塞来昔布调节 miR-129-5p / HMGB1 抑制 TNF-α 诱导类风湿关节炎成纤维样滑膜细胞炎症因子分泌
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青岛大学附属医院,山东 青岛 266001

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R-33


Celecoxib regulates miR-129-5p / HMGB1 to inhibit TNF-α-induced rheumatoid arthritis fibroblast-like synovial cell inflammatory cytokine secretion
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Affiliated Hospital of Qingdao University, Qingdao 266001, China

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    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 40 μmol / 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、10 μmol / 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 10 μmol / 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 0 μmol / L 比较,2. 5、5、10 μmol / 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 40 μmol / 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、10 μmol / 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 10 μmol / 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。

    Abstract:

    Objective To investigate the effect of celecoxib on the secretion of MH7A inflammatory factor in fibroblast-like synoviocytes in rheumatoid arthritis (RA) induced by tumor necrosis factor-α (TNF-α). Methods MH7A 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)-129-5p, and high mobility group box-1 protein (HMGB1) in the supernatant of TNF-α-induced MH7A 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-129-5p and HMGB1, and transfection of miR-129-5p mimics or HMGB1 siRNA into MH7A cells was performed to observe the effect of miR-129-5p overexpression or downregulation of HMGB1 expression on TNF-α-induced inflammatory factor secretion in MH7A cells. In addition, an miR-129-5p inhibitor was transfected into MH7A cells induced by TNF-α to observe the effect of low miR-129-5p expression on TNF-α-induced secretion of inflammatory factors in MH7A cells treated with 10 μmol / L celecoxib. Results Compared with 0 μmol / L, the activity of MH7A cells treated with 2. 5, 5 and 10 μmol / L celecoxib exhibited no significant difference (P> 0. 05). However, the activity of MH7A 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 MH7A cells, as well as expression of HMGB1 protein, and promoted the expression of miR-129-5p in a concentration-dependent manner. miR-129-5p can target and bind to HMGB1 to negatively regulate its expression. After overexpression of miR-129-5p or downregulation of HMGB1 expression, levels of IL-6 and IL-1β in the supernatant of MH7A cells induced by TNF-α were significantly decreased ( P< 0. 05). Low expression of miR-129-5p significantly reversed the inhibitory effects of 10 μmol / L celecoxib on TNF-α-induced IL-6 and IL-1β levels in the supernatant of MH7A cells. Conclusions Celecoxib can inhibit TNF-α-induced inflammatory factor secretion by MH7A cells, and its mechanism may be related to the regulation of miR-129-5p / HMGB1.

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时 萍,陶 野,王晨静,李 欣,柳艳平,曹 玉.塞来昔布调节 miR-129-5p / HMGB1 抑制 TNF-α 诱导类风湿关节炎成纤维样滑膜细胞炎症因子分泌[J].中国比较医学杂志,2022,32(9):82~89.

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  • 收稿日期:2021-12-08
  • 在线发布日期: 2023-01-16
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