黄芪皂苷改善膝关节骨性关节炎大鼠模型炎症及其机制研究

Astragalus saponin alleviates inflammation in rat model of knee osteoarthritis and its mechanism

目的探讨黄芪皂苷对膝关节骨性关节炎(OA)的作用及其潜在的分子机制。方法取60只Wistar大鼠,采用关节腔内注射单碘乙酸盐(MIA)建立大鼠膝关节OA模型,按照随机数字表法分为对照组、OA-1组、OA-5组、OA-10组、OA-20组和OA-28组,每组10只。对照组关节腔内单次注射40μl 0.9%氯化钠注射液;OA-1组、OA-5组、OA-10组、OA-20组和OA-28组大鼠关节腔内单次注射4 mg MIA(溶于40μl 0.9%氯化钠注射液中)。另取30只Wistar大鼠按照随机数字表法分为OA组、OA+0.9%氯化钠注射液组(下称OA+生理盐水组)和OA+黄芪皂苷组,每组10只。OA+黄芪皂苷组大鼠皮下注射0.5 ml/(kg·d)黄芪皂苷;OA+生理盐水组大鼠每天皮下注射0.5 ml 0.9%氯化钠注射液;OA组大鼠不予任何药物处理;干预28 d。采用Western blot法检测上述各组大鼠膝关节软骨组织中蛋白激酶A(PKA)/环状单磷酸腺苷反应元件结合蛋白(CREB)信号通路相关蛋白[PKA、磷酸化PKA(p-PKA)、CREB、磷酸化CREB(p-CREB)]、炎症相关蛋白(TNF-α、IL-6和IL-1β)表达水平。采用免疫组化染色法检测上述各组大鼠膝关节软骨组织中CREB表达水平。结果与对照组比较,OA-28组大鼠CREB水平下调(P<0.05),并且均值最低。与对照组比较,OA-1组、OA-5组、OA-10组、OA-20组和OA-28组大鼠膝关节软骨组织中PKA、p-PKA、CREB和p-CREB蛋白表达水平均降低,TNF-6、IL-6和IL-1β蛋白表达水平均增加,差异均有统计学意义(均P<0.05)。与OA+生理盐水组比较,OA+黄芪皂苷组大鼠膝关节软骨组织中PKA、p-PKA、CREB、p-CREB蛋白表达水平均增加,TNF-α、IL-6和IL-1β蛋白表达水平均降低,差异均有统计学意义(均P<0.05)。结论黄芪皂苷可以通过激活PKA/CREB信号通路抑制OA大鼠炎症相关蛋白的表达,进而发挥抗炎活性。

Objective To investigate the effect of astragalus saponin(AS)on rat knee osteoarthritis(OA)and its mechanism.Methods Sixty male Wistar rats were randomly divided into 6 groups with 10 rats in each group.The rat knee OA model was induced by injection of monoiodoacetate(MIA).The control group was injected with 40μl 0.9%normal saline(NS)in the joint cavity,and rats in OA-1,OA-5,OA-10,OA-20 and OA-28 groups were intra-articularly injected with 4 mg MIA(dissolved in 40μl 0.9%NS),and sacrificed at d1,d5,d10,d20 and d28,respectively.The OA model was also induced in other 30 rats,and the OA rats were randomly divided into 3 groups with 10 rats in each group.Rats in OA group had no special treatment,rats in OA+NS group and OA+AS group were subcutaneously injected with 0.9%NS or 0.5 ml/kg AS daily to 28 d,respectively.The expression levels of protein kinase A(PKA)and cAMP responsive element-binding protein(CKEB)signaling related proteins PKA,p-PKA,CREB,p-CREB,inflammation-related proteins TNF-α,IL-6 and IL-1βwere detected in rat knee cartilage by Western blot.CREB expression levels in the knee cartilage were determined by immunohistochemical staining.Results Compared with the control group,the CREB levels in the OA-28 rats were downregulated(P<0.05),with the lowest mean value.Compared with the control group,the expression levels of PKA,p-PKA,CREB and p-CREB were significantly decreased in OA-1,OA-5,OA-10,OA-20 and OA-28 groups(all P<0.05).Compared with the OA+NS group,the expression levels of PKA,p-PKA,CREB and p-CREB in the OA+AS group were significantly increased,and the expression levels of TNF-α,IL-6 and IL-1βwere significantly decreased(all P<0.05).Conclusion AS can inhibit the expression of inflammation-related proteins in rat knee joint tissues by activating PKA/CREB signaling pathway to exert anti-inflammatory activity in rats with knee OA.