黄芪甲苷通过调控Wnt/β-catenin信号通路对人成纤维细胞凋亡和肌成纤维细胞转化的作用  

Effects of astragaloside-Ⅳon human fibroblast apoptosis and myofibroblast transformation by regulating Wnt/β-catenin signaling pathway

作　　者：肖雪飞 刘石 符艳 游柏稳[2] XIAO Xuefei;LIU Shi;FU Yan;YOU Baiwen(The First Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410007,China;The Second Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410005,China)

机构地区：[1]湖南中医药大学第一附属医院,湖南长沙410007 [2]湖南中医药大学第二附属医院,湖南长沙410005

出　　处：《湖南中医药大学学报》2024年第12期2183-2192,共10页Journal of Hunan University of Chinese Medicine

基　　金：湖南省中医药科研计划项目(2021165)。

摘　　要：目的利用转化生长因子β1(transforming growth factor-β1,TGF-β1)刺激人成纤维细胞HFL-1模拟特发性肺纤维化的病理过程,探讨黄芪甲苷(astragaloside-Ⅳ,AS-Ⅳ)对人成纤维细胞凋亡和肌成纤维细胞转化的作用和机制。方法采用10 ng/mL TGF-β1刺激HFL-1细胞,给予不同浓度(0、1.25、2.5、5、10μmol/L)的AS-Ⅳ溶液干预24 h后,利用RT-PCR检测纤连蛋白(fibronectin,FN)mRNA表达量。随后细胞分为空白组、TGF-β1组、TGF-β1+AS-Ⅳ组、TGF-β1+XAV939组。采用流式细胞术检测细胞凋亡率;采用RT-PCR法检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、FN、重组人胶原蛋白-1(collagenⅠ)、B淋巴细胞瘤2(B-cell lymphoma 2,Bcl-2)及Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)mRNA表达情况;采用免疫荧光法检测α-SMA、上皮细胞钙黏蛋白(E-Cadherin)、collagenⅠ、FN、β-联蛋白(β-catenin)及半胱氨酸蛋白酶蛋白-3(Cleaved Caspase-3)的蛋白表达;Western blot检测FN、β-catenin、Cleaved Caspase-3蛋白表达水平。结果与空白组比较,TGF-β1组细胞凋亡率上升(P<0.05),FN、collagenⅠ、α-SMA和Bcl-2 mRNA表达增加(P<0.05),Bax mRNA表达减少(P<0.05),α-SMA、collagenⅠ、FN、Cleaved Caspase-3及β-catenin蛋白表达增加(P<0.05);与TGF-β1组比较,TGF-β1+AS-Ⅳ组和TGF-β1+XAV939组细胞凋亡率下降(P<0.05),FN、collagenⅠ、α-SMA和Bcl-2 mRNA表达减少(P<0.05),Bax mRNA表达增加(P<0.05),α-SMA、collagenⅠ、FN、Cleaved Caspase-3及β-catenin蛋白表达减少(P<0.05);与TGF-β1+XAV939组相比较,TGF-β1+AS-Ⅳ组细胞凋亡率下降(P<0.05),FN、collagenⅠ、Bcl-2和α-SMA mRNA表达量降低(P<0.05),Bax mRNA表达量增加(P<0.05)。结论AS-Ⅳ可抑制TGF-β1诱导的HFL-1细胞凋亡和肌成纤维细胞转化,该作用可能与Wnt/β-catenin信号通路有关。Objective To simulate the pathological process of idiopathic pulmonary fibrosis(IPF)by stimulating human fibroblast(HFL-1)cells with transforming growth factor-β1(TGF-β1),and to explore the effects and mechanism of astragaloside-Ⅳ(AS-Ⅳ)on human fibroblast apoptosis and myofibroblast transformation.Methods HFL-1 cells were stimulated with 10 ng/mL TGF-β1 and then treated with different concentrations(0,1.25,2.5,5,10μmol/L)of AS-Ⅳsolution for 24 h.The mRNA expression of fibronectin(FN)was determined by RT-PCR.Subsequently,the cells were divided into blank group,TGF-β1 group,TGF-β1+AS-Ⅳgroup,and TGF-β1+XAV939 group.Flow cytometry was used to measure cell apoptosis rate,RT-PCR to examine the mRNA expressions ofα-smooth muscle actin(α-SMA),FN,recombinant human collagen-1(collagenⅠ),B-cell lymphoma 2(Bcl-2),and Bcl-2 associated X protein(Bax),immunofluorescence to determine the protein expressions ofα-SMA,E-Cadherin,collagenⅠ,FN,β-catenin,and Cleaved Caspase-3,and Western blot to check the protein expression levels of FN,β-catenin,and Cleaved Caspase-3.Results The optimal concentration of AS-Ⅳfor inhibiting FN expression was 5μmol/L.Compared with the blank group,the cell apoptosis rate in the TGF-β1 group increased(P<0.05),the mRNA expressions of FN,collagenⅠ,α-SMA,and Bcl-2 were elevated(P<0.05),while Bax mRNA expression decreased(P<0.05).Additionally,the protein expressions ofα-SMA,collagenⅠ,FN,Cleaved Caspase-3,andβ-catenin also increased(P<0.05).Compared with the TGF-β1 group,both the TGF-β1+AS-Ⅳgroup and TGF-β1+XAV939 group showed decreased cell apoptosis rates(P<0.05),reduced mRNA expressions of FN,collagenⅠ,α-SMA,and Bcl-2(P<0.05),increased Bax mRNA expression(P<0.05),and decreased protein expressions ofα-SMA,collagenⅠ,FN,Cleaved Caspase-3,andβ-catenin(P<0.05).Compared with the TGF-β1+XAV939 group,the cell apoptosis rate in the TGF-β1+AS-Ⅳgroup decreased(P<0.05),the mRNA expressions of FN,collagenⅠ,Bcl-2,andα-SMA decreased(P<0.05),while that of Bax increased(

关 键 词：黄芪甲苷 特发性肺纤维化 成纤维细胞 肌成纤维细胞转化 WNT β-联蛋白 凋亡 

分 类 号：R285.5[医药卫生—中药学]