蔓性千斤拔素D通过TLR2/MyD88/NF-κB信号通路调控CIA大鼠炎症反应的相关机制

Mechanism of Flemiphilippinin D Regulating Inflammatory Response in CIA Rats Through TLR2/MyD88/NF-κB Signaling Pathway

目的:观察蔓性千斤拔素D对大鼠胶原诱导型关节炎(CIA)的作用并探讨其作用机制。方法:将40只大鼠随机分为正常组、CIA组、甲氨蝶呤(MTX)组(1.35 mg·kg^(-1))、蔓性千斤拔素D低剂量组(1.5 mg·kg^(-1))及蔓性千斤拔素D高剂量组(3.0 mg·kg^(-1)),每组8只,除正常组外,其余均采用Ⅱ型胶原诱导CIA模型。给药组通过灌胃给予相应药液,正常组给予相应体积生理盐水,MTX组每周1次,蔓性千斤拔素D各组每天1次,共给药28 d。实验中记录各组大鼠关节炎评分和关节肿胀度,苏木素-伊红(HE)染色观察各组大鼠踝关节病理学变化,酶联免疫吸附测定法(ELISA)检测大鼠血清炎症因子白细胞介素(IL)-1β、IL-6、IL-8、肿瘤坏死因子(TNF)-α水平,实时荧光定量聚合酶链式反应(Real-time PCR)检测Toll样受体2(TLR2)、髓样分化因子88(MyD88)、核转录因子-κB(NF-κB) p65 mRNA的表达量,蛋白免疫印迹法(Western blot)检测TLR2、MyD88、NF-κB p65蛋白的表达量。结果:与正常组比较,CIA组大鼠踝关节明显肿胀,关节炎临床评分及关节肿胀度显著升高(P<0.01),踝关节组织结构明显破坏,血清炎性因子IL-1β、IL-6、IL-8、TNF-α含量显著升高(P<0.01),TLR2、MyD88、NF-κB p65mRNA及蛋白表达水平显著升高(P<0.01)。与CIA组比较,各给药组大鼠关节炎临床评分及关节肿胀度均明显降低(P<0.05,P<0.01),踝关节组织结构病理变化明显改善,血清IL-1β、IL-6、IL-8、TNF-α含量均明显降低(P<0.05,P<0.01),踝关节TLR2、MyD88、NF-κB p65 mRNA及蛋白表达水平均明显下降(P<0.05,P<0.01)。结论:蔓性千斤拔素D在一定程度上可降低类风湿关节炎大鼠炎性因子的表达,能起到良好的治疗作用,其作用机制可能是通过抑制TLR2/MyD88/NF-κB信号通路的活化而发挥抗炎作用。

Objective:To observe the effect of Flemiphilippinin D on collagen-induced arthritis(CIA)in rats and explore its mechanism.Method:Forty rats were randomly divided into normal group,CIA group,methotrexate(MTX)group(1.35 mg·kg^(-1)),low-dose Flemiphilippinin D group(1.5 mg·kg^(-1)),and high-dose Flemiphilippinin D group(3.0 mg·kg^(-1)),with eight rats in each group.Except for the normal group,the CIA model was induced by typeⅡcollagen.Each group was given corresponding liquid medicine or normal saline,once a week in the MTX group,and once a day in the Flemiphilippinin D groups for a total of 28 days.The arthritis score and joint swelling degree of rats were experimentally recorded.Pathological changes in the ankle joint of rats were observed by hematoxylin-eosin(HE)staining.Serum levels of inflammatory cytokines interleukin(IL)-1β,IL-6,IL-8,and tumor necrosis factor(TNF)-αwere detected by enzyme-linked immunoabsorbent assay(ELISA),and the mRNA expression of Toll-like receptor 2(TLR2),myeloid differentiation factor 88(MyD88),and nuclear transcription factor-κB(NF-κB)p65 were detected by Realtime fluorescence quantitative polymerase chain reaction(Real-time PCR),and the protein expressions of TLR2,MyD88,and NF-κB p65 were detected by Western blot.Result:Compared with the normal group,the ankle joint of the CIA group was significantly swollen,and the clinical score of arthritis and the degree of joint swelling were significantly increased(P<0.01).The ankle joint tissue structure was significantly damaged,and the levels of inflammatory factors IL-1β,IL-6,IL-8,and TNF-αin serum were significantly increased(P<0.01).The mRNA levels and protein levels of TLR2,MyD88,and NF-κB p65 were significantly increased(P<0.01).Compared with the CIA group,arthritis clinical score and joint swelling of rats in each administration group were significantly reduced(P<0.05,P<0.01),and the pathological changes in the ankle joint were significantly improved.The contents of serum IL-1β,IL-6,IL-8,and TNF-αwere significantly decreased(P<0.05,P<0.01).The mRNA levels and protein levels of TLR2,MyD88,and NF-κB p65 in the ankle joint were significantly decreased(P<0.05,P<0.01).Conclusion:To a certain extent,Flemiphilippinin D can reduce the expression of inflammatory factors in rheumatoid arthritis rats and play a good therapeutic effect.It works perhaps by inhibiting the activation of the TLR2/MyD88/NF-κB signaling pathway and thus shows an antiinflammatory effect.