断藤益母汤调控NF-κB通路减轻LPS诱导的软骨细胞炎症  

Duanteng Yimu Decoction(断藤益母汤) Reduces LPS-induced Chondrocyte Inflammation by Regulating NF-κB Pathway

作　　者：韩隆胤 王强[1] 刘明岭[3] 钱凯 曾丽盈 杜彦仪 黄文广 王辰 李楠 林昌松[3] HAN Longyin;WANG Qiang;LIU Mingling;QIAN Kai;ZENG Liying;DU Yanyi;HUANG Wenguang;WANG Chen;LI Nan;LIN Changsong(Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong,China;School of Traditional Chinese Medicine,Jinan University,Guangzhou 510632,Guangdong,China;The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong,China)

机构地区：[1]广州中医药大学,广东广州510405 [2]暨南大学中医学院,广东广州510632 [3]广州中医药大学第一附属医院,广东广州510405

出　　处：《中华中医药学刊》2021年第6期22-27,I0016,共7页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金(81573930,81774262);中央高校基本科研业务费专项资金(21618335);广东省自然科学基金(2017A030311009,2018A0303130112);广东省医学科学技术研究基金(A2019066);广东省中医药局科研项目(20201072)。

摘　　要：目的研究断藤益母汤对LPS诱导的软骨细胞炎症的影响及其分子机制。方法收集类风湿关节炎(rheumatoid arthritis, RA)患者的膝关节软骨进行体外细胞培养和鉴定;使用CCK8法检测LPS和断藤益母汤对软骨细胞活力的影响;使用实时荧光定量PCR及ELISA检测软骨细胞中白介素-6(IL-6)、白介素-8(IL-8)、基质金属蛋白酶-9(MMP-9)、基质金属蛋白酶-13(MMP-13)的表达;使用免疫印迹法检测NF-κB信号通路中IKK、IκBα、NF-κB蛋白表达及其磷酸化的水平;使用免疫荧光法检测NF-κB p65的入核情况。结果 (10μg/mL)的LPS及断藤益母汤在100、200、400μg/mL的浓度下对RA软骨细胞的活力没有明显的影响(P>0.05);PCR及ELISA检测显示断藤益母汤降低了LPS诱导的软骨细胞中IL-6、IL-8、MMP9、MMP13的表达(P<0.05或P<0.01);免疫印迹分析结果显示LPS增加了软骨细胞中IKK、IκBα及NF-κB蛋白的磷酸化(P<0.01),断藤益母汤抑制了LPS诱导的软骨细胞中IKK、IκBα及NF-κB蛋白的磷酸化(P<0.05或P<0.01);细胞免疫荧光显示断藤益母汤抑制了LPS导致的NF-κB p65入核。结论断藤益母汤通过调控NF-κB通路减少了LPS诱导的软骨细胞炎症。Objective To investigate the effect and molecular mechanism of Duanteng Yimu Decoction(断藤益母汤,DTYMD) on inflammation in chondrocytes induced by LPS. Methods The knee cartilage tissue was collected from rheumatoid arthritis patients for in vitro cell culture and identification. CCK8 method was useed to detect the effect of LPS and DTYMD on the activity of chondrocytes. Real-time quantitative PCR and ELISA were used to detect expressions of IL-6, IL-8, MMP-9 and MMP-13 in chondrocytes. Immunoblot was used to detect the expressions of IKK, IκBα and NF-κB protein and its phosphorylation level in NF-κB signaling pathway. The immunofluorescence was used to detect the nuclear entry of NF-κB p65. Results LPS(10 μg/mL) and concentrations of 100 μg/mL, 200 μg/mL, and 400 μg/mL had no significant effect on the viability of RA chondrocytes(P>0.05). PCR and ELISA tests showed that DTYMD reduced the expressions of IL-6, IL-8, MMP9 and MMP13 in chondrocytes induced by LPS(P<0.05 or P<0.01). The results of Western blot analysis showed that LPS increased the phosphorylation of IKK, IκBα and NF-κB proteins in chondrocytes(P<0.01), and DTYMD inhibited the phosphorylation of IKK, IκBα and NF-κB protein in chondrocytes induced by LPS(P<0.05 or P<0.01). Cellular immunofluorescence showed that DTYMD inhibited the nuclear entry of NF-κB p65 caused by LPS. Conclusion DTYMD reduces LPS-induced chondrocyte inflammation by regulating the NF-κB pathway.

关 键 词：断藤益母汤 类风湿关节炎 软骨细胞 NF-ΚB信号通路 炎症 

分 类 号：R259.932.2[医药卫生—中西医结合]