Study On The Effect Of JEV Infection Testicular Interstitial Cells Against IRF3 Signal Pathway

Japanese encephalitis（JE）,caused by the Japanese encephalitis virus（JEV）infection,is a natural zoonotic infectious disease transmitted by mosquitoes.It was named after the pathogen was first isolated in Japan in 1934 and is mainly transmitted in a human mosquito pig cycle in nature.The clinical manifestations of JEV infection mainly include neurological symptoms such as encephalitis,meningitis,and myelitis,with a mortality rate of up to 30%.30-50%of patients have permanent sequelae of central nervous system injury.Pigs are the most important natural proliferative host of JEVs.When pigs are infected with JEVs,sows may experience miscarriage and stillbirth;Male pigs exhibit unilateral or bilateral testicular enlargement,toxic semen,and loss of breeding ability.However,currently,research on the pathogenic mechanism of JEV mainly focuses on the inflammation of the central nervous system caused by JEV.There is relatively little research on the pathogenic mechanism of JEV on the reproductive system.As the main site for testosterone synthesis and secretion in male animals,the state of testicular interstitial cells is crucial for the health of the reproductive system in male animals.Therefore,this cell is used as an in vitro infection model for relevant research,It is of great significance for the reproductive system disorders caused by JEV infection.Studies have shown that JEV infection can activate RIG-I and TLR3 receptors,but the mechanism by which they trigger innate immunity during JEV infection is still unclear.Therefore,this study used both in vivo and in vitro cell experiments to infect mouse testicles and mouse testicular interstitial cells with JEV GZ strain,activate the RIG-I/TLR3 mediated IRF3 signaling pathway,and induce IFN-βThe secretion provides a theoretical basis for the host antiviral immune response mechanism caused by JEV infection.The main research findings are as follows1.The effect of JEV infection on the IRF3 signaling pathway in testicular tissue of miceIn this study,mice were used as an in vivo model of JEV infection.The JEV GZ strain（full gene Gen Bank number KC9150165）isolated and identified in our laboratory was used for intraperitoneal injection.The dose of inoculation was 100μL10⁴TCID₅₀per mouse.After 5 days of injection,the mice exhibited typical neurological symptoms such as mental depression,bristling,and motor disorders.Collect the testicles of mice with obvious symptoms after JEV vaccination,and perform RT-PCR and Western blot detection.The results showed that JEV NS1 nucleic acid and protein were detected in the testicles of the infected group mice,while the control group mice had no target bands in the testicles.The results indicate that JEV can infect mouse testes through intraperitoneal injection,and in vivo validation experiments can be conducted to activate the IRF3 signaling pathway in mouse testicular interstitial cells.The above results indicate that JEV infection in mice can activate the RIG-I/TLR3-IRF3 signaling pathway,phosphorylate IRF3,and induce IFN-βUpregulation of expression triggers the body’s innate immune antiviral immune response.After typical neurological symptoms appeared in the mice infected with JEV,the mice were euthanized and their testes were collected under sterile conditions.The m RNA transcription levels of RIG-I,TLR3,and IRF3 genes in the testes of the infected and control groups were detected using fluorescence quantitative PCR.Western blot was used to detect the protein expression of RIG-I,TLR3,IRF3,and p-IRF3.The fluorescence quantitative PCR results showed that compared with the control group,JEV infection can cause significant upregulation of the m RNA transcription levels of RIG-I,TLR3,IRF3,and p-IRF3 genes.Compared with the control group,RIG-I and IFN-βThere was a significant difference（P<0.05）;TLR3 and IRF3 showed extremely significant differences（P<0.001）.Western blot results showed a significant increase in the expression of RIG-I,TLR3,and IRF3 proteins（P<0.05）,and an extremely significant upregulation of IRF3 phosphorylation compared to the control group（P<0.01）.The above results indicate that JEV infection in mice can activate the RIG-I/TLR3-IRF3 signaling pathway,phosphorylate