加味补肾壮筋汤对兔膝骨关节炎Wnt/β-catenin信号通路的影响

Effect of Modified Bushen Zhuangjin Decoction on Wnt/β-catenin Signaling Pathway in Rabbit Knee Osteoarthritis

目的:基于Wnt/β-catenin信号通路探讨加味补肾壮筋汤(modified Bushen Zhuangjin decoction,MBZD)治疗兔膝骨关节炎(knee osteoarthritis,KOA)的作用机制。方法:从36只大耳白兔中随机选取8只作为对照组(Control),其余白兔在右膝关节注射木瓜蛋白酶以建立KOA模型。将造模成功的白兔随机分为模型组(KOA)、加味补肾壮筋汤组(MBZD,3.29 g·kg^(-1))及塞来昔布组(celecoxib,CELE,9.33 mg·kg^(-1)),每组8只。MBZD和CELE组白兔按照相应剂量灌胃,Control和KOA组白兔给予等体积生理盐水,每日1次,连续28 d。取右侧膝关节进行Pelletier评分,HE染色观察软骨与滑膜组织病理形态,免疫组织化学法(immunohistochemistry,IHC)检测软骨与滑膜组织中基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)、Ⅱ型胶原α1(collagen typeⅡalpha 1,COL2A1)表达水平,Western blot法检测软骨组织中白细胞介素-1β(interleukin 1β,IL-1β)、MMP-13、COL2A1、Wnt-3a、β-catenin、C-MYC蛋白表达水平,RT-qPCR法检测软骨组织中Wnt-3αmRNA、β-catenin mRNA、C-MYC mRNA表达水平。结果:与Control组比较,KOA组白兔Pelletier评分升高(P<0.05);与KOA组比较,MBZD组和CELE组白兔Pelletier评分降低(P<0.05)。HE染色显示,Control组白兔软骨和滑膜组织形态无明显异常。KOA组白兔软骨严重缺损,滑膜组织异常增生伴炎症细胞浸润。MBZD组和CELE组白兔软骨和滑膜组织损伤明显改善。IHC显示,与Control组比较,KOA组白兔软骨和滑膜组织MMP-13表达水平升高、COL2A1表达水平降低(P<0.05),经MBZD和CELE治疗后得到逆转(P<0.05)。Western blot显示,与Control组比较,KOA组白兔软骨组织IL-1β、MMP-13、Wnt-3α、β-catenin、C-MYC蛋白表达水平升高,COL2A1蛋白表达水平降低(P<0.05),经MBZD和CELE治疗后得到逆转(P<0.05)。RT-qPCR显示,与Control组比较,KOA组白兔软骨组织Wnt-3αmRNA、β-catenin mRNA、C-MYC mRNA水平升高,经MBZD和CELE治疗后得到逆转(P<0.05)。结论:MBZD可改善KOA白兔膝关节炎症反应和软骨细胞退变,其机制可能与调控Wnt/β-catenin信号通路有关。

Objective:Based on Wnt/β-catenin signaling pathway,to explore the mechanism of modified Bushen Zhuangjin Decoction in treatment of knee osteoarthritis(KOA)in rabbits.Methods:Thirty-six big-eared white rabbits were randomly divided into a control group(Control,8)and a model group(KOA,28),and the latter was injected with papain in the right knee joint in order to establish a KOA model.The successful modeled white rabbits were randomly divided into the model group,the modified Bushen Zhuangjin Decoction group(MBZD,3.29 g·kg^(-1))and celecoxib(CELE,9.33 mg·kg^(-1))group,with 8 rabbits in each group.The rabbits in the MBZD and CELE groups were gavaged according to the corresponding dosage,and the Control and KOA groups were given an equal volume of saline once a day for 28 d.The right knee joints were scored by Pelletier′s score,and the pathological morphology of cartilage and synovial tissue was observed by HE staining,and matrix metalloproteinase-13(matrix metalloproteinase-13)was detected in the cartilage and synovial tissue by immunohistochemistry(IHC).The expression levels of matrix metalloproteinase 13(MMP-13)and collagen type II alpha 1(COL2A1)in cartilage and synovial tissues were detected by immunohistochemistry(IHC),and the expression levels of interleukin 1β(IL-1β),MMP-13,COL2A1,MMP-1β,COL2A1,and MMP-1βin cartilage tissues were detected by Western blot.13,COL2A1,Wnt-3a,β-catenin,and C-MYC protein expression levels in cartilage tissues,and Wnt-3αmRNA,β-catenin mRNA,and C-MYC mRNA expression levels in cartilage tissues by RT-qPCR.Results:The Pelletier score of white rabbits in the KOA group was elevated compared with that of the Control group(P<0.05);and the Pelletier score of white rabbits in the MBZD and CELE groups was decreased compared with that of the KOA group(P<0.05).HE staining showed that there were no obvious abnormalities in the morphology of cartilage and synovial tissues of white rabbits in the Control group,and that white rabbits in the KOA group showed severe defects in cartilage and abnormal proliferation with inflammatory cell dipping in the synovial tissues.In the KOA group,the cartilage and synovial tissue of rabbits were severely defective and the synovial tissue was infiltrated with inflammatory cells,while the cartilage and synovial tissue of rabbits in the MBZD and CELE groups showed significant improvement in the damage,and the IHC showed that the expression levels of MMP-13 in the cartilage and synovial tissue of rabbits in the KOA group were elevated and the expression level of COL2A1 was decreased compared with those of the Control group(P<0.05),which was reversed after MBZD and CELE treatments(P<0.05).Western blot showed that the cartilage tissues of white rabbits in the KOA group had elevated levels of IL-1β,MMP-13,Wnt-3α,β-catenin,and C-MYC protein expression and reduced levels of COL2A1 protein expression compared with the Control group(P<0.05),which were reversed after MBZD and CELE treatment(P<0.05).RT-qPCR showed that the levels of Wnt-3αmRNA,β-catenin mRNA,and C-MYC mRNA in cartilage tissues of white rabbits in the KOA group were elevated compared with those in the Control group(P<0.05),which were reversed by MBZD and CELE treatment.Conclusion:Modified Bushen Zhuangjin Decoction can improve the inflammatory response and chondrocyte degeneration in the knee joints of KOA white rabbits,and its mechanism may be related to the regulation of Wnt/β-catenin signaling pathway.