黄芪总苷上调miRNA-744表达抑制肝星状细胞活化的效应机制  

Mechanism of astragalosides on inhibiting the activation of hepatic stellate cells by up-regulating miRNA-744 expression

作　　者：胡永红 任爽 刘伟 陈高峰 慕永平 陈佳美 刘平 HU Yonghong;REN Shuang;LIU Wei;CHEN Gaofeng;MU Yongping;CHEN Jiamei;LIU Ping(Institute of Liver Diseases,Shuguang Hospital of Shanghai University of Traditional Chinese Medicine,Key Laboratory of Liver and Kidney Diseases,Ministry of Education,Shanghai Key Laboratory of Traditional Clinical Chinese Medicine,Shanghai 201203,China;Institute of Interdisciplinaiy Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院肝病研究所肝肾疾病病证教育部重点实验室上海市中医临床重点实验室,上海201203 [2]上海中医药大学交叉科学研究院,上海201203

出　　处：《上海中医药大学学报》2021年第1期32-40,共9页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：国家自然科学基金资助项目(81530101);上海市青年科技启明星计划项目(19QA1408900);中国肝炎防治基金会王宝恩肝纤维化研究基金资助项目(2020010).

摘　　要：目的:探析黄芪总苷抑制肝星状细胞活化的作用机制。方法:(1)以转化生长因子β1(TGF-β1,2.5 ng/ml)诱导人肝星状细胞株LX-2细胞活化,并给予不同浓度黄芪总苷(1.25、2.5、5、10、20、30、40μg/ml)。药物作用24 h后,Edu掺入法检测细胞增殖,筛选适合的药物浓度。(2)将LX-2细胞分为对照组、TGF-β1(2.5 ng/ml)组、TGF-β1+黄芪总苷(20μg/ml)组、TGF-β1+SB431542(10μmol/L)组。各组给予相应干预24 h后,PCR检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(ColⅠ)、TGF-β1、miRNA-744基因表达;Western blot检测ColⅠ、TGF-β受体Ⅰ(TβRⅠ)、Smad2、p-Smad2、Smad7的蛋白表达;免疫荧光检测纤维状肌动蛋白(F-actin)表达。(3)取细胞分别加入miRNA-744激动剂(50 nmol/L)、miRNA-744拮抗剂(100 nmol/L)或阴性对照试剂,构建相应的转染细胞。在3种体系的转染细胞,将细胞分为对照组、TGF-β1(2.5 ng/ml)组、TGF-β1+黄芪总苷(20μg/ml)组、黄芪总苷(20μg/ml)组。各组给予相应干预24 h后,Edu掺入法检测细胞增殖,免疫荧光检测F-actin表达。结果:(1)20、30和40μg/ml黄芪总苷可显著抑制TGF-β1诱导的LX-2细胞增殖,选用20μg/ml的剂量进行后续实验。(2)TGF-β1组细胞F-actin、α-SMA、ColⅠ、TGF-β1和TβRⅠ的表达以及p-Smad2/Smad2蛋白表达比值较对照组显著升高(P<0.05,P<0.01);而与TGF-β1组相比,TGF-β1+黄芪总苷组细胞F-actin、α-SMA、ColⅠ、TGF-β1和TβRⅠ的表达以及p-Smad2/Smad2蛋白表达比值显著降低(P<0.05,P<0.01)。TGF-β1组细胞Smad7和miRNA-744的表达较对照组显著降低(P<0.05,P<0.01),而TGF-β1+黄芪总苷组细胞Smad7和miRNA-744的表达较TGF-β1组显著升高(P<0.01)。(3)与阴性对照组相比,miRNA-744拮抗剂作用可显著升高LX-2细胞的Edu阳性染色细胞占比和F-actin表达(P<0.05),且对TGF-β1诱导的LX-2细胞具有相似效应。在miRNA-744拮抗剂构建的转染细胞,给予黄芪总苷协同干预后,Edu阳性染色细胞占比和F-actin表�Objective: To explore the mechanism of astragalosides(AST) on inhibiting the activation of hepatic stellate cells. Methods:(1)Human hepatic stellate cell line LX-2 was activated by transforming growth factor-β1(TGF-β1,2.5 ng/ml), meanwhile, the cells were treated with AST at different concentrations(1.25, 2.5, 5, 10, 20, 30, 40 μg/ml). After treatment for 24 hours, Edu incorporation method was used to detect the cell proliferation, and the appropriate drug concentration was selected.(2)LX-2 cells were divided into the control group, TGF-β1(2.5 ng/ml) group, TGF-β1+AST(20 μg/ml) group and TGF-β1+SB431542(10 μmol/L) group. Each group was treated with the corresponding intervention for 24 hours. The gene expressions of α-smooth muscle actin(α-SMA), collagen Ⅰ(ColⅠ), TGF-β1 and miRNA-744 were detected by PCR, the protein expressions of ColⅠ, TGF-β receptor Ⅰ(TβRⅠ), Smad2, p-Smad2 and Smad7 were detected by Western blot, and the filamentous actin(F-actin) expression was detected by immunofluorescence staining.(3)The transfected cells were constructed by adding miRNA-744 agonist(50 nmol/L), miRNA-744 antagonist(100 nmol/L) or negative control reagent, respectively. In the three systems of transfection cells, the cells were divided into the control group, TGF-β1(2.5 ng/ml) group, TGF-β1+AST(20 μg/ml) group and AST(20 μg/ml) group. Each group was treated with the corresponding intervention for 24 hours. The cell proliferation was detected by Edu incorporation method, and the expression of F-actin was detected by immunofluorescence staining. Results:(1)20, 30 and 40 μg/ml AST could significantly inhibit the proliferation of LX-2 cells induced by TGF-β1. Therefore, the concentration of 20 μg/ml was selected for the following experiments.(2)The expressions of F-actin, α-SMA, ColⅠ, TGF-β1 and TβRⅠ and the expression ratio of p-Smad2/Smad2 protein in the TGF-β1 group were significantly higher than those in the control group(P<0.05, P<0.01). Compared with the TGF-β1 group, the expressions

关 键 词：黄芪总苷 miRNA-744 TGF-Β/SMAD信号通路 肝星状细胞 

分 类 号：R285[医药卫生—中药学]