activation,expression and regulation of a series of genes.In previous work,using next-generation sequencing technology,we profiled global mRNA expression changes in response to in vitro and in vivo JEV infection.Integration analysis revealed that JEV infection regulated apoptosis-related Foxo signaling pathway.Foxo expression was reduced by JEV infection in vitro and in vivo.Knock down of Foxo promoted apoptosis,while its overexpression reduced apoptosis in JEV-infected Neuro-2a cells.Neuro-2a cells were infected with P3 strain 72 hours at an MOI of 5.Since JEV infection induced apoptosis and regulated Foxo signaling pathway,we next examined the relation between Foxo expression and apoptosis during JEV infection.We examined the expression of apoptosis-related Foxo target genes.Pro-apoptotic protein Bim and anti-apoptotic proteins Bcl-6 and p21 have been reported as downstream genes of Foxo.The expression of Foxo downstream genes including pro-apoptotic protein Bim,anti-apoptotic protein Bcl-6 and p21 was decreased by JEV infection.Knockdown of Foxo decreased the levels of Bim,Bcl-6 and p21 in Neuro-2a cells.Overexpression of Foxo increases the levels of Bim,Bcl-6 and p21 in JEV-infected Neuro-2a cells.Overexpression of Bcl-6 and p21 down-regulates the rate of apoptosis of Neurovirus-infected Neuro-2a cells.These results show that Foxo in JEV-infected Neuro-2a cells predominantly promotes cell survival by anti-apoptotic proteins Bcl-6 and p21 although it also induces the expression of pro-apoptotic Bim.A STAT3 binding site was identified in the 2 kb promoter region upstream of Foxo transcription start site by TFBind software.Reporter and ChIP assays found that JEV infection in Neuro-2a cells reduced the expression of STAT3 and its interaction with Foxo promoter.Moreover,STAT3 knockdown decreased the expression of Foxo in Neuro-2a cells.Therefore,JEV reduced Foxo expression,at least in part,by downregulating STAT3.Taken together,we identifiedforthefirsttimethatJEVinducedapoptosisbyinhibiting STAT3-Foxo-Bcl-6/p21 pathway,which provided new insight into JEV-related encephalitis.We have found that the expression of anti-apoptotic proteins p21 was diversification when JEV induced apoptosis by inhibiting STAT3-Foxo-Bcl-6/p21 pathway.p21 is an anti-apoptotic proteins,also known as marker of senescence.Since JEV infection induced variety expression of p21,we next examined the markers level of senescence during JEV infection.We examined the expression of reactive oxygen species?ROS?and activity of?-galactosidase in JEV-infected Neuro-2a cell.Neuro-2a cells were infected with P3strain 24hours at an MOI of 1.The activity of?-galactosidase and expression of ROS was increased by JEV infection.Some Studies have shown that the endoplasmic reticulum stress response induces the production of ROS via the Ca²⁺pathway.Therefore,we used the calcium ion chelator BAPTA-AM to treat JE virus-infected Neuro-2a cells and found that the endoplasmic reticulum stress response activated and down-regulates the levels of ROS,p21 and p53 proteins in the cells.Thus,Japanese encephalitis virus-infected Neuro-2acellsinducereactiveoxygenspeciesproductionthroughthe Ca²⁺-ROS-p53-p21 pathway in the endoplasmic reticulum stress reaction,then induces cellular senescence.In conclusion,we for the first time find that Japanese encephalitis virus induces cellular senescence by activate Ca²⁺pathway of ER stress when Neuro-2a cells were infected with P3 strain 24hours at an MOI of 1.Furthermore,Japanese encephalitis virus induces apoptosis by inhibiting Foxo-p21/Bcl-6 signaling pathway when Neuro-2a cells were infected with P3 strain 72hours at an MOI of 5,which is,at least in part,mediated by downregulating STAT3 expression.These findings provide novel insight into the pathogenesis of Japanese encephalitis.