清络通痹方调控类风湿关节炎“免疫-骨侵蚀”的机制  

Mechanism of Qingluo Tongbi Formula for regulating immune-bone erosion in rheumatoid arthritis

作　　者：刘天阳[1] 周学平[3] 黄传兵[1] 周玲玲[4] 谌曦[1] 万磊[1] 纵瑞凯[1] 范海霞[1] 孙玥[1] 俞志超 汤忠富 徐耿瑞 周子译 LIU Tianyang;ZHOU Xueping;HUANG Chuanbing;ZHOU Lingling;CHEN Xi;WAN Lei;ZONG Ruikai;FAN Haixia;SUN Yue;YU Zhichao;TANG Zhongfu;XU Gengrui;ZHOU Ziyi(Department of Rheumatology of First Affiliated Hospital,Anhui University of Chinese Medicine,Hefei 230031,China;lGraduate School,Anhui University of Chinese Medicine,Hefei 230031,China;First Clinical College,Nanjing University of Chinese Medicine,Nanjing 210000,China;School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210000,China;Graduate School,Nanjing University of Chinese Medicine,Nanjing 210000,China)

机构地区：[1]安徽中医药大学第一附属医院风湿科,安徽合肥23003 [2]安徽中医药大学研究生院,安徽合肥230031 [3]南京中医药大学第一临床学院,江苏南京210000 [4]南京中医药大学药学院,江苏南京210000 [5]南京中医药大学研究生院,江苏南京210000

出　　处：《南方医科大学学报》2023年第10期1706-1714,共9页Journal of Southern Medical University

基　　金：国家自然科学基金(81973741);国家高水平中医药重点学科中医痹病学(国中医药人教涵[2023]85号);安徽中医药大学第一附属医院临床科研项目(2020zyfyzc25)Supported byNationalNaturalScienceFoundationofChina(81973741).

摘　　要：目的观察清络通痹方对类风湿关节炎(RA)B细胞亚群、B细胞活化因子(BAFF)、骨侵蚀及骨形成指标以及核因子-κB受体活化因子配体(RANKL)/核因子-κB受体活化因子(RANK)/骨保护素(OPG)信号通路的影响,探讨本方调控RA“免疫-骨侵蚀”的机制。方法将64例RA患者随机分为对照组和研究组,32例/组,对照组口服甲氨蝶呤,研究组口服清络通痹方,两组疗程均为12周。采用流式细胞术检测RA患者外周血CD3-CD19+B细胞亚群、CD19+CD27+B细胞亚群和CD19+BAFFR+B细胞亚群百分比,ELISA法检测血清BAFF、RANKL、RANK、OPG水平,ELISA法检测RA患者治疗前后血清β胶原降解产物(β-CTX)、人抗酒石酸酸性磷酸酶5b(TRACP-5b)、血清骨钙素(BGP)、骨碱性磷酸酶(BALP)、Ⅰ型前胶原氨基端原肽(PINP)水平,DXEA双能X线骨密度仪测定骨密度。细胞实验:以RAW264.7细胞作为破骨细胞,分成空白组、模型组、清络通痹方低剂量组(125μg·mL^(-1))、中剂量组(250μg·mL^(-1))、高剂量组(500μg·mL^(-1)),甲氨蝶呤组(2μg·mL^(-1))。干预时间为48 h。采用免疫印迹法检测RANKL、RANK、OPG、Fos原癌基因(C-FOS)蛋白表达情况。结果RA患者B细胞与RANKL/RANK/OPG系统呈相关性,清络通痹方能下调RA患者外周血B细胞亚群百分比、血清BAFF水平,下调血清β-CTX及TRACP-5b水平,上调BGP、BALP、PINP水平,改善腰椎骨密度值(P<0.05),并与调控B细胞表达呈显著相关性(P<0.05);该处方能下调OC中RANKL、RANK、CFOS表达,上调OPG表达(P<0.05)。结论清络通痹方调控RA“免疫-骨侵蚀”的机制可能为通过调控B细胞亚群百分比表达及BAFF表达水平抑制骨破坏,并进一步干预RANKL/RANK/OPG通路抑制破骨细胞分化,从而抑制骨侵蚀的发生。Objective To explore the mechanism of Qingluo Tongbi formula for regulating"immune-bone erosion"in rheumatoid arthritis(RA).Methods Sixty-four RA patients were randomized into two groups to receive treatment with oral methotrexate or Qingluo Tongbi Formula for 12 weeks.Flow cytometry was used to analyze the changes in the percentages of CD3-CD19+,CD19+CD27 and CD19+BAFFR+B cell subpopulations in peripheral blood of the patients,and serum levels of B cell activating factor(BAFF),RANKL,RANK and osteoprotegerin(OPG)levels were detected using ELISA.Before and after the treatment,serum levels ofβ-CTX,TRACP-5b,BGP,BALP,and PINP were measured with ELISA,and bone mineral density was determined with DXEA dual-energy X-ray absorptiometry.In the cell experiment,RAW264.7 cells were induced to differentiated into osteoclasts and treated with Qingluo Tongbi Formula at low-,moderate and high doses(125,250 and 500μg/mL,respectively)or with methotrexate(2μg/mL)for 48 h,and the changes in the expression levels of RANKL,RANK,OPG and c-Fos were detected using Western blotting.Results The B cell subgroups in RA patients were correlated with the RANKL/RANK/OPG system.Treatment with Qingluo Tongbi Formula obviously down-regulated the percentages of the B cell subgroups,lowered serum levels of BAFF,β-CTX and TRACP-5b,increased the levels of BGP,BALP and PINP,and improved lumbar bone density of RA patients(P<0.05);All these changes were significantly correlated with the regulation of B cell expressions(P<0.05).In RAW264.7 cells-derived osteoclasts,Qingluo Tongbi Formula significantly decreased the expressions of RANKL,RANK and c-Fos and increased the expression of OPG(P<0.05).Conclusion Qingluo Tongbi Formula inhibits bone erosion in RA possibly by regulating B cell subset percentages and BAFF expression and inhibiting osteoclast differentiation via the RANKL/RANK/OPG pathway.

关 键 词：类风湿关节炎 清络通痹方 骨侵蚀 

分 类 号：R259[医药卫生—中西医结合]