基于网络药理学与体外实验研究清髓消炎汤治疗骨髓炎的作用机制

Study on the mechanism of Qingsui Xiaoyan decoction in treating osteomyelitis based on network pharmacology and in vitro experiment

通过TCMSP和BATMAN-TCM数据库筛选经验方中的中药化合物及作用靶点;通过GeneCards、DRUGBANK、TTD数据库查找骨髓炎疾病相关靶点,取疾病与中药化合物靶点的交集靶点预测清髓消炎汤治疗骨髓炎的潜在靶点.运用Cytoscape软件构建分析“中药-成分-成分作用靶点-疾病靶点”网络图;利用STRING数据库平台分析作用靶点PPI网络;通过DAVID、微生信数据库对作用靶点进行GO功能和KEGG通路的富集分析.利用AutoDockTools及PYMOL软件对清髓消炎汤中核心靶点与疾病的核心靶点进行分子对接与体外实验验证清髓消炎汤对骨髓炎常见耐药菌MRSA的抑制,探索清髓消炎汤治疗骨髓炎的作用机制.筛选得到清髓消炎汤中289个成分靶点,骨髓炎有944疾病靶点,得到交集靶点83个.通过网络拓扑学分析得到主要有效成分quercetin、luteolin、fisetin、kaempferol、baicalein、naringenin,主要作用靶点TNF、IL-6、IL-10、STAT3、IL-1β、MAPK14等.主要成分与靶点分子对接结果均为负数,均有很好的自由结合能力.清髓消炎汤(Qingsui Xiaoyan Decoction,QSXYD)对MRSA的MIC和MBC分别是31.3和62.5 mg/mL;QSXYD影响MRSA生物被膜(MRSA-BF)内的活菌量和生物被膜的代谢活性,并呈现剂量依赖(P<0.05);SEM显示QSXYD影响细菌的个体大小、结构疏密程度、菌体表面的形态;RT-PCR结果显示sub-MIC浓度的QSXYD可明显抑制MRSA相关毒力基因与生物膜形成相关基因的表达(P<0.05).通过网络药理学和体外实验等方法研究结果显示,清髓消炎汤可以通过多靶点、多通路作用于治疗和预防骨髓炎,为清髓消炎汤临床应用于治疗骨髓炎提供理论依据.

Based on the experience of network pharmacology and in vitro experimental research,the mechanism of Qingsui Xiaoyan decoction in treating osteomyelitis provides theoretical basis for subsequent basic and clinical research.The traditional Chinese medicine compounds and action targets in empirical prescriptions were screened by TCMSP and BATMAN-TCM database,the disease-related targets of osteomyelitis were found by GeneCards,DRUGBANK and TTD databases,and the intersection targets of disease and traditional Chinese medicine compounds were selected to predict the potential targets of Qingsui Xiaoyan decoction in treating osteomyelitis.The network diagram of“traditional Chinese medicine-component-component action target-disease target”was constructed by Cytoscape software,the PPI network of action target was analyzed by STRING database platform,and the GO function and KEGG pathway enrichment of action target were analyzed by DAVID and microbiological information database.The molecular docking and in vitro experiments on the core targets and disease core targets of Qingsui Xiaoyan decoction were carried out by using AutoDockTools and PYMOL software to verify the inhibition of Qingsui Xiaoyan decoction on the common drug resistant bacteria MRSA of osteomyelitis and to explore the mechanism of Qingsui Xiaoyan decoction in the treatment of osteomyelitis.289 component targets and 944 disease targets of osteomyelitis were found in Qingsui Xiaoyan decoction,and 83 intersection targets were obtained.Through network topology analysis,the main active components obtained are quercetin,luteolin,fisetin,kaempferol,baicalein,naringenin,and the main targets of action are TNF,IL-6,IL-10,STAT3,and IL-1β、MAPK14,etc.The docking results of main components and target molecules are negative,and both have good free binding ability.The MIC and MBC of Qingsui Xiaoyan decoction(QSXYD)for MRSA were 31.3 and 62.5 mg/mL,respectively,which affected the amount of viable bacteria in MRSA biofilm(MRSA-BF)and the metabolic activity of the biofilm in a dose-dependent manner(P<0.05),and SEM showed that QSXYD affected the individual size,structural density and surface morphology of bacteria.RT-PCR results showed that QSXYD at the concentration of sub-MIC could significantly inhibit the expression of MRSA-related virulence genes and biofilm formation-related genes(P<0.05).The results of network pharmacology and in vitro experiments show that Qingsui Xiaoyan decoction can treat and prevent osteomyelitis through multi-targets and multi-pathways,which provides a theoretical basis for the clinical application of Qingsui Xiaoyan decoction in the treatment of osteomyelitis.