风湿宁对风寒湿痹证CIA大鼠模型滑膜组织中TLR4/NF-κB信号通路的影响

Effect of Fengshining on TLR4/NF-κB Signal Pathway in Synovial Tissue of Wind-Cold-Dampness Arthralgia Syndrome CIA Rat Model

目的：探讨风湿宁对风寒湿痹证胶原诱导性关节炎（CIA）大鼠模型滑膜组织中Toll样受体4/核转录因子-κB（TLR4/NF-κB）信号通路的影响及相关的作用机制。方法：SPF级雌性Wistar大鼠随机分为6组,分别为正常组,模型组,阳性药组（来氟米特片,2.33 mg·kg^-1）,风湿宁低、中、高剂量组（9.12,18.24,36.48 g·kg^-1）,每组9只。除正常组外,其余各组大鼠采用风寒湿外感致病因素刺激,结合牛Ⅱ型胶原乳化剂诱导的方法,制备风寒湿痹证CIA大鼠模型。造模成功后,各组大鼠给予相应药物灌胃干预,每日1次,连续4周。观察药物对大鼠关节肿胀度,酶联免疫吸附测定法（ELISA）血清类风湿因子（RF）,环瓜氨酶肽（ACPA）,白细胞介素（IL）-1β,IL-10,肿瘤坏死因子-α（TNF-α）含量,实时荧光定量聚合酶链式反应（Realtime PCR）及蛋白免疫印迹法（Western blot）滑膜组织中TLR4,髓样分化因子88（My D88）,NF-κB酶抑制蛋白α（IκBα）,NF-κB mRNA和蛋白表达的情况。结果：与正常组比较,模型组CIA大鼠模型的关节肿胀度明显升高,血清RF,ACPA,IL-1β,TNF-α含量显著升高,IL-10含量显著降低,滑膜组织中TLR4,My D88,IκBα,NF-κB mRNA和蛋白表达显著升高（P〈0.05,P〈0.01）;与模型组比较,风湿宁低、中、高剂量组可明显降低风寒湿痹证CIA大鼠模型的关节肿胀度与血清RF,ACPA,IL-1β,TNF-α含量,升高血清IL-10水平,下调滑膜组织中TLR4,My D88,IκB-α,NF-κB mRNA与蛋白表达量（P〈0.05,P〈0.01）。结论：风湿宁可能通过抑制TLR4/NF-κB信号通路,从而抑制IL-1β,TNF-α的产生,起到治疗RA的作用,且其疗效与药物剂量有一定相关性。

Objective：To investigate the effect and mechanism of Fengshining on wind-cold-dampness arthralgia syndrome CIA rat model in synovial tissue based on Toll-like receptor 4/nuclear factor-κB（TLR4/NF-κB） signal pathway.Method：SPF female Wistar rats were randomly divided into six groups：the blank control group,the model control group,the positive control group（2.33 mg·kg^-1）,the low,medium and highdose Fengshining groups（9.12,18.24,36.48 g·kg^-1）,with nine rats in each group.Except for the blank control group,the rest rats were included into the wind-cold-dampness arthralgia syndrome CIA rat model through the wind-cold-dampness stimulation combing with bovine type Ⅱ collagen.Each group of rats was given the corresponding drug by gavage once a day for four weeks after the successful modeling.Joint swelling degree of the rats was observed.Serum rheumatoid factors（RF）,anti-cyclic citrullinated peptide antibodies（ACPA）,interleukin-1β（IL-1β）,interleukin-10（IL-10）,tumour necrosis factor-α（TNF-α） were detected.TLR4,myeloid differentiation factor88（My D88）,IκBα,NF-κB mRNA and protein expressions in synovial tissue were measured.Result：Compared with the blank control group,the wind-cold-dampness arthralgia syndrome CIA model group showed significant increase in joint swelling degree and serum RF,ACPA,IL-1β,TNF-α content,and significant reduction in IL-10 content.The mRNA and protein expressions of TLR4,My D88,IκB-α,NF-κB in synovial tissue were significantly increased（P〈0.05,P〈0.01）.Compared with the model group,low,medium and high-dose Fengshining groups showed significant decrease in joint swelling degree and serum RF,ACPA,IL-1β,TNF-α content in the wind-cold-dampness arthralgia syndrome CIA rat model,increase in serum IL-10 level,and down-regulation in mRNA and protein expressions of TLR4,My D88,IκBα,NF-κB（P〈0.05,P〈0.01）.Conclusion：Fengshining may play a role in the treatment of RA by inhibiting TLR4/NF-κB signal pathway and the production of IL-1β and TNF-α.Its efficacy is correlated with drug dose to some extent.