基于网络药理学和实验验证探讨三七总皂苷调控铁死亡干预口腔黏膜下纤维化的作用机制

Mechanism of Panax Notoginseng Saponins in Regulating Ferroptosis for Intervention in Oral Submucous Fibrosis Based on Network Pharmacology and Experimental Validation

目的:利用网络药理学和实验验证探究三七总皂苷活性成分调控铁死亡干预口腔黏膜下纤维化的药效物质基础、潜在靶标及作用机制。方法:结合前期研究,通过文献检索确定网络药理学分析的三七总皂苷中的候选有效成分,并通过PubChem(有机小分子生物活性数据库,http://pubchem.ncbi.nlm.nih.gov)检索有效成分,使用GeneCards数据库获得三七总皂苷活性成分的对应靶基因。在GeneCards、OMIM数据库中搜集口腔黏膜下纤维化的相关靶点。将三七总皂苷活性成分与口腔黏膜下纤维化的交集靶点通过Cytoscape 3.7.0软件绘制相应“活性成分-作用靶点”网络,并借助CytoHubba插件获得三七总皂苷活性成分的度值(Degree)排名,并对关键核心靶点进行分析。基因本体论(GO)功能和京都基因与基因组百科全书(KEGG)通路富集分析通过DAVID数据库进行。将三七总皂苷活性成分干预口腔黏膜下纤维化的靶基因与通过FerrDb数据库获取得到的铁死亡过程中的标记基因及调控因子取交集,获得通过调控铁死亡过程干预口腔黏膜下纤维化的三七总皂苷靶基因。利用AutoDockTool1.5.7软件进行化合物和核心靶点的分子对接。人永生化角质形成细胞(HaCaT细胞系)体外培养后分为空白对照组、模型对照组、三七总皂苷12.5、25、50μg/mL组;各组在相应不含药或含药的完全培养液中培养24 h后收集细胞;采用Western blot法检测各组细胞Ⅰ型胶原(COL-I)、低氧诱导因子-1(HIF-1α)、轻链溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶4(GPX4)蛋白表达;透射电镜观察细胞线粒体形态学变化。结果:共筛选出三七总皂苷中5个活性成分,包括人参皂苷Rb1、人参皂苷Rg1、人参皂苷Rd、三七皂苷R1和人参皂苷Re,以及前列腺素内过氧化物合酶2(PTGS2)、一氧化氮合酶2(NOS2)等20个作用靶标。三七总皂苷中的5个活性成分与RNA聚合酶II启动子对pri-miRNA转录的正向调控、细胞对缺氧的反应及转化生长因子受体信号通路等生物过程,核浆、胞质和胞核等细胞组成,酶结合、相同的蛋白质结合和RNA聚合酶ⅱ序列特异性DNA结合转录因子结合等分子功能密切相关;KEGG发现其可通过介导PI3K-AKT信号通路、癌症途径、脂肪细胞因子信号通路、肿瘤中PD-L1表达和PD-1检查点等信号通路调控铁死亡,从而发挥干预口腔黏膜下纤维化的作用。细胞实验结果表明,模型对照组较空白对照组COL-I、HIF-1α蛋白表达显著上调,SLC7A11、GPX4蛋白表达显著下调(P<0.01);与模型对照组比较,三七总皂苷12.5、25、50μg/mL组COL-I、HIF-1α蛋白表达明显下调(P<0.05),SLC7A11、GPX4蛋白表达明显上调(P<0.05)。空白对照组细胞线粒体形态结构正常;模型对照组细胞线粒体形态结构萎缩,线粒体变小,嵴明显减少,呈典型的铁死亡表现;三七总皂苷12.5、25、50μg/mL组细胞线粒体形态结构逐渐恢复正常。结论:三七总皂苷具有多成分、多靶点、多通路干预口腔黏膜下纤维化的作用特点,下调HIF-1α信号通路抑制铁死亡可能是其抗纤维化重要机制之一。

