基于TMT标记联合LC-MS/MS阐释茵芪三黄解毒汤抗肝纤维化的药效作用机制

Explanation on the pharmacodynamic mechanism of Yinqi Sanhuang Jiedu Decoction against hepatic fibrosis based on TMT labeling combined with LC-MS/MS technology

目的:采用串联质谱标签(TMT)标记联合液相色谱串联质谱(LC-MS/MS),从分子水平上探究茵芪三黄解毒汤(YQSH)抗肝纤维化的作用机制。方法:24只小鼠随机分为空白组、模型组、YQSH组、阳性药组,每组6只。除空白组外,其余组小鼠予10%四氯化碳橄榄油溶液腹腔注射,建立肝纤维化模型,给药治疗后,摘眼球取血,取肝组织。观察各组小鼠肝功能、肝组织病理,提取空白组、模型组、YQSH组肝组织蛋白质进行蛋白质组学分析。结果:YQSH能显著改善肝纤维化模型小鼠的肝功能(P<0.01)和肝组织病理损害。肝组织中共鉴定出6 214个蛋白,与模型组比较,经YQSH干预后,共有127个差异蛋白,这些差异蛋白的生物过程主要富集于细胞外基质(ECM)、核细胞的前复制复合体组装、质膜修复等。分子功能主要涉及相同的蛋白结合、肌动蛋白绑定、肌动蛋白结合等。细胞组成主要定位于ECM、膜筏、应力纤维等。KEGG分析显示,YQSH的调控作用涉及PI3K-AKT、Rap1、NOD样受体等信号通路。PPI结果显示,白蛋白(Alb)、波形蛋白(Vim)、凝溶胶蛋白(GSN)、膜联蛋白A5(ANXA5)等可能是YQSH抗肝纤维化的核心作用靶点。结论:YQSH可能通过调控多个靶点蛋白及PI3K-AKT、Rap1、NOD样受体等重要信号通路抑制肝星状细胞活化、减少胶原纤维增生与沉积、减轻炎症反应,从而发挥抗肝纤维化作用。

Objective:To explore the mechanism of Yingqi Sanhuang Jiedu Decoction(YQSH)against hepatic fibrosis at the molecular level by using tandem mass tags(TMT)labeling combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS).Methods:Experimental mice were randomly divided into blank group,model group,YQSH group and positive drug group,with 6 mice in each group.Except for blank group,mice in other groups were intraperitoneally injected with 10%carbon tetrachloride olive oil solution to establish hepatic fibrosis model.After treatment,blood was collected from eyeballs and liver tissues were collected.The liver function and histopathology of mice in each group were observed,and the proteins in liver tissues of blank group,model group and YQSH group were extracted for proteomic analysis.Results:YQSH could significantly improve the liver function and pathological damage of liver fibrosis model mice(P<0.01).A total of 6214 proteins were identified in liver tissues.Compared with the model group,127 different proteins were identified after YQSH intervention.The biological processes of these different proteins were mainly concentrated in extracellular matrix(ECM),nuclear cell pre-replication complex assembly,and plasma membrane repair.The molecular functions mainly involve the same protein binding,actin binding,actin binding and so on.The cell composition was mainly located in ECM,membrane raft,stress fiber and so on.KEGG analysis showed that the regulation of YQSH involved PI3K-AKT,Rapl,NOD-like receptor and other signaling pathways.PPI results showed that albumin(Alb),vimentin(Vim),coagulsol protein(GSN)and annexin A5(ANXA5)may be the core targets of YQSH anti-hepatic fibrosis.Conclusion:YQSH may inhibit the activation of hepatic stellate cells,reduce the proliferation and deposition of collagen fibers,and alleviate inflammation by regulating several target proteins and important signaling pathways such as PI3K-AKT,Rapl and NOD-like receptors,thereby playing an anti-hepatic fibrosis role.