复方杜仲健骨颗粒治疗骨关节炎的抗炎潜在药效物质及作用机制研究

Anti-osteoarthritis components and mechanism of Fufang Duzhong Jiangu Granules

骨性关节炎是一种以关节软骨退行性病变为特征的中老年常见病。复方杜仲健骨颗粒具有滋补肝肾、养血荣筋、通络止痛的功效,是临床上用于治疗骨性关节炎的经典方药。该研究利用分子模拟技术结合分子生物学手段探讨并验证了复方杜仲健骨颗粒治疗骨关节炎潜在药效物质及分子作用机制。花生四烯酸代谢通路为典型的抗炎通路,其中分泌型磷脂酶A2-ⅡA(secretory phospholipase A2 groupⅡA,sPLA2-ⅡA)、5-脂氧合酶(5-lipoxygenase, 5-LOX)、环氧化酶-2(cyclooxygenase-2,COX-2)及白三烯A4水解酶(leukotriene A4 hydrolase, LTA4H)是该通路的关键靶标。因此,该研究首先基于花生四烯酸代谢通路4个关键靶标的药效团及分子对接模型,对复方杜仲健骨颗粒12味中药共计1 522个化学成分进行虚拟筛选,进一步结合方剂配比,对虚拟筛选结果进行加权分析。结果显示,该制剂中73个成分对于上述4个靶点作用活性较强,提示其可作为复方潜在抗炎药效物质。综合考虑成分的预测药效、易得性及其复方配伍环境,选择阿魏酸松柏酯、橄榄树脂素、黄芩苷作为验证化合物。研究进一步利用脂多糖(lipopolysaccharide, LPS)诱导的RAW264.7炎症细胞模型对其抗炎活性进行验证,结果显示3个成分均可有效抑制NO的释放,在一定程度上验证了药效物质选择的准确性。此外,结果表明,靶点5-LOX、COX-2及LTA4H的作用活性较高,提示上述3个靶标可能为复方杜仲健骨颗粒治疗骨关节炎的关键作用靶点;以潜在药效物质追溯来源中药,方中杜仲、牛膝、人参、枸杞、黄芪的活性值远高于其他中药,表明上述5味中药可能为复方杜仲健骨颗粒作用于花生四烯酸通路发挥抗炎作用的主要有效中药。该研究获得了复方杜仲健骨颗粒治疗骨关节炎的抗炎潜在药效物质,阐释了复方杜仲健骨颗粒作用于花生四烯酸代谢途径中的LOX、COX通路从而发挥治疗骨关节炎的作用机制,为复方杜仲健骨颗粒的药效物质及分子作用机制研究提供了参考。

Osteoarthritis is a common disease characterized by degenerative lesions of articular cartilage in the elderly.Fufang Duzhong Jiangu Granulues(FDJG), a classical prescription for the treatment of osteoarthritis, has the effects of nourishing liver and kidney, nourishing blood and sinew, and dredging collaterals and relieving pain.In this study, molecular simulation technology was combined with molecular biology methods to explore and verify the potential pharmacodynamic substances and molecular mechanism of FDJG in the treatment of osteoarthritis.Arachidonic acid(AA) metabolic pathway is a typical anti-inflammatory pathway, and secretory phospholipase A2 group ⅡA(sPLA2-ⅡA), 5-lipoxygenase(5-LOX), cyclooxygenase-2(COX-2), and leukotriene A4 hydrolase(LTA4 H) are the key targets of the pathway.Therefore, in this study, based on the pharmacophores and molecular docking models of the four key targets in AA pathway, a total of 1 522 chemical components in 12 medicinals of FDJG were virtually screened, followed by weighted analysis of the screening results in combination with the proportions of the medicinals in the prescription.The results showed that mainly 73 components in the preparation could act on the above four targets, suggesting they might be the potential anti-osteoarthritis components of FDJG.Considering the predicted effectiveness, availability, and compatibility of the medicinals, coniferyl ferulate, olivil, and baicalin were selected for further verification.Specifically, lipopolysaccharide(LPS)-induced RAW264.7 inflammatory cell model was used to verify the anti-inflammatory activity of the three components.The results showed that the three can effectively inhibit the release of NO, supporting the above selection.In addition, targets 5-LOX, COX-2, and LTA4 H had high activity, which suggested that they may be the key anti-osteoarthritis targets of FDJG.The comprehensive activity values of Eucommiae Cortex, Achyranthis Bidentatae Radix, Ginseng Radix et Rhizoma, Lycii Fructus, and Astragali Radix were much higher than that of other medicinals in the prescription, indicating that they may be the main effective medicinals in FDJG acting on the AA pathway.In this study, the potential anti-osteoarthritis components of FDJG were obtained.Moreover, it was clarified that the anti-osteoarthritis mechanism of FDJG was to act on LOX and COX pathway in AA metabolic pathway, which provided a reference for the study of pharmacodynamic substances and molecular mechanism of FDJG.