| Objective:Osteoporosis fracture is the main complication of osteoporosis(OP),which seriously endangers the quality of life of patients.In order to repair the bone defects caused by it,incorporating anti osteoporosis drugs into tissue engineering scaffolds to promote bone regeneration is an effective way to solve this problem.However,currently used drugs have significant side effects and other issues,limiting their clinical use.Chinese herbs for tonifying the kidney and activating blood circulation can effectively treat osteoporotic fractures in clinical practice,with high safety.Due to the unclear mechanism of action and dose-effect relationship of these traditional Chinese medicines,their further application is hindered.Therefore,according to the pathogenesis theory of "kidney deficiency and blood stasis" of traditional Chinese medicine,we selected Rhizoma Drynariae and Panax Notoginseng as the research objects,aiming to reveal the target-mechanism-dose relationship of their effects at the cellular and molecular levels,so as to provide experimental evidence for the subsequent clinical application of materials carrying drugs.Methods:1.Modern network pharmacology analysis techniques combined with literature information are applied to predict relevant targets for drug treatment of diseases,and preliminary validation is performed with the help of molecular docking techniques.The blood-entering components of PNS and OTF were obtained from literature search and online database,and their potential targets were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and Swiss Target Prediction.The OP targets were obtained by means of searching Online Mendelian Inheritance in Man(OMIM)and Gene Cards.The common targets of the drug and disease were screened by Venn.Cytoscape was used to construct a " drugcomponent-target-disease" network,and the core components were screened according to the node degree.The protein-protein interaction(PPI)network of the common targets was constructed by STRING and Cytoscape,and the core targets were screened according to the node degree.GO and KEGG enrichment analysis of potential therapeutic targets were carried out by R language.Molecular docking was used to determine the binding activity of some active components to key targets by Auto Dock Vina.Finally,HIF-1 signaling pathway was selected for further in vitro experimental verification according to the results of KEGG pathway analysis.2.To explore the effects of drugs on the proliferation and differentiation of osteoblasts through an in vitro osteoblast model.MC3T3-E1 cell were cultured in vitro and the effect of drugs on cell survival was detected using CCK-8,screening out the concentration of administration.After the osteogenic induction model was established,the induced MC3T3-E1 were intervened with drugs seven days in the PNS,OTF,and PNS-OTF groups.Subsequently,the differentiation ability of cells was judged based on the depth of ALP staining,and the final dose concentration was selected based on this experiment.Wound healing test was used to observe the effect of drugs on cell migration,and RT-QPCR test was used to detect the expression level of HIF-1α-related mRNA.3.HUVEC cells were cultured in vitro to explore the effect of drugs on angiogenesis.CCK-8 method was used to investigate the cytotoxicity of the dr ug to cells and select the appropriate dose concentration.Angiogenesis experim ents were used to observe cellular angiogenesis and investigate the role of PN S-OTF in promoting angiogenesis.RT-QPCR test was used to detect the expres sion level of HIF-1α-related mRNA.Results:1.The results of network pharmacology showed that there were 45 active in gredients such as leachianone A,kurarinone,20(R)-protopanaxatriol,20(S)-protopan axatriol,kaempferol,and 103 therapeutic targets such as IL6,AKT1,TNF,VEGF A and MAPK3.The results of molecular docking showed that the core components had good binding ability to the core targets.Target protein enrichment pathways include PI3K-AKT signaling pathway,HIF-1 signaling pathway,TNF signaling pathway,etc.HIF-1 signaling pathway were selected for subsequent experimental verification.2.CCK8 experiment showed that PNS could promote the proliferation of MC3T3-E1 cells,and the compatibility group had no effect on cell survival.T here was no cytotoxicity when the concentration of OTF was 6.25～25ug/m L,s o 12.5ug/m L OTF was selected for subsequent experiments.The ALP staining results showed that the compatibility group had better differentiation promoting effects than single drugs,and the drug concentration that could promote differentiation was selected based on the ALP staining results in the early stage.The Wound healing test results showed that PNS,OTF,and PNS-OTF could promote the migration of MC3T3-E1 cells,but the effect of the compatibility group on promoting migration was not as good as that of panax notoginseng saponins alone.RT-q PCR results showed that the RNA levels of HIF-1α and ERK ge nes increased in PNS,OTF and PNS-OTF groups,but the expression promotion effect of the compatible group was not as good as that of OTF.3.The CCK8 experiment results showed that there was no cytotoxicity in the OTF group at 3.125～25 ug/m L,so the OTF group selected 12.5ug/m L for subsequent experiments.PNS and PNS-OTF groups can promote the proliferatio n of HUVEC cells.The results of angiogenesis experiments show that PNS-OT F has a certain degree of promoting angiogenesis.RT-q PCR results showed that PNS-OTF group increased the mRNA level expression of HIF-1α and VEGF A genes compared to single drug group.Conclusion:PNS、OTF、PNS-OTF have the effects of promoting osteogenesis and angi ogenesis.The mechanism of action is related to the drug action on HIF-1α,VE GFA,ERK and other target genes and regulation of HIF-1 signaling pathway. |
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