乙肝病毒新基因HBwx通过外泌体介导的线粒体自噬在肝星状细胞活化中的作用

Liver fibrosis is a common pathological change in many liver diseases, in China, hepatitis B virus (HBV) infection is the predominant cause. Hepatic stellate cells (HSCs) activation plays a central role in liver fibrosis, during which two characteristics exist: 1) HBV cannot infect HSCs directly, suggesting ways existed during pathogenesis of hepatitis B cirrhosis; 2) Apoptosis is NOT evidenced even mitochondria are functional overloaded, suggesting mechanisms exited in eliminating those overloaded mitochondria. 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 & 81570528), 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 hepatitis B cirrhosis. Secondly, HBwx was found in exosomes from blood samples of hepatitis B cirrhosis patients. Thirdly, HBwx expressing in liver cells inhibited apoptosis induced by overloaded mitochondria. Fourth，molecular function analysis revealed interaction of HBwx and mitochondrial proteins. Moreover, we found mitophagy in activated HSCs...We then hypothesis that HBwx functions in hepatitis B cirrhosis through regulation mitophagy via exosomes. Our central hypothesis will be tested by 3 specific aims: 1. We will elucidate whether HBwx regulate mitophagy and HSCs activation via exosomes. We will also explore the role of exosomes by specific inhibitors or genetic manipulating with CRISPR/Cas9 gene editing systerm; 2. As we have already utilized genetically manipulated mice expressing HBwx in liver, we will use these mice to explore function of HBwx in liver cirrhosis model and elucidate the molecular pathway; 3. To confirm the HBwx-exosome-mitophagy-HSCs activation pathway by testing the related molecular in fresh liver tissue from patients with hepatitis B cirrhosis. This contribution will uncover molecular fuction of HBwx, better our understanding of the molecular basis of liver cirrhosis.

肝纤维化是多种肝脏疾病共同的病理改变，在我国HBV感染仍是首要病因。肝星状细胞（HSCs）活化是肝纤维化的中心环节，HSCs活化过程存在两个突出特点：HBV不直接感染HSCs；HSCs线粒体超负荷但不发生凋亡。HBV X基因上游存在168bp的前X，前X和X合称全X（HBwx）。先前我们研究发现HBwx：表达于肝纤维化组织、存在于乙肝肝硬化患者血清外泌体、抑制线粒体损伤所致肝细胞凋亡、与线粒体相关蛋白相互作用，并在活化HSCs中检测到线粒体自噬。基于上述结果，我们提出“HBwx-外泌体-线粒体自噬-HSCs”途径促进肝纤维化发展的假说。本课题将1在细胞模型明确HBwx通过外泌体途径调节线粒体自噬和HSCs活化的作用，并探究作用机制；2在肝纤维化动物模型研究HBwx通过外泌体调节线粒体自噬和HSCs活化的作用；3在乙肝肝硬化患者肝组织验证上述通路。以阐明HBwx的功能，丰富肝硬化的发病理论。

本研究首先关注了HBwx通过外泌体促进肝星状细胞（Hepatic stellate cells, HSCs）炎症反应，以及HSCs炎症反应在HBV诱导的纤维化和HBV相关慢加急性肝衰竭（HBV-Acute on chronic liver failure, HBV-ACLF）发病机制中的作用。我们通过免疫组织化学检测肝纤维化及肝衰竭患者肝组织HBwx和α-SMA的表达，研究从HBV诱导的纤维化、HBV-ACLF患者和健康对照组收集外泌体，研究同时在细胞学和动物学水平通过Westernblot、rt-PRC和ELISA进行相关检测。研究发现HBwx通过外泌体促进HSCs炎症，并在HBV诱导的纤维化发病过程中诱导HSC激活，在HBV-ACLF发病过程中促进肝细胞凋亡。调节HBwx可能成为治疗HBV诱导的纤维化和HBV-ACLF的新方法。.氧化应激已被证实在ACLF发病过程中介导肝脏炎症反应。本研究的第二部分我们发现根据入院时血浆SOD水平可将ACLF患者分为高风险组和低风险组。与单独应用血清SOD水平和MELD评分相比综合血清SOD水平及MELD评分与ACLF患者病情及预后关系更加密切。综合血清SOD水平和MELD评分可用于预测ACLF患者预后。HEV病毒促进HEV相关肝衰竭（急性肝衰竭、慢加急性肝衰竭）发病机制中机体氧化应激水平。早期检测血清SOD水平可作为评价HEV相关肝衰竭预后的指标。调节机体氧化应激可能成为治疗ACLF、HEV相关肝衰竭的新途径。.氧化应激与炎症反应在肺纤维化的发病过程中起着关键作用。本研究的第三部分我们探索了Thymosin β4（Tβ4）与氧化应激、炎症反应在肺纤维化中的作用，研究发现Tβ4参与减轻氧化损伤、促进线粒体自噬、减轻炎症反应。Tβ4可减轻LPS诱导的小鼠肺损伤、炎症反应和肺纤维化。调节Tβ4可能发展为治疗肺纤维化的新途径。