基于XBP1s在肝脏胰岛素抵抗中促进脂质合成的作用探讨祛湿化瘀方抗非酒精性脂肪肝的机理

Qushi Huayu Decoction, a traditional Chinese formula, has been observed to ameliorate the non-alcoholic fatty liver disease (NAFLD) in clinical and inhibit hepatic de novo lipogenesis (DNL) and expression of acetyl-coenzyme A carboxylase (ACC), a target gene of X-box binding protein 1 (XBP1), in NAFLD rat. Recently, the pathogenesis investigation of NAFLD supported that, in NAFLD, IRE1-alpha was activated by phorsphorylation, to splice mRNA of XBP1, the spliced XBP1 (XBP1s), as a transcriptional factor, promote the transcription of the enzymes involved in DNL, such as ACC and stearyl coenzyme A desaturase 1 (SCD1), by binding to the promoter of the target genes, which contributes to the lipid accumulation in the liver. Based on the inhibitory effects of Qushi Huayu Decoction on hepatic DNL and ACC in NAFLD, we proposed that inhibition on hepatic DNL is the major foundation of Qushi Huayu Decoction decreasing hepatic lipid in NAFLD, which is probably related to the IRE1-alpha - XBP1s pathway. The effect of Qushi Huayu Decoction on hepatic DNL and IRE1-alpha - XBP1s pathway will be observed in high-fructose diet induced NAFLD mice in vivo and insulin-induced DNL hepatocyte in vitro. The effect of Qushi Huayu Decoction on XBP1s will be observed in XBP1s knocking down and over expressing hepatocyte in vitro. The purpose of this project is to find out the underlying mechanisms of Qushi Huayu Decoction decreasing hepatic DNL in NAFLD based on the IRE1-alpha - XBP1s pathway, so that provide the scientific explain for the pathogenesis cognition and the principle of treatment of NAFLD in traditional Chinese medicine.

在临床证实祛湿化瘀方有效防治非酒精性脂肪肝(NAFLD)、抑制NAFLD大鼠肝脏脂质从头合成(DNL)及X盒结合蛋白1(XBP1)靶基因乙酰辅酶A羧化酶(ACC)的基础上，结合新近“NAFLD肝脏胰岛素抵抗时，IRE1剪切XBP1mRNA为XBP1s，启动DNL关键酶转录而促进DNL”的研究进展，提出“IRE1-XBP1s介导的DNL是NAFLD肝脏脂质沉积的主要因素之一，抑制DNL是祛湿化瘀方减少肝脏脂质的主要效应基础，可能与IRE1-XBP1s途径密切相关”的假说。采用1)高果糖诱导NAFLD模型及胰岛素刺激肝细胞DNL模型，观察祛湿化瘀方对肝脏DNL及IRE1-XBP1s通路的作用；2)XBP1s敲减及过表达的肝细胞胰岛素刺激模型，观察祛湿化瘀方对XBP1s的作用。以期阐明祛湿化瘀方抗NAFLD的主要机制，为NAFLD“湿热痰凝，瘀阻血络”的病机认识和“祛湿化瘀”治法提供科学依据。

本项目在前期工作基础上，结合新近“非酒精性脂肪性肝病（NAFLD）肝脏胰岛素抵抗时，肌醇依赖酶（IRE）1剪切X盒结合蛋白1(XBP1)mRNA为XBP1s，启动脂质从头合成(DNL)关键酶转录而促进DNL”的研究进展，提出“IRE1-XBP1s介导的DNL是NAFLD肝脏脂质沉积的主要因素之一，抑制DNL是祛湿化瘀方减少肝脏脂质的主要效应基础，可能与IRE1-XBP1s途径密切相关”的假说。主要研究内容：1)高果糖诱导NAFLD模型及衣霉素刺激肝细胞DNL模型，观察祛湿化瘀方对肝脏DNL及IRE1-XBP1s通路的作用；2)XBP1s敲减及过表达的肝细胞模型，观察祛湿化瘀方对XBP1s的作用。重要结果：1高果糖饮食可诱导肝组织脂质含量增加、肝脏XBP1s表达明显上调、DNL的关键酶及转录因子基因表达显著增加，并在2周末达到峰值。2祛湿化瘀方显著减轻高果糖饮食诱导的NAFLD小鼠肝脏DNL及XBP1s活化而对经典的启动DNL的转录因子CHREBP和固醇调节原件结合蛋白（SREBP）1无明显影响。3祛湿化瘀方药物血清对衣霉素体外肝细胞XBP1s活化有明确抑制作用，其入血成分栀子苷、绿原酸和虎杖苷是抑制XBP1s启动DNL的主要化合物。4祛湿化瘀方药物血清显著抑制XBP1s过表达的肝细胞XBP1s及其靶基因SCD1、ACC2的表达，栀子苷是其中主要的效应物质。科学意义：本项目的研究工作不同于既往启动DNL的经典转录因子（CHREBP、SREBP1）通路，从“XBP1s启动肝脏DNL”的角度揭示了祛湿化瘀方治疗NAFLD的机制。为NAFLD“湿热痰凝，瘀阻血络”的病机认识和“祛湿化瘀”治法的科学内涵提供依据。