基于Toll样受体信号通路威灵仙总皂苷对类风湿关节炎大鼠踝关节病理学改变影响

Effect of total saponins of Clematis on pathological changes in the ankle-joint of rheumatoid arthritis rats based on Toll-like receptor signaling pathway

目的基于Toll样受体信号通路探讨威灵仙总皂苷(TSC)对类风湿性关节炎(RA)大鼠踝关节病理学改变的影响。方法将60只雄性SD大鼠随机分为6组,分别为空白对照组、RA模型组、阳性药物对照组以及TSC低、中、高剂量组,每组各10只。除空白对照组外,其余5组大鼠均采用足跖皮内注射弗氏完全佐剂复制RA模型,建模成功后,TSC低、中、高剂量组分别给予50、100、200 mg/kg/d TSC灌胃,阳性药物对照组给予甲氨蝶呤0.051 mg/ml灌胃,空白对照组及RA模型组均给予等体积生理盐水灌胃。灌胃15 d后,观察各组大鼠踝关节滑膜、关节腔、软骨及周围软组织病理变化情况。采集大鼠腹主动脉血,酶联免疫吸附测定法检测血清白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)等炎症因子水平;Western blot法定量分析各组大鼠关节滑膜组织内Toll样受体4(TLR4)、髓样分化蛋白(MyD88)、激活核转录因子-κB(NF-κB)p65蛋白表达量及NF-κB p65磷酸化(p-NF-κB p65)蛋白表达水平;实时荧光定量PCR法检测各组大鼠关节滑膜组织内TLR4、MyD88、NF-κB p65的mRNA表达水平。结果与空白对照组大鼠比较,RA模型大鼠踝关节周长、足垫厚度以及关节炎指数均上升,踝关节腔出现明显RA病理改变,血清IL-6、IL-1β及TNF-α水平升高,踝关节组织内MyD88、NF-κB p65及TLR4的mRNA及蛋白表达量均上升,p-NF-κB p65蛋白表达上升,差异均有统计学意义(P<0.05)。与RA模型组比较,TSC低、中、高剂量组大鼠踝关节周长、足垫厚度、关节炎指数降低,踝关节病理改变减轻,血清IL-6、IL-1β及TNF-α水平及踝关节组织内MyD88、NF-κB p65及TLR4 mRNA及蛋白表达量均下降,p-NF-κB p65蛋白表达下降,以上改变均呈剂量依耐性降低,差异均有统计学意义(P<0.05)。结论TSC可能通过抑制TLR信号通路(TLR4/MyD88/NF-κB p65),降低机体炎症反应,减轻RA大鼠踝关节病理改变及关节肿胀度。

Objective To investigate the effect of total saponins of Clematis(TSC)on pathological Changes in the ankle-joint of rheumatoid arthritis(RA)rats based on Toll-like receptor signaling pathway.Methods Sixty male SD rats were randomly divided into 6 groups,including blank control group,RA model group,positive drug control group,low,medium and high dose TSC groups,with 10 rats in each group.Except for the blank control group,the other five groups of rats were injected intradermal with Freund′s complete adjuvant to create RA model.After successful modeling,the low,medium and high dose TSC groups were given 50,100 and 200 mg/kg/d TSC by intragastric administration,while the positive drug control group was given 0.051 mg/ml methotrexate by intragastric administration.Blank control group and RA model group were given equal volume of normal saline gavage.After 15 days of intragastric administration,the pathological changes of synovium,articular cavity,cartilage and surrounding soft tissue of ankle joints were observed.Abdominal aortic blood was collected from rats.The levels of interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and other inflammatory factors in serum were determined by enzyme-linked immunosorbent assay.The expression levels of Toll-like receptor 4(TLR4),myeloid differentiation protein(MyD88)and activated nuclear transcription factor-κB(NF-κB)p65 and the protein levels of phosphorylation of NF-κB p65(p-NF-κB p65)in synovial tissues of joints of rats were quantitatively analyzed by Western blot.The mRNA expression levels of TLR4,MyD88 and NF-κB p65 in synovial tissues of joints were detected by real-time quantitative PCR.Results Compared to blank control group,RA model rats showed various abnormal changes including increased ankle circumference,foot pad thickness and arthritis index,pathological changes in the ankle joint cavity,elevated levels of serum IL-6,IL-1βand TNF-α,upregulated mRNA and protein expression of MyD88,NF-κB p65 and TLR4,and upregulated protein expression of p-NF-κB p65,with statistical difference(P<0.05).Compared with RA model group,ankle circumference,foot pad thickness,arthritis index,pathological changes of ankle joint,serum IL-6,IL-1βand TNF-αlevels,mRNA and protein expressions of MyD88,NF-κB p65 and TLR4 and expression of p-NF-κB p65 protein in the low,medium and high dose TSC groups were decreased,and the above changes were dose dependent,and the differences were statistically significant(P<0.05).Conclusion TSC may alleviate the body inflammatory response and attenuate the pathological changes and joint swelling in ankle joint of RA rats via inhibiting the TLR signaling pathway(TLR4/MyD88/NF-κB p65).