不同引经药配伍二妙方对大鼠实验性骨关节炎作用的比较研究

Effect of Ermiao Fang Combined with Different Medicinal Guides in Rat Osteoarthritis Model

摘要:

目的：比较不同引经药配伍二妙方（二妙）对骨关节炎（osteoarthritis，OA）大鼠的治疗作用，并探讨相关机制。方法：健康雄性SD大鼠60只，随机分成5组，即假手术、模型、二妙（1.6 g·kg^-1·d^-1）、二妙+细辛（1.6 g·kg^-1·d^-1+0.3 g·kg^-1·d^-1） 和二妙+独活（1.6 g·kg^-1·d^-1+0.3 g·kg^-1·d^-1）。除假手术外，其余各组前交叉韧带切断及部分半月板切除建立OA模型，造模同时分别给予相应药物灌胃，假手术组和模型组给予等体积的蒸馏水，连续给药3周和6周后分批取材。观察对膝关节软骨和滑膜的组织病理学改变，软骨细胞凋亡，以及对关节金属蛋白酶（matrix metalloproteinase-13，MMP-13）、金属蛋白酶抑制剂（tissue inhibitor of metalloproteinase-1，TIMP-1）的表达，血清白介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、一氧化氮（NO）和诱导型一氧化氮合酶（iNOS）含量的影响。结果：与假手术组比，模型组关节软骨、滑膜炎症病理积分和软骨细胞凋亡率明显增加（P<0.01），关节MMP-13表达增高而TIMP-1表达下降，血清IL-1β，TNF-α，NO，iNOS水平提高。与模型组比较，二妙组3周起能明显改善关节软骨和滑膜病变，减少软骨细胞凋亡，降低血清TNF-α水平，6周时能抑制MMP-13并提高TIMP-1在软骨的表达，减少血清IL-1β，NO，iNOS的水平。与二妙组比较，加引经药后能进一步改善关节组织病理学，降低软骨细胞凋亡（P<0.01），抑制MMP-13和提高TIMP-1的表达（P<0.05），降低血清各炎性因子的水平；尤其加独活后，在3周时便能明显降低Makin积分（P<0.05），6周时能显著提高二妙组对滑膜和IL-1β，TNF-α的抑制水平（P<0.05）。结论：二妙方能减缓OA大鼠的病理进程，配伍引经药细辛或独活后可不同程度提高疗效，其中独活相对作用更明显；其机制可能与减少软骨细胞凋亡、调节MMP-13和TIMP-1平衡及减少炎症细胞因子有关。

Abstract:

Objective: To compare the the effects of Ermiao Fang (EM) with different medicinal guides in osteoarthritis(OA) rats, and explore the related mechanism. Method: Sixty male Sprague-Dawley rats were randomly divided into sham-operated, model, EM (1.6 g·kg^-1·d^-1), EM plus Angelicae Pubescentis Radix (1.6 g·kg^-1·d^-1+0.3 g·kg^-1·d^-1) and EM plus Asari Radix et Rhizoma (1.6 g·kg^-1·d^-1+0.3 g·kg^-1·d^-1), evenly 12 in each group. Except the sham group, underwent surgical destabilization (anterior cruciate ligament transection and partial medial meniscectomy) of the right knee to induce OA.Meanwhile distilled water and corresponding drugs were administrated by gavage respectively, and materials were drawn at the third and sixth week. To observe the histopathological changes of knee articular cartilage and synovium by HE staining. And Chondrocytes apoptosis was tested by TUNEL assay. Matrix metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured by immunohistochemical assay. Interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in serum were detected by ELISA or radioimmunoassay. Result: Compared with the sham group, the pathological score of articular cartilage and synovium and the chondrocytes apoptosis rate in the model guoup were higher (P<0.01), and it was decreased MMP-13 and augmented TIMP-1 expression in the cartilage, and the levels of IL-1β, TNF-α, NO and iNOS in serum were rised. Compared with the model group, the EM could obviously improve the articular cartilage andsynovial lesions, reduce the apoptosis of cartilage cells, and reduce the level of TNF-α in serum at the third week. At the sixth week, it obviously decreased MMP-13 and augmented TIMP-1 expression in the cartilage, inhibited release of IL-1β, NO and iNOS. Compared with the EM group, the medicinal guide groups could further improve the joint pathology, reduce the cartilage cell apoptosis (P<0.01), regulate the expression of MMP-13 and TIMP-1(P<0.05), and reduce the levels of the inflammatory cytokines. Especially at EM plus with Angelicae Pubescentis Radix group, it could significantly decrease the Makin score from the third week(P<0.05), and improve the inhibition levels of the synovium and IL-1β, TNF-α of the EM group at the sixth week. Conclusion: The EM could slow the pathological process in osteoarthritis rat model. Combined with Angelicae Pubescentis Radix or Asari Radix et Rhizoma, it could further improve the curative effect with different levels. Especially, the effect of Angelicae Pubescentis Radix was more remarkable. And the mechanism may be related with the reduction of chondrocytes apoptosis, the regulation of MMP-13 and TIMP-1 equilibrium and reducing the inflammatory cytokine.