雷公藤内酯醇对类风湿性关节炎模型大鼠血管新生和PTEN/PI3K/AKT通路的影响

Effects of triptolide on angiogenesis and PTEN/PI3K/AKT pathway in model rats with rheumatoid arthritis

目的:探讨雷公藤内酯醇对类风湿性关节炎大鼠血管新生和第10号染色体缺失的磷酸酯酶及张力蛋白同源物基因/磷酸肌醇-3-激酶/蛋白激酶B(PTEN/PI3K/AKT)通路的影响,阐明雷公藤内酯醇治疗类风湿性关节炎的可能机制。方法:将80只大鼠随机分为对照组、模型组、阳性药物组和治疗组,每组20只。采用Ⅱ型胶原(ColⅡ)诱导建立风湿性关节炎大鼠模型。阳性药物组大鼠给予双氯芬酸钠缓释片治疗,治疗组大鼠给予雷公藤内酯醇治疗。治疗后,检测各组大鼠体质量和足爪厚度,评估关节炎指数积分。HE染色检测各组大鼠关节滑膜组织病理形态表现,免疫组织化学染色测定关节滑膜组织中微血管密度(MVD)和血管内皮生长因子(VEGF)表达水平,ELISA法检测各组大鼠血清VEGF水平,Western blotting法检测各组大鼠滑膜组织中PTEN、PI3K、AKT和磷酸化-AKT(p-AKT)蛋白水平。结果:与对照组比较,模型组大鼠体质量降低(P<0.05),足爪厚度升高(P<0.05),滑膜组织中MVD和VEGF表达水平升高(P<0.05),滑膜组织中PTEN蛋白表达水平降低(P<0.05),PI3K、AKT和p-AKT蛋白表达水平升高(P<0.05)。与模型组比较,阳性药物组和治疗组大鼠体质量升高(P<0.05),足爪厚度降低(P<0.05),滑膜组织中MVD和VEGF表达水平降低(P<0.05),滑膜组织中PTEN蛋白表达水平升高(P<0.05),PI3K、AKT和p-AKT蛋白表达水平降低(P<0.05)。与阳性药物组比较,治疗组大鼠滑膜组织中MVD和VEGF表达水平降低(P<0.05),滑膜组织中PTEN蛋白表达水平升高(P<0.05),PI3K、AKT和p-AKT蛋白表达水平降低(P<0.05)。类风湿性关节炎大鼠足爪厚度和关节炎指数与VEGF(r=0.564,r=0.492)及p-AKT(r=0.561,r=0.468)表达水平呈正相关关系(P<0.05),与PTEN(r=-0.437,r=-0.521)呈负相关关系(P<0.05);MVD与VEGF(r=0.587)、PI3K(r=0.567)、p-AKT(r=0.601)表达水平呈正相关关系(P<0.05),与PTEN水平(r=-0.502)呈负相关关系(P<0.05)。结论:雷公藤内酯醇可抑制类风湿性关节炎大鼠血管新生和PTEN/PI3K/AKT通路激活,从而发挥对类风湿性关节炎的治疗作用。

Objective To investigate the effect of triptolide on the angiogenesis and phosphatase and tensin homolog delected on chromosome/phosphoinositide-3-kinase/protein kinase B(PTEN/PI3K/AKT)pathway in the rats with rheumatoid arthritis,and to elucidate the possible mechanism of triptolide in the treatment of rheumatoid arthritis.Methods:A total of 80 rats were randomly divided into control group,model group,positive drug group and treatment group;there were 20 rats in each group.The rheumatoid arthritis model was established with typeⅡcollagen(ColⅡ)induction.The rats in positive drug group were treated with diclofenac sodium sustained-release tablets,and the rats in treatment group were treated with triptolide.After treatment,the body weights,paw thickness and arthritis index scores of the rats in various groups were measured;HE staining was used to determine the pathomorphology of joint synovial tissue of the rats in various groups;immunohistochemical staining was used to determine the microvessel density(MVD)of vascular endothelial growth factor(VEGF)expression level in synovial tissue of the rats in various groups;ELISA method was used to determine the serum levels of VEGF of the rats in various groups;Western blotting method was used to determine the expression levels of PTEN,PI3K,AKT and phosphorylated-AKT(p-AKT)proteins in synovial tissue of the rats in various groups.Results:Compared with control group,the body weight of rats in model group was decreased(P<0.05),the paw thickness was increased(P<0.05),the MVD and expression level of VEGF in synovial tissue were increased(P<0.05),the expression level of PTEN protein in synovial tissue was decreased(P<0.05),and the expression levels of PI3K,AKT and p-AKT proteins were increased(P<0.05).Compared with model group,the body weights of the rats in positive drug group and treatment group were increased(P<0.05),the paw thickness was decreased(P<0.05),the MVD and the expression levels of VEGF in synovial tissue were decreased(P<0.05),the expression levels of PTEN protein were decreased(P<0.05),and the expression levels of PI3K,AKT and p-AKT proteins were decreased(P<0.05).Compared with positive drug group,the MVD and expression levels of VEGF in synovial tissue of the rats in treatment group were decreased(P<0.05),the expression level of PTEN protein in synovial tissue was increased(P<0.05),and the expression levels of PI3K,AKT and p-AKT proteins were decreased(P<0.05).The paw thickness and arthritis index of rheumatoid arthritis rats were positively correlated with VEGF(r=0.564,r=0.492)and p-AKT(r=0.561,r=0.468)(P<0.05),and negatively correlated with PTEN(r=-0.437,r=-0.521)(P<0.05);MVD was positively correlated with VEGF(r=0.587),PI3K(r=0.567),and p-AKT(r=0.601)(P<0.05),and negatively correlated with PTEN(r=-0.502)(P<0.05).Conclusion:Triptolide can inhibit the angiogenesis and PTEN/PI3K/AKT pathway activation in the rheumatoid arthritis rats,thereby exerting its therapeutic effect on rheumatoid arthritis.