日本脑炎病毒(JEV)感染睾丸间质细胞对TLRs信号通路的影响及其调控炎症因子的分泌  

Effects of JEV infection on TLRs signaling pathway and its regulation on secretion of inflammatory factors in Leydig cells

作　　者：何松 晏仁潭 汤德元[1] 曾智勇[1] 王彬[1] 毛茵茗 周飘 廖正波 陈旭 袁盛林 胡雯雯 周敏 HE Song;YAN Rentan;TANG Deyuan;ZENG Zhiyong;WANG Bin;MAO Yinming;ZHOU Piao;LIAO Zhengbo;CHEN Xu;YUAN Shenglin;HU Wenwen;ZHOU Min(College of Animal Science,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025

出　　处：《中国兽医学报》2024年第11期2409-2417,共9页Chinese Journal of Veterinary Science

基　　金：贵州大学重点资助项目(黔科合平台人才[2018] 5781-8)

摘　　要：为了探究日本脑炎病毒(JEV)在感染睾丸间质细胞过程中对TLRs信号通路的影响及其调控炎症因子的分泌情况,本研究以1 MOI剂量的JEV接种睾丸间质细胞后,使用qPCR方法检测不同时间段TLR3、TLR7、TLR8、TRIF和MyD88基因的mRNA水平,Western blot方法检测JEV感染睾丸间质细胞6 h时TLR3、TLR7、TRIF和MyD88蛋白表达水平,ELISA检测不同时间段(6、12、24 h)IL-1β、IL-6和TNF-α的表达情况。结果显示:JEV感染睾丸间质细胞6 h后显著上调TLR3、TLR7、TRIF和MyD88基因mRNA水平(P<0.05),下调TLR8基因mRNA水平(P<0.05);Western blot结果显示,JEV感染睾丸间质细胞6 h时TLR3、TLR7、TRIF和MyD88蛋白表达均显著上调(P<0.05),与相应mRNA转录水平结果一致;TLR8蛋白表达变化不显著。ELISA检测结果显示,JEV感染睾丸间质细胞6 h后IL-6极显著增加(P<0.01),IL-1β和TNF-α表达无显著变化。利用siRNA分别对TLR3、TLR7、TLR8、TRIF和MyD88进行基因沉默,对沉默后的细胞接种JEV 6 h时取上清液ELISA检测IL-6表达水平。结果显示,沉默TLR3、TLR7、TLR8、TRIF和MyD88后均可显著减轻JEV感染引起的IL-6分泌增加(p<0.05),表明JEV感染睾丸间质细胞后可通过激活TLR3、TLR7和TLR8信号通路诱导炎症因子IL-6的表达。本研究为深入阐明JEV感染导致的繁殖障碍机制提供了参考。This study aims to investigate the effects of Japanese encephalitis virus(JEV)on TLRs signaling pathway and its regulation of the secretion of inflammatory factors during the infection of testicular interstitial cells,In this study,the mRNA levels of TLR3,TLR7,TLR8,TRIF and MyD88 genes were detected by qPCR after 1 MOI dose of JEV was inoculated into testicular stromal cells at different time periods.Western blot assay was used to detect the expression levels of TLR3,TLR7,TRIF and MyD88 protein at 6 h after JEV infection,and ELISA was used to detect the expression levels of IL-1β,IL-6 and TNF-αat different time periods(6,12 and 24 h).The results showed as follows:After 6 h of JEV infection,the mRNA levels of TLR3,TLR7,TRIF and MyD88 genes were significantly up-regulated(P<0.05),and the mRNA levels of TLR8 genes were down-regulated(P<0.05).Western blot results showed that the protein expressions of TLR3,TLR7,TRIF and MyD88 were significantly up-regulated when JEV infected testicular stromal cells for 6 h(P<0.05),which was consistent with the corresponding mRNA transcription levels.There was no significant change in TLR8 protein expression.ELISA results showed that 6 h after JEV infection of testicular stromal cells,IL-6 was significantly increased(P<0.01),and the expressions of IL-1βand TNF-αwere not changed.TLR3,TLR7,TLR8,TRIF and MyD88 were silenced by siRNA,and the silenced cells were inoculated with JEV for 6 h,and IL-6 expression levels were detected by ELISA.The results showed that silenced TLR3,TLR7,TLR8,TRIF and MyD88 could significantly reduce the increase of IL-6 secretion induced by JEV infection(P<0.05).These results indicated that JEV could induce the expression of inflammatory factor IL-6 by activating TLR3,TLR7 and TLR8 signaling pathway after infection of testicular stromal cells.This study provides a reference for further elucidating the mechanism of reproductive disorders caused by JEV infection.

关 键 词：日本脑炎病毒 睾丸间质细胞 TOLL样受体 炎症因子 

分 类 号：S852.65[农业科学—基础兽医学]