磷酸化Atg13介导的线粒体自噬在肝星状细胞活化中的机制

It was well-established that hepatic stellate cells(HSCs) activation functions a central role in liver fibrosis in response to all kinds of insults. During the process of liver fibrosis, both hepatocytes and HSCs are exposed simultaneously to hepatic toxins, the former is injured even subjected to apoptosis or necrosis, whereas the latter demonstrate activation and proliferation, suggesting a survival mechanism is involved. Moreover, apoptosis is NOT evidenced even mitochondria are functional overloaded. It's remains unclear how those overloaded mitochondria are eliminated, and dose it contribute to HSC activation? Previously, we found HSC activation was regulated by autophagy, a lysosomal degradation system. Strikingly, we founded Atg13, a protein involved in mitophagy, displayed hyper-phosphorylation during HSCs activation by screening all those autophagy related proteins, suggesting a player in such process. Based on literature and ours' findings, we hypothesized that HSC activation were regulated by mitophagy via Atg13 phosphorylation. In this study we sought to identify previously unknown players in mitophagy particularly required in HSC activation. Firstly, the exact site(s) of Atg13 phosphorylation and upstream kinase(s) will be explorer during HSC activation by in vivo kinase assay, in vitro kinase assay, kinase inhibition experiment, and site-directed mutation analysis in HSCs cell model, followed by generation of antibody specific for phosphorylation site(s) to facilitate downstream investigation. Secondly, we sought to establish the in vivo relationship and connectionship among Atg13 phosphorylation, mitophagy and HSC activation in human fibrotic liver tissue. Subsequently, in HSC cell model and fibrotic animal model, reveal the function of Atg13 phosphorylation on mitophagy and explorer the underlying signaling transduction by using siRNA and site-directed mutation techniques. In addition, the effect of mitophagy on HSC activation as well as on hepatic fibrosis well be revealed by inhibitor specific against mitophagy. We propose a key role for Atg13 phosphorylation in selective mitophagy, underscoring the importance of mitochondrial maintenance in HSC activation.

肝星状细胞（HSCs）活化是各种病因所致肝纤维化的中心环节。肝纤维化过程中，暴露于相同的肝损因素，肝细胞发生损伤，而HSCs活化增殖，提示后者具有特殊的存活机制；此外，HSCs线粒体荷载但并未发生凋亡，损伤线粒体如何被清除，在HSCs活化中扮演什么角色？我们前期发现自噬调控HSCs活化，具有线粒体自噬功能的蛋白Atg13被磷酸化。我们推测磷酸化Atg13通过自噬清除损伤线粒体进而促进HSCs活化。本课题先在细胞及分子水平确定特异Atg13磷酸化位点及上游激酶；然后在纤维化患者肝组织明确特异位点的Atg13磷酸化、线粒体自噬与HSCs活化的关系；进一步在HSCs及纤维化动物模型中用通过siRNA及定点突变抑制特异位点Atg13磷酸，观察该位点磷酸化对线粒体自噬的影响及机制；最后通过阻断线粒体自噬，研究线粒体自噬对HSCs活化水平及肝纤维化的影响。这将了解HSCs活化分子机制，为治疗提供靶点。

肝衰竭是病毒、酒精、药物、缺血再灌注等多种因素导致肝脏损害的最严重形式，尽管抗病毒、人工肝、肝移植等治疗措施显著提高救治成功率，但存活率仍不高于 60%。肝衰竭是我国重要的疾病负担，导致病患家庭致贫返贫，也是临床医师面临的救治难点。肝衰竭的早期病理表现为：①肝细胞的大块或亚大块坏死：②肝星状细胞增殖活化；③炎症细胞浸润。课题组前期研究发现：在肝星状细胞活化过程中，自噬和炎症发挥重要的调控作用。据此，本课题以急性肝衰竭为疾病背景，探索该疾病的可靠预后指标；肝星状细胞在肝衰竭疾病进程中的重要作用；自噬和炎症如何调节肝星状细胞活性；为急性肝衰竭的诊疗提供新思路。. 研究发现：①急性肝衰竭患者血清中SOD水平升高，在疾病进展期尤为显著；且SOD升高预示着患者预后不良。急性肝衰竭患者血清中NO水平升高，在疾病进展期尤为显著；且NO升高预示着患者预后不良；②在肝星状细胞（HSCs）中，LPS通过ROS抑制HSCs线粒体自噬，激活肝星状细胞的炎症反应，在急性肝衰竭疾病进程中发挥重要的调控作用；③在肝星状细胞、肝癌细胞（HepG2）中，一氧化氮（NO）抑制自噬并促进凋亡，且自噬与凋亡相互调节，成为急、慢性肝病中的重要调控机制；. 在本期国科金支持下，课题组围绕多种急、慢性肝病，以肝星状细胞，自噬及线粒体自噬，氧化应激为轴心，探索肝病进程中的具体分子机制，发现了急性肝衰竭的可靠预后指标，为多种急、慢性肝病的诊断和治疗提供了可能的作用靶点。