The Establishment Of The Model Of JEV Infection In Leydig Cells And Its Effect On Testosterone Secretion

Japanese encephalitis virus（JEV）,a positive-sense,single-stranded RNA virus,belonging to the genus Flavivirus of the family Flaviviridae,JEV is a zoonotic,vector-borne virus,spread primarily by mosquitoes,JE is a legally reported infectious disease in China.The clinical manifestation of human infected JEV is viral encephalitis,patients are characterized by high fever,headache,stiffness of neck muscles,disorientation,seizures,paralysis,coma,and eventually death.It causes clinical symptoms in humans,including high fever,headache,stiffness of neck muscles,disorientation,seizures,paralysis,coma,and eventually death.Permanent neurologic or psychiatric sequelae can occur in 30-50%of cases.Pigs are the major virus-amplifying host and nature host for virus,JEV infects pigs can cause high viraemia.Mosquitoes can be infected after biting pigs with viremia,and then transmit the virus to humans or pigs through the bites.Therefore,the circulation mode of pig-mosquito-human,pig-mosquito-pig is often presented during the spread of JE.Therefore,the prevention and control of JE in pigs is not only a requirement for the healthy development of the breeding industry,but also a major requirement for safeguarding human health.Inflammation underlies a wide variety of physiological and pathological processes.Orchitis is the most obvious feature of the clinical symptoms of JEV-infected boars,and it is also an important reason for the decline of boar semen quality and the inability to breed.However,the molecular mechanism of JEV-induced orchitis is still unclear.In this study,the establishment of the Leydig cell model of JEV infection,the effect of JEV infection on Leydig cells on inflammatory factors and testosterone secretion,and the effect of JEV infection on RIG-Ⅰ signaling pathway will be investigated to explore JEV infection.Molecular mechanisms of boar orchitis and its effect on testosterone secretion.This study is divided into the following three parts:1.Establishment of JEV infected TM3 cell modelVirus infection cell model is the most commonly used to study the pathogenesis of viruses.In this study,we used the JEV GZ strain（Gen Bank number:KC9150165）inoculated TM3 at 12 h,24 h,36 h,48 h,and 60 h to observe the cytopathic effect（CPE）.Rounding and detachment of cells in infected TM3 was noticed at 36 h of inoculation,the CPE phenomenon aggravated with the inoculation time:a large number of cells became round and shrunken,shedding,voids appeared between cells,and cells died by lysis.JEV-specific NS1 proteins were detected by RT-PCR and Western blot by collecting cell suspension and cell lysate proteins.These results suggest that JEV could productively infect TM3 cells.JEV infected TM3 cells（MOI=1）,collected supernatants at different time points for TCID₅₀ determination.It was found that JEV replicates in TM3 cells to produce new virions.The highest virus titer in the liquid reached 10^-5.8 TCID₅₀/m L,and then the virus titer gradually decreased.The results showed that:JEV can replicate and proliferate in TM3 cells.The in vitro model of JEV infection of TM3 cells was successfully established.2.Effects of JEV infection on inflammatory factors and testosterone secretion in TM3cellsElevated inflammatory factors in testicular tissue are the main features of orchitis.In order to explore the effect of JEV infection on TM3 cells on their inflammatory factors and testosterone secretion.We used JEV to infect TM3 cells（MOI=1）collected the cell supernatants and cell lysates at 6 h and 12 h to detect the contents of IL-6,IL-1βand TNF-αby ELISA.The results showed that:JEV infected TM3 cells could increase IL-6 and decrease testosterone secretion in the cell supernatant,which was significantly different from the control（P<0.01）TNF-αand IL-6 did not change,moreover JEV did not cause changes in the three inflammatory factors in the lysate.The results showed that JEV could induce cellular inflammatory and suppress testosterone secretion.3.Study on the effect of JEV on RIG-Ⅰsignaling pathway after infecting TM3 cellsA large number of studies have reported that the RIG-Ⅰ innate immune signaling pathway is closely related to the pathogenesis and infection of JEV.In order to explore the effect of JEV infection on TM3 cells on the RIG-Ⅰ signaling pathway,we used JEV infected TM3cells（MOI=1）extracted total RNA and protein,q-PCR and Western blot detected transcription level and protein of RIG-Ⅰchange.The results showed that JEV could up-regulate the transcription and expression of RIG-Ⅰ after infection of TM3 cells.The lysates of TM3 cells were collected to detect the activation of NF-κB signaling pathway downstream of RIG-Ⅰ,and the results showed that JEV infection could increase the level of p-IκBαprotein and the NF-κB signaling pathway was activated,indicating that The RIG-Ⅰ-NF-κB signaling pathway is involved in the inflammatory response of TM3 cells induced by JEV infection.ELISA detected IL-6 and testosterone secretion after JEV challenge in si RNA-treated cells,proved silencing of RIG-Ⅰ could significantly down-regulate the expression of IL-6 induced by JEV infection（P<0.05 compared with the challenge group）,and restore the level of testosterone secretion（P<0.05 compared with the challenge group）,but the expression levels of both were comparable to those in the control group still has a very significant difference P<0.01.The activation of RIG-Ⅰ-NF-κB signaling pathway was detected by Western blot,and the results showed that the protein expressions of RIG-Ⅰ and p-IκBαdecreased after silencing RIG-Ⅰ.These results indicate that the inflammation induced by JEV was regulated by RIG-Ⅰ-NF-κB,and silencing of RIG-Ⅰ receptor can inhibit the inflammation induced by JEV infection of Leydig cells and weaken the inhibitory effect of JEV on Leydig cells testosterone secretion.