基于Wnt信号通路研究表没食子酸儿茶素没食子酸酯对肝星状细胞活化的作用及机制  

Effect and mechanism of epigallocatechin gallate on activation of hepatic stellate cells based on Wnt signaling pathway

作　　者：石明亮 王晓磊[1] 李江琳[1] 段文飞[1] SHI Ming-liang;WANG Xiao-lei;LI Jiang-lin;DUAN Wen-fei(Department of General Surgery,The First Affiliated Hospital of Henan University,Kaifeng 475000,China)

机构地区：[1]河南大学第一附属医院普外一科,河南开封475000

出　　处：《中草药》2022年第3期758-766,共9页Chinese Traditional and Herbal Drugs

基　　金：河南省2017年科技发展计划项目(172102310284)。

摘　　要：目的基于Wnt信号通路探究表没食子酸儿茶素没食子酸酯(epigallocatechin gallate,EGCG)对人肝星状细胞LX-2活化的影响及其作用机制。方法体外培养LX-2细胞,CCK-8法筛选EGCG的实验浓度;取对数生长期的LX-2细胞,设置对照组(正常培养)、模型组[10 ng/mL转化生长因子-β1(transforming growth factor-β1,TGF-β1)]、EGCG低剂量组(12.5μmol/L EGCG+10 ng/mL TGF-β1)、EGCG高剂量组(25.0μmol/L EGCG+10 ng/mL TGF-β1)、EGCG+siRNA-NC组[小窝蛋白-1(Caveolin-1,Cav-1)-siRNA阴性对照+25.0μmol/L EGCG+TGF-β1]和EGCG+Cav-1-siRNA组(Cav-1-siRNA+25.0μmol/L EGCG+TGF-β1),CCK-8法检测各组细胞存活率;流式细胞术检测细胞凋亡率和细胞周期分布情况;吖啶橙/溴乙锭(AO/EB)染色法观察细胞凋亡形态;qRT-PCR法检测细胞α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原蛋白(CollagenⅠ)和基质金属蛋白酶组织抑制剂-1(tissue inhibitors of matrix metalloproteinase-1,TIMP-1)mRNA表达情况;Western blotting检测细胞Cav-1和Wnt信号通路相关蛋白表达情况。结果EGCG降低LX-2细胞存活率,呈剂量相关性。与对照组相比,模型组LX-2细胞存活率、S期和G2/M期的细胞比例、细胞α-SMA、CollagenⅠ、TIMP-1 mRNA表达和Wnt1、Wnt5a、β-连环蛋白(β-catenin)、细胞周期蛋白Cyclin D1、原癌基因c-Myc蛋白表达水平均显著升高(P<0.05),细胞凋亡率、G0/G1期的细胞比例、凋亡细胞比例、Cav-1蛋白表达水平均显著降低(P<0.05);与模型组相比,EGCG低、高剂量组LX-2细胞存活率、S期和G2/M期的细胞比例、细胞α-SMA、CollagenⅠ、TIMP-1 mRNA表达和Wnt1、Wnt5a、β-catenin、Cyclin D1、c-Myc蛋白表达水平均显著降低(P<0.05),细胞凋亡率、G0/G1期的细胞比例、凋亡细胞比例、Cav-1蛋白表达水平均显著升高(P<0.05);且在EGCG干预的基础上,沉默Cav-1的表达可显著上调Wnt1、Wnt5a蛋白表达,减弱EGCG对Wnt信号通路的抑制作用。结论EGCG可能通过上调Cav-1表Objective To explore the effect and mechanism of epigallocatechin gallate(EGCG)on the activation of hepatic stellate cells LX-2 based on Wnt pathway.Methods LX-2 cells were cultured in vitro,and the experimental concentration of EGCG was screened by CCK-8 method.LX-2 cells in the logarithmic growth phase were divided into control group(normally cultured),model group[10 ng/mL transforming growth factor-β1(TGF-β1)],low-dose EGCG group(12.5μmol/L EGCG+10 ng/mL TGF-β1),highdose EGCG group(25.0μmol/L EGCG+10 ng/mL TGF-β1),EGCG+siRNA-NC group[Caveolin-1(Cav-1)-siRNA negative control+25.0μmol/L EGCG+TGF-β1]and EGCG+Cav-1-siRNA group(Cav-1-siRNA+25.0μmol/L EGCG+TGF-β1),CCK-8 method was used to detect cell survival rate of each group;Flow cytometry was used to detect cell apoptosis and cell cycle distribution;Acridine orange/ethidium bromide(AO/EB)staining method was used to observe the morphology of cell apoptosis;qRT-PCR was used to detectα-smooth muscle actin(α-SMA),CollagenⅠand tissue inhibitor of matrix metalloproteinase-1(TIMP-1)mRNA expressions of cells;Western blotting was used to detect the expressions of Cav-1 and Wnt signaling pathway related proteins of cells.Results EGCG reduced survival rate of LX-2 cells in a concentration-dependent manner.Compared with control group,LX-2 cells survival rate,ratios of S phase and G2/M phase cells,mRNA expressions ofα-SMA,CollagenⅠand TIMP-1,and protein expressions of Wnt1,Wnt5a,β-catenin,Cyclin D1,c-Myc were significantly increased in model group(P<0.05),apoptosis rate,ratio of G0/G1 phase cells,ratio of apoptotic cells,and Cav-1 protein expression were significantly reduced(P<0.05).Compared with model group,LX-2 cells survival rate,ratios of S phase and G2/M phase cells,mRNA expression ofα-SMA,CollagenⅠ,TIMP-1,and protein expressions of Wnt1,Wnt5a,β-catenin,Cyclin D1,c-Myc in low-,high-dose EGCG groups were significantly decreased(P<0.05),apoptosis rate,ratio of G0/G1 phase cells,ratio of apoptotic cells,and Cav-1 protein expression were significantly

关 键 词：表没食子酸儿茶素没食子酸酯 肝星状细胞 凋亡 WNT信号通路 小窝蛋白-1 

分 类 号：R285.5[医药卫生—中药学]