补肾通络方对大鼠膝骨关节炎膝关节软骨细胞外基质代谢的影响

Effect of Bushen Tongluo Formula on the extracellular matrix metabolism in knee cartilage of the rats with knee asteoarthritis

目的探讨补肾通络方对膝骨关节炎大鼠的膝关节软骨中基质金属蛋白酶-3/13(matrix metalloproteinase-3/13,MMP-3/13)、组织金属蛋白酶抑制剂(tissue inhibitor of metalloproteinase-1,TIMP-1)m RNA和蛋白聚糖酶-1(aggrecanase-1即ADAMTs-4)蛋白的表达影响。方法将42只雄性Wistar大鼠随机分为7组:正常组,模型组,西药组,成药组,补肾通络方低、中、高剂量组,向大鼠双侧后肢膝关节腔内分别注射4%木瓜蛋白酶生理盐水溶液0.2 mL,于第1、4、7天注射以建立膝骨关节炎(Knee osteoarthritis,KOA)模型。造模成功后,分别以蒸馏水、美洛昔康水溶液、抗骨增生胶囊水溶液、补肾通络颗粒方低、中、高浓度水溶液对大鼠进行灌胃给药干预,干预过程中观察大鼠的一般状态,于灌胃给药干预4周后处死,并刮取双膝关节内胫骨平台及股骨内、外侧髁软骨组织,置于冻存管中备用,用实时定量反转录酶-聚合酶链锁反应(Real-time reverse transcript polymerase chain reaction,RTPCR)法测定软骨中MMP-3/13和TIMP-1在m RNA水平表达的变化及Western blot法测定ADAMTs-4蛋白表达情况。结果 MMP-3和MMP-13的m RNA表达情况在模型组最高(P<0.05),正常组表达最低(P<0.05),不同药物对其影响不同,其中补肾通络方的中剂量组表达水平在各给药组中最小,成药组表达含量在各给药组中最大,补肾通络方中剂量组与高剂量组相比,中剂量组表达低于高剂量组,然二者之间差异无统计学意义(P>0.05)。TIMP-1的m RNA含量在模型组中最低(P<0.05),正常组中表达最多(P<0.05),在各给药组中补肾通络方高剂量组含量接近正常组,而低剂量组表达最少。软骨ADAMTs-4蛋白表达情况,模型组含量最高,正常组含量最低,各给药组之间含量不同,但差异无统计学意义(P>0.05)。结论补肾通络方治疗KOA疗效明显,其作用机制可能是通过抑制MMP-3/13和ADAMTs-4的水平,增加TIMP-1的表达,减轻膝关节软骨的破坏情况,改善KOA的症状,从而达到治疗KOA的作用。

Objective To study Bushen Tongluo Formula for MMP-3/13,TIMP-1 mRNA and aggrecanase-1 expression in knee joint cartilage rats with knee osteoarthritis.Methods 42 Wistar rats as experimental animals,were randomly divided into 7 groups： normal group,model group,western medicine group,proprietary Chinese medicine group,low,middle and high dose group of Bushen Tongluo Formula,4% papain physiological saline solution was injected into the knee joint of the bilateral hind limbs of the rat,and 0.2 m L was injected to establish the model of osteoarthritis of the knee on first,fourth,seventh days.After building,respectively gastric drug intervening rats with distilled water,meloxicam solution,prevent bone hyperplasia capsule solution and Bushen Tongluo Formula with dosages of low,medium,high solutions,to observe the general condition of rats during the process of intervention.After 4 weeks of intragastric administration,the rats were killed and the tibial plateau and the internal and lateral condyle cartilage in the knee joint were scraped and placed in the cryopreservation tube,to determine mRNA expression levels of MMP-3/13 and TIMP-1 with Real-time PCR and ADAMTs-4 with Western blotting.Results the expression of MMP-3/13 mRNA is the highest content in model group（P〈0.05）,the lowest in normal group（P〈0.05）.The effects of different drugs on them were different.The expression level of the middle dose group of Bushen Tongluo Formula was the smallest among all the administration groups,and the expression level of the drug group was the largest in each proprietary group,compared with the high dose group,the middle dose group was lower than the high dose group,but there was no statistical difference between the two groups（P〉0.05）.Content of TIMP-1 mRNA is the lowest one in model group（P〈0.05）,while highest expressed in normal group（P〈0.05）.ADAMTs–4 in cartilage protein expression,the highest level is in model group and normal group is the lowest,each dose group of content is different,but there is no statistically significant difference among them（P〉0.05）.Conclusion Bushen Tongluo Formula has obvious effect in the treatment of KOA,and its mechanism may be through inhibiting the level of MMP-3/13 and ADAMTs-4,increasing the expression of TIMP-1,reducing the destruction of articular cartilage and improving the symptoms of KOA,so as to achieve the role of KOA treatment.