“易层”贴敷对膝骨关节炎大鼠滑膜纤维化的改善作用及机制

Improvement effect and mechanism of“Layers adjusting external application”paste on synovial fibrosis in rats with knee osteoarthritis

目的探究“易层”贴敷对膝骨关节炎(KOA)大鼠滑膜纤维化(SF)的改善作用及潜在机制。方法将雄性SD大鼠随机分为假手术组、KOA组和易层组,每组8只。KOA和易层组大鼠以前交叉韧带离断法复制KOA模型。造模14 d后,易层组大鼠于膝关节均匀涂抹“易层”贴敷[三色散(每100 cm^(2)涂抹8 g)和复方三黄油膏(每100 cm^(2)涂抹5 g)],每天贴敷8 h,连续28 d。末次给药后,观察各组大鼠的滑膜炎症程度及纤维化程度并进行Krenn评分,检测其滑膜组织中Ⅰ型胶原蛋白(collagenⅠ)、高迁移率族蛋白B1(HMGB1)、磷酸化核因子κB p65(p-NF-κB p65)蛋白的表达,血清中白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)的含量,以及滑膜组织中纤维化及HMGB1/Toll样受体4(TLR4)/NF-κB通路相关蛋白及mRNA的表达。结果与假手术组比较,KOA组大鼠滑膜衬里细胞大量增生且排列紊乱,炎症细胞浸润和胶原纤维沉积明显;collagenⅠ、HMGB1、p-NF-κB p65阳性表达细胞均明显增加,血清中IL-1β、IL-6、TNF-α含量,滑膜组织中纤维化相关蛋白(转化生长因子β、collagenⅠ、金属蛋白酶组织抑制物1、α-平滑肌肌动蛋白)及mRNA的表达,以及HMGB1、TLR4蛋白及mRNA,p-NF-κB p65蛋白及NF-κB p65 mRNA的表达均显著升高(P<0.01)。与KOA组比较,易层组大鼠滑膜组织的病理改变明显改善,上述定量指标均显著逆转(P<0.05或P<0.01)。结论“易层”贴敷可显著改善KOA大鼠的SF,其作用可能与抑制HMGB1/TLR4/NF-κB信号通路激活有关。

OBJECTIVE To investigate the improvement effect and potential mechanism of“Layers adjusting external application”paste on synovial fibrosis(SF)in rats with knee osteoarthritis(KOA).METHODS Male SD rats were randomly divided into sham operation group,KOA group and Layers adjusting external application group,with 8 rats in each group.KOA model was induced by the anterior cruciate ligament disruption method in KOA group and Layers adjusting external application group.Fourteen days after modeling,the Layers adjusting external application group was given“Layers adjusting external application”paste[Sanse powder(8 g for every 100 cm^(2)),Compound sanhuang ointment(5 g for every 100 cm^(2))]on the knee joint,8 h every day,for 28 d in total.After the last administration,the degree of synovitis and fibrosis in rats was observed,and Krenn scoring was performed in each group.The expressions of collagenⅠ,high mobility group protein B1(HMGB1)and phosphorylated nuclear factor-κB p65(p-NF-κB p65)were detected in the synovial membrane;the contents of interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α)in serum as well as the expressions of fibrosis-related and HMGB1/Toll-like receptor 4(TLR4)/NF-κB signaling pathway-related proteins and mRNA were detected in synovial tissue.RESULTS Compared with the sham operation group,the synovial lining cells in the KOA group showed significant proliferation and disordered arrangement,the inflammatory cell infiltration and collagen fiber deposition were obvious;the positive expressing cells of collagenⅠ,HMGB1 and p-NF-κB p65 were increased significantly;the contents of IL-1β,IL-6 and TNF-αin serum,the expressions of fibrosis-related protein(transforming growth factor-β,collagenⅠ,tissue inhibitor of metalloproteinase 1,α-smooth muscle actin)and their mRNA as well as the expressions of HMGB1,TLR4 protein and their mRNA,the expressions of p-NF-κB p65 protein and NF-κB p65 mRNA were all increased significantly in synovial tissues of rats(P<0.01).Compared with the KOA group,the pathological changes in the synovial tissue of rats in Layers adjusting external application group were significantly improved,and the above quantitative indicators were significantly reversed(P<0.05 or P<0.01).CONCLUSIONS“Layers adjusting external application”paste could significantly improve SF in KOA rats,the mechanism of which may be associated with the inhibition of the activation of HMGB1/TLR4/NF-κB signaling pathway.