S1PR2调节非酒精性脂肪肝的分子机制研究

Nonalcoholic fatty liver disease (NAFLD) is identify as a global problem due to increased incidence of obesity , which is characterized by increased fat depots in the liver, could precede more severe diseases such as non-alcoholic steatohepatitis (NASH), cirrhosis, and in some cases hepatocellular carcinoma. The mechanism of NAFLD is associate with the increased accumulation of lipid in the liver caused by impairment of fatty acid beta oxidation, and lipid accumulation in the liver is also linked with the progression of endoplasmic reticulum stress (ER stress), mitochondria stress, and impaired autophagy, resulting in the condition known as lipotoxicity. This latter event can cause the immune response in the Kupffer cells and hepatic stellate cells, which leads to the progression of NASH, hepatic cirrhosis, and hepatocellular carcinoma. In recent years, other scientists and our previous studies indicate that 1-phosphate - sphingosine receptor 2 (S1PR2) play an important role in NAFLD development . But how it accomplish this has not been fully elucidated. This research is plans to use gene silencing, transfection techniques, immunohistochemistry, QPCR, Western-Blot, ELISA and other molecular biology methods, microarray bioinformatics analysis to study the mechanism by which S1PR2 regulate the development of NAFLD. Proven scientific hypothesis "S1PR2 / ERK / PCSK9 / LDLR pathway trigger excessive lipid accumulation in liver cells; S1PR2 / IL6 / JAK / STAT3 pathway regulating inflammatory hepatocellular damage and apoptosis." This study will provide useful new targets for the prevention and treatment of NAFLD.

非酒精性脂肪肝（NAFLD）在全世界范围发病率逐年上升，且随着病程进展可并发代谢综合症、肝硬化、肝癌等多种致命性疾病。其发病机制目前公认为脂质沉积过多、胆固醇代谢紊乱及免疫炎症。近年来文献及申请人前期研究表明：1-磷酸-鞘氨醇受体2（ S1PR2）参与调控上述多个病理过程，在NAFLD发生发展中起重要作用。本课题拟应用基因沉默、转染技术，免疫组化、QPCR、Western-Blot、ELISA等分子生物学方法，基因芯片结合生物信息学分析等，分别在动物、细胞、分子不同水平深入研究S1PR2调控NAFLD发生发展的机制。证明科学假说“S1PR2/ERK/PCSK9/LDLR通路促发肝细胞内过度脂质堆积和胆固醇代谢紊乱， S1PR2/IL6/JAK/STAT3通路调控肝细胞免疫炎症损伤和凋亡病理过程。”为NAFLD防治提供新的有益靶标。

本课题围绕1-磷酸-鞘氨醇（S1P）、其膜受体（S1PR2）、前蛋白转化酶枯草溶菌素9（PCSK9）及下游信号通路对非酒精性脂肪肝（NAFLD）肝脏损伤的调控及可能机制展开研究，并在整体和离体水平阐述通过S1PR2调节肝脏脂质沉积过程，可能成为NAFLD防治新的、有针对性的靶标。本研究主要有以下发现：1）揭示了S1PR2参与调节肝细胞异常脂质沉积过程，在NAFLD小鼠模型中肝脏S1PR2表达减少、PCSK9表达增加，小鼠原代肝细胞和L02细胞中S1PR2基因缺失或与抑制剂结合均可导致细胞内脂质沉积增加；2）抑制S1PR2对NAFLD脂质沉积的增加作用可能是通过该受体向胞核易位并促进其下游MAPK信号通路激活，调节PCSK9的表达实现：在L02细胞中S1PR2抑制剂可促进ERK磷酸化，增加PCSK9表达。3）构建S1PR2干扰、过表达质粒，应用基因芯片技术寻找S1PR2潜在关联基因，相较于对照组，27种基因在干扰、过表达时均有改变，其中包括CCR10、EGR1编码基因等；4）初步检测了MCD造模小鼠中筛选出具有明显表达差异的基因EGR1的表达情况，其对肝细胞的异常脂质沉积信号通路的调节在NAFLD发生发展中的作用有待重复和进一步实验探索。这些发现为探索NAFLD的临床治疗提供了新的思路与候选药物。本课题研究已发表SCI文章4篇，另外1篇正在完善、整理投稿。对照课题计划书，已完成研究任务。