Zhou Pengcheng, Ma Jianli, Yu Wei, Chen Keling, Zhang Wensheng, Zhou Jiang
Department of Respiratory Medicine, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, China.
Clinical Medical School, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, China.
Evid Based Complement Alternat Med. 2023 Feb 11;2023:4359616. doi: 10.1155/2023/4359616. eCollection 2023.
To predict and determine the mechanism through which Tiao-Bu-Fei-Shen (TBFS) formula improves glucocorticoid resistance in chronic obstructive pulmonary disease (COPD), using network pharmacology, molecular docking technology, and studies.
The main active components and associated targets of TBFS were screened using the systems pharmacology database of traditional Chinese medicine database (TCMSP). The main COPD targets were retrieved from the Human Gene (GeneCards) and DrugBank databases. A protein-protein interaction (PPI) network was constructed using the protein interaction platform STRING and Cytoscape 3.6.1. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genome Pathway (KEGG) analyses were performed using the biological information annotation database Metascape. Molecular docking was performed using the AutoDock Vina software. THP-1 monocytes were treated with TBFS-containing serum and cigarette smoke extract (CSE) for 48 h, and cell proliferation in each group was determined using cell counting kit-8 (CCK-8). A COPD cell model was constructed by stimulating THP-1 monocytes with CSE for 12 h. A lentivirus vector for RNA interference of histone deacetylase 2 (HDAC2) gene was constructed and transfected into the THP-1 monocytes, and the transfection efficiency was verified using quantitative polymerase chain reaction (qPCR) and western blotting (WB). The expression of HDAC2 in each group of cells was detected using qPCR, and the expression of HDAC2, phosphoinositide-3 kinase (PI3K) p85, glucocorticoid receptor (GR), and P-AKT1 in each group of cells was detected through WB.
A total of 344 TBFS active components, 249 related drug targets, 1,171 COPD target proteins, and 138 drug and disease intersection targets were obtained. Visual analysis of the PPI network map revealed that the core COPD targets of TBFS were AKT1, IL-6, TNF, TP53, and IL1-. KEGG pathway enrichment analysis resulted in the identification of 20 signaling pathways as the main pathways involved in the action of TBFS against COPD, including the PI3K-Akt, TNF, and IL-17 signaling pathways. Molecular docking experiments revealed a strong binding capacity of kaempferol, luteolin, and quercetin to the ATK1 protein in TBFS, with quercetin performing the best. PCR results showed that treatment with TBFS significantly increased the expression levels of HDAC2 in the COPD model. WB results showed that TBFS treatment significantly increased the expression levels of GR and HDAC2 in the COPD model, while reducing the expression levels of P-AKT1.
TBFS treatment improves glucocorticoid resistance observed in COPD through downregulation of the PI3K-Akt signaling pathway and promotion of GR expression.
运用网络药理学、分子对接技术及实验研究，预测并确定调补肺肾方（TBFS）改善慢性阻塞性肺疾病（COPD）糖皮质激素抵抗的机制。
利用中药系统药理学数据库（TCMSP）筛选TBFS的主要活性成分及相关靶点。从人类基因（GeneCards）和药物银行（DrugBank）数据库检索COPD主要靶点。使用STRING蛋白质相互作用平台和Cytoscape 3.6.1构建蛋白质-蛋白质相互作用（PPI）网络。利用生物信息注释数据库Metascape进行基因本体（GO）富集分析和京都基因与基因组百科全书通路（KEGG）分析。使用AutoDock Vina软件进行分子对接。将含TBFS血清和香烟烟雾提取物（CSE）处理THP-1单核细胞48小时，每组细胞增殖情况采用细胞计数试剂盒-8（CCK-8）测定。通过CSE刺激THP-1单核细胞12小时构建COPD细胞模型。构建组蛋白去乙酰化酶2（HDAC2）基因RNA干扰慢病毒载体并转染至THP-1单核细胞，采用定量聚合酶链反应（qPCR）和蛋白质免疫印迹法（WB）验证转染效率。用qPCR检测每组细胞中HDAC2的表达，通过WB检测每组细胞中HDAC2、磷脂酰肌醇-3激酶（PI3K）p85、糖皮质激素受体（GR）和磷酸化AKT1（P-AKT1）的表达。
共获得344个TBFS活性成分、249个相关药物靶点、1171个COPD靶蛋白和138个药物与疾病交集靶点。对PPI网络图进行可视化分析显示，TBFS治疗COPD的核心靶点为AKT1、IL-6、TNF、TP53和IL1-。KEGG通路富集分析确定20条信号通路为TBFS治疗COPD的主要作用通路，包括PI3K-Akt、TNF和IL-17信号通路。分子对接实验表明，山奈酚、木犀草素和槲皮素与TBFS中的ATK1蛋白具有较强结合能力，其中槲皮素效果最佳。PCR结果显示，TBFS处理可显著提高COPD模型中HDAC2的表达水平。WB结果显示，TBFS治疗可显著提高COPD模型中GR和HDAC2的表达水平，同时降低P-AKT1的表达水平。
TBFS治疗通过下调PI3K-Akt信号通路和促进GR表达改善COPD中的糖皮质激素抵抗。
