基于网络药理学和实验验证探讨石榴皮对非酒精性脂肪性肝炎的作用机制

Exploring Mechanism of Pomegranate Peel on Non-alcoholic Steatohepatitis Based on Network Pharmacology and Experimental Verification

关键词:  石榴皮  网络药理学  非酒精性脂肪性肝炎  细胞实验  分子机制

Key words:  pomegranate peel  network pharmacology  non-alcoholic steatohepatitis  cell experiments  molecular mechanism

摘要:

目的 基于网络药理学和细胞实验验证的方法探讨石榴皮改善非酒精性脂肪性肝炎(non-alcoholic steatohepatitis，NASH)作用机制。方法 利用中药系统药理学数据库(TCMSP)进行条件检索，获取石榴皮的活性成分及其对应的作用靶点。利用人类基因数据库(GeneCards)等5个数据库获取NASH相关的靶点。将获得的石榴皮和NASH靶点进行筛选，通过韦恩图得到共同靶点。使用蛋白相互作用数据库(STRING)构建蛋白质-蛋白质相互作用网络，并用Cytoscape 3.7.1建立“石榴皮-成分-靶点-NASH”网络。利用Metascape软件进行基因本体(gene ontology，GO)富集分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)通路分析。最后借助人肝癌细胞HepG2观察石榴皮中主要活性成分对NASH的影响。结果 石榴皮活性成分有7个，获得靶点基因191个，NASH靶点1 818个，两者交集靶点98个，拓扑学分析显示，石榴皮治疗NASH的核心成分为槲皮素、山奈酚和木犀草素，核心靶点为蛋白激酶B (protein kinase B，Akt1)、白细胞介素-1β(interleukin 1β，IL-1β)、白细胞介素-6(interleukin 6，IL-6)、肿瘤坏死因子(tumor necrosis factor，TNF)等。KEGG通路分析结果显示石榴皮治疗NASH主要涉及磷脂酰肌醇-3-激酶(phosphatidylinositol 3 kinase，PI3K)/Akt、核因子κB (nuclear factor kappa-B，NF-κB)等信号通路。体外细胞试验结果显示，与对照组相比，模型组磷酸化蛋白激酶B (phosphorylated-Akt^Thr308，p-Akt^Thr308)、IL-6等蛋白表达水平升高(P<0.05)；与模型组相比，石榴皮活性成分可显著降低p-Akt^Thr308、IL-6等蛋白的表达水平(P<0.05)，也能够降低IL-6、TNF-α基因的mRNA表达水平(P<0.05)。结论 石榴皮可通过多成分、多靶点、多通路发挥抗NASH的作用，其机制可能与石榴皮中活性成分槲皮素、山奈酚和木犀草素影响Akt1等核心靶点及调控PI3K/Akt、NF-κB等信号通路，进而抑制相关炎症因子表达有关。

Abstract:

OBJECTIVE To explore the mechanism of pomegranate peel in improving non-alcoholic steatohepatitis (NASH) based on network pharmacology and cell experiments verification. METHODS Using the Traditional Chinese Medicine System Pharmacology Database(TCMSP) to obtain the active components of pomegranate peel and their corresponding targets. NASH-related disease targets were obtained from five disease databases, including the Human Gene Database(GeneCards), etc. To screen the targets of pomegranate peel and NASH and obtain the common targets through Venn diagrams. The protein-protein interaction network of pomegranate peel-NASH was constructed using the protein interaction database(STRING), and the “pomegranate peel-component-target-NASH” network was established with Cytoscape 3.7.1. Gene ontology(GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were performed using Metascape software. Finally, the effect of the main active components in pomegranate peel on NASH was observed with human hepatoma cells(HepG2). RESULTS There were 7 active ingredients in pomegranate peel, 191 target genes, 1 818 NASH targets, and 98 intersection targets. Topological analysis showed that the core components of pomegranate peel in the treatment of NASH were quercetin, kaempferol, and luteolin, and the core targets were protein kinase B(Akt1), interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor(TNF). KEGG pathway analysis predicted that pomegranate peel treatment of NASH mainly involved phosphatidylinositol-3-kinase(PI3K)/Akt, nuclear factor kappa B(NF-κB), and other signaling pathways. The results of in vitro cell experiments showed that the expression levels of phosphorylated protein kinase B(p-Akt^Thr308), IL-6 and other proteins were elevated in the model group compared with the control group(P<0.05). Compared with the model group, the active ingredients of pomegranate peel could significantly reduce the expression level of p-Akt^Thr308 and IL-6(P<0.05), as well as the mRNA expression level of IL-6 and TNF-α(P<0.05). CONCLUSION Pomegranate peel can exert anti-NASH effects through multiple components, multiple targets, and multiple pathways. The mechanism may be related to the active components quercetin, kaempferol, and luteolin in pomegranate peel affecting core targets such as Akt1 and regulating PI3K/Akt, NF-κB, and other signaling pathways, thereby inhibiting the expression of related inflammatory factors.