Network Pharmacology Method To Investigate The Osteogenic Differentiation Of Mesenchymal Stem Cells Induced By Asperosaponin ?

Objective:Predicting the osteogenic differentiation target proteins of human umbilical cord mesenchymal stem cells?UCMSCs?by Asperosaponin ??ASA ??by network pharmacology,and exploring the target proteins and their signaling pathways.Possible role of bone differentiation.Methods:1.Network pharmacology methods.The PUBCHEM database was used to query the ASA ? 2D structure;the protein bound by the PHARMMAPPER database was predicted,and the binding protein with a score of>0.4 was selected;the osteogenesis-related signaling pathway was obtained by literature search;the proteins in these signaling pathways were searched by the KEGG database,and predicted.The ASA ? binding protein is crossed,and the protein obtained by the intersection is a possible target protein of ASA ? in the osteogenic signaling pathway;molecular docking verification of ASA ? and target protein.2.Human umbilical cord mesenchymal stem cells were isolated from the umbilical cord of postpartum pregnant women and identified by antibodies against mesenchymal stem cell surface markers CD44 and CD73.Human umbilical cord mesenchymal stem cells were treated with 10-5 mol/L ASA ? to establish an osteogenic differentiation cell model.The expression of alkaline phosphatase was observed by alkaline phosphatase calcium and cobalt method.The expression levels of osteogenic marker genes OPN,OPG,TGF-?and RUNX2 mRNA were detected by QPCR.The ASA ? target protein predicted by network pharmacology was also detected.mRNA levels;ASA ? osteogenic differentiation cell model was treated with insulin signaling pathway inhibitor?BMS-754807?and estrogen signaling pathway inhibitor?Fulvestrant?,and osteogenic related gene expression levels were observed by QPCR.Results:1.620 ASA ? in vivo binding proteins were predicted;these proteins were cross-linked with proteins in the osteogenic differentiation-related signaling pathway to obtain the target protein.The target protein is directly docked with ASA ?,and compared with the endogenous ligand of the target protein,the target proteins that ASA ? may bind to include MAP3K3,ESR2,IGF1R,KDR,ERBB4,RAC1,HCK,LYN,MMP14,MMP2,CAMK4,PTPRE.2.The UCMSCs were successfully isolated.The UCMSCs were treated with 10^-5mol/L ASA ?.Compared with the negative control,the expression levels of osteogenic marker genes OPN,OPG,TGF-?and RUNX2 increased significantly after3d and 5d.p<0.01.The cells were positive for alkaline phosphatase staining after 14days of ASA ? induction.After induction with 10^-5mol/L ASA ? for 3 days,the expression levels of predicted target proteins MMP2,IGF1R,MMP14,MAP3K3,CAMK4,LYN and ESR2 were significantly increased compared with the negative control group,p<0.01,of which LYN,The expression levels of MMP2,ESR2 and IGF1R mRNA were significantly increased,reaching 12.6,12.2,8.7 and 8.2 times,respectively.The expression levels of RAC1 and THBS1 mRNA were not increased,p<0.01.Compared with the ASA ?-inducing group alone,the osteogenic differentiation marker genes OPN,OPG,TGF-?,and RUNX2 mRNA expression levels were significantly decreased when the insulin signaling pathway inhibitor?BMS-754807?was added,p<0.01,predicted.The expression level of IG? target protein IGF1R mRNA was decreased in insulin signaling pathway,p<0.01.Compared with the ASA ?-inducing group alone,the addition of the estrogen signaling pathway inhibitor?Fulvestrant?constitutes a significant decrease in the expression levels of the osteogenic marker genes OPN,OPG,TGF-?,and RUNX2 mRNA,p<0.01,predicted estrogen signal.The expression levels of ESR2 and MMP2 mRNA in the pathway of ASA ? were significantly decreased,p<0.01.Conclusion:1.The predicted increase of target protein MAP3K3 mRNA expression level indicates that it may be an important protein for ASA ? to induce osteogenic differentiation of UCMSCs.2.The expression of osteogenic differentiation marker gene was significantly decreased in the insulin signaling pathway inhibition group?BMS-754807?,and the expression level of IGF1R was significantly decreased.The network pharmacology predicted that the protein was ASA ? binding target protein,and ASA ? may be activated by binding to IGF1R.Insulin signaling pathways provide evidence for involvement in osteogenic differentiation.3.The estrogen signaling pathway inhibitor composition significantly reduced the expression of bone differentiation marker gene,and the expression level of ESR2was significantly decreased.The network pharmacology predicted that the protein is ASA ? binding target protein,and ASA ? may activate estrogen signaling pathway by binding ESR2.Participation in osteogenic differentiation provides evidence.4.The expression levels of target protein MMP2,IGF1R,MMP14,MAP3K3,CAMK4,LYN and ESR2 were significantly increased,suggesting that the signaling pathway may be involved in ASA ?-induced osteogenic differentiation of UCMSCs,including estrogen,AMPK,FoxO,No similar reports have been reported on the neurotrophin signaling pathway.The expression levels of LYN,MMP2,ESR2 and IGF1R mRNA were significantly increased,suggesting that these target proteins may play an important role in the osteogenic differentiation of UCMSCs induced by ASA ?.