淫羊藿苷联合富血小板血浆对骨性关节炎的软骨保护作用和分子机制研究

Cartilage Protective Effect and Molecular Mechanism of Icariin Combined with Platelet-Rich Plasma on Osteoarthritis

目的:体外和体内探讨淫羊藿苷(ICA)联合富血小板血浆(PRP)对骨性关节炎(OA)的软骨保护作用和分子机制。方法:(1)软骨细胞经白细胞介素1β(IL-1β)处理建立体外OA模型,分为ICA+PRP组、ICA组、PRP组和IL-1β组,观察软骨细胞增殖[细胞计数试剂盒-8(CCK-8)]和凋亡(流式细胞术)情况,采用酶联免疫吸附试验检测肿瘤坏死因子α(TNF-α)水平。(2)选取新西兰大白兔30只,取6只作为正常组;手术建立新西兰大白兔膝关节OA模型,分为模型组、ICA+PRP组、ICA组和PRP组,每组6只。采用蛋白质印迹法分析Wnt1、β-catenin和糖原合成酶激酶3β(GSK-3β)的表达变化。结果:(1)与对照组相比,ICA+PRP组、ICA组和PRP组细胞TNF-α水平明显降低,差异均有统计学意义(P<0.05)。ICA+PRP组细胞的增殖活性明显优于ICA组、PRP组,细胞的凋亡率明显低于ICA组、PRP组,差异均有统计学意义(P<0.05)。ICA组细胞的增殖活性、凋亡率明显优于PRP组,差异均有统计学意义(P<0.05)。(2)ICA+PRP组兔的国际骨关节炎研究学会(OARSI)评分明显优于ICA组、PRP组,差异均有统计学意义(P<0.05)。与正常组相比,模型组兔软骨β-catenin、Wnt1的mRNA和蛋白表达水平升高,GSK-3β的mRNA和蛋白表达水平降低,差异均有统计学意义(P<0.05);与模型组比较,ICA+PRP组和ICA组兔软骨Wnt、β-catenin的mRNA和蛋白表达水平均显著降低,GSK-3β的mRNA和蛋白表达水平显著升高,差异均有统计学意义(P<0.05);且ICA+PRP组的效果(上述指标)优于ICA组。结论:ICA联合PRP可通过促进软骨细胞增殖、抑制炎症损伤和细胞凋亡来发挥抗OA的作用,其中Wnt/β-catenin信号通路密切参与此过程。

OBJECTIVE:To probe into the cartilage protective effect and molecular mechanism of icariin(ICA)combined with platelet-rich plasma(PRP)on osteoarthritis(OA)in vitro and in vivo.METHODS:(1)Chondrocytes were treated with interleukin 1β(IL-1β)to establish the OA model in vitro,and were divided into the ICA+PRP group,ICA group,PRP group and IL-1βgroup.Chondrocyte proliferation(CCK-8)and apoptosis(flow cytometry)were observed.The levels of tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay.(2)Thirty New Zealand white rabbits were selected,and 6 were selected as the normal group;the New Zealand white rabbit knee joint OA model was established by surgery,and the rabbits were divided into the model group,ICA+PRP group,ICA group and PRP group,with 6 cases in each group.The expressions of Wnt1,β-catenin and glycogen synthase kinase 3β(GSK-3β)were analyzed by western blotting.RESULTS:(1)Compared with the control group,the levels of TNF-αin ICA+PRP group,ICA group and PRP group decreased significantly,the differences were statistically significant(P<0.05).The proliferation activity of ICA+PRP group was significantly better than that of ICA group and PRP group,and the apoptosis rate of ICA+PRP group was significantly lower than that of ICA group and PRP group,with statistically significant differences(P<0.05).The proliferation activity and apoptosis rate of ICA group were significantly better than those of PRP group,the differences were statistically significant(P<0.05).(2)The Osteoarthritis Research Society International(OARSI)score of ICA+PRP group was significantly better than that of ICA group and PRP group,and the differences were statistically significant(P<0.05).Compared with the normal group,the mRNA and protein expressions ofβ-catenin and Wnt1 in cartilage of the model group increased,while the mRNA and protein expressions of GSK-3βdecreased,with statistically significant differences(P<0.05).Compared with the model group,the mRNA and protein expressions of Wnt andβ-catenin in ICA+PRP group and ICA group decreased significantly,and the mRNA and protein expressions of GSK-3βincreased significantly,with statistically significant differences(P<0.05);and the effect of ICA+PRP group was better than that of ICA group.CONCLUSIONS:ICA combined with PRP can play an anti-OA effect by promoting chondrocyte proliferation,inhibiting inflammatory injury and cell apoptosis,and Wnt/β-catenin signaling pathway is closely involved in this process.