通络生骨胶囊对骨关节炎的治疗作用及其分子机制研究

目的:利用碘乙酸诱的SD大鼠骨关节炎模型,研究通络生骨胶囊对骨关节炎(OA)的治疗作用;利用IL-1β诱导的人软骨细胞(SW1353)损伤模型,研究通络生骨胶囊对软骨细胞保护作用的分子机制。方法:SD大鼠膝关节注射碘乙酸(3 mg/只)诱导形成OA模型,造模3 d后分为对照组、模型组和不同浓度的通络生骨溶液组(0.5、1、2 g/kg)。给药3周后检测血清中IL-1β的含量,膝关节软骨细胞中MMP-13的mRNA表达水平以及c-Fos蛋白表达水平。检测经通络生骨胶囊(1、5、10、50、100μg/mL)处理48 h后的IL-1β(20 ng/mL)损伤SW1353细胞8 h后的细胞活率;荧光定量PCR法检测Wnt4a、β-catenin、GSK-3β、MMP-13、ADAMTS4、Nrf2、GCLC和NQO-1的mRNA表达水平,Western-Blot的方法检测β-catenin和MMP-13的蛋白含量。结果:不同浓度(0.5、1、2 g/kg)的通络生骨胶囊溶液均可以有效降低碘乙酸诱导的IL-1β含量,抑制软骨细胞中的MMP-13和c-Fos的表达,呈剂量效应关系。在IL-1β刺激的SW1353损伤模型中,不同浓度(10、50、100μg/mL)的通络生骨胶囊溶液均能显著增强细胞活率,抑制IL-1β诱导的Wnt4a、β-catenin、MMP-13、ADAMTS4和GSK-3β的mRNA表达水平上调作用,增强IL-1β抑制的Nrf2、GCLC和NQO-1的mRNA表达水平。结论:通络生骨胶囊可以通过降低IL-1β等炎症因子的分泌,抑制IL-1β诱导的Wnt/β-catenin信号通路的活化、降低MMP-13、ADAMTS4等基质金属蛋白酶的分泌,增强Nrf2/HO-1信号通路,并从多方面提高软骨组织的抗损伤能力。

Therapeutic Effect and Molecular Mechanism of Tongluo Shenggu Capsule in Treating OA

Objective:To study the therapeutic effect of Tongluo Shenggu Capsule on osteoarthritis(OA)in SD rats induced by iodoacetic acid,and to study the molecular mechanism of protective effect of Tongluo Shenggu Capsule on chondrocytes in the model of SW1353 chondrocyte injury induced by IL-1β.Methods:OA model was induced by iodoacetic acid injection into SD rats knee joint.After 3 days of modeling,the rats were randomly divided into the control group,the model group and Tongluo Shenggu Capsule groups(0.5 g/kg,1 g/kg and 2 g/kg).Serum level of IL-1β,MMP-13 mRNA expression and c-Fos protein expression of the chondrocytes in knee joint were detected 3 weeks after medication.After 48 h intervention by Tongluo Shenggu Capsule(1μg/mL,5μg/mL,10μg/mL,50μg/mL and 100μg/mL)in advance,SW1353 cells were damaged by IL-1β(20 ng/mL)for another 8 h then the cell viability was measured.The mRNA expression levels of Wnt4a,β-catenin,GSK-3β,MMP-13,ADAMTS4,Nrf2,GCLC and NQO-1 were detected by fluorescence quantitative PCR,and the protein contents ofβ-catenin and MMP-13 were measured by Western-Blot method.Results:Tongluo Shenggu Capsule solution with different concentrations(0.5 g/kg,1 g/kg and 2 g/kg)could effectively reduce the contents of IL-1βinduced by iodoacetic acid,and inhibit the expressions of MMP-13 and c-Fos in chondrocytes,which showed a dose-effect relationship.In the damage model induced by IL-1β,Tongluo Shenggu Capsule solution with different concentrations(10μg/mL,50μg/mL and 100μg/mL)significantly enhanced cell viability,inhibited the mRNA expression levels of Wnt4a,β-catenin,MMP-13,ADAMTS4 and GSK-3βinduced by IL-1β,and enhanced the mRNA expression levels of Nrf2,GCLC and NQO-1 inhibited by IL-1β.Conclusion:Tongluo Shenggu Capsule can reduce the secretion of inflammatory factors,such as IL-1β.It can inhibit the activation of the Wnt/β-catenin induced by IL-1β,it can reduce the secretion of matrix metalloproteinases,such as MMP-13 and ADAMTS4,as well as enhance the Nrf2/HO-1 signaling pathway to improve the anti-injury ability of cartilage in many aspects.