Bioinformatics Analysis Of The Intervention Of Gubenzenggu Pills On The Mechanical Sensitive Signals Of Osteoblasts Under Low Mechanical Stress

Objective:To excavate the mechanical sensitive signals of osteoblasts based on the bioinformation platform,analyze the effective active components of the intervention of the mechanical sensitive signals of osteoblasts by Gubenzengu pills with the help of bioinformation technology,and verify them through molecular docking and molecular dynamics simulation,so as to explore the mechanism of the intervention of the mechanical sensitive signals of osteoblasts by Gubenzengu pills.Methods:Chip data set GSE42874 was obtained from GEO database,and the differentially expressed genes were screened by R software.The active components and targets of Gubenzenggu pills were retrieved from TCMSP database,and labeled with Uniprot database.The target genes of effective components of Gubenzenggu pills were intersected with differentially expressed genes to obtain the target genes,and the key genes and effective components were determined.The selected target genes were enriched by GO and KEGG through the DAVID database,and the enrichment results of the selected target genes were visualized by Weisheng.Download the 2D structure of the small molecule ligand of the key active ingredients from the Pub Chem database and convert it to mol2 format with the help of Chem3 D software as a small molecule ligand.Then the 3D structural information corresponding to the target protein was retrieved in the PDB database,and then Pymol was used to dehydrate and deligand small molecular ligands to determine the target macromolecular receptors.Finally,Autodock software was used to determine the active pocket,and then Vina software was used to perform molecular docking of ligand-receptor,and the minimum free energy of the combination was calculated,and then the molecular docking results were visualized by Pymol and Discovery Studio2019.Gromacs software was used to simulate the dynamics of the combined conformation.Firstly,the energy of 5.0×104 steps was optimized by the steepest descent method.After the energy was minimized,the temperature of the system was balanced with NVT ensembles at 300 K and 100 ps,and then the pressure balance was simulated with 1bar and 100 ps NPT ensembles.After the simulation system was completely pre-balanced,the molecular dynamics simulation was performed for 50 Ns under the conditions of 300 K temperature and 1 atmosphere pressure.Results:A total of 4230 mechanosensitive genes of osteoblasts were obtained from the chip data set of GEO database,and 54 differentially expressed genes were screened.Include LCN2,F8,PYHIN1,AVIL,TAF7 L,AKR1B7,MS4A4 D,CSN3,SERPINB1 B,CAR6,SRGN,COX6A2,CYP4B1,ENM4,TRP53INP1,IFI44 L,H2-M10.1,PVRL2,and IF I27L2 A,LY6A,EGR1,CXCL2,CXCL1,FOS,EGR2,CXCL5,IFNB1,ALDH3A1,PLAT,SERPINE1,RGCC,CCL2,CCL7,CYR61,CRABP1,AK4,PLK2,PTGER4,MT1,IFNA4,FOSB,ID3,GSTA3,LIF,MGST3,ZFP36,TNFSF11,IER3,PDK1,CCRN4 L,PCOLCE2,COL5A3,KLF2,RIPK2,etc.Eighty-one active components of Gubenzenggu pills were detected in TCMSP,includingβ-sitosterol,stigmasterol,chrysanthin,fructose A,luteolin,taraxolol,stigmaster-7-enol,methylmonocytin,isofluranone,kaempferol,quercetin,arachidonic acid,sitosterol,kaempferol,and so on,which acted on 211 target genes.There are 5 common target genes of effective active components of Gubenzenggu pills and differential genes of mechanical sensitive signal of osteoblasts,including FOS,CCL2,PLAT,SERPINE1 and CXCL2.Quercetin is the key active component acting on FOS,CCL2,PLAT,SERPINE1 and CXCL2.There are 17 GO functions involved in target genes and 14 signaling pathways.The binding energy scores of quercetin with FOS,CCL2,PLAT,SERPINE1 and CXCL2 were-7.4kcal/mol,-7.0kcal/mol,-7.9kcal/mol,-6.8kcal/mol and-6.8kcal/mol,respectively.The RMSD,RMSF and Rg values of CCL2,PLAT,SERPINE1,CXCL2 and quercetin complex were small and fluctuated less.Conclusion:(1)The intervention of Gubenzenggu pills on the mechanical sensitive signals of osteoblasts has the action mechanism of multiple pathways and targets,so as to achieve the purpose of overall regulation;(2)Quercetin was the key effective component of the target gene of Gubenzenggu Pill to interfere with the mechanical sensitive signal of osteoblasts;(3)The target genes of Gubenzenggu pills to interfere with the mechanical sensitive signals of osteoblasts are: FOS,CCL2,PLAT,SERPINE1,CXCL2,these targets may exert biological effects through cellular response to lipopolysaccharide,inflammatory response,negative regulation of plasminogen activation,negative regulation of fibrinolysis,fibrinolysis,chemokine-mediated signaling pathways,etc.It involves IL-17,TNF signaling,fluid shear stress and atherosclerosis,the role of AGE-RAGE signaling pathways in diabetes complications,Yersinia infection,Apelin,Nod-like receptors,chemokines and other signaling pathways.(4)Through molecular docking and molecular dynamics simulation,quercetin has good binding activity with FOS,CCL2,PLAT,SERPINE1 and CXCL2,and the conformation of quercetin with CCL2,PLAT,SERPINE1 and CXCL2 is stable.