摘要
目的探讨羟氯喹(HCQ)对活化的类风湿关节炎成纤维样滑膜细胞(RA-FLS)炎症因子白细胞介素(IL)-6表达的影响及分子机制研究。方法 RA-FLS细胞系MH7A细胞用10 ng/ml的肿瘤坏死因子α(TNF-α)诱导活化后,采用不同浓度的HCQ(2.5,5,10μmol/L)处理,使用MTS的方法检测细胞活力;采用酶联免疫吸附试验(ELISA)方法检测培养液中IL-6的浓度,采用实时定量聚合酶链反应(qPCR)检测IL-6 mRNA的表达水平。分子机制研究方面,采用蛋白质印迹法检测核因子(NF)-κB信号通路关键分子p-p65,p65蛋白的表达情况,qPCR检测NF-κB抑制剂对IL-6mRNA表达的影响。结果与空白对照组相比,TNF-α刺激MH7A后IL-6的分泌和mRNA表达显著升高(P<0.01),且p65磷酸化水平明显升高。用不同浓度的HCQ处理(2.5,5,10μmol/L)后,IL-6的分泌和mRNA表达均下降,呈浓度依赖性;同时p65蛋白的磷酸化水平明显降低,同样呈浓度依赖性。提前给予NF-κB激动剂佛波醇酯(PMA)预处理可逆转HCQ对MH7A细胞IL-6表达的抑制作用。结论 HCQ主要通过NF-κB信号通路抑制TNF-α诱导的MH7A细胞IL-6分泌和mRNA表达。
Objective To investigate the effect of hydroxychloroquine(HCQ) on the expression of inflammatory factor IL-6 in activated rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS) and its molecular mechanism.Methods RA-FLS cell line MH7 A cells were induce-activated by 10 ng/ml tumor necrosis factor alpha(TNF-α) and then treated with different concentrations of HCQ(2.5, 5, and 10 μmol/L). Cell viability was detected by MTS assay.The concentration of inflammatory factor IL-6 in culture medium was detected by ELISA, and the expression of IL-6 mRNA by real-time quantitative PCR. In the molecular mechanistic study, the protein expression levels of p-p65 and p65 in the NF-κB signaling pathway were detected by Western blotting. The effect of NF-κB inhibitor on IL-6 mRNA expression was detected by qPCR. Results Compared with the blank control group, TNF-α stimulation in MH7 A cells resulted in significantly increases in IL-6 secretion and mRNA expression(P<0.01), and in phosphorylation of p65. After treatment with different concentrations of HCQ(2.5, 5, and 10μmol/L), IL-6 secretion and mRNA expression decreased in a concentration-dependent manner, so were the phosphorylation levels ofp65 protein. Pretreatment with NF-κB agonist PMA was shown to regress the inhibitory effect of HCQ on IL-6 expression in MH7 A cells. Conclusion HCQ mainly inhibits TNF-α-induced IL-6 secretion and mRNA expression in MH7 A cells via NF-κB signaling pathway.
作者
刘国钰
郑雅丹
戴嘉婧
王庆文
方征宇
Liu Guoyu;Zheng Yadan;Dai Jiajing;Wang Qingwen;Fang Zhengyu(Shenzhen Medical Center Peking University-Hong Kong University of Science and Technology, Guangdong 518036, China)
出处
《中国药物与临床》
CAS
2019年第15期2538-2540,共3页
Chinese Remedies & Clinics
基金
深圳市基础研究计划（JCYJ20170307112009204
JCYJ20170306161757367）
