SOX9对非酒精性脂肪性肝病的作用及机制研究

Non-alcoholic fatty liver disease (NAFLD) is a growing threat to human health, but the knowledge and treatment to NAFLD are still relatively limited at present. Sex-determining region Y box protein 9 (SOX9) is an important transcription factor that controls cell fate decision during the development and homeostasis of a broad range of tissues as a member of SRY family.Besides,SOX9 involves in the lipid metabolism in various cells. However, the effect and mechanisms of SOX9 in lipid metabolism in hepatocytes and NAFLD remain unclear. Our previous studies revealed that SOX9 can significantly attenuate oleic acid –induced steatosis in HepG2 cells. In addition, hepatocyte-specific overexpression of SOX9 could ameliorate HFD-induced NAFLD, while hepatocyte-specific knockout of SOX9 aggravated NAFLD in mice. And SOX9 could increase the activation of AMPK signaling pathway. In this study, we will further clarify the relationship between SOX9 expression in hepatocyte and NAFLD. Moreover, hepatocyte-specific knockout or overexpression of SOX9 will be performed in mice to elucidate the effect of SOX9 on NAFLD. Finally, the molecular mechanism of SOX9 affecting NAFLD through LKB1/AMPK signal pathway will be further illustrated. This study will provide novel theory on pathogenesis of NAFLD and novel targets for the prevention and treatment of NAFLD.

非酒精性脂肪性肝病对人类健康的威胁越来越严重，但目前对其认识和治疗手段仍相对有限。SOX9是SOX家族中重要的转录因子，在胚胎发育，维持细胞干性及组织稳态中具有重要意义，且在多种细胞中参与脂质代谢。但SOX9对肝细胞脂质代谢和NAFLD发生发展的作用及机制尚不明确。我们前期研究发现，SOX9可以减轻油酸诱导的HepG2细胞脂肪变性；在小鼠NAFLD模型中发现，特异性上调肝细胞SOX9表达减轻NAFLD，而特异性下调肝细胞SOX9表达则加重NAFLD；同时还发现SOX9可以促进AMPK信号通路活化。本研究将在此基础上进一步明确肝细胞SOX9的表达与NAFLD的关系，明确特异性调控肝细胞SOX9表达对小鼠NAFLD的作用，阐明SOX9调控LKB1/AMPK信号通路影响NAFLD的分子机制；为NAFLD的发生发展提出新的理论，并为其防治提供新的靶点。

研究目的：在明确调控SOX9表达后对肝细胞脂肪变性的影响并初步探究其机制，为NAFLD的发生发展提出新的理论，并为其防治提供新的靶点。.研究方法：选取HepG2细胞，利用腺病毒AdSOX9上调和小干扰RNA下调HepG2细胞中SOX9的表达，再加入棕榈酸油酸诱导细胞发生脂肪变性，比较调控SOX9表达后细胞脂肪变性情况和脂质代谢相关基因变化。在动物实验中，我们利用腺相关病毒AAV-TBG-Cre和Sox9f/f小鼠构建肝细胞特异性敲除SOX9的Sox9HKO小鼠，给予Sox9HKO小鼠和Sox9f/f小鼠胆碱蛋氨酸缺乏饮食（MCD）诱导小鼠发生脂肪性肝病，对比两组小鼠肝细胞脂肪变的严重程度。更进一步，我们利用腺病毒AAV8-TBG-SOX9和C57小鼠构建肝细胞特异性过表达SOX9的Sox9OE小鼠，给予Sox9OE小鼠和Sox9f/f小鼠MCD饮食后，观察两组小鼠肝细胞脂肪变的变化。利用RT-PCR、Western blotting等检测各组小鼠肝组织SOX9、脂质代谢指标、炎症指标等的表达变化。并进一步在体内外探究SOX9影响肝细胞脂肪变性的机制。.研究结果：上调SOX9表达可减少HepG2细胞脂质沉积，并能抑制脂质合成基因（FASN、ACC1、SREBP）和脂质转运基因（CD36、FABP1）的表达；而下调SOX9则增加脂质沉积，促进脂质合成基因（FASN、ACC1）和脂质转运基因（CD36、FABP1）的表达。在MCD模型小鼠中发现，Sox9HKO 小鼠相较对照组小鼠肝脏脂质沉积加重、肝细胞脂肪变、气球样变、小叶炎症以及纤维化程度均较对照小鼠加重；RT-PCR显示Sox9 HKO 小鼠肝脏脂肪从头合成相关基因（Srebp1）、脂质转运相关基因（Cd36、Fabp1）、炎症相关基因（Ccl2、Il6）相较对照小鼠转录水平表达上调，而脂解相关基因（Ces1）转录水平表达下调，纤维化相关基因（Col1a1、Acta2）转录水平表达上升。特异性上调肝细胞中SOX9的表达减轻小鼠肝细胞脂肪变性（结果整理中）。并进一步发现，SOX9可以调控AMPK信号通路影响，可能通过该信号通路影响肝细胞脂质代谢。.研究结论：体内外研究表明，SOX9可能通过活化AMPK信号通路减轻肝细胞脂肪变性。