VSIG4抑制MHV-3病毒感染诱发的暴发型肝衰竭的分子机制研究

Macrophages (Mφs) and their secreted proinflammatory factor can aggravate the pathogenesis of fulminant hepatic failure (FH) induced by murine hepatitis virus 3(MHV-3), which can be delayed by inhibiting the inflammation caused by macrophages. V-set and Ig domain-containing 4(VSIG4) is a co-inhibitory molecule specifically expressed on the surface of tissue macrophages, such as peritoneal macrophages and kupffer cells. In our previous study, we found that VSIG4 deficiency exacerbates MHV-3 induced liver injury and mortality and which is depend on IL-1β. VSIG4 deficiency macrophages produce more reactive oxygen species (ROS) which increase NLRP3 inflammasom and secrete more IL-1β than WT. These results indicated that VSIG4 maybe contribute to delay the pathogenesis of FH by down-regulating the expression of ROS. However，the molecular mechanism about VSIG4 regulate ROS is not yet clear. In this study， we want to infect p47phox knockout mice with MHV-3 to analyze the role of ROS in FH induced by MHV-3, transfect RAW264.7 cells with VSIG4 expression vector to study the mechanism about VSIG4 regulate ROS using qPCR, Western Blot and EMSA, screen the proteins interacting with VSIG4 using TAP-chip and co-IP to identify the new effector molecules of VSIG4 targeting. In addition, the probably therapeutic effect of VSIG4 signal pathway should be evaluated by using VSIG4 agonist antibodies or ROS inhibitors treatment in vivo. This study will clarify the mechanism of VSIG4 involved in regulating the process of FH, which provide a new strategy to the therapy of FH.

巨噬细胞(Mφs)及其分泌的炎症因子可加剧鼠肝炎病毒株-3(MHV-3)诱发的暴发型肝衰竭（FH）。VSIG4是特异表达于Mφs表面的免疫分子。前期研究我们发现， MHV-3可刺激VSIG4缺陷Mφs产生过量活性氧（ROS）、导致NLRP3炎症小体活化及IL1β分泌,最终引发VSIG4 KO小鼠的快速死亡。但VSIG4调控ROS分泌的机制尚未知。本研究拟:使用p47phox KO小鼠感染MHV-3，明确ROS对MHV-3所致FH的影响；转染RAW264.7细胞表达VSIG4，利用qPCR，WB等分析VSIG4调节ROS分泌的分子机制；使用Co-IP、TAP-质谱技术鉴定VSIG4调控NAPDH氧化酶活性可能的新效应分子；在体给予ROS阻断剂或VSIG4激动型抗体，探讨基于VSIG4信号对FH的干预效果。本研究将阐明VSIG4抑制FH病理进程的作用机制，并为FH的临床免疫干预提供新策略。

巨噬细胞(Mφs)及其分泌的炎症因子可加剧鼠肝炎病毒株-3(MHV-3)诱发的暴发型肝衰竭（FH）。VSIG4是特异表达于Mφs表面的免疫分子。前期研究发现， MHV-3可刺激VSIG4缺陷Mφs产生过量活性氧（ROS）、导致NLRP3炎症小体活化及IL1β分泌,最终引发VSIG4 KO小鼠的快速死亡。但VSIG4调控ROS分泌的机制尚未知。本研究通过建立NADPH氧化酶p47亚基（p47phox）KO小鼠的MHV-3感染模型，比较ROS分泌及生存曲线等的变化，发现P47phoxKO小鼠感染MHV-3病毒后ROS减少，生存率提高，肝脏病变减轻；为明确VSIG4调节巨噬细胞介导炎症的分子机制，我们构建了稳定表达VSIG4的RAW264.7细胞系并分析其LPS刺激下的极化及代谢情况，结果表明VSIG4可以通过重编程巨噬细胞丙酮酸代谢及mtROS的产生进而抑制LPS诱导的IL-1β、IL-6、TNF等M1细胞因子及CD40的表达；为了明确VSIG4是如何影响丙酮酸及mtROS，我们检测了影响丙酮酸代谢的关键酶—丙酮酸脱氢酶（PDH）的表达及与其活性密切相关的PDH的磷酸化水平，结果显示与野生型对照小鼠相比，VSIG4 KO小鼠BMDMs上PDK2的mRNA表达水平明显下调，而PDK家族的其它3个分子PDK1、PDK3、PDK4在野生型小鼠和VSIG4敲除小鼠中没有差异；为明确VSIG4在体内的功能，我们给予MHV-3易感的C57BL/6野生型小鼠尾静脉注射过表达VSIG4的慢病毒和对照慢病毒，然后两组小鼠同时腹腔注射MHV-3病毒，感染后72h后检测肝脏炎症因子、FGL2、PDK2等相关分子的表达水平。结果表明，VSIG4过表达小鼠PDK2表达和PDH磷酸化水平明显增高，TNF-α、IL-6、IL-1beta的表达以及FGL2在肝脏的成绩明显受到抑制，肝脏病理明显减轻，小鼠生存率明显提高。综上，在巨噬细胞的活化过程中，VSIG4通过PI3K/Akt-STAT3 信号级联上调丙酮酸脱氢酶激酶2（pyruvate dehydrogenase kinase-2，PDK2）的表达，抑制丙酮酸脱氢酶（pyruvate dehydrogenase，PDH）依赖的线粒体ROS的产生，负调巨噬细胞的M1极化。VSIG4信号能够缓解FH的进程，为FH的治疗提供新靶点。