红景天苷调控miR-20a-5p/TIMP2轴对类风湿关节炎成纤维样滑膜细胞增殖和迁移的影响  

Effects of salidroside on proliferation and migration of fibroblastoid synovial cells in rheumatoid arthritis by regulating miR-20a-5p/TIMP2 axis

作　　者：朱光昭 方璐 严婕 李琴[1] Zhu Guangzhao;Fang Lu;Yan Jie;Li Qin(Dept of Rheumatology,Qinghai Hospital of Traditional Chinese Medicine,Xining 810000;Medical College of Qinghai University,Xining 810016)

机构地区：[1]青海省中医院风湿病科,西宁810000 [2]青海大学医学部,西宁810016

出　　处：《安徽医科大学学报》2024年第5期803-809,共7页Acta Universitatis Medicinalis Anhui

基　　金：青海省卫生健康委重点课题(编号:2022-wjzd-04)。

摘　　要：目的探讨红景天苷调控miR-20a-5p/金属蛋白酶组织抑制剂-2(TIMP2)轴对人类风湿关节炎成纤维样滑膜细胞(HFLS-RA)功能和活化的影响。方法以HFLS-RA细胞为研究对象,将HFLS-RA细胞分为肿瘤坏死因子-α(TNF-α)组、对照组、红景天苷组、miR-20a-5p抑制物阴性对照(inhibitor NC)组、miR-20a-5p抑制物组、红景天苷+miR-20a-5p模拟物阴性对照(mimic NC)组、红景天苷+miR-20a-5p模拟物组。实时荧光定量聚合酶链反应(qRT-PCR)检测HFLS-RA细胞中miR-20a-5p表达;酶联免疫吸附试验(ELISA)检测HFLS-RA细胞上清液中白细胞介素(IL)-1β、IL-6水平;细胞计数试剂盒8(CCK-8)法、5-乙炔基-2’-脱氧尿苷(EdU)染色检测HFLS-RA细胞增殖;划痕实验检测HFLS-RA细胞迁移;免疫印迹实验(Western blot)检测HFLS-RA细胞中TIMP2、细胞周期素D1(CyclinD1)、基质金属蛋白酶(MMP)-9蛋白表达;双荧光素酶验证miR-20a-5p与TIMP2的关系。结果与对照组比较,TNF-α组miR-20a-5p表达、IL-1β、IL-6水平、吸光度(OD_(450))值、EdU阳性细胞率、划痕愈合率及CyclinD1、MMP-9蛋白上调,TIMP2蛋白下调(P<0.05);与TNF-α组相比,红景天苷组miR-20a-5p表达、IL-1β、IL-6水平、OD_(450)值、EdU阳性细胞率、划痕愈合率及CyclinD1、MMP-9蛋白下调,TIMP2蛋白上调(P<0.05);与inhibitor NC组、TNF-α组相比比较,miR-20a-5p抑制物组miR-20a-5p表达、IL-1β、IL-6水平、OD_(450)值、EdU阳性细胞率、划痕愈合率及CyclinD1、MMP-9蛋白下调,TIMP2蛋白上调(P<0.05);与红景天苷+mimic NC组、红景天苷组相比,红景天苷+miR-20a-5p模拟物组miR-20a-5p表达、IL-1β、IL-6水平、OD_(450)值、EdU阳性细胞率、划痕愈合率及CyclinD1、MMP-9蛋白表达升高,TIMP2蛋白表达降低(P<0.05)。miR-20a-5p与TIMP2存在靶向调控关系。结论红景天苷可能通过调控miR-20a-5p/TIMP2抑制TNF-α诱导的HFLS-RA细胞增殖、迁移及炎症反应。Objective To investigate effect of salidroside on the function and activation of rheumatoid arthritis fibroblast-like synoviocyte(HFLS-RA)by regulating the miR-20a-5p/tissue inhibitor of metalloproteinase-2(TIMP2)axis.Methods HFLS-RA cells were used as the research object.HFLS-RA cells were separated into control group,tumor necrosis factor-α(TNF-α)group,salidroside group,inhibitor NC group,miR-20a-5p inhibitor group,salidroside+mimic NC group,and salidroside+miR-20a-5p mimic group.qRT-PCR was applied to detect the expression of miR-20a-5p in HFLS-RA cells;enzyme-linked immunosorbent assay(ELISA)was applied to detect the levels of interleukin-1β(IL-1β)and IL-6 in the supernatant of HFLS-RA cells;cell counting kit-8(CCK-8)method and 5-ethynyl-2′-deoxyuridine(EdU)staining were applied to detect HFLS-RA cell proliferation;scratch experiment was applied to detect HFLS-RA cell migration;Western blot was applied to detect the expression of TIMP2,CyclinD1,and matrix metalloproteinase(MMP)-9 proteins in HFLS-RA cells;double lucifer-ase was applied to verify the relationship between miR-20a-5p and TIMP2.Results Compared with the control group,the expression of miR-20a-5p,the levels of IL-1βand IL-6,OD_(450) value,EdU positive cell rate,scratch healing rate,and the expression of CyclinD1 and MMP-9 proteins in the TNF-αgroup increased,the expression of TIMP2 protein decreased(P<0.05);compared with the TNF-αgroup,the expression of miR-20a-5p,the levels of IL-1βand IL-6,OD_(450) value,EdU positive cell rate,scratch healing rate,and CyclinD1 and MMP-9 proteins expression decreased,the expression of TIMP2 protein increased in salidroside group(P<0.05);compared with the TNF-αgroup and inhibitor NC group,the expression of miR-20a-5p,the levels of IL-1βand IL-6,OD_(450) value,EdU positive cell rate,scratch healing rate,and the expression of CyclinD1 and MMP-9 proteins in the miR-20a-5p inhibitor group decreased,the expression of TIMP2 protein increased(P<0.05);compared with the salidroside group and the salidroside+mim

关 键 词：红景天苷 miR-20a-5p 金属蛋白酶组织抑制剂-2 类风湿关节炎 成纤维样滑膜细胞 增殖 迁移 

分 类 号：R285.5[医药卫生—中药学]