基于Wnt/β-catenin信号通路探究推拿对KOA大鼠软骨降解和软骨细胞终末分化的影响

Effects of Tuina on cartilage degradation and chondrocyte terminal differentiation in rats with knee osteoarthritis(KOA)via the Wnt/β-catenin signaling pathway

目的探讨推拿对膝骨关节炎(KOA)模型大鼠的疗效及其对Wnt/β-catenin信号通路相关蛋白的影响。方法从32只无特定病原体级Sprague-Dawley大鼠中随机选取8只作为对照组。剩余24只采用4%木瓜蛋白酶0.2 mL关节腔注射建立KOA模型。造模后随机分为模型组、推拿组和阳性药物组,每组各8只。采用Lequesne评分评估造模情况。造模成功后,对照组不进行任何干预;推拿组采用膝关节局部顺时针环形拇指揉法,纵向压力为3 N,频率控制为每分钟120-140次,每次15 min;最后屈伸关节10次。阳性药物组每日予以塞来昔布(每公斤体重24 mg)灌胃。各组干预每日1次,每周6 d,连续4周。治疗结束后对大鼠再次进行Lequesne评分评估;苏木素-伊红(HE)染色、番红O-固绿染色观察软骨形态结构;改良Mankin评分进行病理评分;酶联免疫吸附法检测软骨II型胶原C端肽(CTX-II)和寡聚基质蛋白(COMP);免疫印迹法检测软骨组织Wnt信号蛋白4、β-连锁蛋白、基质金属蛋白酶13(MMP-13)和骨形态发生蛋白2(BMP-2);免疫组化检测软骨细胞X型胶原(ColX)阳性细胞率。结果推拿组和阳性药物组Lequesne评分均优于模型组(P<0.01),HE染色、番红O-固绿染色形态结构及改良Mankin评分同样优于模型组(P<0.01)。与对照组相比,模型组血清中的CTX-II和COMP含量显著上升(P<0.01);软骨组织中Wnt4、β-catenin、MMP-13和BMP-2蛋白表达水平显著上升(P<0.01);软骨细胞ColX阳性细胞率显著上升(P<0.01)。与模型组相比,推拿组和阳性药物组CTX-II和COMP含量显著下降(P<0.01);Wnt4、β-catenin、MMP-13和BMP-2蛋白表达显著下降(P<0.01);软骨细胞ColX阳性细胞率显著下降(P<0.01)。与阳性药物组相比,推拿组CTX-II、COMP含量和Wnt4、β-catenin、MMP-13和BMP-2蛋白表达下降(P<0.05或P<0.01);软骨细胞ColX阳性细胞率显著下降(P<0.01)。结论推拿可以减缓KOA的退变,其机制可能与通过下调Wnt/β-catenin信号通路降低MMP-13和BMP-2蛋白表达,减少软骨细胞外基质降解,减缓细胞终末分化有关。

Objective To investigate the therapeutic effects of Tuina(Chinese therapeutic massage)in a knee osteoarthritis(KOA)rat model and its influence on proteins associated with the Wnt/β-catenin signaling pathway.Methods A total of 32 specific-pathogen-free grade Sprague-Dawley rats were used.Eight rats were randomly selected as the control group(CG).The remaining 24 rats underwent intra-articular injections with 0.2 mL of 4%papain to prepare the KOA rat models.After the model was established,the 24 rats were randomly and equally assigned to 3 groups,including a model group(MG),a Tuina group(TG),and a positive medicine group(PMG),with 8 rats in each group.The Lequesne score was applied to evaluate the success of model development.After the model was successfully established,the CG did not receive any intervention,and the TG was treated with local,clockwise annular Rou-Kneading around the knee joint with the thumbs.The pressure in the longitudinal direction was 3 N,and the frequency was designed to be 120-140 times/min for 15 min,followed by flexing the joint 10 times.The PMG was intragastrically administered with celecoxib[24 mg/(kg·bw)] every day.These interventions were performed once a day,6 d per week,for a total of 4 weeks.After treatment,the Lequesne score was applied again to assess the severity of the KOA in the rats;hematoxylin-eosin(HE)staining and a mixture of equal volumes of aqueous solutions of safranin O-fast green were used to stain and observe the cartilage morphology and structure;the modified Mankin score was applied to evaluate the pathology;enzyme-linked immunosorbent assay method was used to quantify the C-telopeptide fragments of type II collagen(CTX-II)and cartilage oligomeric matrix protein(COMP);Western blotting was then applied to quantify Wnt4,β-catenin,matrix metalloproteinase 13(MMP-13),and bone morphogenetic protein 2(BMP-2)protein expression;immunohistochemistry was conducted to determine the percentage of collagen type X(ColX)-positive cells.Results The Lequesne score of the TG and PMG was both lower than that of the MG(P<0.01);the HE staining,safranin O-fast green stained morphology and structure,and modified Mankin scores of the TG and the PMG were also better than those in the MG(P<0.01).Compared with the CG,the amounts of CTX-II and COMP in the serum were significantly increased(P<0.01);the expression of Wnt4,β-catenin,MMP-13,and BMP-2 proteins in the cartilage tissue was significantly increased(P<0.01),and the percentage of ColX-positive chondrocytes was significantly increased(P<0.01)in the MG.In comparison with those in the MG,the amounts of CTX-II and COMP were significantly decreased(P<0.01),the expression of Wnt4,β-catenin,MMP-13,and BMP-2 proteins was significantly decreased(P<0.01),and the percentage of ColX-positive chondrocytes was significantly decreased(P<0.01)in the TG and PMG.Compared with the PMG,the contents of CTX-II and COMP and the expression of Wnt4,β-catenin,MMP-13,and BMP-2 proteins were decreased(P<0.05 or P<0.01);the percentage of ColX-positive chondrocytes was significantly decreased(P<0.01)in the TG.Conclusion Tuina can relieve the degeneration of KOA,and the mechanism may be related to the down-regulation of the Wnt/β-catenin signaling pathway,the decrease in MMP-13 and BMP-2 protein expression,the reduction in chondrocyte extracellular matrix degradation,and slowing down the terminal cell differentiation.