非酒精性脂肪性肝炎肝郁脾虚证TLR4/TRIF通路与代谢组学机制研究

Our previous studies have inproved that insulin resistance occurs throughtout the process of the development and progression of nonalcoholic steatohepatitis (NASH), and lipopolysaccharide (LPS) was the trigger factor of IR. The earlier studies have revealed that a variety of metabolites changes can be observed in liver disease. There is a delicate balance between inflammation and metabolism signal, but the specific mechanism is still unclear. In traditional Chinese medicine theory, the liver govern free flow of qi, and the basic pathogenesis of adiposis hepatica is stagnation of liver qi and spleen deficiency. With NASH, we speculate that the banlance between the characteristics of metabolism and TLR4 mediated by LPS with its inflammatory signal transduction pathway may be the molecular basis of the stagnation of liver qi and spleen deficiency. This research intervene the rat model with the combination of disease and syndrome of stagnation of liver qi and spleen deficiency, with the method of soothing the liver and strengthening the spleen . To observe the balance relationship between the TLR4/TRIF signaling pathway and characteristics of metabolism which both in the model rat liver and kupffer cells with animal experiment and cell culture. By in-depth studies at multi-level as body, organ, cell, molecule and gene levels, clarifying the mechanism of pathogenesis of the syndrome of stagnation of liver qi and spleen deficiency for NASH, and reveal part of the biologic connotation of "the liver govern free flow of qi".

课题组前期研究证实，胰岛素抵抗（IR）贯穿于非酒精性脂肪性肝炎（NASH）发生发展的始终，脂多糖（LPS）是IR的触发因子。研究发现，肝脏病可出现多种代谢物的变化，炎症和代谢信号存在微妙的平衡，但具体机制尚不清楚。中医理论认为"肝主疏泄"，脂肪肝发病的基本病机为"肝郁脾虚"。我们推测NASH中LPS介导的TLR4及其下游的炎性信号转导通路和NASH的代谢组学特征之间的平衡是肝郁脾虚的分子基础。本课题通过建立NASH肝郁脾虚证病证结合的动物模型，并以疏肝健脾法予以反证，分别进行动物实验和细胞培养，观察各组大鼠肝脏和库普弗细胞的TLR4/TRIF通路及其与代谢组学特征之间的平衡关系。在充分体现中医理论的整体观念和辨证论治特点以及方证相应的基础上，拟从整体、器官、细胞、分子和基因水平进行多层次的深入研究，阐明非酒精性脂肪性肝炎肝郁脾虚证的发病机制，揭示"肝主疏泄"的部分生物学内涵。

非酒精性脂肪性肝病（NAFLD）的发病率增长迅速并呈现低龄化趋势，已成为全球重要的公共健康问题之一。部分非酒精性脂肪性肝炎（NASH)患者可进展为肝硬化，甚至可继续发展为肝细胞癌，给人们带来重大危害。肝郁脾虚证是NASH发病的基本病机，其生理基础为“肝主疏泄”。我们推测NASH中LPS介导的TLR4及其下游的炎性信号转导通路和NASH的代谢组学特征之间的平衡是脂肪肝肝郁脾虚的分子基础。本课题首先通过建立NASH肝郁脾虚证病证结合的动物模型，为NASH的证候相关研究提供了模型基础，同时利用该病证结合大鼠模型，通过形态学以及分子生物学技术证实NASH肝郁脾虚证病证的发病机制不仅与TLR4/TRIF信号通路的活性变化有关，而逍遥散可以通过抑制该信号通路活性，从而发挥“疏肝健脾”之功效；同时通过代谢组学技术，证实NASH肝郁脾虚证发病前后以及逍遥散治疗前后，大鼠血浆、尿液及粪便中存在有不同的差异代谢产物（分别找到9种、12种、10种差异代谢物，这些物质主要从氨基酸代谢紊乱、脂质代谢紊乱、糖代谢紊乱、胰岛素抵抗、免疫调节紊乱等方面参与NASH的发生发展），为阐明NASH肝郁脾虚证证候的发病机制及疏肝健脾法的治疗机制，揭示“肝主疏泄”的部分生物学内涵提供了实验依据。随后，我们通过LPS刺激J774A.1细胞以建立巨噬细胞炎症模型，从离体层面模拟NASH的发病机制，并通过逍遥散含药血清进行干预后，通过形态学及分子生物学技术手段从离体角度证实逍遥散防治NASH的机制与抑制TLR4/TRIF信号通路的活性有关，不仅阐释疏肝健脾法的部分抗炎机制，而且进一步揭示中药复方逍遥散抗炎的作用，为临床上推广应用提供实验依据。