基于纤溶酶途径介导的软骨细胞外基质损伤机制研究温针灸治疗膝骨性关节炎的作用机理

目的通过观察温针灸干预后膝骨性关节炎(KOA)兔关节软骨尿激酶型纤溶酶原激活物(u PA)、基质金属蛋白酶-1(MMP-1)、基质金属蛋白酶-13(MMP-13)因子表达的变化,探索温针灸治疗KOA的作用机制。方法将40只新西兰兔随机分为空白组、模型组、温针灸组、u PA抑制剂组,每组10只,采用右后肢石膏管型固定法制备KOA模型,空白组不予处理,模型组每天于兔架上固定15 min,温针灸组予以温针灸治疗,u PA抑制组每两日于关节腔注射u PA抑制剂稀释液1 m L/kg,干预结束后取兔关节软骨行TUNEL、RT-PCR、Western blot检测。结果 TUNEL细胞凋亡检测发现:较空白组,模型组软骨细胞凋亡率明显增高,差异有统计学意义(P <0. 05);较模型组,温针灸组及u PA抑制剂组软骨细胞凋亡率明显下降,差异有统计学意义(P <0. 05)。RT-PCR检测结果发现:较空白组,模型组软骨中u PA、MMP-1、MMP-13 mRNA表达明显升高,差异有统计学意义(P <0. 05);较模型组,温针灸组u PA、MMP-1、MMP-13 mRNA表达显著降低,差异有统计学意义(P <0. 05);u PA抑制剂组软骨中u PA、MMP-1、MMP-13 mRNA表达显著降低,差异有统计学意义(P <0. 05);Western blot检测发现:较空白组,模型组软骨中u PA、MMP-1、MMP-13蛋白表达显著升高,差异有统计学意义(P <0. 05);较模型组,温针灸组u PA、MMP-1、MMP-13蛋白表达显著降低,差异有统计学意义(P <0. 05) u PA抑制剂组软骨中u PA、MMP-1、MMP-13蛋白表达显著降低,差异有统计学意义(P <0. 05);相关性分析显示u PA与MMP-1、MMP-13均由相关关系。结论 (1)温针灸可抑制软骨细胞的过度凋亡;(2)温针灸可有效抑制软骨细胞中u PA、MMP-1、MMP-13的表达,且可通过抑制u PA的生成进而阻断MMP-1、MMP-13的活化,发挥保护关节软骨最终达到治疗KOA的作用。

Mechanism of Warming Acupuncture on KOA Based on Fibrinolytic Pathway Mediated Chondrocyte Extracellular Matrix Injury Mechanism

Objective To investigate the mechanism of warming acupuncture in the treatment of KOA by observing the changes of expressions of uPA,MMP-1 and MMP-13 in articular cartilage of KOA rabbits which were treated by warming acupuncture.Methods Forty New Zealand rabbits were randomly divided into blank group,model group,warming acupuncture group and uPA inhibitor group.The KOA model was made by right hind limb gypsum tube fixation.The blank group were not treated.The rabbits in the warming acupuncture group were treated with warming acupuncture.The uPA inhibitor group was injected with 1 mg/kg of the uPA inhibitor dilution in the joint cavity every two days.TUNEL,RT-PCR and Western blot were used to detect the articular cartilage.Results The apoptosis rate of chondrocytes in the model group was significantly higher than that in the blank group(P<0.05).The apoptosis rate of the chondrocytes in the warming acupuncture group and the uPA inhibitor group was significantly lower than that in the model group(P<0.05).RT-PCR showed that the expressions of uPA,MMP-1 and MMP-13 mRNA in the model group were significantly higher than those in the blank group(P<0.05).The expressions of uPA,MMP-1 and MMP-13 mRNA in the cartilage of warming acupuncture group were significantly lower than those in the model group(P<0.05).The uPA,MMP-1 and MMP-13 mRNA in the uPA inhibitor group were significantly lower than those in the model group(P<0.05).Western blot showed that the expressions of uPA,MMP-1 and MMP-13 protein in the cartilage of warm acupuncture group were significantly lower than those in the model group(P<0.05).The uPA,MMP-1 and MMP-13 protein in the cartilage of uPA inhibitor group were significantly lower than those in the model group(P<0.05).Correlation analysis showed that uPA was correlated with MMP-1 and MMP-13.Conclusion①Warming acupuncture can effectively inhibit the excessiveapoptosis of chondrocytes.②Wanning acupuncture can inhibit the expressions of uPA,MMP—1 and MMP-13 in chondrocytesand block the activations of MMP-1 and MMP-13 through inhibiting the fomiation of uPA,and play a protective articular cartilage to treat KOA.