基于计算机辅助药物设计方法探究敦煌消痹止痛酊治疗骨关节炎的物质基础

Deciphering of the Material Basis of Dunhuang Xiaobi Zhitong Tincture in the Treatment of Osteoarthritis Based on Computer Aided Drug Design Method

关键词:  敦煌消痹止痛酊  骨关节炎  皮肤渗透性  酶活性测定  分子对接  分子动力学模拟  软骨外植体

Key words:  Dunhuang Xiaobi Zhitong tincture  osteoarthritis  transdermal penetration  enzyme activity assay  molecular docking  molecular dynamics simulation  cartilage explants

摘要:

目的 采用皮肤渗透性参数计算、复杂网络分析、分子对接、分子动力学模拟等计算机辅助药物设计结合化学分析、体外生物试验的“干湿”结合策略探究敦煌消痹止痛酊治疗骨关节炎(osteoarthritis，OA)的物质基础。方法 选择被实验证实的中药来源的可透皮化合物，利用Schrödinger软件Qikprop模块进行皮肤渗透性参数计算，建立透皮参数范围标准，并筛选敦煌消痹止痛酊中满足条件的化合物。对敦煌消痹止痛酊进行功效分组，通过靶点反向预测-复杂网络分析-KEGG通路富集分析探讨不同功效组治疗OA的潜在靶点及KEGG通路。采用分子对接的方法对方中符合皮肤渗透性参数范围的成分进行虚拟筛选，挑选化合物进行酶活性测定、ADMET计算、皮肤渗透性测定、细胞试验、外植体试验研究。结果 全方得到潜在透皮成分为69个，复杂网络分析结果显示活血组治疗OA的特有靶点主要为VEGFA、IGF1R及HIF-1α等，主要KEGG通路为Ras、RAP1等。清热组治疗OA的特有靶点主要为TLR9、IL-6及ADAMTS5等，主要KEGG通路涉及TNF、NF-κB等信号通路；分子对接-酶活性-细胞试验结果显示，升麻素、欧前胡素和香草酸对COX-2具有抑制活性，可以抑制软骨细胞PGE2的合成。在软骨外植体模型中，升麻素可以有效缓解软骨损伤；体外透皮试验显示，升麻素12 h内皮肤累积渗透量达到128.1 μg·cm^-2，透皮吸收百分率为28.2%。分子动力学模拟结果显示，升麻素与COX-2的氨基酸残基TYR 355、LEU 384、SER 530形成较为稳定的氢键作用，与PHE 518形成较为稳定的疏水相互作用。结论 本研究采用计算机辅助药物设计联合多种化学生物实验技术的“干湿”结合策略初步阐明了敦煌消痹止痛酊治疗OA的物质基础，为外用中药的研究提供参考。

Abstract:

OBJECTIVE To investigate the material basis of Dunhuang Xiaobi Zhitong tincture in the treatment of osteoarthritis(OA) using a “dry and wet” strategy, consisting of computer aided drug design such as transdermal permeability calculation, complex network, molecular docking, molecular dynamics simulation, combined with chemical analysis, enzyme inhibition assay and experiments in vitro. METHODS Select transdermal compounds from traditional Chinese medicine that had been verified by experiments. The skin permeability parameters were calculated by using Qikprop module of Schrödinger software. The range of transdermal parameters was established and the qualified components in Dunhuang Xiaobi Zhitong tincture were screened. Efficacy groups were divided according to Dunhuang Xiaobi Zhitong Tincture treatment efficacy, and potential targets and KEGG pathways for OA treatment in different efficacy groups were explored through reverse prediction of targets, complex network analysis and KEGG pathway enrichment analysis. The molecular docking method was used to virtually screen the components in the target COX-2 partner that met the range of skin permeability parameters, and the compounds were selected for enzyme activity assay, ADMET calculation, skin permeability assay, cell experiments, and explant experiments. RESULTS The 69 potential transdermal compounds were obtained. Complex network analysis showed that the targets of OA treatment in the promoting blood circulation group were mainly VEGFA, IGF1R and HIF-1α. The main KEGG signaling pathways were Ras and RAP1 signaling pathways, etc. The targets of OA treatment in the resolving heat group were mainly TLR9, IL-6 and ADAMTS5. The main KEGG signaling pathways were TNF, NF-κB signaling pathway, etc. The results of macromolecular docking-enzyme activity-cell experiment showed that, cimifugin, imperatorin, and vanillic acid could inhibit COX-2 activity and inhibit the synthesis of PGE2 in chondrocytes. Cimifugin could effectively relieve the damage of cartilage damage in cartilage explant models. The transdermal absorption experiment showed that the cumulative permeability per unit area of cimifugin in 12 h was 128.1 μg·cm^-2. The average transdermal accumulation rates was 28.2%. The result of molecular dynamics simulation showed that cimifugin formed a relatively stable hydrophobic interaction with PHE 518 and formed stable hydrogen bond with the residues TYR 355, LEU 384 and SER 530. CONCLUSION In this study, a “dry and wet” combination strategy of computer aided drug design combines with a variety of chemical and biological experiment are used to elucidate the material basis of Dunhuang Xiaobi Zhitong tincture in the treatment of OA. This work may provide methodological reference for the study of external preparation of traditional Chinese medicine.