天津医药

天津医药 ›› 2021, Vol. 49 ›› Issue (3): 252-257.doi: 10.11958/20201131

• 实验研究 • 上一篇    下一篇

丹皮酚对IL-1β诱导HFLS-RA的保护作用及相关机制研究

李梅1，欧大明1，黄丽芳1，谢立虎1，张济2   

  1. 1南华大学附属第一医院风湿免疫科（邮编421001），2风湿免疫实验室
  • 收稿日期:2020-04-26 修回日期:2020-12-31 出版日期:2021-03-15 发布日期:2021-03-15
  • 作者简介:李梅（1983），女，硕士，主治医师，主要从事风湿免疫相关研究。E-mail：157843277@qq.com
  • 基金资助:
    湖南省卫生健康委项目（20200050）

The protective effect of paeonol on IL-1β induced inflammatory response of human fibroblast-like synoviocytes and rheumatoid arthritis

LI Mei1, OU Da-ming1, HUANG Li-fang1, XIE Li-hu1, ZHANG Ji2   

  1. 1 Department of Rheumatology and Immunology, 2 Rheumatic Immunology Laboratory, the First Affiliated Hospital of South China University, Hengyang 421001, China
  • Received:2020-04-26 Revised:2020-12-31 Published:2021-03-15 Online:2021-03-15

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摘要： 目的　探讨丹皮酚对白细胞介素（IL）-1β诱导人类风湿关节炎成纤维样滑膜细胞（HFLS-RA）的保护作用及机制。方法　将HFLS-RA细胞分为空白对照组，10 μg/L IL-1β组及0.1、1、10、100 μmol/L丹皮酚组，采用MTT法检测细胞增殖情况；酶联免疫吸附测定试验（ELISA）法检测HFLS-RA中肿瘤坏死因子（TNF）-α、IL-6水平。采用免疫荧光法观察空白对照组、10 μg/L IL-1β组及10 μmol/L丹皮酚组HFLS-RA中核因子（NF）-κB p65细胞核移位情况；Western blot法检测空白对照组，10 μg/L IL-1β组，0.1、1、10 μmol/L丹皮酚组及阴性对照组（仅10 μmol/L丹皮酚处理）的基质金属蛋白酶（MMP）-1、MMP-3和Toll样受体-4（TLR4）蛋白表达。将24只雄性DBA/1小鼠随机均分为对照组、CIA组和治疗组，对小鼠进行四肢累计评分，HE染色观察关节组织病理学变化，ELISA法检测滑膜组织中TNF-α、IL-1β水平。结果　丹皮酚对IL-1β诱导HFLS-RA细胞增殖无明显抑制作用。与空白对照组相比，10 μg/L IL-1β组TNF-α、IL-6、NF-κB p65核移位、MMP-1、MMP-3和TLR4蛋白表达水平均升高，而经丹皮酚干预后可逆转这些改变（P＜0.05）。与CIA组相比，治疗组小鼠四肢累计评分明显下降（P＜0.05）；HE染色结果示丹皮酚治疗48 d时踝关节病理变化有所改善，其中炎性细胞明显下降，软骨破坏减轻。治疗组小鼠滑膜组织中TNF-α、IL-1β水平低于CIA组。结论　丹皮酚具有减轻IL-1β诱导HFLS-RA炎性因子表达和缓解CIA小鼠关节炎症状的作用，其机制可能与抑制TLR-4-NF-κB p65通路，减轻炎症反应有关。

关键词: 关节炎, 类风湿；牡丹皮；白细胞介素1β；NF-κB；基质金属蛋白酶类；丹皮酚；人成纤维样滑膜细胞

Abstract: Objective　To explore the protective effect of paeonol on IL-1β induced inflammatory response of human fibroblast-like synoviocytes and rheumatoid arthritis (HFLS-RA) and its possible mechanism. Methods　The HFLS-RA cells were divided into control group, 10 μmol/L paeonol group and 0.1, 1, 10 and 100 μmol/L HFLS-RA groups. The effect of paeonol on the proliferation of HFLS-RA was detected by MTT. The levels of TNF-α and IL-6 in HFLS-RA were detected by enzyme linked immunosorbent assay (ELISA). The nuclear translocation of NF-κB p65 was observed by immuno-fluorescence in blank control group, 10 μg/L IL-1β group and 10 μmol/L paeonol group. The expressions of MMP-1, MMP-3 and TLR4 groups in blank control group, 10 μg/L IL-1β group, 0.1, 1, 10 μmol/L paeonol and negative control group (only treated with 10 μmol/L paeonol) were detected by Western blot assay. Twenty-four male DBA/1 mice were randomly divided into control group, CIA group and treatment group. The limb cumulative score was evaluated. The pathological changes of joint tissues were observed by HE staining. The contents of TNF -α and IL-1β in synovial tissue were detected by ELISA. Results　Paeonol showed no inhibitory effect on IL-1β induced HFLS-RA proliferation. Compared with the blank control group, the expression levels of TNF-α, IL-6, NF-κB p65 nuclear translocation, MMP-1, MMP-3 and TLR4 protein were increased in 10 μg/L IL-1β group, and these changes could be reversed by paeonol intervention (P＜0.05). Compared with the CIA group, the limb cumulative score decreased significantly in the treatment group (P＜0.05). HE observation showed that the pathological changes of ankle joint were improved after 48 days of paeonol treatment, in which the inflammatory cells decreased significantly and the cartilage damage was alleviated. The levels of TNF-α and IL-1β in synovial tissue were lower in treatment group than those of CIA group. Conclusion　Paeonol can reduce the IL-1β induced expression levels of inflammatory factors in HFLS-RA and relieve arthritis symptoms in CIA mice, which may be related to the down regulation of TLR4-NF-κB p65 pathway.

Key words: arthritis, rheumatoid, cortex moutan, interleukin-1beta, NF-kappa B, matrix metalloproteinases, paeonol, human fibroblast like synoviocytes