闹羊花毒素Ⅲ通过调控Wnt1/Dvl1/β-catenin通路影响成纤维样滑膜细胞增殖凋亡的实验研究  

Rhodojaponin Ⅲ Affected Fibroblast-like Synovial Cell Proliferation and Apoptosis by Regulating Wnt1/Dvl1/β-catenin Pathway: an Experimental Research

作　　者：刘笑蓉[1] 刘湘丹[1] 王智[1] 曾娟 周日宝[1] LIU Xiao-rong;LIU Xiang-dan;WANG Zhi;ZENG Juan;ZHOU Ri-bao(College of Pharmacy,Hunan University of Chinese Medicine,Changsha,410208)

机构地区：[1]湖南中医药大学药学院,长沙410208

出　　处：《中国中西医结合杂志》2022年第10期1199-1206,共8页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：湖南省教育厅科学研究项目(No.19C1384);湖南省中医药管理局科研计划项目(No.2021161);湖南中医药大学中药学一级学科开放基金项目(No.2020ZYX01);湖南中医药大学青苗计划(No.校行人字[2017]25);湖南省一流学科中药学(No.校行科字[2018]3);2020年湖南省一流本科专业建设点(No.湘教通[2020]248号);2020年国家级一流本科专业建设点(No.教高厅函[2021]7号)。

摘　　要：目的探讨闹羊花毒素Ⅲ对类风湿关节炎(RA)的治疗作用及其机制。方法采用肿瘤坏死因子-α(TNF-α)诱导RA成纤维样滑膜细胞(RA-FLS细胞)作为RA的细胞模型。以雷公藤多苷作为阳性对照药物,过表达蓬乱蛋白1(Dvl1)后加入闹羊花毒素Ⅲ研究其作用机制。细胞增殖毒性检测法(CCK-8法)检测细胞活力,蛋白质免疫印迹(Western Blot)检测B淋巴细胞瘤-2蛋白(Bcl2)、Bcl2相关X蛋白(Bax)、无翅型MMTV整合位点家族成员1(Wnt1)、Dvl1、β-连环蛋白(β-catenin)的表达量,实时荧光定量聚合酶链式反应(RT-qPCR)检测Wnt1、Dvl1、β-catenin mRNA水平,酶联免疫吸附法(ELISA)检测细胞上清中IL-6、IL-8、IL-10、IL-4、CC趋化因子配体2(CCL2)、CC趋化因子配体5(CCL5)的含量。结果RA细胞模型构建成功,随着闹羊花毒素Ⅲ浓度的增加,RA-FLS细胞增殖减缓(P<0.05),凋亡增加(P<0.01);闹羊花毒素Ⅲ与雷公藤多苷降低了RA-FLS细胞中IL-6、IL-8、CCL2、CCL5并且增加了IL-10、IL-4的含量(P<0.05);降低了Wnt1、β-catenin、Dvl1蛋白与mRNA的表达量(P<0.01)。闹羊花毒素Ⅲ可以逆转由过表达Dvl1所造成的RA-FLS细胞增殖的增加(P<0.05)、促炎因子(IL-6、IL-8)与趋化因子(CCL2、CCL5)的增加(P<0.05),并逆转Wnt通路中Wnt1,Dvl1,β-catenin蛋白与mRNA表达的增加(P<0.01)。结论闹羊花毒素Ⅲ通过调控Wnt1/Dvl1/β-catenin通路影响TNF-α诱导的RA-FLS细胞增殖凋亡及炎症因子等的变化,可能通过这种机制治疗RA。Objective To observe the therapeutic effect and mechanism of rhodojaponin Ⅲ on rheumatoid arthritis(RA). Methods The cell model of RA was induced by treating rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS) cells with tumor necrosis factor-α(TNF-α). Tripterygium polyglycosides were used as the positive control. Rhodojaponin Ⅲ was added after the overexpression of Dishevelled 1(Dvl1) to study the mechanism. Cell Counting Kit-8(CCK-8) assay was performed to detect cell vitality. Western Blot was used to detect the expressions of B-Cell CLL/lymphoma 2(Bcl2), BCL2-associated X protein(Bax), winglesstype MMTV integration site family member 1(Wnt1), Dishevelled 1(Dvl1), and beta catenin(β-catenin). Realtime fluorescence quantitative polymerase chain(RT-qPCR) was conducted to test the expressions of Wnt1, Dvl1, and β-catenin mRNA. ELISA was used to analyze the concentrations of interleukin 6(IL-6), IL-8, IL-10, and IL-4, C-C motif chemokine ligand 2(CCL2), and C-C motif chemokine ligand 5(CCL5). Results The cell model of RA was successfully established. With the increase of rhodojaponin Ⅲ, RA-FLS cell vitality decreased(P<0.05), and cell apoptosis increased(P<0.01). Both Rhodojaponin Ⅲ and tripterygium polyglycosides downregulated IL-6, IL-8, CCL2, and CCL5 concentrations(P<0.05), up-regulated IL-10 and IL-4 contents(P<0.05), and down-regulated the expressions of Wnt1, β-catenin and Dvl1(P <0.01). Rhodojaponin Ⅲ reversed increased Dvl1 induced RA-FLS proliferation(P<0.05), increased proinflammatory factors IL-6 and IL-8(P<0.05) and chemokines CCL2 and CCL5(P<0.05), and reversed increased expressions of Wnt1, Dvl1, and β-catenin in RA-FLS(P<0.01). Conclusion Rhodojaponin Ⅲ had an effect on proliferation, apoptosis, and inflammatory factors in TNF-α-induced RA-FLS cells by regulating the Wnt1/Dvl1/β-catenin pathway, and might alleviated RA by this mechanism.

关 键 词：类风湿关节炎 闹羊花毒素Ⅲ 蓬乱蛋白1 无翅型MMTV整合位点家族成员1 β-连环蛋白 

分 类 号：R285[医药卫生—中药学]