白芍总苷介导TLR4/NF-κB信号通路对类风湿关节炎模型大鼠的治疗作用及其机制

Therapeutic effect of total glucosides of paeony in model rats with rheumatoid arthritis by mediating TLR4/NF-κB signaling pathway and its mechanisim

目的:探讨白芍总苷(TGP)通过调控Toll样受体4 (TLR4)/核因子κB (NF-κB)信号通路对类风湿关节炎(RA)大鼠关节情况、炎症因子及凋亡因子的影响,并阐明其作用机制。方法法:利用牛Ⅱ型胶原蛋白溶液和完全弗氏佐剂构建胶原诱导性关节炎(CIA)大鼠模型,将CIA模型大鼠随机分为CIA组、低剂量(25 mg·kg~(-1)) TGP组(TGP-LD组)、中剂量(50 mg·kg~(-1)) TGP组(TGP-MD)和高剂量(100 mg·kg~(-1)) TGP组(TGP-HD组),每组10只;另选取10只健康SD大鼠作为对照组。记录各组大鼠体质量、关节炎指数及关节肿胀度;采用酶联免疫吸附试验(ELISA)法检测各组大鼠血清中肿瘤坏死因子α(TNF-α),白细胞介素6 (IL-6)和白细胞介素1β(IL-1β)水平;采用实时荧光定量PCR (RT-qPCR)和Western blotting法检测各组大鼠关节滑膜组织中TLR4、NF-κB、活化的含半胱氨酸的天冬氨酸蛋白水解酶3 (Cleaved caspase-3)、 B细胞淋巴瘤2 (Bcl-2)、Bcl-2相关X蛋白(Bax) mRNA和蛋白表达水平。结果:与对照组比较,CIA组大鼠体质量和关节滑膜组织中Cleaved caspase-3和Bax mRNA和蛋白表达水平明显降低(P<0.05),关节炎指数、关节肿胀度、关节滑膜组织中Bcl-2、TLR4、NF-κB p65 mRNA及蛋白表达水平,血清中TNF-α、IL-6和IL-1β水平明显升高(P<0.05)。与CIA组比较,不同剂量TGP组大鼠体质量和关节滑膜组织中Cleaved caspase-3及Bax mRNA和蛋白表达水平明显升高(P<0.05),关节炎指数、关节肿胀度、关节滑膜组织中Bcl-2、TLR4、NF-κB p65 mRNA和蛋白表达水平,血清中TNF-α、IL-6和IL-1β水平明显降低(P<0.05)。与TGP-LD组比较,TGP-MD组和TGP-HD组大鼠体质量和关节滑膜组织中Cleaved caspase-3及Bax mRNA和蛋白表达水平明显升高(P<0.05),关节炎指数、关节肿胀度、关节滑膜组织中Bcl-2、TLR4、NF-κB p65 mRNA和蛋白表达水平,血清中TNF-α、IL-6、IL-1β水平明显降低(P<0.05)。结论:TGP可通过抑制TLR4/NF-κB信号通路,改善CIA症状,降低炎症反应,并促进关节滑膜组织细胞的凋亡。

Objective:To investigate the effects of total glucosides of paeony(TGP)on joint condition,inflammatory factors and apoptosis factors in the rheumatoid arthritis(RA)rats by regulating the Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway,and to clarify its mechanism.Methods:The collagen-induced arthritis(CIA)rat models were established with bovine typeⅡcollagen and complete Freund’s adjuvant,and the CIA model rats were randomly divided into CIA group,low dose(25 mg·kg-1)of TGP(TGP-LD)group,middle dose(50 mg·kg-1)of TGP(TGP-MD)group,and high dose(100 mg·kg-1)of TGP(TGP-HD),with 10 rats in each group.Another 10 healthy SD rats were selected as control group.The body weights,arthritis indexes and joint swelling degrees of the rats in each group were measured,and the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in serum of the rats in each group were detected by ELISA method.The mRNA and protein expression levels of TLR4,NF-κB,Cleaved caspase-3,B cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)in joint synovium tissue of the rats in each group were detected by Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods.Results:Compared with control group,the body weight and the expression levels of Cleaved caspase-3 and Bax mRNA and proteins in joint synovium tissue of the rats in CIA group were significantly decreased(P<0.05),while the arthritis index,the joint swelling degree,the expression levels of Bcl-2,TLR4,and NF-κB p65 mRNA and proteins in joint synovium tissue,and the TNF-α,IL-6,and IL-1βlevels in serum of the rats in CIA group were significantly increased(P<0.05).Compared with CIA group,the body weights and the expression levels of Cleaved caspase-3 and Bax mRNA and protein in joint synovium tissue of the rats in different doses of TGP groups were significantly increased(P<0.05),while the arthritis indexes,the joint swelling degrees,the expression levels of Bcl-2,TLR4,and NF-κB p65 mRNA and proteins in joint synovium tissue,and the TNF-α,IL-6,IL-1βlevels in serum of the rats were significantly decreased(P<0.05).Compared with TGP-LD group,the body weights and the expression levels of Cleaved caspase-3 and Bax mRNA and proteins in joint synovium tissue of the rats in TGP-MD group and TGP-HD group were significantly increased(P<0.05),while the arthritis indexes,the joint swelling degrees,the expression levels of TLR4,NF-κB,p65 and Bcl-2 in joint synovium tissue,and the TNF-α,IL-6,and IL-1βlevels in serum of the rats were significantly decreased(P<0.05).Conclusion:TGP can improve the symptoms of CIA,reduce the inflammatory reaction and promote the apoptosis of synovial tissue by inhibiting TLR4/NF-κB signal pathway.