柔肝方通过抑制纤维化蛋白抗肝纤维化的机制研究  

Study on machanism of Rougan Formula in anti-hepatic fibrosis by inhibiting fibrosin

作　　者：李茜 吴惠春[1] 谭家鑫 黄凌鹰[1] 高月求[1] 李曼[1] LI Qian;WU Huichun;TAN Jiaxin;HUANG Lingying;GAO Yueqiu;LI Man(Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai Key Laboratory of Traditional Chinese Clinical Medicine&Laboratory of Cellular Immunology,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院上海市中医临床重点实验室,细胞免疫学实验室,上海201203

出　　处：《中国免疫学杂志》2022年第3期263-269,共7页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(81673767,81673938,81874436,81973773,82074155,82074372);国家科技重大专项(2018ZX10725-504);上海市临床重点专科——中医肝病(shslczdzk01201);国家重大疑难疾病中西医临床协作试点项目[ZY(2018-2020)-FXTW-2004];上海市教育委员会“曙光计划”(18SG39);上海市科委优秀学术带头人计划(20XD1423500);上海市三年行动计划项目(SHDC2020CR3089B)资助。

摘　　要：目的:柔肝方是上海市名中医王灵台教授治疗肝纤维化的临床有效验方,本研究旨在探讨柔肝方通过抑制纤维化蛋白(FBRS)抗肝纤维化的分子机制。方法:质谱法鉴定柔肝方的化学成分及其入血成分。BALB/c小鼠随机分对照组、模型组、柔肝方高剂量组、柔肝方中剂量组和柔肝方低剂量组。通过尾静脉注射ConA(12.5 mg/kg),每周1次,共10周,建立肝纤维化小鼠模型。体外应用重组FBRS蛋白刺激人肝星状细胞系(LX2细胞),并用柔肝方大鼠药物血清干预。肝组织HE染色、天狼猩红染色和Masson染色。生化法检测血清ALT、AST、肝纤四项(LN、HA、PⅢNP、Col-Ⅳ)表达水平。Western blot法检测肝组织α-SMA,Western blot法和ELISA法检测Ⅰ型胶原(Col-Ⅰ)、TGF-β1、FBRS的表达水平。结果:各剂量柔肝方均可减少ConA肝纤维化模型小鼠肝脏的胶原沉积,且呈剂量依赖性抑制;柔肝方可明显降低模型小鼠肝组织α-SMA、TGF-β1、Col-Ⅰ和FBRS的表达水平;体外应用重组FBRS蛋白可上调LX2细胞的α-SMA、TGF-β1、Col-Ⅰ和FBRS的表达,柔肝方药物血清可抑制FBRS介导的LX2细胞的活化及胶原表达。质谱分析鉴定淫羊藿苷、丹酚酸B、绿原酸是柔肝方中含量较高的活性成分。结论:柔肝方通过下调肝星状细胞FBRS的表达抑制其活化及胶原沉积,发挥抗肝纤维化作用。Objective:Rougan Formula is a clinical effective formula for treating hepatic fibrosis,which is invented by Professor WANG Lingtai,who is a famous doctor of traditional Chinese medicine in Shanghai.Ths study was performed to investigate the molecular mechanism of Rougan Formula treating hepatic fibrosis by inhibiting fibrosin.Methods:Analysis of chemical components and blood components of Rougan Formula were carried out by mass spectrometry.BALB/c mice were randomly divided into control group,model group,high-dose Rougan Formula group,middle-dose Rougan Formula group and low-dose Rougan Formula group.ConA was injected by the tail vein once a week for 10 weeks(12.5 mg/kg).Recombinant FBRS protein was used to activate hepatic stellate cell line(LX 2 cells),and rat serum containing Rougan Formula was applied to explore the effects of Rougan Formula on LX 2 cells.HE staining,Sirius red staining and Masson staining were performed on histological sections of liver tissues.The serum levels of ALT,AST and four items detection of liver fibrosis on the liver fibrosis degree(LN,HA,PⅢNP,Col-Ⅳ)were detected by biochemical method.The expression levels ofα-SMA in liver tissues were detected by Western blot.The expression levels of Col-Ⅰ,TGF-β1 and FBRS in liver tissues and LX 2 cells were detected by Western blot and ELISA.Results:Liver collagen deposition in ConA-induced hepatic fibrosis model mice was decreased by Rougan Formula in a dose-dependent manner.In addition,the expression levels ofα-SMA,TGF-β1,Col-Ⅰand FBRS in liver tissue were significantly reduced by Rougan Formula.In vitro,α-SMA,TGF-β1,Col-Ⅰand FBRS levels in LX 2 cells were increased by recombinant FBRS protein,and rat serum containing RGF can inhibit the activation of LX 2 cells induced by recombinant FBRS protein.Component analysis showed that icariin,salvianolic acid B,chlorogenic acid were the high content active components in Rougan Formula.Conclusion:Rougan Formula can inhibit the activation of hepatic stellate cells and collagen deposi⁃t

关 键 词：柔肝方 纤维化蛋白 肝纤维化 肝星状细胞 

分 类 号：R575.5[医药卫生—消化系统]