文献类型：期刊文献

中文题名：基于网络药理学和细胞实验探讨参苓白术散对肝纤维化中TGF-β通路的作用机制

英文题名：Mechanism Exploration of Shenling Baizhu Powder on TGF-β Pathway in Treating Hepatic Fibrosis Based on Network Pharmacology and Cell Experiment

作者：孙墨晗[1];靳玉秋[1];赵哲[1];田萌媛[1];胡蓉[1];陈光顺[1]

第一作者：孙墨晗

机构：[1]甘肃中医药大学,甘肃兰州730000

第一机构：甘肃中医药大学

年份：2023

卷号：40

期号：1

起止页码：19

中文期刊名：中医药信息

外文期刊名：Information on Traditional Chinese Medicine

基金：国家自然科学基金项目(81660772);甘肃省教育科技创新项目(2021CXZX-772)。

语种：中文

中文关键词：参苓白术散;肝纤维化;网络药理学;HSC-T6;TGF-β1

外文关键词：Shenling Baizhu Powder;Hepatic fibrosis;Network pharmacology;HSC-T6;TGF-β1

摘要：目的:基于网络药理学预测参苓白术散抗肝纤维化(HF)的潜在作用靶点及可能相关的信号通路,同时通过体外细胞实验验证其潜在的分子机制。方法:运用TCMSP和BATMAN-TCM数据库初步获取参苓白术散的活性成分和作用靶点,借助Gene Card和OMIM数据库收集疾病相关的靶点,两者取交集绘制韦恩图,采用Cytoscape 3.7.2软件建立“药物-活性成分-靶点”网络图,并通过STRING数据库获得PPI网络,通过GO及KEGG富集分析筛选出主要作用通路和靶点蛋白。根据预测情况通过细胞实验对生物学分析进行验证。建立大鼠肝星状细胞(HSC-T6)模型,设置空白组,低、中、高剂量组分别给予100、200、400μg/mL参苓白术散处理,阳性对照组给予200μg/mL秋水仙碱处理。MTT法检测不同组别细胞增殖的抑制率,划痕实验检测用药后HSC-T6的迁移能力,ELISA法检测给药后各组细胞内α-SMA蛋白含量,Western blot法检测各组细胞中TGF-β1、Smad2和Smad7蛋白的表达水平。结果:经筛选最终获得参苓白术散潜在靶点970个,疾病靶点513个,药物与疾病交集靶点117个,主要作用于Akt1、IL-6、TGF-β1、TNF等核心靶点,涉及脂质与动脉粥样硬化、糖尿病并发症中的AGE-RAGE信号通路、肿瘤坏死因子信号通路、乙型肝炎通路、转化生长因子-β信号通路等通路。与空白组相比,MTT检测显示,参苓白术散能够抑制HSC-T6增殖,差异具有统计学意义(P <0.05);细胞划痕实验显示,给药后可以减弱HSC-T6迁移能力,差异具有统计学意义(P <0.05);ELISA实验显示,参苓白术散可以降低α-SMA蛋白含量表达,差异具有统计学意义(P <0.05);Western blot实验显示,参苓白术散能够下调TGF-β1、Smad2和Smad7蛋白表达水平,差异具有统计学意义(P <0.05)。结论:参苓白术散抗肝纤维化具有多成分、多靶点、多通路的特点,其作用机制可能通过参与细胞增殖、凋亡、免疫和炎症反应等生物过程,同下调TGF-β1、Smad2和Smad7蛋白有关。
Objective: To predict the potential targets and possible related signaling pathways of Shenling Baizhu Powder in treating hepatic fibrosis based on network pharmacology, and to verify its potential molecular mechanism through in vitro cell experiments. Methods: Active components and action targets of Shenling Baizhu Powder were preliminarily obtained using TCMSP and BATMAN-TCM databases. The Gene Card and OMIM were applied to collect disease-related targets and draw a Venn diagram by taking the intersection. Cytoscape 3.7.2 software was used to build a ’drug-active ingredient-target’ network map, and the PPI network was obtained through the STRING database. The main pathways and target proteins were identified through GO and KEGG enrichment analysis screening. Biological assays were validated by cellular experiments based on predictions. Rat hepatic stellate cell(HSC-T6) model was established. The lowdose group, the medium-dose group and the high-dose group were administered with Shenling Baizhu Powder at the concentrations of 100 μg/mL, 200 μg/mL and 400 μg/mL, respectively. The positive control group was treated with Colchicum Alkaline at the dose of 200 μg/mL. The inhibition rate of Shenling Baizhu Powder on the proliferation of HSC-T6 was detected by MTT method, the migration ability of HSC-T6 was detected by scratch test, and the intracellular α-SMA content in each group was detected by ELISA method. Western blot method was used to detect the protein expressions of TGF-β1, Smad2 and Smad7 in cells of each group. Results: After screening, 970 potential targets of Shenling Baizhu Powder, 513 disease targets, and 117 drug-disease intersection targets were obtained, mainly acting on core targets of Akt1, IL-6, TGF-β1 and TNF, involved in lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, hepatitis B pathway and TGF-β signaling pathways. Compared with the blank group, Shenling Baizhu Powder could inhibit the proliferation of HSC-T6 in MTT assay, and the difference was statistically significant(P<0.05). The cell scratch test results showed that the HSC-T6 migration was significantly inhibited after the interventions of Shenling Baizhu Powder(P<0.05). The ELISA experiment showed that Shenling Baizhu Powder could reduce the protein expression of α-SMA(P<0.05). The Western blot experiment showed that Shenling Baizhu Powder could down-regulate the protein expressions of TGF-β1, Smad2 and Smad7(P<0.05). Conclusion: Shenling Baizhu Powder has the characteristics of multi-component, multi-target and multi-pathway in treating hepatic fibrosis. Its action mechanism may be involved in biological processes of cell proliferation, apoptosis, immunity and inflammatory response, and may be related to down-regulating the proteins of TGF-β1, Smad2 and Smad7.