| Objective:We used proteomics techniques to screen serum differential proteins for postmenopausal osteoporosis to investigate the molecular mechanism of postmenopausal osteoporosis;used network pharmacology and molecular docking techniques to predict the potential mechanism of action of deer antler for postmenopausal osteoporosis;and investigated the biological mechanism of deer antler extract intervention in postmenopausal osteoporosis model mice.Methods:1.12 sera each from postmenopausal patients with normal bone density and postmenopausal osteoporosis were collected from the Affiliated Hospital of Changchun University of Traditional Chinese Medicine,and 4 samples from each group were randomly selected for proteomic analysis to remove the high abundance proteins of the samples and obtain the low abundance proteins of the samples.Then the low abundance proteins were extracted and enzymatically digested,and the obtained peptides were analyzed by mass spectrometry to screen out the differential proteins in the two groups of serum samples.The GO database was used to analyze the functional annotation of the proteins,the KEGG database was used to perform the pathway enrichment analysis,and the protein interaction network was constructed.2.The active ingredients and targets of antler were screened in the BATMAN-TCM database.Then,the targets related to postmenopausal osteoporosis were screened in the Gene Cards database and OMIM database,and the targets related to antler and PMOP were intersected.Cytoscape 3.7.2 software was used to construct "disease-drug-component-target" network diagrams and protein interaction networks(PPI)through the STRING database to screen out core targets;then R language software was used to analyze the common targets between antler and PMOP for GO enrichment analysis and KEGG pathway annotation analysis.In addition,we used the professional software Maestro 11.0 for molecular docking to validate the network pharmacology-based predictive analysis.3.8-week-old C57BL/6 female mice were selected as the study subjects,and the postmenopausal osteoporosis mouse model was established and divided into blank group,sham-operated group,OVX group,and antler administration group,in which the blank group,sham-operated group and OVX group were gavaged with RO water,and the antler group was gavaged with deer antler extract.After 3 weeks of continuous gavage,the serum-related indexes of mice were detected,and the femur was subjected to HE staining and Micro-CT imaging to observe the improvement effect of deer antler extract on mice with postmenopausal osteoporosis model.Transcriptomic analysis of mouse femur was performed by RNA-seq technique.Results:1.87 differential proteins,including 43 up-regulated and 44 down-regulated proteins,were identified in the sera of postmenopausal osteoporosis patients.The differential proteins mainly have functions such as binding,catalytic activity,and molecular function regulators,and are involved in cellular processes,biological regulation,stress response,multicellular biological processes,cellular localization,and metabolic processes,and the differential proteins constitute a complex network with each other.2.The potential active ingredients of deer antler mainly include 17β-estradiol,adenosine triphosphate and estrone.These components act on key target genes such as AKT1,IL6,MAPK3,TP53,EGFR,SRC,TNF,etc.They are involved in a series of processes such as cell differentiation,apoptosis,metabolism and inflammation,and regulate PI3K/Akt signaling pathway,MAPK signaling pathway,thyroid hormone signaling pathway,estrogen signaling pathway and tight junctions.Molecular docking showed negative binding fractions for 17β-estradiol,adenosine triphosphate and estrone and key targets,indicating that the ligand molecules can spontaneously bind to the receptor proteins.3.Through the establishment of postmenopausal osteoporosis model and the treatment of deer antler extract intervention,the detection of E2,IL6,PINP and CTX serum indexes were improved.HE staining showed that the number of femoral stem trabeculae distribution increased in the deer antler group compared with the OVX group,and they were connected to each other in a larger grid;the trabeculae were locally thicker,the trabeculae spacing was reduced,and the proportion of the trabeculae distribution area in the observed field of view increased.Micro-CT imaging showed more bone trabeculae and improved BV/TV in the antler group compared to the OVX group.138 differentially expressed genes were obtained from the transcriptome database analysis of the femur treated with antler extract,and GO functional enrichment showed that in the GO annotated cellular component category,the differential genes were mainly located in the pericellular,organelle,and collagen trimer regions.In the molecular function category,differential genes were mainly involved in structural molecular activities.In the biological process category,differential genes were mainly involved in metabolic and multicellular biological processes.k EGG pathway enrichment analysis of differential genes significantly enriched in signaling pathways mainly included thyroid hormone signaling pathway,5-hydroxytryptaminergic synapses and tight junctions.Conclusion:1.Differential proteins in postmenopausal patients with osteoporosis are involved in cellular processes,bioregulation,stress response,multicellular biological processes,cellular localization,and metabolic processes.Pathways such as insulin resistance,vascular smooth muscle contraction,phospholipase D signaling pathway,and tight junctions are involved.The development of osteoporosis in postmenopausal women may be related to differentially expressed proteins promoting osteoclast activity,inhibiting osteoblast differentiation,and promoting inflammation.2.Deer antler extract has therapeutic effects on postmenopausal osteoporosis model mice.Deer antler plays a role in the treatment of postmenopausal osteoporosis model mice by participating in bone development and structural molecular activity,regulating signaling pathways such as thyroid hormone signaling pathways,5-hydroxytryptaminergic synapses and tight junctions,reducing inflammation,stimulating bone formation and inhibiting bone resorption.Deer antler may be used as an supplementary medicine in the treatment of postmenopausal osteoporosis. |
