Unraveling The Mechanisms Of MRFHRV Induce Neural Tube Defects Based On Network Pharmacology And Molecular Docking Analysis

Background:Neural tube defects(NTDs)are serious congenital malformations of the central nervous system caused by abnormal closure of the neural tube in the early embryonic development.It can lead to varying degrees of disability or even death in children and burden families and society.Nutrients during pregnancy,such as folic acid,inositol,and vitamin A,whose metabolic balance is crucial to neural tube development,intervening key targets in nutrient metabolic pathways is a common modeling method for inducing NTDs animal models.In our previous study,we used methotrexate(MTX),raltitrexed(RTX),5-fluorouracil(5-FU),and hydroxyurea(HU)to intervene the key targets in folic acid metabolic pathways and established NTDs mouse models.In addition,it has been reported that retinoic acid(RA)and valproic acid(VPA)induce NTDs in mice by interfering with critical targets in vitamin A and inositol metabolic pathways.In this study,MTX,RTX,5-FU,HU,RA,and VPA(MRFHRV)can induce NTDs,but whether MRFHRV cause NTDs through common targets and interaction networks has yet to be reported.Objective:This study uses network pharmacology and molecular docking to study the common mechanism of NTDs induced by MRFHRV.This study also provides new ideas for further elucidating the molecular mechanism and prevention of NTDs and more medication guidance information for women of childbearing age and pregnant women.Method:In this study,the pathogenic targets of NTDs were screened using Gene Cards,NCBI,and OMIM databases.The targets of MRFHRV were screened using Pubchem,Swisstargets,and Uniprot databases.Enter the screened MRFHRV targets and NTDs pathogenic targets into the Venn diagram to obtain the common targets,input the common targets into the STRING database to construct the PPI network,use the Cytoscape software Network analyzer for analysis,and screen core targets of MRFHRV induce NTDs according to the Degree value.Using the kyoto Encyclopedia of Genes and Genomes(KEGG)for the analysis of the signaling pathways implicated in common targets.We imported the common targets and signaling pathways of MRFHRV and NTDs into Cytoscape software to draw and visualize the network diagram.In order to further explore the binding capacity between the screened core target and MRFHRV,the core target,and MRFHRV were molecularly docked using the Auto Dock docking program.Targets and signaling pathways have been screened out to suggest the regulation of MRFHRV on cell proliferation and differentiation.The mouse embryonic stem cells(m ESCs)and mouse models are used for verification.CCK8(cell counting kit 8)was used to verify the effect of MRFHRV on the proliferation of m ESCs.In our previous study,RTXinduced mouse NTDs were established.The optimal teratogenic dose of RTX of 11.5mg/kg was chosen in C57BL/6 pregnant mice injected intraperitoneally and dissected them at GD11.5 and GD13.5,respectively;Stereoscopic observation and HE staining confirmed the NTDs mouse model.Using immunofluorescence to detect RTXinduced GD11.5 and GD13.5 NTDs mouse models of astrocyte differentiation-glial fibrillary acidic protein(glial fibrillary acidic protein,GFAP)to explore the effect of RTX on neuroepithelial cell differentiation in mouse embryos.Results:1.Through database retrieval,we obtained 347 MRFHRV targets,3952 NTDsrelated pathogenic targets,and 188 common targets between MRFHRV and NTDs.The top 10 core targets were screened using the STRING database and Cytoscape software,including tumor protein p53(TP53),mitogen-activated protein kinase 1(MAPK1),heat shock protein shock protein 90AA1(HSP90AA1),estrogen receptor1(ESR1),growth factor receptor bound protein 2(GRB2),histone deacetylase 1(HDAC1),epidermal growth factor receptor(EGFR),phosphatidylinositol-4,5-bisphosphate-3-kinase catalytic subunit alpha(PIK3CA),retinoid X receptor alpha(RXRA),and tyrosine kinase Fyn(FYN).According to the KEGG pathway enrichment analysis,there are 114 KEGG pathways involved in the common targets of NTDs and MRFHRV.The main pathways include the neuroactive ligand-receptor interaction pathway,Lipid and atherosclerosis pathway,and PI3K-Akt signaling pathway.Combining the P value,the number of covered genes,and the reported correlation with neural tube development,it is suggested that MRFHRV may induce the occurrence of NTDs by regulating the PI3K-Akt signaling pathway.Based on the above information,the MRFHRV-gene-signaling pathway-NTDs network was constructed using Cytoscape software.2.To evaluate the binding ability of MRFHRV and the 10 core targets,molecular docking is used to evaluate its binding energy value.In the docking,MTX had the strongest binding affinity with EGFR,FYN,HDAC1,MAPK1,RXRA,and TP53 is the strongest,which are-7.9 kcal/mol,-7.6 kcal/mol,-7.6 kcal/mol,-7.9kcal/mol,-9.1 kcal/mol and-8.5 kcal/mol,respectively.The strongest binding force of RTX with ESR1,GRB2,HSP90AA1,and PIK3 CA,which are-7.0 kcal/mol,-6.7kcal/mol,-8.9 kcal/mol,and-6.5 kcal/mol,respectively.Among the core targets,PIK3 CA is an important component of the PI3K-Akt signaling pathway.It has a strong binding ability to MRFHRV,especially RTX,and the binding site is located in the p85 B domain of the PIK3 CA regulatory subunit.3.The CCK8 experiment verified the effect of MRFHRV on inhibiting cell proliferation.When m ESCs were exposed to MTX,RTX,5-FU,RA,HU,and VPA for 24 h,the significant doses that inhibited m ESCs proliferation were 0.1 μM,0.05μM,0.05 μM,0.25 μM,0.25 μM,and 1 m M(P < 0.05).When the exposure time was extended to 48 h or 72 h,the doses to inhibit cell proliferation by MTX,RTX,5-FU,RA,HU,or VPA were 0.05 μM,0.05 μM,0.05 μM,0.25 μM,0.25 μM,and 0.5 m M,respectively(P < 0.05).4.The RTX-induced NTDs group and the control group were detected by immunofluorescence staining.It was found that the GFAP-positive cells in the RTXinduced NTDs embryonic brain tissue increased significantly at GD11.5 and GD13.5(P < 0.05),indicating that RTX exposure promoted neuroepithelial cell differentiation into astrocytes.Conclusion:The core targets of MRFHRV-induced NTDs are TP53,MAPK1,GRB2,HDAC1,EGFR,RXRA,FYN,PIK3 CA,HSP90AA1,and ESR1.MRFHRV mainly affects the activity of the PI3K-Akt signaling pathway by binding to the PIK3 CA regulatory subunit p85 B,which may affect the proliferation and differentiation of neuroepithelial cells leading to NTDs.