当归拈痛汤对佐剂性关节炎大鼠滑膜组织内外凋亡途径关键因子的影响

Effect of Danggui Niantongtang on Key Factors of Extrinsic and Intrinsic Apoptotic Pathway in Synovial Tissue of Adjuvant Arthritis Rats

目的:观察当归拈痛汤对佐剂性关节炎(AA)大鼠滑膜组织内、外凋亡途径中关键调控因子表达水平的影响,进一步探讨该方防治类风湿关节炎的作用机制。方法:将40只SPF级SD大鼠随机分为正常组、模型组、当归拈痛汤组(11.34 g·kg^(-1))和雷公藤组(9.45 mg·kg^(-1)),除正常组外,其余各组采用灭活结核分支杆菌佐剂建立佐剂性关节炎大鼠模型,于免疫当天开始按分组每日灌胃给药,正常组与模型组大鼠给予等体积生理盐水,持续28 d。观察大鼠一般情况和体质量改变,足趾容积测量仪检测受试大鼠足趾容积的变化,苏木素-伊红(HE)染色观察大鼠膝关节滑膜组织形态学改变,蛋白免疫印迹法(Western blot)和实时荧光定量聚合酶链式反应(Real-time PCR)检测各组大鼠滑膜组织肿瘤坏死因子受体超族成员6(Fas),Fas相关死亡域蛋白(FADD),B淋巴细胞瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax)及其共同下游效应分子半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)mRNA和蛋白的表达水平。结果:与正常组比较,模型组大鼠足趾容积显著增加(P<0.01),滑膜细胞增生显著,滑膜组织Fas,FADD,Bax和Caspase-3 mRNA和蛋白表达水平明显降低(P<0.05,P<0.01),Bcl-2 mRNA和蛋白表达水平明显升高(P<0.01);与模型组比较,当归拈痛汤组和雷公藤组大鼠的足趾肿胀度显著减轻(P<0.01),滑膜增生情况明显改善,Fas,FADD,Bax和Caspase-3 mRNA和蛋白表达水平明显升高(P<0.05,P<0.01),Bcl-2 mRNA和蛋白表达水平明显下降(P<0.05,P<0.01)。结论:当归拈痛汤可以有效减轻AA大鼠关节肿胀情况,改善滑膜组织异常增生,其作用机制可能与调控内、外在凋亡途径相关因子Fas,FADD,Bax,Bcl-2,Caspase-3表达,促进滑膜细胞凋亡相关。

Objective: To observe the effect of Danggui Niantongtang on the protein and mRNA expression of key regulatory factors of the extrinsic and intrinsic apoptotic pathway in synovial tissue of adjuvant arthritis(AA)rats,and to further explore the mechanism of Danggui Niantongtang in the prevention and treatment of rheumatoid arthritis. Method: The general condition of AA rats,including its body weight,were observed. The changes of toe volume were detected by toe volume meter. Histopathological changes of synovium of knee joint were observed by hematoxylin-eosin(HE)staining. The mRNA and protein expression levels of tumor necrosis factor receptor super family 6(Fas),Fas-associating protein with a novel death domain(FADD),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax)and cysteinyl aspartate specific proteinase Caspase-3(Caspase-3)were detected by Real-time fluorescence quantitative polymerase chain reaction(Realtime PCR)and Western blot. Result: Compared with the normal group,the toe volume of the model group increased significantly(P<0.01),with significantly proliferated synovial cells,significantly reduced mRNA and protein expression levels of Fas,FADD,Bax and Caspase-3 in synovial tissues(P<0.05,P<0.01),and significantly increased Bcl-2 level(P<0.01). Compared with the model group,the swelling degree of toes in Danggui Niantongtang group and Tripterygium group was significantly alleviated(P<0.01),with significantly improved synovial hyperplasia,significantly increased mRNA and protein expression levels of Fas,FADD,Bax and Caspase-3(P<0.05,P<0.01),and significantly decreased expression levels of bcl-2 mRNA and protein(P<0.05, P<0.01). Conclusion: Danggui Niantongtang can effectively reduce joint swelling and abnormal proliferation of synovial tissue in AA rats. Its mechanism may be related to regulating the expression of Fas,FADD,Bax,Bcl-2 and Caspase-3,and promoting the apoptosis of synovial cells.