MPST/H2S调控胰岛素抵抗促进NAFLD发生发展的作用及其机制研究

Nonalcoholic fatty liver disease (NAFLD) is one of the chronic liver diseases with high morbility, but its pathogenesis remains poorly understood. The role of hydrogen sulfide (H2S) in NAFLD has been received great attention in recent years. Our previous studies revealed that the protein expression of 3-mercaptopyruvate sulfurtransferase (MPST), one of H2S-generating enzymes, was markedly increased in free fatty acid (FFA)-induced L02 cells and livers from high fat diet (HFD) fed mice as well as NAFLD patients. Mechanistically, MPST promoted steatosis via inhibition of cystathionine-γ-lyase (CSE) /H2S pathway and subsequent upregulation of the SREBP-1 pathway, JNK phosphorylation, and hepatic oxidative stress. In this research project, we aim to investigate the direct effect of H2S on insulin resistance in NAFLD cell models and mouse models including MPST knockout mice. Furthermore, by using laser confocal microscopy, flow cytometry, target gene overexpression and siRNA or recombinant virus mediated gene knockdown, we try to research and determine the roles and mechanisms of MPST on insulin resistance in NAFLD, which involves H2S-mediated regulatory effect on JNK-inhibited insulin receptor signaling pathway, MCP-1 evoked inflammation, H2O2-induced mitochondria dysfunction and apoptosis. The findings of this project will provide new insights into the mechanism of NAFLD and bring new clues for new therapeutic strategies for NAFLD.

非酒精性脂肪性肝病（NAFLD）是高发慢性肝病，其发病机制尚未明了。硫化氢（H2S）在NAFLD中的作用近年来逐渐受到关注。我们前期研究发现，内源性H2S产生酶之一——3-巯基丙酮酸转移硫酶（MPST）的蛋白表达在NAFLD中显著上调，通过负调控CSE/H2S通路，继而激活脂肪合成关键蛋白SREBP-1相关通路，增强JNK磷酸化水平及加剧肝脏氧化应激状态，在NAFLD发生发展中起重要作用。本研究拟在已有基础上，建立NAFLD细胞和小鼠模型并结合MPST基因敲除小鼠，在观察H2S对脂肪肝胰岛素抵抗直接影响的同时，采用siRNA、重组腺病毒、激光共聚焦、流式细胞术等技术，进一步探讨并确定MPST通过H2S调节JNK/IRS1/PI3K/AKT信号通路、MCP-1介导炎症反应、诱导线粒体功能障碍、促进细胞凋亡而调控胰岛素抵抗参与NAFLD发生发展的作用及分子机制，具有较高创新性及临床意义。

背景：非酒精性脂肪性肝病（NAFLD）发病机制至今仍未完全明了，治疗策略尚且局限。而我们前期研究已证实硫化氢代谢主要酶3-巯基丙酮酸转硫酶（MPST）在NAFLD发生发展中起到重要作用，近年研究提示硫化氢参与胰岛素抵抗调节机制，且我们新近研究提示抑制MPST与AKT磷酸化为下游的胰岛素信号通路调控有关。主要研究内容：MPST调控胰岛素抵抗参与NAFLD发生发展的机制。重要结果：与野生型原代细胞相比，抑制MPST可上调AKT及GSK3β磷酸化水平，介导参与细胞水平胰岛素抵抗改善的机制。在雌鼠中，与野生型小鼠组相比，MPST+/-组小鼠糖耐量试验（GTT）AUC有下降趋势，无统计学差异；胰岛素耐量试验（ITT）结果显示，与野生型小鼠组相比，注射胰岛素30min后MPST-/-组小鼠血糖显著下降，注射胰岛素60min后，MPST+/-组小鼠血糖显著下降，MPST+/-与MPST-/-组AUC有下降趋势，无统计学差异。在雄鼠中，与野生型小鼠组相比，MPST+/-组小鼠GTT及ITT AUC均有下降趋势，但无统计学差异。因此NAFLD体内模型中MPST对胰岛素抵抗的调节在雌雄鼠间存在差异。在细胞水平分子机制研究提示，与野生型原代细胞相比，抑制MPST可上调PCK1，G6PC，FBP1，PC的mRNA水平，于转录水平调控糖异生途径。通过Western Blot技术检测发现，与野生型原代细胞相比，抑制MPST可上调G6PC，FBP1，PC的蛋白水平，提示MPST部分参与蛋白水平调控糖异生途径。在NAFLD细胞模型中阴性对照转染组相比，抑制MPST可显著降低LDH mRNA及蛋白水平，提示MPST参与调控糖酵解途径。意义：MPST对糖代谢存一定在调控作用，胰岛素抵抗并非MPST调控NAFLD的主要介导机制，MPST对糖脂代谢的调控作用分子机制仍有待进一步深入研究。