Objective:To explore the pharmacodynamic material basis,potential targets and mechanism of Panax notoginseng saponins in regulating ferroptosis for intervention in oral submucous fibrosis by network pharmacology and experimental research.Methods:Combined with previous studies,the candidate active components of Panax notoginseng saponins for network pharmacological analysis were identified through literature search,and the active ingredients were retrieved by Pub Chem(organic small molecule bioactivity database,http://pubchem.ncbi.nlm.nih.gov).The target genes of Panax notoginseng saponins were obtained from GeneCards database.The related targets of oral submucosal fibrosis were collected from GeneCards and OMIM databases.The intersection targets of Panax notoginseng saponins and oral submucous fibrosis were visualized as an"active ingredient-target"network using Cytoscape 3.7.0 software.The degree of active components of Panax notoginseng saponins was obtained by CytoHubba plug-in,and the key core targets were analyzed.Gene Ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were conducted through the DAVID database.The target genes of Panax notoginseng saponins active components that intervene in oral submucous fibrosis intersected with the marker genes and regulatory genes in the process of ferroptosis obtained through FerrDb database,and the target genes of Panax notoginseng saponins interfering in oral submucous fibrosis by regulating the process of ferroptosis were obtained.Molecular docking of the compounds and core targets was performed using AutoDockTool1.5.7 software.HaCa T cells were cultured in vitro and divided into normal control group,model control group,Panax notoginseng saponins 12.5μg/mL,25μg/mL and 50μg/mL groups.After 24 hours of incubation in the respective culture media with or without Panax Notoginseng Saponins,cells were collected.The protein expression levels of collagen-Ⅰ(COL-I),hypoxa-inducing faction-1(HIF-1α),solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)in each group were detected by Western blot.Morphological changes of cell mitochondria were observed by transmission electron microscopy.Results:Five active components of Panax notoginseng saponins were screened,including ginsenoside Rb1,ginsenoside Rg1,ginsenoside Rd,Panax notoginseng saponin R1,and ginsenoside Re,along with 20 target genes,such as prostaglandin peroxidase synthase 2(PTGS2),and nitric oxide synthase 2(NOS2).The five active components of Panax notoginseng saponins are closely related to the biological processes such as the positive regulation of pri-miRNA transcription by RNA polymerase II promoter,the cell response to hypoxia,the transforming growth factor receptor signaling pathway,the Cellular components such as nucleoplasm,cytoplasm,and nucleus,and the molecular functions such as enzyme binding,the same protein binding,RNA polymeraseⅡsequence-specific DNA binding transcription factor binding.KEGG revealed that these components may regulate ferroptosis by mediating PI3K-AKT signaling pathway,cancer pathway,adipocytokine signaling pathway,PD-L1 expression in tumors and PD-1 checkpoint and other signaling pathways,thereby playing a role in interfering in oral submucous fibrosis.The results of cell experiments showed that compared with the normal control group,the protein levels of COL-I and HIF-1αwere significantly upregulated and the protein levels of SLC7A11 and GPX4 were significantly downregulated in the model control group(P<0.01).Compared with the model control group,the Panax notoginseng saponin 12.5μg/mL,25μg/mL and 50μg/mL groups exhibited a gradient decrease in COL-I and HIF-1αprotein expression(P<0.05)and a gradient increase in SLC7A11 and GPX4 protein expression(P<0.05).Mitochondrial morphology and structure was normal in the normal control group,while in the model control group,mitochondria structure demonstrated shrinkage,smaller size,dramatically reduced cristae,with typical manifestations of ferroptosis.In the Panax notoginseng saponins 12.5μg/mL,25μg/mL and 50μg/mL groups,the mitochondria structure gradually returned to normal,in a drug concentration-dependent manner.Conclusion:Panax notoginseng saponins have the characteristic of multi-components,multi-targets and multi-pathway intervention in oral submucous fibrosis.The Downregulation of the HIF-1αsignaling pathway to inhibit ferroptosis may be one of the important anti-fibrosis mechanisms.