Study On The Mechanism Of RIG-1 Signal Pathway In Japanese Encephalitis Virus Induced Cell Apoptosis

Japanese encephalitis virus(JEV)can cause damage to the central nervous system in humans and animals,and reproductive disease in pigs.JEV crosses the blood-brain barrier and causes Japanese encephalitis(JE),which is characterized by neuronal damage and poses a serious threat to public health and safety.The Japanese encephalitis(JE)poses a serious threat to public health and safety.Apoptosis is a genetically regulated form of cell death that plays a critical role in the development of the organism and in the maintenance of its homeostasis.Apoptosis is involved in JEV-induced neuronal damage.Therefore,it is important to investigate the mechanism of JEV-induced apoptosis to alleviate brain tissue damage.In this study,we used RNA-Seq technology to find the differences in gene expression at the transcriptional level in brain tissues of JEV-infected mice,screened the differentially expressed genes and signaling pathways related to apoptosis,analyzed the regulatory effects of the screened signaling pathways on apoptosis in mouse microglia at the cellular level,and explored the regulatory mechanisms of signaling pathways on apoptosis in the organism,in order to elucidate the molecular mechanisms of JEV-induced brain injury for providing a theoretical basis for the elucidation of the molecular mechanism of JEV-induced brain injury.Main findings:1.Transcriptome differential analysis of JEV-infected brain tissueIn this study,the 6-week-old BALB/C female rats were infected with 105 PFU of JEV P3 strain,and the pathological changes of brain tissue were observed by HE staining,which revealed significant inflammatory responses such as neuronal degeneration and necrosis,glial cell proliferation and perivascular inflammatory cell aggregation in the brain.Immunohistochemical staining technique was used to observe most of the neuronal cells positive for JEV antigen.Using QPCR techniques found that Bax m RNA expression in the JEV infection group is higher than the control group and Bcl-2 m RNA expression is lower than the control group.Transcriptome analysis screened a total of 2468 differentially expressed genes,mostly associated with signal transduction and immune response.By GO functional enrichment analysis,these genes were involved in biological processes related to viral infection,innate immunity and host defense responses.By KEGG enrichment analysis,the enriched pathways were directly or indirectly associated with antiviral responses,mainly focusing on JAK-STAT signaling pathway,Toll-like receptor signaling pathway,NF-κB signaling pathway,TNF signaling pathway and cytokine-cytokine receptor interactions.The QPCR validation results of several selected genes showed highly significant correlation with RNA-Seq.It indicates that JEV infection activates innate immune response and inflammatory response.2.Effect of JEV infection on apoptosis-associated proteins in microglia cellUsing KEGG enrichment analysis,11 apoptosis-related signaling pathways were identified in vivo in response to JEV infection,including protein processing in the endoplasmic reticulum,NF-κB,JAK-STAT,chemokines,RIG-1,PI3K/Akt,p53,MAPK,Foxo,Ras,and c AMP signaling pathways.Light microscopy showed that JEV infection of BV2 cells caused significant cytopathic lesions.Using indirect immunofluorescence,JEV was found to increase the activation level of microglia.Nuclear consolidation and darkening of staining were found in JEV-infected cells by Hoechst 33342 staining.Western blot technique revealed that Bcl-2 protein expression was reduced and Bax protein expression was increased in the JEV-infected group,while JEV increased the release of cytochrome c and upregulated apoptosis-related proteins cleaved caspase-3 and cleaved caspase-9.These results suggest that JEV can cause mitochondria in BV2 cells at late stages of infection dependent apoptotic response in BV2 cells at late stages of infection.3.Effect of interfering with RIG-1 expression on viral replication and microglial cell apoptosisUsing QPCR and Western blot technique,we found that RIG-1,MAVS,IKK?,p-TBK1,p-NF-κB(p-p65)and p-IRF3 in JEV-infected BV2 cells were elevated at 24 h and 36 h and then gradually decreased.Viral protein levels increased at 48 h and 60 h and were not significantly expressed at 24 h and 36 h.p-Akt and p m TOR were elevated and then decreased.The level of p62 protein was increased,the level of MDM2 protein expression was decreased,and the level of p53 protein was increased in the late stage of infection.This indicates that JEV infection can activate the RIG-I signaling pathway,activate the p53 signaling pathway and activate the Akt-m TOR pathway in the early stage of infection.The expression of RIG-1 protein in BV2 cells was interfered by RNA interference,and the expression of pro-apoptotic proteins Bax,cleaved caspase-3 and cleaved caspase-9 was decreased,the expression of anti-apoptotic protein Bcl-2 was increased,and the expression of viral proteins was decreased by Western blot technique.This suggests that interference with RIG-1expression can inhibit viral replication and ensure cell survival by preventing mitochondrial dysfunction.Based on the results of the study,the following conclusions can be drawn.1.JEV infection causes apoptosis,which is involved in the typical neurological symptoms in mice.2.JEV activate the Akt/m TOR pathway in mouse microglia early in infection,triggering survival signals.3.The RIG-1 signaling pathway is involved in JEV-induced apoptosis through a mitochondria-dependent pathway,and interference with RIG-1 expression leads to reduced replication of JEV.