M型磷脂酶A2受体通路介导线粒体稳态失衡在非酒精性脂肪性肝病的作用及机制研究

Mitochondria homeostasis disruption, charactered by hypo energy metabolism and over production of ROS, may be closely associated with the etiology and pathogenesis of non-alcoholic fatty liver disease (NAFLD). Abnormal mitochondrial membrane metabolism of phospholipid is one of the most important mechanism of Mitochondria homeostasis disruption. M type phospholipase A2 receptor (MsPLA2R) related pathway was identified as a major signal in metabolism of phospholipid，which can promote the action and translocation of cPLA2 to the inner mitochondrial membrane ,therefore catalyzes the hydrolysis of phospholipids and generates lysophospholipids and fatty acids. Our preliminary data showed higher levels of ligands of MsPLA2R in patients with NAFLD and hepatocytes sitimulated with its ligands present abnormal energy production. So we hypothesis that activation of MsPLA2R has an important role in the development of NAFLD. In this study, we plan to analyze the changes of monichondria membrane phospholipids, oxygen consumption, activities of respiration chain enzymes and ROS in NAFLD models in vitro and vivo by means of liquid Chromatograph Mass Spectrometer, Seahorse XF Cell Energy Phenotype Test, enzyme linked immunosorbent assay and Western blot. We will identify those proteins who mediate the activities and translocation of cPLA2 by co-immunoprecipitation with Mass Spectrometer, by which we can further elucidate the mechanism of membrance lipid changes to cause mitochondria defect. We aim to find out a new therapeutic target and provide theoretical basis for it.

线粒体内膜磷脂代谢紊乱介导的以低能量和高活性氧生成为特征的线粒体稳态失衡是非酒精性脂肪肝（NAFLD）发病关键环节。M型磷脂酶A2受体（MsPLA2R）通路作为肝脏磷脂代谢的关键途径，其激活胞浆型磷脂酶A2（cPLA2）活化转位至线粒体内膜降解其磷脂成分。我们前期研究发现NAFLD患者MsPLA2R配体水平异常升高，且体外模型激活该受体观察到线粒体能量产生显著下降，因此该通路过度激活可能是NAFLD线粒体异常新机制。本项目拟运用超高效液相色谱联用质谱、生物能量代谢检测、酶联免疫及蛋白印迹法在细胞器、细胞、转基因动物和临床肝标本分析激活和抑制MsPLA2R通路对肝线粒体膜磷脂成分、线粒体氧耗量、呼吸链酶活性及活性氧水平变化，应用免疫共沉淀联用蛋白质谱鉴定介导cPLA2变化的分子，观察敲减和过表达该分子对cPLA2活性和向线粒体膜转向线粒体膜转位的影响，阐明线粒体磷脂异常引起NAFLD的机制。

非酒精性脂肪性肝病(NAFLD)，已成为全球第一大慢性肝病。肥胖与NAFLD密切相关，既往研究大多基于肥胖人群开展，但最近的多项流行病学调查报告显示非肥胖NAFLD的患病率呈逐年上升趋势，成为不容忽视的患者群体。与肥胖NAFLD相比，非肥胖患者的代谢紊乱和组织学病变及远期预后存在巨大的争议。已有的研究显示代谢紊乱、遗传易感性、不良的生活方式、腹型肥胖等均在非肥胖NAFLD的发病过程中起关键作用，但具体机制尚不明确。本研究首先应用多中心临床病例血清标本进行基于超高效液相色谱飞行时间串联质谱仪（UHPLC-QTOF-MS）及气相色谱串联质谱仪（GC-MS）的代谢组学分析，探讨非肥胖NAFLD合并颈动脉粥样硬化患者的代谢组学特点，初步证实磷脂代谢相关产物含量异常是非肥胖NAFLD合并CAS发生的独立危险因素，基于脂肪酸从头合成指数(16:0/18:2n-6)，磷脂酰乙醇胺 (20:2/16:0) 和磷脂酰甘油 (18:0/20:4) 的血清代谢物预测模型能够显著提高非肥胖NAFLD合并亚临床颈动脉粥样硬化的预测能力。第二部分进一步分析磷脂酰甘油代谢关键基因多态性与非肥胖NAFLD易感性及临床表型的关系，鉴定出4个与NAFLD易感性相关的SNP位点变异，神经酰胺合酶(CERS4) rs17160348位点变异与肥胖和非肥胖NAFLD易感性均相关，M型磷脂酶A2受体(PLA2R1) rs35771982仅与非肥胖NAFLD相关，而对氧磷脂酶1(PON1) rs854560和PLA2R1 rs3749117均与肥胖NAFLD易感性关系密切，并进一步发现与CAS及病理学改变相关的SNP位点，PLA2R1 rs35771982是非肥胖NAFLD发生中重度脂肪变及脂肪性肝炎的危险因素。因此在第三部分，深入探究磷脂代谢过程中关键信号分子分泌型磷脂酶A2（sPLA2）激活肝细胞代谢的关键受体PLA2R1在NAFLD发病的作用及机制，得出NAFLD状态下高水平的sPLA2与PLA2R1结合启动下游的JAK2/STAT3通路激活了细胞质型磷脂酶A2活性，通过介导线粒体膜磷脂重构从而改变了线粒体膜脂区的微环境成分，引发膜电位下降，影响呼吸链的电子传递，造成了线粒体膜损伤的生物学效应，最终影响氧化磷酸化导致ATP合成减少和活性氧(ROS)释放从而干扰线粒体稳态，在NAFLD发病过程中起着重要作用