摘要
目的 探讨塞来昔布对肿瘤坏死因子-α(TNF-α)诱导类风湿关节炎(RA)成纤维样滑膜细胞(FLSs)MH7A炎症因子分泌的影响及其机制。方法 以0、2.5、5、10、20和40μmol/L塞来昔布处理MH7A细胞24 h后,采用噻唑蓝(MTT)法检测细胞活力以筛选无毒性作用浓度;采用酶联免疫吸附测定(ELISA)法、实时荧光定量PCR(RT-qPCR)法和Western blot法分别检测2.5、5、10μmol/L塞来昔布处理TNF-α诱导的MH7A细胞上清液中炎症因子白细胞介素(IL)-6、IL-1β水平和微小RNA(miR)-129-5p表达水平以及高迁移率族蛋白1(HMGB1)表达水平;双荧光素酶报告基因实验检测miR-129-5p和HMGB1的靶向关系;转染miR-129-5p mimics或HMGB1-siRNA至MH7A细胞中,观察miR-129-5p过表达或下调HMGB1表达对TNF-α诱导的MH7A细胞炎症因子分泌的影响;另外,将miR-129-5p inhibitor转染至TNF-α诱导的MH7A细胞中,观察miR-129-5p低表达对10μmol/L塞来昔布作用下TNF-α诱导MH7A细胞炎症因子分泌的影响。结果 与0μmol/L比较,2.5、5、10μmol/L塞来昔布处理后MH7A细胞活性差异无统计学意义(P>0.05),但20和40μmol/L塞来昔布处理后MH7A细胞活性明显降低(P<0.05)。在TNF-α诱导下,2.5、5、10μmol/L塞来昔布可呈浓度依赖性抑制MH7A细胞上清液中IL-6、IL-1β水平和细胞中HMGB1蛋白表达并促进miR-129-5p表达;miR-129-5p可与HMGB1靶向结合,且miR-129-5p可负向调控HMGB1蛋白表达;miR-129-5p过表达或下调HMGB1表达后,TNF-α诱导的MH7A细胞上清液中IL-6、IL-1β水平明显降低(P<0.05);并且,miR-129-5p低表达可明显逆转10μmol/L塞来昔布对TNF-α诱导的MH7A细胞上清液中IL-6、IL-1β水平抑制作用。结论 塞来昔布可抑制TNF-α诱导MH7A细胞炎症因子分泌,其作用机制可能与调控miR-129-5p/HMGB1有关。
Objective To investigate the effect of celecoxib on the secretion of MH7 A inflammatory factor in fibroblast-like synoviocytes in rheumatoid arthritis(RA) induced by tumor necrosis factor-α(TNF-α).Methods MH7 A cells were treated with 0,2.5,5,10,20 or 40 μmol/L celecoxib for 24 h before detecting cell viability by MTT assay to screen non-toxic concentration.Enzyme-linked immunosorbent assay,real-time fluorescent quantitative PCR,and Western blot were used to detect levels of interleukin(IL)-6,IL-1β,microRNA(miR)-129-5 p,and high mobility group box-1 protein(HMGB1) in the supernatant of TNF-α-induced MH7 A cells treated with 2.5,5 and 10 μmol/L celecoxib.A double-luciferase reporter gene assay was used to detect the targeting relationship between miR-129-5 p and HMGB1,and transfection of miR-129-5 p mimics or HMGB1 siRNA into MH7 A cells was performed to observe the effect of miR-129-5 p overexpression or downregulation of HMGB1 expression on TNF-α-induced inflammatory factor secretion in MH7 A cells.In addition,an miR-129-5 p inhibitor was transfected into MH7 A cells induced by TNF-α to observe the effect of low miR-129-5 p expression on TNF-α-induced secretion of inflammatory factors in MH7 A cells treated with 10 μmol/L celecoxib.Results Compared with 0 μmol/L,the activity of MH7 A cells treated with 2.5,5 and 10 μmol/L celecoxib exhibited no significant difference(P>0.05).However,the activity of MH7 A cells decreased significantly after 20 and 40 μmol/L celecoxib treatment(P<0.05).Under induction by TNF-α,2.5,5 and 10 μmol/L celecoxib inhibited IL-6 and IL-1β levels in the supernatant of MH7 A cells,as well as expression of HMGB1 protein,and promoted the expression of miR-129-5 p in a concentration-dependent manner.miR-129-5 p can target and bind to HMGB1 to negatively regulate its expression.After overexpression of miR-129-5 p or downregulation of HMGB1 expression,levels of IL-6 and IL-1β in the supernatant of MH7 A cells induced by TNF-α were significantly decreased(P<0.05).Low expression of miR-129-5 p significantly reversed the inhibitory effects of 10 μmol/L celecoxib on TNF-α-induced IL-6 and IL-1β levels in the supernatant of MH7 A cells.Conclusions Celecoxib can inhibit TNF-α-induced inflammatory factor secretion by MH7 A cells,and its mechanism may be related to the regulation of miR-129-5 p/HMGB1.
作者
时萍
陶野
王晨静
李欣
柳艳平
曹玉
SHI Ping;TAO Ye;WANG Chenjing;LI Xin;LIU Yanping;CAO Yu(Affiliated Hospital of Qingdao University,Qingdao 266001,China)
出处
《中国比较医学杂志》
CAS
北大核心
2022年第9期82-89,共8页
Chinese Journal of Comparative Medicine
作者简介
时萍(1975-),女,本科,研究方向:药物试验。E-mail:sp82911767@163.com;通信作者:曹玉(1974-),男,博士,主任药师,研究方向:药物试验。E-mail:Jidi1767@126.com。
