基于网络药理学探讨川陈皮素改善慢性鼻窦炎伴鼻息肉的分子机制

Exploring the Molecular Mechanism of Nobiletin in Improving Chronic Rhinosinusitis with Nasal Polyps Based on Network Pharmacology

摘要:

目的 基于网络药理学探讨川陈皮素改善慢性鼻窦炎伴鼻息肉（CRSwNP）的关键靶点并设计实验加以验证。方法 川陈皮素靶点通过TCMSP、ETCM、CTD、HERB 2.0及SymMap数据库进行筛选，CRSwNP靶点通过GEO数据库进行筛选，通过R包获得差异表达基因及共有靶点，对共有靶点进行基因本体（GO）及京都基因与基因组百科全书（KEGG）通路分析，基于Cytoscape软件进行关键靶点筛选，采用CB-Dock2平台进行分子对接验证，CCK-8法检测川陈皮素对人鼻黏膜上皮细胞（HNEpC）的最佳无害浓度，采用LPS诱导HNEpC建立炎症模型，ELISA法检测细胞上清液中IL-6、IL-8、IL-1β及TNF-α水平，ROS试剂盒检测氧化应激，Western blot检测缺氧诱导因子-1α（HIF-1α）及血红素加氧酶1（HMOX1）蛋白表达。结果 共获得39个共有靶点，GO及KEGG分析显示主要涉及HIF-1信号通路等，Cytoscape筛选出MMP9、IL1β、TGFβ1、CXCL8、HMOX1、CCK为关键靶点，分子对接显示川陈皮素与HMOX1结合能为-8.8 kcal/mol；CCK-8法确定80 μmol/L为最佳无害浓度；ELISA结果显示川陈皮素可显著降低IL-6、IL-8、IL-1β及TNF-α水平；ROS检测显示川陈皮素可改善细胞氧化应激；Western blot显示川陈皮素可下调HIF-1α及HMOX1蛋白表达。结论 川陈皮素可有效抑制LPS诱导的鼻黏膜上皮细胞炎症反应及氧化应激损伤；HMOX1被确定为川陈皮素潜在的关键靶点，抑制HIF-1α/HMOX1正反馈环路发挥抗CRSwNP作用。

Abstract:

Abstract: Objective To investigate the key targets of nobiletin in improving chronic rhinosinusitis with nasal polyps (CRSwNP) based on network pharmacology and to design experiments to verify them. Methods Nobiletin targets were screened by TCMSP, ETCM, CTD, HERB 2.0 and SymMap databases. CRSwNP targets were screened by GEO database. Differentially expressed genes and common targets were obtained by R package. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on common targets. Cytoscape software was used to screen key targets. Molecular docking was verified by CB-Dock2 platform. CCK-8 method was used to detect the optimal non-toxic concentration of nobiletin on human nasal epithelial cells (HNEpC). LPS induced HNEpC was used to establish an inflammation model. ELISA was used to detect the levels of IL-6, IL-8, IL-1β and TNF-α. ROS kit was used to detect oxidative stress. Western blot was used to detect the expression of hypoxia-inducible factor-1α (HIF-1α) and heme oxygenase-1 (HMOX1). Results A total of 39 common targets were obtained. GO and KEGG analysis showed that they were mainly involved in HIF-1 signaling pathway, etc. MMP9, IL1β, TGFβ1, CXCL8, HMOX1 and CCK were screened as key targets by Cytoscape. Molecular docking showed that the binding energy of nobiletin and HMOX1 was -8.8kcal/mol. CCK-8 method determined that 80 μmol/L was the optimal non-toxic concentration. ELISA results showed that nobiletin could significantly reduce the levels of IL-6, IL-8, IL-1β and TNF-α. ROS detection showed that nobiletin could improve cellular oxidative stress. Western blot showed that nobiletin could downregulate the expression of HIF-1α and HMOX1 proteins. Conclusions Nobiletin can effectively inhibit LPS induced inflammatory response and oxidative stress damage in nasal epithelial cells. Nobiletin exerts anti-CRSwNP effects by binding to HMOX1 and inhibiting the HIF-1α/HMOX1 positive feedback loop, HMOX1 was identified as a potential key target.