Mechanism of Panax Notoginseng Saponins in Regulating Ferroptosis for Intervention in Oral Submucous Fibrosis Based on Network Pharmacology and Experimental Validation
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 notog-inseng 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 fi-brosis by regulating the process of ferroptosis were obtained.Molecular docking of the compounds and core targets was performed using AutoDockTooll.5.7 software.HaCa T cells were cultured in vitro and divided into normal control group,model control group,Panax notogin-seng saponins 12.5 μg/mL,25 μg/mL and 50 μg/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 no-toginseng saponins were screened,including ginsenoside Rbl,ginsenoside Rgl,ginsenoside Rd,Panax notoginseng saponin Rl,and ginsen-oside 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 Cellu-lar components such as nucleoplasm,cytoplasm,and nucleus,and the molecular functions such as enzyme binding,the same protein binding,RNA polymerase Ⅱ sequence-specific DNA binding transcription factor binding.KEGG revealed that these components may regulate ferropto-sis by mediating PI3K-AKT signaling pathway,cancer pathway,adipocytokine signaling pathway,PD-L1 expression in tumors and PD-1 check-point 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-1α were 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 Pa-nax notoginseng saponin 12.5 μg/mL,25 μg/mL and 50 μg/mL groups exhibited a gradient decrease in COL-Ⅰ and HIF-1α protein expres-sion(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 12.5 μg/mL,25 μg/mL and 50 μg/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-1α signaling pathway to inhibit ferroptosis may be one of the important anti-fibrosis mechanisms.
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