Mechanism Of Astragalus Against Oral Submucosal Fibrosis Based On Network Pharmacology And Experimental Verification

Objective:Oral submucous fibrosis(OSF)is a chronic progressive oral mucosal disease characterized by massive accumulation of collagen fibers in the submucosa,which tends to become cancerous,but lacks effective and ideal treatment drugs.Astragalus(AST)is a traditional Chinese medicine with a long history of use.Its main active ingredients,such as polysaccharides,flavonoids and saponins,all have anti-fibrosis effects.The main mechanism of action includes regulating the expression of fibrosis related gene proteins,affecting epithelial mesenchymal transformation and regulating inflammatory response.These mechanisms are closely related to the pathogenesis of OSF,suggesting that AST has the potential to against OSF.This study through the network pharmacology methods to analyze the AST potential effective active ingredient of OSF,core targets and main biological function,signal pathways,using molecular docking techniques to analyze the possibility of composition-target binding,and molecular cell experiments not only to verify the accuracy of the above analysis.Moreover,the potential mechanism of AST against OSF was preliminarily explored,providing theoretical and cellular molecular experimental basis for AST treatment of OSF.Methods:Part 1: Network pharmacology analysis of ASI against OSFThe active components of AST were screened from TCMSP,and uploaded to Swiss Target Prediction database to match corresponding targets,and the AST related targets were summarized.The potential therapeutic targets of OSF were retrieved from Gene Cards and OMIM databases,and the potential therapeutic targets of OSF were obtained after summing up and weight removal.The AST and OSF target information was analyzed by Venny to obtain the "AST-OSF" intersection target data.The data information of drugs,components,targets and diseases were matched by Cytoscape software in pairs,and the network diagram of "AST-component-target-OSF" was constructed.PPI network analysis was performed after the intersection gene information was analyzed by STRING database.GO functional annotation and KEGG pathway analysis were performed on the intersection targets by Metascape database.The first five of the analyzed drug active ingredients and potential targets were simulated by Pymol,OBGUI,Auto Dock Tools-1.5.6 and other software to verify the possibility of binding.Part 2: Cell and molecular experiments of ASI against OSFPreparation of different concentrations of Arecoline and Astragalus injection(ASI),acting on oral mucous fibroblasts(OMF)for 24 hours,CCK-8 to detect drug toxicity.OMF was cultured in medium with different concentrations of ARE(0μg/ml,10μg/ml,20μg/ml,30μg/ml,40μg/ml)for 24 hours.The expression level of actin alpha 2(ACTA2),a marker protein of myofibroblasts,was detected by RT-QPCR to determine the optimal concentration of OMF converted into myofibroblasts stimulated by ARE,and the myofibroblast model was established by using the optimal concentration.The mRNA expression of core proteins EGFR and VEGFA were detected to determine the promoting effect of ARE on them.After cell model was established,ARE stimulation was removed,and culture medium containing different concentrations of ASI(100 mg/ml,200 mg/ml,300 mg/ml,400mg/ml)was used for 24 hours.RT-PCR was used to detect the mRNA expression levels of marker protein ACTA2 and predicted core proteins EGFR and AEGFA,further verifying the accuracy of pharmacological data of the network from the cellular molecular perspectives,and preliminarily exploring the potential molecular mechanism of AST against fibrosis.Results:Part 1: Network pharmacology analysis of ASI against OSFAST contains 21 potential active components that can play an anti-OSF role through68 action targetsThe main active components of AST were Jaranol,3,9,-di-o-methylnissolin,isorhamnetin,(6a R,11 a R)-9,10-dimethoxy-6a,11a-dihydro-6H-benzofurano[3,2-c]chro men-3-ol,Kaempferol by analyzing the network diagram of "AST-ingredient-target-OSF".PPI analysis of AST mainly plays an anti-OSF role through EGFR,VEGFA,MAPK3,HRAS,JUN and other targets.The GO function mainly involves 178 biological processes(BP),65 cell components(CC)and 63 molecular functions(MF),including response to wounding,the regulation of protein serine/threonine kinase activity,extracellular matrix,phosphotransferase activity.KEGG pathway enrichment was associated with 138 pathways,mainly affecting PI3K-Akt,JAK-STAT and other signaling pathways.The main active components of AST may bind to core targets.Part 2: Cell and molecular experiments of ASI against OSFAfter stimulation of OMF with different concentrations of ARE and ASI for 24 hours,drug toxicity was detected by CCK-8 method.The results showed that the two drugs inhibited OMF cell viability in a concentration-dependent manner.The IC50 value of ARE is 120.0μg/ m L,and that of ASI is 553.3mg/ml.RT-PCR results showed that the mRNA expression levels of myofibroblast marker ACTA2 and core proteins EGFR and VEGFA were significantly increased with the increase of ARE concentration.The mRNA expressions of ACTA2,EGFR and VEGFA were significantly increased at 20μg/ m L and 30μg/ m L and 40μg/ m L respectively(P <0.001).These results suggest that ARE can successfully induce OMF transformation into myofibroblasts and promote EGFR and VEGFA mRNA expression.RT-PCR results after ASI treatment showed that the mRNA expression of ACTA2,EGFR and VEGFA induced by ARE could be inhibited with the increase of ASI concentration.ASI at 300mg/ m L and 400mg/ m L significantly down-regulated ACTA2 mRNA expression(P < 0.05).However,100mg/ml ASI significantly down-regulated EGFR and VEGFA mRNA expressions(P < 0.05),and the inhibition effect increased with the increase of ASI concentration.Conclusion:1.Network pharmacologic found that Astragalus contains 25 potential active components such as Jaranol,3,9,-di-o-methylnissolin,isorhamnetin,Kaempferol,which can act on 68 potential targets such as EGFR and VEGFA.It affects the regulation of response to wounding,the regulation of protein serine/threonine kinase activity and other biological functions,and regulates PI3K-AKT and other signaling pathways to play an anti-OSF role.2.ARE can induce the activation of OMF into myofibroblasts and up-regulate the mRNA expression of EGFR and VEGFA,promoting the occurrence of OSF.ASI can inhibit OMF activation induced by ARE,down-regulate core targets EGFR and VEGFA play an anti-OSF role.3.ASI may be an effective drug against OSF through multi-component,multi-target and multi-pathway.