Study On The Mechanism And Clinical Serum Proteomics Verification Of Postmenopausal Osteoporosis Treated By QiangGuYin Based On Bioinformatics

Objective:To investigate the molecular basis of Qiangguyin(QGY)and its predictive targets,serum molecular markers of o steoporosis,serum target of QGY,to clarify the multi-targets characteristics of QGY,and its mechanism and targets in the treatment of Postmenopausal osteoporosis.Methods:First,the method of system biology was used to determine the known compounds corresponding to the herbs by the Chinese medicine comprehensive database(TCMID)according to the rules of compatibility of QGY.The name of the compound was determined by NCBI data into the unique ID number.And then we put the data of the unique ID number into the molecular mechanism of the Chinese medicine molecular mechanism bioinformatics analysis tool(BATMAN-TCM)to predict the drug-related gene/protein targets,and then use the bioinformatics method to predict the Gene ontology(GO),signal pathway and protein-protein interaction network of the targets.The common western medicines for the treatment of osteoporosis are collected and then imported into the DrugBank database to retrieve their associated targets that would be compared to the QGY targets.The gene/protein targets associated with osteoporosis are collected by retrieving the DisGeNET database and text mining,which also would be compared with QGY targets.Then,in order to further verify the predictability of the target,etc,this study will screen the clinical targets of QGY using proteomics techniques.In order to provide background data for the identification of QGY targets,first,20 serum protein samples were recruited(10 cases of postmenopausal patients with osteoporosis and 10 cases of postmenopausal women without osteoporosis)and the high abundance ratios protein was removed,differentiation protein was extracted and labeled with TMT reagent.Then,mass spectrometric detection,data analy sis of differentially expressed proteins,and analysis of biological information were carried out.At last,20 Serum protein samples were recruited(ten patients with primary type I osteoporosis before and after QGY treatment)and the high abundance ratios protein was removed,two serum samples were extracted and labeled with TMT reagent.Then,mass spectrometric detection,identification of differentially expressed proteins and bioinformatics analysis of differentially expressed proteins were carried out by combining with the study results of the first step and the second step,looking for effective targets of serum differential proteomics of QGY.Results:In the first part of the study,815 corresponding compounds were retrieved in the TCMID database,from 12 herbs.A total of 435 compounds were included in the scope of this study,of which 391 were unique compounds belonging to a single herb;the remaining 44 compounds were co-contained with different herbs,and herbal compounds were fed into BATMAN-TCM for retrieval.2487 targets were retrieved.2487 targets were further enriched in 138 GO terms,such as Nucleic Acid Binding Transcription Factor Activity,Signal Transducer Activity,Oxidoreductase Activity,P<0.05.32 signal system,211 signal pathways Was significantly enriched,P<0.05.68 protein targets were retrieved in the Drugbank database,in addition to a small molecule hydroxyapatite target and a Zinc ion binding target,with a total of 70 targets.Through the DisGENET database and literature mining,687 osteoporosis-related reliable disease genes were collected.In the second part of this study,87 significantly differentially expressed proteins were screened from the differentiated protein expression profile by LC-ESI-MS/MS combined with TMT labeling.While 50 proteins were up-regulated,and 37 proteins were down-regulated.Differentially expressed proteins were analyzed by GO annotation,these proteins are mainly involved in 15 kinds of biological processes,7 kinds of cellular component,6 kinds of molecular function.RAB7A,TSP1,GAS6,SPP24 were screenedas candidate proteins which were related to mechanism of bone remodeling of osteoporosis.By STRING10.0 protein interaction network analysis tools,RAB7A,TSP1,GAS6 were located in the center of the interaction network.SPP24 was located at edge of the network,but it is directly related to the protein BMP-2 of bone remodeling.RAB7A,TSP1,GAS6,SPP24 may be associated with the pathogenesis of postmenopausal osteoporosis.In the third part of this study,results showed that a total of 60 proteins were identified,within a 99%confidence interval,to be differentially regulated of which,34 proteins were upregulated and 26 proteins were downregulated.Differentially expressed proteins analyzed by Gene Ontology(GO)annotation mainly get involved in 12 different biological processes,7 types of cellular components and,6 kinds of molecular functions.Angiotensinogen(AGT),Stromelysin-1(MMP3),Heparanase(HPSE)and Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)were screened as candidate protein targets of QGY treatment which were related to metabolic mechanism of bone remodeling and/or bone collagen of osteoporosis.By the utilization of the protein interaction network analysis tool named STRING10.0,it can be confirmed that AGT,MMP3,HPSE and GAPDH were located in the key node of the PPIs network.Furthermore,AGT,MMP3,HPSE and GAPDH were found that they were directly related to BMP family,MAPK family,WNT family,SMAD family and Tumor necrosis factor ligand superfamily member 11(TNFSF11)family.Based on the differentially expressed proteins in the second and third parts,32 proteins were thought to be associated with the protection of bone mass.AGT,ALB,FGA,GAPDH,MPO,ACTN3,ANGPT1,TUBA1C,HP,CFB were identified as the core targets,based on the serum targets and predictive targets of QGY.Conclusions:1.The herbs,compounds and targets related to compounds of QGY is an effective research carrier for QGY.2.The prevention and treatment of osteoporosis with QGY is based on multiple target strategies.3.QGY by coordinating the body’s metabolic balance is preventing and treating osteoporosis for the cause of the disease.4.These results provide that the proteomics analysis by using TMT combined with LC-MS/MS was a feasible method for screening the potential therapeutic targets associated with QGY treatment.It suggests that AGT,MMP3,HPSE and GAPDH may be candidate protein targets of QGY treatment which can be used in therapeutic effect monitor and early diagnosis of primary type I osteoporosis.5.AGT,ALB,FGA,GAPDH,MPO,ACTN3,ANGPT1,TUBA1C,HP,CFB were identified as the core targets,based on the serum targets and predictive targets of QGY.