The Mechanism Of QUE In Treating GIOP Based On Network Pharmacology

Objective:The theoretical data of quercetin(QUE)in the treatment of glucocorticoid-induced osteoporosis(GIOP)were obtained by the method of network pharmacology,and then the GIOP cell model was established to observe the effect of QUE on the osteogenic differentiation ability of the model,in order to verify the authenticity and scientific nature of the theoretical data,and provide new insights for QUE in the treatment of GIOP.Methods:1.Target points of QUE were obtained by TCMSP、Swiss Target Prediction website,and disease targets of GIOP were obtained by Genecards OMIM data.2.Import the intersection target into the Metascape database,and get the GO enrichment analysis and KEGG pathway analysis data results.3.Use PDB data and Maestro software to dock QUE with the protein molecules with the higher Degree value in the PPI network to prove that the prediction results of network pharmacology are accurate and reliable.4.Induce MC3T3-E1 cells by DEX stem,and use CCK-8,Annexin V-FITC/PI method,ALP staining,Alizarin red staining,q-PCR,Western Blot and other methods to evaluate whether the GIOP cell model is successfully constructed.5.Use CCK-8,Annexin V-FITC/PI method,ALP staining,Alizarin red staining,qPCR,Western Blot and other methods to evaluate the effect of QUE on the osteogenic differentiation of GIOP cell model.In order to further explore the mechanism of QUE in the treatment of GIOP,Western Blot detects changes in the protein levels of PI3K/AKT pathway and MAPK pathway.Result:1.152 QUE targets and 1479 GIOP targets were obtained.2.There are 83 targets at the intersection of QUE and GIOP.The proteins with the highest Degree value in the PPI network are AKT,IL-6,TNF,VEGFA,JUN,CASP3,and MAPK.3.GO enrichment analysis showed that the treatment of GIOP by Que mainly involved biological processes such as transcription factor activity,endopeptidase activity and cytokine receptor binding,etc.KEGG pathway analysis showed that the treatment of GIOP by Que mainly involved cancer-related pathways,PI3K/ Akt pathway and MAPK pathway.4.The molecular docking results showed that QUE binds well to the protein 3D structure of AKT,MAPK,and CASP3.5.100μM DEX induced MC3T3-E1 cells can successfully construct a GIOP cell model.6.CCK-8,Annexin V-FITC/PI method,ALP staining,Alizarin red staining,q-PCR,Western Blot and other results showed that 1μM QUE was the best intervention concentration for GIOP cell model.Subsequent experiment CCK-8 results showed that the concentration of 1μM QUE could promote the proliferation of GIOP cell model(P<0.05);Annexin V-FITC/PI method showed that 1μM QUE can inhibit the apoptosis of GIOP cell model.7.The results of ALP staining and Alizarin Red staining showed that 1μM QUE could promote ALP activity and the number of mineralized nodules in the GIOP cell model.8.The results of q-PCR and Western Blot showed that 1μM QUE could promote the expression of osteogenic markers ALP,BMP-2,COL1 and RUNX2 in the GIOP cell model,and this effect is that QUE promotes the phosphorylation of PI3K/AKT pathway and inhibits MAPK.Pathway phosphorylation is achieved.Conclusion:1.The theoretical data of QUE treatment of GIOP is scientific and authentic.2.QUE has a protective effect on the GIOP cell model.3.The PI3K/AKT pathway and MAPK pathway are involved in the proliferation,apoptosis and differentiation of GIOP cell models.