IL-22介导自噬信号通路抑制NLRP3炎性小体活化在药物性肝损伤中发挥肝保护效应的作用及机制研究

The incidence rate of drug-induced liver injury (DILI) is increased year by year. DILI has a great impact on public health and attracted much attention of clinicians. Some studies demonstrated NLRP3 inflammasome could promote and aggravate DILI by activating pro-inflammatory cytokines and inflammatory cytokine cascades. Meanwhile, inflammasome activation are restrained and regulated by autophagy. The update studies shown that IL-22 could activate autophagy, have an effect on endoplasmic reticulum stress and alleviate the injury from reactive oxygen species (ROS). Our previous study indicates that IL-22 plays a protective role in DILI not only by ROS suppression but inducing autophagy activation. However the precise mechanisms of which, remain unknown. Therefore, it is speculated that IL-22 could induce autophagy to restrain inflammasome activation, alleviate the liver injury by promote phosphorylation of STAT3, which could interaction with protein kinase R(PKR). Wild type, IL-22 gene knockout, liver–specific STAT3 gene knock-out mice will be used for drug-induced liver injury model, as well as ex vivo experiments would be adopted into the our study to explore the association among IL-22, autophagy and NLRP3 inflammasome. Furthermore, it would be clarified that the mechanisms of IL-22 induced the phosphorylation of STAT3 involved in autophagy regulating inflammasome in DILI.

药物性肝损伤(DILI)发病人数日益增多，严重威胁人类健康，备受临床关注。研究表明NLRP3炎性小体通过活化促炎因子发挥级联放大炎症反应介导DILI发生，同时NLRP3炎性小体的活化受到细胞自噬的调控。最新研究发现IL-22可介导下游自噬信号通路活化，影响内质网应激，减轻氧应激损伤。我们前期研究发现IL-22不仅通过抑制氧应激诱导的炎症反应发挥肝保护效应，而且可以增强自噬，但确切调控机制尚未完全明了。据此，我们推测IL-22可能通过STAT3的磷酸化，调节与下游信号蛋白PKR相互作用，激活自噬通路，减轻炎性小体活化进而发挥肝保护。本课题拟通过野生型、IL-22基因敲除、肝脏特异性STAT3基因敲除小鼠诱导DILI模型并结合体外实验了解IL-22、自噬和NLRP3炎性体之间的关联性，进一步阐明IL-22介导自噬信号通路抑制NLRP3炎性小体活化在保护DILI中的作用及相关机制。

药物性肝损伤发病人数日益增多，严重威胁人类健康，备受临床关注。本课题组前期研究发现IL-22在药物性肝损伤小鼠模型中，可抑制活性氧（ROS）的表达并减轻肝细胞损伤，但具体机制有待进一步阐明。本课题旨在探讨APAP介导的药物性肝损伤中，IL-22可能通过活化自噬通路下调炎性因子及炎性小体的表达，从而发挥肝细胞保护效应的具体机制。本研究首先采用CBA多因子检测测量血浆中IL-1β、TNF中的含量，以及qPCR检测对照组，APAP造模后6h组、APAP造模后24h组三组小鼠肝内炎症小体相关分子（ASC、IL-18、IL-1β、IL-33）的表达。APAP造模后小鼠肝内炎症小体相关分子IL-1β、ASC均有明显上调，提示小鼠药物性肝损伤模型中肝内炎症小体激活，发现IL-22干预组肝内IL-1β的表达较APAP组有明显下调。在前期PCR Array的研究基础上，发现IL-22处理组自噬蛋白p62的基因表达较APAP组有所升高。通过电镜、免疫组化及qPCR检测等方法，进一步发现APAP组和IL-22处理组肝内自噬小体的数量、LC3剪切体的表达及LC3B、p62的RNA表达较对照组均有明显升高，APAP联合IL-22处理组升高最为明显。上述结果进一步提示IL-22增强自噬的活化。为进一步评估肝内IL-22的表达及其来源，课题组通过流式分析发现，APAP造模后，小鼠肝内IL-22+细胞明显升高，初步研究揭示APAP造模小鼠肝内IL-22的升高来自于T淋巴细胞的激活和分泌。通过转染腺病毒 mRFP-GFP-LC3，观察IL-22与APAP和体外细胞共孵育后，对自噬的活化调控作用。通过测定IL-22处理组、APAP组小鼠肝内谷胱甘肽的含量，进一步阐明IL-22在对APAP的肝内代谢无明显调控作用。这些研究提示IL-22可增强自噬的活化下调炎性因子及炎性小体的表达，从而减轻药物性肝损伤中肝细胞的损伤坏死，产生肝脏保护效应，为研发药物性肝损伤的治疗药物提供新的靶点。