SIRT3-ATG4B介导的线粒体自噬在二氢杨梅素改善NASH中的作用及其机制研究

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide and the challenges for the prevention and treatment of this disease, represents an enormous public health challenge worldwide. Nonalcoholic steatohepatitis (NASH), an inflammatory phenotype of NAFLD, can proceed to cirrhosis and even hepatocellular carcinoma, and even worse, there are still no approved and effective therapies to conquer this disease. Recently, many studies have confirmed dihydromyricetin (DHM), a flavonoid derived mainly from natural plants such as vine tea, has antioxidative and hepatoprotective effect. Our previous clinical trial indicated that DHM could attenuate hepatocellular injury and inflammatory markers in patients with NAFLD. We also found that DHM could improve the mitochondrial respiratory chain function and redox homeostasis in vivo and in vitro. Those suggested that DHM might be an ideal phytochemicals in the prevention of NASH，though the potential mechanisms of these effects remained elusive, which requires further study urgently. Mitochondrial dysfunction and impaired redox homeostasis is a central feature of simple steatosis to NASH transition. Mitophagy, the specific autophagic elimination of damaged mitochondria, is a critical mechanism to regulate mitochondrion function and maintains quality control of this important organelle. SIRT3 has been shown to be involved in the regulation of autophagy. SIRT3 knockout mice fed a high-fat diet (HFD) exhibited aggravated NASH symptom, however, DHM treatment could increase SIRT3 expression and activity. Based on the current knowledge and our preliminary study, we propose a hypothesis that “DHM could improve the expression and activity of SIRT3, and regulate SIRT3-mediated mitophagy, thereby enhance mitochondrial function and inhibit hepatocellular injury. We intended to verify the hypothesis on NASH model in vitro and in vivo, to investigate the exact mechanism of DHM on ameliorating NASH development through modulation of SIRT3 signaling. Meanwhile, to provide scientific evidence on NASH intervention through dietary approaches.

非酒精性脂肪性肝病（NAFLD）已成为全球性公共卫生问题。非酒精性脂肪性肝炎（NASH）是NAFLD的一种炎症阶段，至今尚无公认有效的治疗手段。研究表明二氢杨梅素（DHM）具有抗氧化和护肝的效应，我们的前期研究也发现DHM可显著降低NAFLD患者的肝脏炎症水平。因此，我们推测DHM可能成为预防NASH的理想植物化学物，但具体机制尚不明确。肝细胞线粒体功能障碍及氧化应激损伤是NASH发生发展的中心环节，而线粒体自噬是维持线粒体功能的调控机制。SIRT3被认为是重要的自噬调控因子，SIRT3基因敲除小鼠容易发生NASH。据此，我们提出“DHM可能通过上调SIRT3表达，促进线粒体自噬，改善肝细胞线粒体功能、抑制肝细胞损伤从而有效预防NASH”的科学假说。本项目拟通过建立NASH动物和细胞模型，围绕肝细胞线粒体自噬和线粒体功能调节，探讨DHM通过SIRT3预防NASH发生发展的效应及其分子机制。

非酒精性脂肪性肝病（nonalcoholic fatty liver disease，NAFLD）是目前全球范围内肝脏疾病最主要的病因。非酒精性脂肪性肝炎（NASH）是NAFLD的一种炎症阶段，至今尚无公认有效的治疗手段。研究表明，肝细胞线粒体功能障碍及氧化应激损伤是NAFLD发生发展的中心环节，而线粒体自噬是维持线粒体功能的调控机制。结合既往研究结果，我们推测二氢杨梅素（DHM）可能通过上调SIRT3表达，促进线粒体自噬，改善肝细胞线粒体功能、抑制肝细胞损伤从而有效预防NASH。. 通过PA处理HepG2和HHL-5建立NASH细胞模型；采用ATG4B敲出的HepG2细胞验证ATG4B在SIRT3介导的自噬激活中的作用。. 结果显示，DHM显著改善PA诱导的HepG2和HHL-5细胞总活性氧（ROS）、线粒体活性氧（mtROS）增加和线粒体膜电位降低（MMP），改善线粒体损伤，增强线粒体自噬水平。DHM对PA诱导的肝细胞自噬阻滞和氧化应激的改善效应被SIRT3抑制剂和自噬抑制剂部分消除；此外，ATG4B基因消除也部分消除了DHM对PA诱导的肝细胞自噬阻滞和氧化应激的改善效应。最后我们发现DHM处理增加了肝细胞中SIRT3的蛋白表达和ATG4B的SIRT3依赖性去乙酰化。. 综上所述，本研究证明DHM通过上调肝细胞SIRT3蛋白表达，增强SIRT3介导的ATG4B去乙酰化，促进肝细胞线粒体自噬，进而减轻棕榈酸诱导的氧化应激，从而有效预防NASH。