瘀血痹片对大鼠胶原诱导性关节炎的干预作用及抗炎机制  

Effect and Mechanism of Yuxuebi Tablet Against Collagen-Induced Arthritis of Rats

作　　者：袁蓓 苏晓慧[2] 郭婉怡 汪倩 刘春芳[2] 韩岚 林娜[2] YUAN Bei;SU Xiao-hui;GUOWan-yi;WANG Qian;LIU Chun-fang;HAN Lan;LIN Na(School of Pharmcy,Anhui University of Chinese Medicine,Hefei 230012,China;Institute of Chinese Materia Medico,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]安徽中医药大学药学院,合肥230012 [2]中国中医科学院中药研究所,北京100700

出　　处：《中国实验方剂学杂志》2021年第11期52-62,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家“重大新药创制”科技重大专项(2019ZX09731002);中国中医科学院中药研究所技术研发项目(20190316,20200326)。

摘　　要：目的:观察瘀血痹片对胶原诱导性关节炎(CIA)大鼠的干预作用并探索抗炎相关机制。方法:建立大鼠CIA模型,给药组和阳性药组分别灌胃给予低、中、高剂量瘀血痹片(0.1,0.2,0.4 g·kg^(-1))和甲氨蝶呤(0.4 mg·kg^(-1)),每3 d检测发病率、机械痛敏阈值及冷刺激痛敏;给药30 d后取材,外周血检测血小板计数(PLT)及纤维蛋白原(FIB)含量;苏木素-伊红染色法分析CIA大鼠关节组织病理变化;免疫组化法(IHC)和蛋白免疫印迹法(Western blot)分别检测CIA大鼠关节组织中白细胞介素(IL)-1β,IL-8,核转录因子-κB(NF-κB)p65,磷酸化NF-κB p65(p-NF-κB p65),大鼠肉瘤(Ras)及Raf-1蛋白表达情况。此外,采用10μg·L^(-1)的肿瘤坏死因子-α(TNF-α)诱导人类类风湿关节炎成纤维样滑膜细胞(RA-FLS),转移小室法检测RA-FLS迁移及侵袭能力。Western blot检测RA-FLS中Ras,Raf-1,p-NF-κB p65蛋白表达水平。结果:与正常组比较,CIA模型组发病率升高,机械痛阈值明显降低(P<0.05,P<0.01),冷刺激反应评分显著增高(P<0.01),外周血中PLT和FIB含量,大鼠关节组织病理评分,RA-FLS迁移和侵袭细胞个数及炎性相关因子的表达水平均显著升高(P<0.01);与模型组比较,瘀血痹片低、中、高剂量组均能降低CIA大鼠发病率,提高其机械痛阈值并降低冷刺激痛敏反应评分(P<0.05,P<0.01);降低CIA大鼠外周血中PLT和FIB含量(P<0.05,P<0.01),改善关节滑膜增生、骨及软骨破坏等病理变化(P<0.05,P<0.01),并抑制RA-FLS的迁移及侵袭。此外,瘀血痹片低、中、高剂量组均能不同程度地著抑制CIA大鼠关节组织中IL^(-1)β,IL-8,Ras,Raf-1和p-NF-κB p65的含量,以及RA-FLS细胞中Ras,Raf-1及p-NF-κB p65等蛋白表达水平(P<0.05,P<0.01)。结论:瘀血痹片具有降低大鼠CIA发病率、关节炎临床症状和改善关节组织病理变化、抑制滑膜生成等作用,并且这一作用可能与其对Ras/Raf-1/NF-κB信号通路的抑制有关。Objective:To explore the intervention effect of Yuxuebi tablet(YXB)on collagen-induced arthritis(CIA)in rats and its anti-inflammatory mechanism. Method: Following CIA modeling,the rats in the drug administration groups were separately treated with intragastric administration of YXB(0.1,0.2,and 0.4 g·kg^(-1))and methotrexate(MTX,0.4 mg·kg^(-1)),once a day. The incidence of CIA,mechanical pain threshold(MPT)and cold pain threshold(CPT)were evaluated once every three days. After continuous administration for30 days,the peripheral blood of rats was collected for the determination of platelet(PLT)count and fibrinogen(FIB)content. The hematoxylin-eosin(HE)staining was conducted to analyze the pathological changes in joint tissues. The protein expression levels of interleukin(IL)-1β,IL-8,nuclear transcription factor-κB(NF-κB)p65,phosphorylated NF-κB(p-NF-κB)p65,Ras,and Raf-1 in joint tissues of CIA rats were detected by immunohistochemistry(IHC)and Western blot. The rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS)were induced by tumor necrosis factor-α(TNF-α,10 μg·L^(-1))in vitro and then subjected to transwell migration/invasion assay,followed by the detection of protein expression levels of Ras,Raf-1,and p-NF-κB p65 in RAFLS by Western blot. Result: Compared with the control group,the model group exhibited an increased incidence of CIA,significantly decreased MPT(P<0.05,P<0.01),elevated CPT(P<0.01)and PLT and FIB in the peripheral blood,worsened histopathological score of joints,enhanced RA-FLS migration and invasion,and up-regulated inflammatory factors(P<0.01). The comparison with the model group revealed that YXB at different doses obviously reduced the incidence of CIA,increased MPT,down-regulated CPT and PLT and FIB in the peripheral blood(P<0.05,P<0.01),ameliorated the pathological changes like synovial hyperplasia and bone and cartilage destruction(P<0.05,P<0.01),and inhibited RA-FLS migration and invasion. Besides,the low-,medium-,and high-dose YXB reversed the IL^(-1)β,IL-8,Ras,Raf-1,an

关 键 词：类风湿关节炎(RA) 瘀血痹片 成纤维样滑膜细胞(FLS) 炎症因子 Ras/Raf-1/核转录因子-κB(NF-κB) 

分 类 号：R2-0[医药卫生—中医学] R22