摘要
目的 探讨吴茱萸碱(Evo)对膝骨关节炎(KOA)大鼠软骨损伤的影响及其作用机制.方法 将60只大鼠随机分为对照组、模型组、Evo低剂量组、Evo中剂量组、Evo高剂量组、激活剂(Shh/Gli1信号通路激活剂Purmorphamine)+Evo高剂量组,每组10只.构建大鼠KOA模型,并对各组给予相应药物干预.对各组大鼠进行行为学观察及Lequesne MG评分,苏木素伊红(HE)染色和番红O-固绿染色观察大鼠软骨组织病理变化及Mankin评分,酶联免疫吸附法(ELISA)检测白细胞介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)水平,dUTP缺口末端标记(TUNEL)法检测软骨细胞凋亡,免疫组织化学检测Ⅱ型胶原蛋白(CⅡ)、基质金属蛋白酶13(MMP13)蛋白表达,Western blotting检测Shh、Smo、Ptch1、Gli1蛋白表达.结果 与对照组相比,模型组大鼠出现活动障碍、软骨组织损伤严重,Lequesne MG评分、Mankin评分、IL-1β、IL-18、TNF-α水平、软骨细胞凋亡率、MMP13蛋白表达及Shh、Smo、Ptch1、Gli1蛋白水平显著升高,CⅡ蛋白表达降低(P<0.05);与模型组相比,Evo低、中、高剂量组大鼠活动障碍、软骨组织受损减轻,Lequesne MG评分、Mankin评分、IL-1β、IL-18、TNF-α水平、软骨细胞凋亡率、MMP13蛋白表达及Shh、Smo、Ptch1、Gli1蛋白水平显著下降,CⅡ蛋白表达升高(P<0.05);Shh/Gli1信号通路激活剂逆转Evo高剂量对KOA大鼠软骨损伤的改善作用.结论 Evo可能通过抑制Shh/Gli1信号通路减轻KOA大鼠炎症反应和软骨细胞凋亡,改善软骨损伤.
Abstract
Objective To investigate the effect of evodiamine(Evo)on cartilage damage in knee osteoarthritis(KOA)model rats and analyze its mechanism of action.Methods Sixty rats were randomly divided into control,model,low-dose Evo,medium-dose Evo,high-dose Evo,and activator(Shh/Gli1 signaling pathway activator purmorphamine)+high-dose Evo groups,with 10 rats per group.A rat KOA model was constructed,and the corresponding drug interventions were administered to each group.Behavioral observations and Lequesne MG scores were conducted among rats in each group.Hematoxylin&eosin staining and safranine O-fixed green staining were applied to observe the pathological changes and Mankin score of rat cartilage tissue.Enzyme linked immunosorbent assays(ELISA)were applied to detect the levels of interleukin-1β(IL-1β),IL-18,and tumor necrosis factor-α(TNF-α),while dUTP nick end labeling was applied to detect chondrocyte apoptosis.Immunohistochemistry was applied to detect the expression of type Ⅱ collagen(CⅡ)and matrix metalloproteinase 13(MMP13)proteins,and Western blotting was applied to detect the expression of Shh,Smo,Ptc1,and Gli1 proteins.Results The rats in the model group showed movement disorders and severe cartilage tissue damage compared with the control group.The Lequesne MG score,Mankin score,IL-1β,IL-18,and TNF-α levels,chondrocyte apoptosis rate,MMP13 protein expression,and Shh,Smo,Ptc1,and Gli1 protein levels obviously increased,while the CⅡ protein expression decreased(P<0.05)compared with the model group.The activity disorders and cartilage tissue damage in rats in the low,medium,and high dose Evo groups were alleviated,the Lequesne MG score,Mankin score,IL-1β,IL-18,TNF-α levels,chondrocyte apoptosis rate,MMP13 protein expression,and Shh,Smo,Ptc1,and Gli1 protein levels were obviously decreased,the CⅡprotein expression increased(P<0.05);while the Shh/Gli1 signaling pathway activator reversed the improvement effect of high-dose Evo on cartilage damage in KOA rats.Conclusion Evodiamine may reduce the inflamma-tory response and chondrocyte apoptosis in KOA rats by inhibiting the Shh/Gli1 signaling pathway,thereby improving cartilage damage.
维普
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