跳骨片抑制大鼠膝骨关节炎Wnt/β-catenin信号转导通路介导炎症反应的机制研究

Tiaogu Tablets suppress the inflammatory response mediated by the Wnt/β-catenin signaling pathway in rat knee osteoarthrisis

目的探讨跳骨片对大鼠膝骨关节炎(OA)Wnt/β-catenin信号转导通路介导炎症反应的机制。方法将30只8周龄雄性SD大鼠随机分为空白组、模型组、跳骨片组,每组10只。空白组不予任何处理,模型组、跳骨片组均采用改良Hulth法建立OA动物模型。建模成功后跳骨片组每天予跳骨片0.32 g/kg灌胃,空白组和模型组每天予等量生理盐水灌胃,干预12周后分别取3组大鼠双侧膝关节股骨髁、关节软骨和滑膜,将股骨髁常规固定、脱钙、包埋及切片,对切片分别进行苏木精-伊红(HE)、甲苯胺蓝染色,光镜下观察关节软骨组织形态学变化;酶联免疫吸附测定(ELISA)检测滑膜中白细胞介素(IL)-1β、基质金属蛋白酶(MMP)-13、肿瘤坏死因子(TNF)-α含量;蛋白质免疫印迹检测关节软骨β-catenin、Wnt-4蛋白表达。结果模型组滑膜中,IL-1β表达量明显高于空白组和跳骨片组(P<0.01),MMP-13表达量高于空白组和跳骨片组(P<0.05),TNF-α表达量明显高于空白组(P<0.01)、高于跳骨片组(P<0.05);模型组关节软骨中,β-catenin蛋白表达量明显高于空白组和跳骨片组(P<0.01),Wnt-4蛋白表达量高于空白组和跳骨片组(P<0.05)。HE染色显示跳骨片组软骨破坏程度低于模型组,甲苯胺蓝染色显示跳骨片组软骨细胞蛋白多糖含量高于模型组(P<0.01)。结论跳骨片能抑制关节软骨Wnt/β-catenin信号转导通路,降低滑膜中IL-1β、MMP-13、TNF-α表达,抑制炎症反应介导的软骨基质降解,延缓关节软骨退变。

Objective To observe the effect of Tiaogu Tablets on the inflammatory response mediated by the Wnt/β-catenin signaling pathway in rat knee osteoarthritis.Methods A total of 30 healthy male SD rats of 8 weeks old were randomly divided into a sham group,a model group and a Tiaogu Tablet group,10 rats in each.Rats in the model and Tiaogu Tablet groups were induced into knee osteoarthritis by the modified Hulth method.The Tiaogu Tablet group was given Tiaogu Tablets 0.32 g/kg every day,while the sham group and the model group were given the same dose of saline.After treatment for 12 weeks,the bilateral knee joints of the rats in all 3 groups were taken,fixed,decalcified,embedded and sliced;then hematoxylin-eosin（HE）staining and toluidine blue staining were used to observe the morphology of the tissue.The levels of interleukin（IL）-1β,matrix metalloproteinases（MMP）-13 and tumor necrosis factor（TNF）-αin the synovium were measured by enzyme linked immunosorbent assay（ELISA）.The protein level ofβ-catenin and Wnt-4 was detected by Western blot.Results In the synovium,the levels of IL-1β,MMP-13 and TNF-αin the model group were significantly increased（P0.05）compared with the sham group and the Tiaogu Tablet group.Compared with the sham group and Tiaogu Tablet group,the protein levels ofβ-catenin and Wnt-4in the model group were significantly increased（P0.05）.HE staining showed that cartilage destruction in Tiaogu Tablet group was less severe than that in the model group.The toluidine blue staining showed the proteoglycan content in chondrocytes in the Tiaogu Tablet group was significantly higher than that in the model group（P0.01）.Conclusion Tiaogu Tablets suppress the expression of inflammatory cytokines in the synovium,inhibit the inflammation-mediated cartilage matrix degradation,and delay the degradation of articular cartilage by down-regulating the Wnt/β-catenin signaling pathway.