乙肝病毒新基因HBwx介导的炎症体反应和肝细胞焦亡在肝衰竭中的作用及机制

Liver failure is a life-threatening clinical syndrome with high mortality. In China, the predominant cause is hepatitis B virus(HBV) infection. The precise mechanism underlying liver failure remains unclear. Liver failure (herein referred to as acute, subacute and acute-on-chronic liver failure) characterized by massive/submassive hepatic necrosis and dysregulated inflammation. The HBV DNA genome is about 3.2 kb and contains four open reading frames (ORFs). A novel ORF with 168 nucleotides upstream of the starting code of the X gene has been identified and termed as Pre-X. The novel ORF Pre-X has transcriptional activity and can be translated with the X gene in frame to form the HBV whole-X gene (HBwx). The molecular function and involvement in diseases of the HBwx remains uncharacterized. In the previous funding cycle(NSFC 30671862), our lab generated a substantial amount of data characterized the molecular function of HBwx. In vivo and in vitro studies failed to link HBwx to liver cancer. Further explorative studies shown a robust HBwx staining in liver tissues from patients with liver failure. Secondly, HBwx expressing cells were more sensitive to ADM and LPS induced necrosis. Thirdly, a synergistic inflammatory effect were observed in HBwx expressing cells treated with LPS. Moreover, co-immunoprecipitation revealed a directed interaction between HBwx and HSP90, a chaperone protein and modulator of NLRP3 inflammasome. Aberrant activation of inflammasome has been linked to the pathogenesis of ischemia-reperfusion liver injury and fibrosis..We hypothesis that HBwx functions in liver failure through NLRP3 inflammasome response and pyroptosis in a HSP90 dependent manner. Our central hypothesis will be tested by four specific aims: (1)Map the binding domains required to mediate interacting each other; explorer whether the binding ability will be strengthen or attenuated in the presence of inflammatory promoting factors or HSP90 inhibitors. (2)We will elucidate whether HBwx induced inflammasome activation and pyroptotic cell death plays a role in liver failure. We will also explore the mechanism of HBwx by testing inflammasome assembly, maturation and releasing of pro-inflammatory factors and pyroptosis by specific inhibitors or genetic manipulating with CRISPR/Cas9 gene editing system.(3)We will utilize genetically manipulated mice expressing HBwx in hepatocytes to explore HBwx function in liver failure mouse model and elucidate the molecular pathway by which HBwx inducing inflammasome and pyroptosis. (4) To confirm the HBwx-HSP90-NLRP3 inflammasome/pyroptosis pathway by testing the related molecular in fresh liver tissue from patients diagnosed with liver failure. This contribution will uncover molecular function of HBwx, re-evaluate inflammatory function of hepatocytes, better our understanding of the molecular basis of liver failure and be widely applicable to other HBV related inflammasomopathies.

肝衰竭是临床常见症候群，病死率极高，在我国首要病因是乙型肝炎病毒（HBV）感染。肝衰竭发病机制尚不清，其特点是肝细胞坏死和炎症风暴。HBV X基因上游存在168bp的前X，与X无终止密码子，前X和X合称whole x（HBwx）。前一期国科金资助下我们发现HBwx：不致瘤、在肝衰竭肝组织高表达、促进肝细胞死亡、增强LPS诱导的炎症、结合HSP90，后者调节NLRP3炎症体和焦亡。基于上述结果，我们提出HBwx-HSP90-NLRP3炎症体/焦亡途径促进肝衰竭发展。本课题①定位HBwx/HSP90结合位点；②在细胞模型明确HBwx对炎症体组装、前炎症因子释放和焦亡作用，用抑制剂和基因敲除探索HBwx诱导炎症体和焦亡的机制；③在肝衰竭动物模型研究HBwx诱导的炎症体和焦亡的作用；④在肝脏组织验证上述通路。以阐明HBwx功能，再认识肝细胞的炎症功能，丰富肝衰竭发病理论，为其他炎症疾病提供思路。

研究背景：肝衰竭是病毒性、酒精性、药物性以及缺血再灌注等多种因素引起的严重肝脏损害.随着内科综合治疗的提升和人工肝、肝移植等治疗手段的普及，肝衰竭患者的存活率较前显著提高，但仍不高于60%，肝衰竭是临床治疗和基础研究的难点与热点。肝衰竭发病过程中，肠道粘膜屏障破坏导致肠道通透性增加，肠内多种细菌产物和炎症介质经门静脉移位至肝脏，激活肝内免疫细胞产生炎症因子，诱发肝脏炎症反应，造成肝细胞损伤；而肝星状细胞（HSCS）的NLRP3炎症小体能被多种炎症介质激活，产生并释放炎症因子。据此，课题组以肝衰竭为疾病背景，研究炎症与自噬（自噬蛋白Atg13）在肝衰竭中的作用及其相互调控机制；重点在细胞水平展开机制研究，进而在人体水平验证。.研究结果：①LPS抑制自噬/线粒体自噬促进HSCs炎症，进而诱导肝细胞凋亡, 参与肝衰竭疾病进程；②LPS通过p38诱导Atg13磷酸化（Ser416），磷酸化Atg13从Atg13-Ulk1复合体解离，抑制自噬促进HSCs炎症，即LPS通过“p38-Atg13-自噬”促进HSCs炎症；③LPS通过ROS抑制线粒体自噬促进HSCs炎症，即LPS通过“ROS-线粒体自噬”促进HSCs炎症；④ACLF患者血清LPS水平升高，高LPS水平与患者不良预后相关；LPS抑制自噬促进HSCs炎症，进而诱导肝细胞凋亡，参与肝衰竭的疾病进程。.结论：肝衰竭患者血清LPS水平升高，高LPS水平与患者不良预后相关；LPS通过ROS抑制线粒体自噬促进HSCs炎症，即LPS通过“ROS-线粒体自噬-HSCs炎症”途径参与肝衰竭的发病过程，提示调控氧化应激或线粒体自噬可能成为肝衰竭治疗的新的研究方向。