基于GEO芯片联合网络药理学、孟德尔随机化和实验验证探究木犀草素治疗心力衰竭的作用机制

Based on GEO chip, network pharmacology, Mendelian randomization and experimental validation, the mechanism of luteolin in the treatment of heart failure was explored

关键词:  木犀草素  心力衰竭  网络药理学  生物信息学  分子对接

Key words:  luteolin  heart failure  network pharmacology  bioinformatics  molecular docking

摘要:

目的 基于生物信息学、网络药理学、分子对接、体外实验和孟德尔随机化探究木犀草素治疗心力衰竭的机制。 方法 采用生物信息学和网络药理学分析木犀草素改善心力衰竭的关键靶点和信号通路。使用分子对接技术对木犀草素与关键靶点进行分子对接。在细胞水平上建立心肌细胞损伤模型，使用CCK-8检测细胞活性；qRT-PCR 检测细胞心肌损伤标志物BNP、关键靶点ESR1、PTGS2、AR和炎性细胞因子IL-18、IL1-β、IL-6的mRNA表达；流式细胞术检测细胞凋亡率和ROS水平；Western blot检测细胞凋亡相关蛋白和SRC/PI3K/AKT信号通路相关蛋白表达。使用孟德尔随机化探究关键靶点和心力衰竭的因果关系。 结果 得到60个木犀草素在心力衰竭中差异表达基因。GO和KEGG富集分析发现木犀草素主要通过PI3K/AKT等信号通路及SRC、AKT1、ESR1、PTGS2、AR、KDR等核心靶点通过蛋白质磷酸化、对氧化应激的反应发挥生物功能。分子对接验证了木犀草素与关键靶点具有较好的结合活性。实验结果表明木犀草素可以有效缓解心肌细胞损伤，减轻氧化应激，降低细胞凋亡率，抑制炎性细胞因子的分泌，并且抑制SRC/PI3K/AKT通路相关蛋白磷酸化的表达。孟德尔随机化研究表明基因SRC（IVW：OR = 1.050；95%CI：1.011～1.091；P = 0.011）可能是心力衰竭进展过程中的重要标志物，与发病风险之间具有潜在的因果关系。 结论 木犀草素对心肌损伤的保护作用是通过调节SRC/PI3K/AKT信号通路，可为后续木犀草素治疗心力衰竭的深入研究提供科学依据。

Abstract:

OBJECTIVE To explore the mechanism of luteolin treatment in heart failure based on bioinformatics, network pharmacology, molecular docking, in vitro experiments, and Mendelian randomization. METHOD Bioinformatics and network pharmacology analysis analyze key targets and signaling pathways of luteolin in improving heart failure. Molecular docking of luteolin to key targets by using molecular docking technology. A myocyte injury model was established at the cellular level, and cell activity was measured by CCK-8; qRT-PCR for mRNA expression of BNP, key targets ESR1, PTGS2, AR, and inflammatory cytokines IL-18, IL 1- β, IL-6; apoptosis rate and ROS level by flow cytometry, and apoptosis-related protein and SRC / PI3K / AKT signaling related protein expression by Western blot. Exploring the causality of key targets and heart failure using Mendelian randomization. RESULTS And obtained 60 genes differentially expressed in luteolin in heart failure. GO and KEGG enrichment analysis found that luteolin mainly plays biological functions through PI3K / AKT and core targets such as SRC, AKT1, ESR1, PTGS2, AR, and KDR through protein phosphorylation and response to oxidative stress. Molecular docking verified the better binding activity of luteolin to the key targets. The experimental results show that luteolin can effectively alleviate cardiomyocyte injury, reduce oxidative stress, reduce the rate of apoptosis, inhibit the secretion of inflammatory cytokines, and inhibit the expression of the phosphorylation of proteins related to SRC / PI3K / AKT pathway. Mendelian randomization studies indicate that gene SRC (IVW: OR = 1.050; 95%CI: 1.011~1.091; P=0.011) may be an important marker in the progression of heart failure, with a potential relationship between the risk of disease. CONCLUSION The protective effect of luteolin on myocardial injury is to regulating SRC / PI3K / AKT signaling pathway, which can provide scientific basis for the further study of luteolin in the treatment of heart failure.