归龟壮骨片治疗膝骨关节炎的作用机制研究

Mechanism of Guigui Zhuanggu Tablet on the Treatment of Knee Osteoarthritis

目的:探讨归龟壮骨片治疗膝骨关节炎模型大鼠的相关作用机制。方法:选取64只雄性SD大鼠并适应性饲养1周,根据手术顺序随机分为干预28 d和56 d假手术组、模型组、硫酸氨基葡萄糖组、归龟壮骨片组,每组各8只。除假手术组和模型组采用生理盐水灌胃,其余各组分别予硫酸氨基葡萄糖和归龟壮骨片水溶剂灌服。采用内侧半月板横断术对大鼠右侧膝关节进行膝骨关节炎造模,术中大鼠内侧半月板明显分离即确认造模成功。各组大鼠分别在造模前及造模后第0,7,14,21,28,35,42,49,56天进行足底机械痛阈值评估,并在造模第28天和第56天后,检测关节灌洗液中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)的浓度,及胫骨平台软骨中MMP-13、ADAMTS-5蛋白表达,并观察大鼠膝关节冠状面病理情况。选取10只雄性新西兰兔制备正常兔血清和含药兔血清,选取12只SD乳鼠获取原代软骨细胞,观察归龟壮骨片含药兔血清不同浓度和作用时间对软骨细胞活力的影响。结果:模型组大鼠足底机械痛阈值明显低于假手术组,差异有统计学意义(P<0.01);与模型组总效应比,归龟壮骨片组大鼠的足底机械痛阈值显著高于模型组,差异有统计学意义(P<0.001)。归龟壮骨片组大鼠关节灌洗液中IL-1β和TNF-α的浓度与模型组差异无统计学意义(P>0.05);仅第28天时,归龟壮骨片组大鼠关节灌洗液中IL-1β较模型组显著降低,差异有统计学意义(P<0.01)。相对于模型组,归龟壮骨片组大鼠的膝关节软骨表面大部分光滑整齐,部分软骨表层不规则,软骨细胞有弥散性增多,部分骨髓间充质发生成纤维化改变,但边缘骨赘增厚明显;归龟壮骨片干预后国际膝骨关节炎研究学会(OARSI)全关节总分较模型组显著降低,差异有统计学意义(P<0.05)。归龟壮骨片组大鼠第28天的胫骨平台软骨中MMP-13和ADAMTS-5蛋白表达水平较模型组差异无统计学意义(P>0.05);归龟壮骨片组大鼠第56天的胫骨平台软骨中的MMP-13和ADAMTS-5水平明显降低,差异有统计学意义(P<0.05)。归龟壮骨片含药兔血清能促进原代软骨细胞活力(P<0.01),呈时序性但不呈浓度梯度依赖性。结论:归龟壮骨片可以通过抑制MMP-13和ADAMTS-5表达并促进软骨细胞的活力,减轻内侧半月板横断术诱导的软骨退变,起到保护关节软骨的作用,还能提高膝骨关节炎大鼠的足底机械痛阈值。

Objective:To investigate the mechanism of Guigui Zhuanggu tablet(GZT)on the treatment of knee osteoarthritis(KOA)in rat models and chondrocytes.Methods:After adapting for 1 week,64 male SD rats were randomly divided into the sham group,model group,Glucosamine Sulfate capsule(GSC)group,and GZT group according to the surgical sequence at treated days 28 and 56.Each group consisted of 8 rats.Except for the sham group and model group were treated with normal saline by intragastric administration,the other groups were given GSC and GZT solution.The medial meniscus transection(MMT)was used to induce KOA in the right knee joint of the rats,and the successful modeling was confirmed by the clear separation of the rat's medial meniscus during the surgery.Each group of rats was subjected to foot mechanical pain threshold assessment before and after modeling at 0,7,14,21,28,35,42,49 and 56 d.The concentration of IL-1βand TNF-αin the joint lavage fluid and MMP-13 and ADAMTS-5 protein expression level in the tibial plateau cartilage were detected after 28 d and 56 d of modeling.The pathological changes in the coronal plane of the rat's knee joint were also observed.The study observed the effects of GZT-containing rabbit serum with different concentrations and treatment durations on the viability of primary chondrocytes from 12 SD rat pups,using normal serum and drug-containing serum from 10 male New Zealand rabbits.Results:The mechanical pain thresholds of rats in the model group were significantly lower than those in the sham group,with a statistically significant difference(P<0.01).Compared with the overall effect in the model group,the mechanical pain thresholds of rats in the GZT group were significantly higher,with a statistically significant difference(P<0.001).The concentrations of IL-1βand TNF-αin the joint lavage fluid of rats in the GZT group did not differ significantly from those in the model group(P>0.05),only at 28 d,the concentration of IL-1βin the joint lavage fluid of rats in the GZT group was significantly reduced compared to that in the model group,with a statistically significant difference(P<0.01).Compared with the model group,most of the surface of the knee joint cartilage in the GZT group rats was smooth and orderly,with irregularities in some parts of the cartilage,diffuse increase in chondrocytes,and fibrotic changes in some bone marrow stromal cells,however,the osteophyte was significantly thickened.The OARSI total joint scores in the GZT group rats were significantly lower than that in the model group after intervention,with a statistically significant difference(P<0.05).There was no significant difference in the expression levels of MMP-13 and ADAMTS-5 in the tibial plateau cartilage of GZT group rats at 28 d compared to those in the model group(P>0.05).The expression levels of MMP-13 and ADAMTS-5 in the tibial plateau cartilage of GZT group rats at 56 d were significantly reduced,with a statistically significant difference(P<0.05).The GZT-containing serum could promote the viability of primary chondrocytes(P<0.01),showing temporal but not concentration-dependent characteristics.Conclusion:GZT can protect joint cartilage by inhibiting the expression of MMP-13 and ADAMTS-5 and promoting the vitality of chondrocytes,thus reducing cartilage degeneration induced by MMT surgery.Furthermore,GZT can increase the mechanical pain threshold of rats with knee osteoarthritis.