基于Notch与Wnt/β-catenin通路探讨黄芪甲苷对人成纤维细胞间质转化的作用  

Study on Effect of Astragaloside Ⅳ on Mesenchymal Transition of Human Fibroblasts Based on Notch and Wnt/β-catenin Pathway

作　　者：龚晓红 李桓[1,2] 陆超群 武上雯 邢清桦 李松伟[2,3] GONG Xiaohong;LI Huan;LU Chaoqun;WU Shangwen;XING Qinghua;LI Songwei(Henan University of Chinese Medicine,Zhengzhou 450000,Henan,China;The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000,Henan,China;Henan Provincial Hospital of Traditional Chinese Medicine,The Second Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000,Henan,China)

机构地区：[1]河南中医药大学,河南郑州450000 [2]河南中医药大学第一附属医院,河南郑州450000 [3]河南省中医院,河南中医药大学第二附属医院,河南郑州450000

出　　处：《中华中医药学刊》2024年第4期60-68,I0019,共10页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81573952,81874465);河南省自然科学基金项目(222300420228);河南省科技攻关项目(2221023103922);河南省科技计划联合基金项目(222301420089);河南省中医药科学研究专项(20-21ZYZD16,2022ZY1015);河南省中医药拔尖人才项目(2022ZYBJ10);河南省中医药临床研究基地专项(2022ZY1015);河南中医药大学科研创新项目(2021KYCX003)。

摘　　要：目的基于Notch与Wnt/β-catenin通路,探讨黄芪甲苷(astragalosideⅣ,AS-Ⅳ)对类风湿关节炎相关间质性肺病(rheumatoid arthritis associated interstitial lung disease,RA-ILD)体外模型的作用机制。方法用Luminex检测法对RA-ILD模型鼠支气管肺泡灌洗液中细胞因子进行检测,筛选出关键细胞因子;共培养人髓系白血病单核细胞(human myeloid leukemia mononuclear cells,THP-1)与正常人肺成纤维细胞(normal human lung fibroblast,NHLF)两种细胞系,加入关键细胞因子模拟RA-ILD体外模型,给予不同干预措施。通过RT-PCR、Western blot、COIP、EdU、ELISA以及双荧光素酶报告基因活性检测方法观察Snail、ZEB-1及E-Cadherin的启动子活性及mRNA表达、Notch通路中的关键蛋白表达、β-catenin的核转位水平、NHLF细胞增殖速率、ColⅠ、ColⅢ及Fibronectin的分泌水平以及β-catenin与NICD、β-catenin与LEF、NICD与CSL的相互作用。结果使用细胞因子处理NHLF细胞后,Snail、ZEB-1的启动子活性及mRNA表达明显增高,Notch通路中的关键蛋白表达水平显著上调,β-catenin的核转位明显增加,EdU阳性细胞明显增加;细胞上清中ColⅠ、ColⅢ及Fibronectin分泌水平显著上调,同时β-catenin与NICD、β-catenin与LEF、NICD与CSL的蛋白结合明显增加;使用Notch抑制剂DAPT和Jagged1 shRNA时结果与上述结论相反;AS-Ⅳ能够通过抑制Jagged1进而下调Notch及β-catenin信号通路的过度活化,抑制β-catenin与NICD、β-catenin与LEF、NICD与CSL的蛋白结合,能够剂量依赖性地抑制Snail及ZEB-1并上调E-Cadherin的启动子活性、mRNA和蛋白表达水平,抑制NHLF细胞的过度增殖及Col I、ColⅢ、Fibronectin的分泌。结论AS-Ⅳ可能通过抑制Notch和Wnt/β-catenin信号通路,抑制NHLF细胞增殖,减少ECM过度沉积,从而减缓RA-ILD肺间质纤维化进展。Objective To investigate the mechanism of astragalosideⅣ(AS-Ⅳ)in an in vitro model of rheumatoid arthritis-associated interstitial lung disease(RA-ILD)based on Notch and Wnt/β-catenin pathways.Methods Inflammatory factors were detected in the bronchoalveolar lavage fluid of RA-ILD model rats by Luminex assay to screen out key inflammatory factors.Two cell lines,THP-1 and NHLF,were co-cultured,and inflammatory factors were added to simulate RA-ILD in vitro model with different interventions.The promoter activity and mRNA expressions of Snail,ZEB-1 and E-Cadherin,expressions of key proteins in Notch pathway,nuclear translocation level of β-catenin and the proliferation of NHLF cells,the secretion levels of ColⅠ,ColⅢ and Fibronectin,and the interaction of β-catenin with NICD,β-catenin with LEF,and NICD with CSL were observed by RT-PCR,Western blot,COIP,EdU,ELISA and dual luciferase reporter gene activity assays.Results After NHLF cells were treated with inflammatory factors,the promoter activity and mRNA expressions of Snail and ZEB-1 were significantly increased,the expression levels of key proteins in the Notch pathway were significantly up-regulated,the nuclear translocation of β-catenin was significantly increased,and EdU-positive cells were significantly increased.The secretion levels of ColⅠ,ColⅢ and Fibronectin in cell supernatants were significantly up-regulated,while the protein binding of β-catenin with NICD,β-catenin with LEF and NICD with CSL was significantly increased.The results were the opposite when using the Notch inhibitors DAPT and Jagged1 shRNA.AS-Ⅳ was able to down-regulate the overactivation of Notch and β-catenin signaling pathways by inhibiting Jagged1 and thus AS-Ⅳwas able to inhibit the excessive activation of Notch and β-catenin signaling pathways,inhibit the protein binding of β-catenin with NICD,β-catenin with LEF and NICD with CSL,dose-dependently inhibit Snail and ZEB-1 and up-regulate the promoter activity,mRNA and protein expression levels of E-Cadherin,in

关 键 词：类风湿关节炎 间质性肺病 成纤维细胞 黄芪甲苷 NOTCH信号通路 Wnt/β-catenin信号通路 

分 类 号：R284[医药卫生—中药学]