解偶联蛋白家族在非酒精性脂肪性肝病慢性化过程中的作用及其机制研究

Nonalcoholic fatty liver disease (NAFLD) is considered as a common chronic liver disease with highest global prevalence. The spectrum of NAFLD consists of simple steatosis, nonalcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis and may even progress to liver cancer. Mitochondrial abnormality plays a pivotal role in the pathogenesis of NAFLD, where uncoupling protein (UCP) induced uncoupling of oxidative phosphorylation is actively involved. Previous study showed the level of liver specific uncoupling protein-HDMCP was significantly increased in steatosis stage of NAFLD and inhibited lipid accumulation through decreasing ATP and hydrogen peroxide levels. Nevertheless, it is still unclear whether there exits other hepatic UCPs and their potential role in chronic progression of NAFLD. Therefore, we firstly aimed to systematically assess the ability of UCP2 and HDMCP in inducing hepatocyte proton leak under normal physiological condition, through constructing mice model of UCP2 and HDMCP double knockout. If those proteins are not fully responsible for measured proton leak, other potential mitochondrial proteins with uncoupling activity were also searched and qualified, using methods of proteomics, bioinformatics and uncoupling ability assessment. Secondly, we planned to investigate the effects of UCP2, HDMCP and other potential mitochondrial proteins with uncoupling activity in the chronic progression of NAFLD, by RNA interference, gene knockout and gene overexpression at in vitro and in vivo levels. The potential underling mechanisms associated with energy metabolism, inflammatory reaction and oxidative stress were also studied.

非酒精性脂肪性肝病（NAFLD）是全球发病率最高的慢性肝病。线粒体功能异常在NAFLD的慢性化进程中发挥了重要作用，这可能与解偶联蛋白（UCP）介导的氧化磷酸化解偶联相关。前期研究发现肝脏特异性解偶联蛋白HDMCP在NAFLD的脂质沉积阶段代偿性增高，并通过降低细胞内ATP和过氧化氢水平对肝脏脂质沉积起抑制作用，但是否存在其他具有解偶联功能的蛋白及在NAFLD慢性化进程中作用如何仍不明确。本课题组拟通过UCP2和HDMCP基因敲除的方法，评估上述蛋白介导肝细胞生理状态下质子渗漏的能力，必要时进一步寻找并鉴定潜在具有解偶联能力的线粒体蛋白。然后对HDMCP、UCP2及可能找到的具有解偶联能力的线粒体蛋白进行体内与体外的干扰或过表达，研究其对NAFLD慢性化进程中的生物学作用，并探讨其相关机制。

背景：NAFLD的危险因素众多，其致病机制仍不明朗，治疗策略有限，解偶联蛋白（UCP）在NAFLD中起到重要的生物学作用。主要研究内容：NAFLD模型中可能具解偶联功能的线粒体蛋白筛选，评价以筛选出可能具解偶联能力的线粒体蛋白为靶点对NAFLD 的治疗作用，阐明其在NAFLD 慢性化进程中的分子作用机理，鉴定其是否具有解偶联功能。重要结果：通过差异凝胶电泳共发现了36个在线粒体上差异表达的多肽片段，进一步通过生物信息学的方法和多序列比对，发现MPST (3-mercaptopyruvate sulfurtransferase, 3-巯基丙酮酸转移硫酶)、HADHA (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, 线粒体三功能蛋白亚基A)及ATP5A1 ATP synthase subunit alpha, ATP合成酶亚基α)与已知的UCP结构最为接近，MPST对线粒体呼吸功能有一定的影响，但其并不具有解偶联功能。FFA的刺激诱导MPST在人脂变肝细胞中表达上调，并部分依赖于NF-κB/p65。在HFD诱导的小鼠NAFLD模型及临床NAFLD患者肝脏中，MPST蛋白表达水平显著升高。应用MPST-shRNA重组腺病毒尾静脉高压注射介导小鼠肝脏MPST表达部分敲低，和使用MPST基因敲除杂合子小鼠介导遗传性的MPST表达部分缺失，均能显著改善HFD诱导的肝脏脂肪变性程度。和体内水平一致地，在L02细胞中抑制MPST表达后，FFA诱导的肝细胞脂变显著改善，而过表达MPST后则脂变加剧。部分敲低MPST会显著上调H2S水平，相反地，过表达MPST则显著下调H2S水平。CoIP实验揭示MPST可以通过直接蛋白-蛋白相互作用而负调控CSE。H2S在MPST对脂肪肝的调节过程中起重要介导作用，与脂肪合成关键蛋白SREBP-1相关通路抑制，JNK磷酸化减弱及肝脏氧化应激改善有关。HADHA对线粒体呼吸功能的作用具有细胞特异性，在人肝癌细胞中HADHA会对线粒体呼吸功能产生一定影响，而在人正常肝细胞中HADHA对线粒体呼吸功能无显著作用，且其并不具有解偶联功能。意义：为基于 MPST 为潜在靶点的 NAFLD 新药研发提供了新的理论依据。