基于网络药理学与实验验证研究荜茇抗肺纤维化的作用机制  

Effect of Piperlongum L against pulmonary fibrosis based on network pharmacology and in vitro studies

作　　者：郭晶晶 甄华[2] 张胜巍 菅若男 GUO Jingjing;ZHEN Hua;ZHANG Shengwei;JIAN Ruonan(Baotou Medical College,Baotou 014040,Neimenggu,China;The First Hospital of Hohhot,Hohhot 010000,Neimenggu,China;The First Affiliated Hospital of Baotou Medical College,Baotou 014040,Neimenggu,China)

机构地区：[1]内蒙古科技大学包头医学院药学院,内蒙古包头014040 [2]呼和浩特市第一医院药剂科,内蒙古呼和浩特010000 [3]包头医学院第一附属医院,内蒙古包头014040

出　　处：《中国临床药理学与治疗学》2024年第10期1120-1133,共14页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：内蒙古自治区首府地区公立医院高水平临床专科建设科技项目(2024SGGZ137);内蒙古高等教育厅基金项目(NJZY23091);包头市青年创新人才项目(202229);内蒙古自治区自然科学基金项目(2023QN08049);包头医学院科学研究基金项目-青苗计划(BYJJ-ZRQM 202304);包头医学院科学研究基金项目(BYJJ-KCRH202418)。

摘　　要：目的:通过网络药理学及分子对接技术初步预测荜茇抗肺纤维化的活性成分、作用靶点及信号通路,并通过体外实验进行初步验证荜茇抗肺纤维化的作用机制。方法:从TCMSP、Swiss Target Prediction和Pub Chem等数据库中获取药物的有效成分及对应靶点。使用Gene Cards和OMIM数据库收集疾病的相关靶点。利用jvenn在线工具筛选出药物与疾病的交集靶点,通过String数据库及Cytoscape3.9.1软件构建“药物-成分-靶点”与PPI网络图。使用微生信平台对交集靶点进行GO及KEGG富集分析,将富集KEGG前20条通路、核心靶点、药物成分,构建“成分-靶点-通路”网络并进行可视化。利用分子对接技术将药物成分与靶点进行对接,并对其部分对接结果可视化。培养HFL-1细胞,采用MTT法检测各浓度对细胞活力的影响,计算抑制率,从而筛选出最佳的药物浓度。将体外培养的HFL-1细胞分为4组,即对照组、TGF-β1组、TGF-β1+LD组(联合LD组)、TGF-β1+HD组(联合HD组)。采用CCK-8试剂检测24、48、72 h的细胞增殖活性。采用平板克隆形成实验来观察药物对HFL-1细胞克隆形成能力的影响。采用RT-qPCR和Western blot实验观察各组细胞α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(COI-Ⅰ)、Ⅲ型胶原蛋白(COI-Ⅲ)的m RNA和PI3K-Akt信号通路蛋白表达水平。结果:共获得荜茇与抗肺纤维化交集靶点为197个。经拓扑学分析获得核心PPI网络,包含29个节点与199条边;GO功能分析显示,荜茇治疗肺纤维化(PF)生物学过程包括:凋亡过程负调控、信号转导、蛋白质磷酸化等。细胞成分包括:细胞质、核浆、等离子体膜。分子功能包括相同的蛋白结合、丝氨酸/苏氨酸/酪氨酸激酶活性、蛋白结合等。KEGG信号通路被155条富集,而其中已被证实,与PF密切相关为PI3K-Akt信号通路,富集排名第四“。成分-靶点-通路”网络中PIK3CA、MAPK3、MAPK1、MTOR、SRC、CCND1、EGFR�AIM:To predict the active components and targets of Piperlongum L.and the associated signaling pathways involved in pulmonary fibrosis using network pharmacology and molecular docking technique and evaluate the mechanism of Piperlongum L against pulmonary fibrosis by in vitro experiments.METHODS:The active ingredients and targets were retrieved from TCMSP,Swiss Target Prediction and PubChem databases.The disease-related targets were retrieved from Gene Cards and OMIM databases.The intersection targets of the drugs and disease-related targets were identified using jvenn online tool.String database was used to construct the"drug-component-target"and PPI network and the networks were visualized using Cytoscape 3.9.1 software.GO and KEGG enrichment analysis were performed on the intersection targets using the DAVID tool.The top 20 KEGG pathways,core targets and drug components were used to construct a"component-target-pathway"network and the network visualization was performed using Cytoscape 3.9.1 software.The interactions between drug compounds and the targets were evaluated by molecular docking,and the docking results were visualized using Discovery studio.HFL-1 cells were cultured and the effect of the drug compounds on cell viability was determined by MTT assay.The inhibition rate was then calculated to determine the optimal drug concentration.HFL-1 cells were cultured in vitro and were assigned into 4 groups:control group,TGF-β1 group,TGF-β1+LD group(LD group),TGF-β1+HD group(HD group).CCK-8 kit was used to evaluate the antiproliferative activity of the drug compounds against HFL-1 cells at 24,48 and 72 h.Plate clone formation assay was performed to evaluate the effect of drugs on the colony formation ability of HFL-1 cells.RT-qPCR and western blot were conducted to determine the effect of the compounds on the mRNA and protein expression levels ofα-smooth muscle actin(α-SMA),collagen type Ⅰ(COI-Ⅰ),and collagen type Ⅲ(COI-Ⅲ)in each group.RESULTS:A total of 197 intersection targets of Piperlongum L a

关 键 词：荜茇 肺纤维化 网络药理学 分子对接 HFL-1细胞 PI3K-AKT信号通路 

分 类 号：R322.35[医药卫生—人体解剖和组织胚胎学]