IRF3,and induce IFN-βUpregulation of expression triggers the body’s innate immune antiviral immune response.2.The effect of JEV infection on the IRF3 signaling pathway in mouse testicular interstitial cellsThis study used testicular stromal cells（TM3）as an in vitro JEV infection cell model,and JEV GZ strain was used to challenge TM3 cells.After 24 hours of exposure,significant cytopathic changes were observed,with cells exhibiting shrinkage,shedding,and ultimately lysis and death.RT-PCR and indirect immunofluorescence methods were used to detect NS1 protein and nucleic acid in testicular interstitial cells.The results showed that the nucleic acid electrophoresis and sequencing results after RT-PCR showed that the target band of JEV NS1 gene could be detected,while indirect immunofluorescence methods could detect JEV NS1 protein fluorescence signals in TM3 cells after exposure.The results indicate that JEV can infect TM3 cells and produce typical cytopathic effects.After infecting TM3 cells with JEV at a dose of 1 MOI,fluorescence quantitative PCR was used to detect the m RNA transcription of RIG-I,TLR3,and IRF3.The results showed that JEV infection upregulated the m RNA transcription levels of each gene,and the difference was most significant at 12 hours of infection（P<0.05）.Western blot results showed that compared with the control group,JEV infection could significantly increase the expression of RIG-I,TLR3,and IRF3 proteins（P<0.05）,and IRF3phosphorylation was significantly upregulated（P<0.001）.Nuclear protein and cytoplasmic protein extraction kits were used to extract nuclear protein and cytoplasmic protein from JEV infected TM3 cells,and Western blot was performed to detect the expression of IRF3 protein in the nucleus and cytoplasm.Indirect immunofluorescence method was also used to observe the cellular localization of IRF3 protein in TM3 cells after JEV infection.The results showed that after JEV infection with TM3,there was a significant upregulation of IRF3 protein expression in the nucleus,which is consistent with the trend of upregulation of IRF3 protein expression in the total cell protein.The indirect immunofluorescence results showed that the IRF3 fluorescence signal in the nucleus of JEV infected TM3 was significantly enhanced.The results showed that JEV infected TM3 could activate the RIG-I/TLR3-IRF3 signaling pathway,and the activated RIG-I and TLR3 could stimulate IRF3phosphorylation and induce IRF3 entry into the nucleus.3.JEV activates the IRF3 signaling pathway in TM3 cells and affects interferonβEffect of secretionTo confirm the activation effect of JEV on the IRF3 signaling pathway,si-RNA was used to silence the pattern recognition receptors RIG-I and TLR3 upstream of IRF3in TM3 cells.si-RNA targeting RIG-I and TLR3 was transfected into TM3 cells,interfering with the m RNA transcription of RIG-I and TLR3 to inhibit the expression of RIG-I and TLR3 proteins.After transfection with si-RNA,Western blot was used to detect the protein expression of RIG-I and TLR3.The results showed that after 36 hours of transfection with si-RNA,the expression of RIG-I and TLR3 proteins was significantly downregulated（P<0.01）,indicating that si-RNA can effectively inhibit the protein expression of RIG-I and TLR3.To investigate the effect of JEV on the expression of IRF3 protein and its downstream interferon after silencing RIG-I and TLR3βThe effect of secretion was evaluated by Western blot and ELISA methods after transfection with si-RNA and targeting TM3 cells at a dose of 1 MOIβConduct testing.Western blot results showed that silencing RIG-I and TLR3 significantly reduced the expression and phosphorylation of IRF3 protein（P<0.01）,but still showed extremely significant differences compared to the control group that was not challenged（P<0.01）.ELISA method for detecting the impact of JEV infection on IFN-βThe effect of secretion,the results show:IFN after JEV infection-βThe expression was significantly increased（P<0.001）,but silencing RIG-I and TLR3 significantly inhibited IFN-βSecretion of（P<0.001）.The results indicate that JEV infection with TM3 can activate the RIG-I/TLR3-IRF3 signaling pathway,stimulate upregulation of IRF3 protein expression and phosphorylation by activating RIG-I and TLR3,and induce IFN-βIncreased secretion.