Study On The Role And Mechanism Of H3k27me3 Modification In Neuroinflammation Induced By Japanese Encephalitis Virus

Japanese encephalitis virus(JEV)is a neurotropic pathogen of zoonotic infectious diseases.It infects human mainly through mosquitoes,which can invade the central nervous system and cause severe viral encephalitis.Infection of pigs can lead to breeding disorders and neonatal piglet encephalitis.The "inflammatory storm" induced by the central nervous system is the main pathological feature after Japanese encephalitis virus infection,but the regulatory mechanism of its induction of neuroinflammatory response remains to be further clarified.Research have shown that histone methylation modification plays an important role in the pathogenesis of neuroinflammatory bowel disease,and histone methylation enzyme EZH2-mediated H3K27 trimethylation is closely related to the control of inflammatory response,but its regulation mediated JEV infection also plays a role in neuroinflammation,the specific mechanism remains unclear.Thence,this study attempts to explore the role of EZH2-mediated histone modifications in the microglia in the process of inflammation induced by JEV.The primary contents as follows:1.Up-regulation of H3K27 tri-methylation upon JEV infectionThe protein level of H3K27me3 after JEV infection was measured by immunoblotting in microglia(BV2)and primary microglia cells(PM).The results manifested that JEV infection could induce significant upregulation of H3K27me3.In addition,the brain tissues of mice infected with JEV P3 were collected,after that the relationship between H3K27me3 level and JEV infection in vivo was tested by immunoblotting.The results explained that JEV infection could also induce significant upregulation of H3K27me3 levels in rat brain tissue.These studies suggest that JEV infection can up-regulate the level of H3K27 trimethylation.2.EZH2-mediated H3K27me3 plays a positive role in JEV-mediated inflammatory responseTo analyze the the mechanism of the up-regulation of H3K27me3 by JEV,we infected BV2 cells and mouse primary glial cells with JEV,respectively,and then detected the expression level of methyltransferase EZH2 of H3K27me3 by q RT-PCR and Western Blot.The results showed that the level of EZH2 was slightly up-regulated but not significantly up-regulated with that of H3K27me3,so we inferred that EZH2 might play a mediated role in this reaction.According to this,CRISPR-Cas9 technology was employed to construct knockdown cell lines of EZH2 gene in microglia cells.After JEV was infected with this cell line,Western Blot was used to verify the effect of EZH2 reduction on H3K27me3 level.The results proved that EZH2 mediated and participated in the reaction of JEV upregulation of H3K27me3.Then,we explored the effect of EZH2-mediated H3K27me3 on JEV-induced neuroinflammation,and detected the expression of JEV-induced inflammatory cytokines in EZH2 knockdown cell lines constructed above by q RT-PCR.The results showed that EZH2 knockdown inhibited the expression of H3K27me3 by JEV,thus inhibiting the level of JEV-induced inflammatory cytokines.On this basis,we use EZH2 specific inhibitors of GSK-343 processing microglia and the generation of glial cells,and then infect JEV and use the q RT PCR,Western Blot method to detect the levels of H3K27me3 and the expression of inflammatory factor,it is found that EZH2 specific inhibitors of GSK-343 can significantly inhibit JEV promote H3K27me3 levels,and can inhibit the expression of inflammatory factor levels.These experiments indicate that EZH2-mediated H3K27me3 can positively regulate JEV-induced neuroinflammatory response.3.EZH2-mediated H3K27me3 regulates JEV-induced inflammatory response by targeting RNF19ATo further reveal the molecular mechanism of H3K27me3 regulating JEV-induced inflammatory response,we analyzed it by Ch IP-seq and found that RNF19 a may be the target gene modified by H3K27me3.When EZH2 was knocked down,the inhibitory effect of H3K27me3 on RNF19 A was also weakened,which proved that EZH2-mediated H3K27me3 modified RNF19 A through methylation and thus inhibited its transcription level.At the same time,we inhibited and overexpressed RNF19 A at the cellular level,and found that inhibition of RNF19 A can promote JEV-induced inflammatory response.However,overexpression of RNF19 A can significantly inhibit JEV-induced inflammatory response.The above experiments showed that EZH2-mediated H3K27me3 regulates JEV-induced inflammatory response by targeting RNF19 A.In conclusion,the data presented herein demonstrate that histone methylation modification in the process of JEV infection to mediate the action of inflammatory response,revealing that EZH2-mediated H3K27me3 by targeting RNF19 A plays an important regulatory mechanism in inflammation,preliminary illustrates the JEV by histone modification the molecular mechanisms regulating nerve inflammation.