独活寄生汤对膝骨关节炎模型大鼠中Wnt/β-catenin信号通路相关蛋白表达的影响

Effect of Duhuo Jisheng Decoction on Wnt3a/β-catenin Pathway Related Protein Expression Levels in Knee Osteoarthritis Model Rats

目的观察独活寄生汤(DHJSD)对膝骨关节炎(KOA)模型大鼠中Wnt/β-连环蛋白(β-catenin)信号通路相关蛋白表达的影响,探讨DHJSD治疗骨关节炎(OA)的机制。方法选取32只SD大鼠,按随机数字表法分为DHJS组、模型(M)组、硫酸氨基葡萄糖(GS)组和正常(NC)组。除NC组外,其余3组予双膝关节腔内注射0.2 mL 4%木瓜蛋白酶法建立KOA模型。造模后DHJS组予DHJSD灌胃,GS组予GS灌胃,NC、M组给予等体积纯净水灌胃。连续给药8周后进行双膝关节X线扫描,观察大鼠膝关节影像学变化并评分,ELISA法检测血清基质金属蛋白酶-13(MMP-13)、白介素-1β(IL-1β)含量,HE染色、番红O固绿染色法观察膝关节软骨病理改变并进行Mankin评分,采用实时聚合酶链式反应(RT-PCR)、蛋白免疫印迹(Western Blot)及免疫组化法检测软骨组织中Wnt/β-catenin通路相关蛋白的mRNA及蛋白表达水平。结果与NC组比较,M组大鼠膝关节间隙狭窄,软骨层损耗,X线评分、Mankin评分升高(P<0.05),DHJS组和GC组上述改变减轻,X线评分及Mankin评分下降(P<0.05);DHJS组血清及软骨MMP-13含量减少(P<0.05);下调软骨β-catenin、Wnt3a mRNA水平以及β-catenin、IL-1β蛋白表达水平(P<0.05),上调骨形态发生蛋白-2(BMP-2)、糖原合成酶激酶-3β(GSK-3β)m RNA和蛋白表达水平(P<0.05)。结论DHJSD能通过下调Wnt3a/β-catenin信号通路相关蛋白的表达水平,抑制MMP-13表达、促进BMP-2表达,从而改善大鼠KOA的影像学及病理学改变,发挥软骨保护作用。

Objective To observe the effect of Duhuo Jisheng Decoction(DHJSD)on the expressions of Wnt/β-catenin pathway related proteins in knee osteoarthritis(KOA)rat model,and to explore its mechanism for osteoarthritis(OA).Methods Totally 32 SD rats were divided into DHJS group,model(M)group,Glucosamine Sulfate(GS)group,and normal control(NC)group according to random number table.Except the NC group,rats in the rest of 3 groups were injected papain(0.2 mL 4%)into bilateral knee joint cavity to establish KOA model.After the KOA modeling rats in DHJS group were intragastrically administered with DHJSD.Rats in GS group were intragastrically administered with GS.Equal volume of purified water was given to rats in NC group and M group by gastrogavage.After 8 weeks continuously administration,the imaging changes of the rats were observed and scored by double knee X-ray scan.Serum levels of matrix metalloprotein(MMP-13)and IL-1βwere detected by ELISA.HE staining and safranin O staining were used to observe the pathological changes of articular cartilage of knee and to score by Markin.Real-time PCR,Western Blot,and immunohistochemical method were used to detect mRNA and protein expression levels of related proteins of Wnt/β-catenin signal pathway in cartilage tissues.Results Compared with the NC group,rats in the M group had narrow knee space,cartilage layer loss,and increased X-ray score and Mankin score(P<0.05).Compared with the M group,the above changes were relieved,and X-ray score and Mankin score decreased in the DHJS group and GC group(P<0.05).Serum and cartilage MMP-13 contents decreased in the DHJS group(P<0.05).mRNA levels ofβ-catenin and Wnt3 a in cartilage,and protein expressions ofβ-catenin and IL-1βin cartilage were downregulated(P<0.05).mRNA and protein expression levels of bone morphogenetic protein-2(BMP-2)and glycogen synthase kinase-3β(GSK-3β)were also up-regulated(P<0.05).Conclusion DHJSD inhibited the expression of MMP-13 and promoted the expression of BMP-2 possibly by down-regulating Wnt3 a/β-catenin pathway related protein expression levels,thus improving the imaging and pathological changes of KOA rats,and playing a role of cartilage protection.