Study On The Biological Mechanism Of Strengthening Deer’s Sinews And Bones Based On The Theory Of "Balance Of Sinews And Bones"

Objective: To explore the biological mechanism of bone strengthening by collagen of deer sinew.Methods: The method of acid enzyme binding was used to establish the extraction and preparation process of deer sinew collagen.The effect of collagen of deer tendon on proliferation of osteoblasts was observed by CCK-8 method.Transcriptome sequencing technique was used to observe the gene expression changes of deer sinew collagen on osteoblasts.To reveal the biological mechanism of bone strengthening effect of deer sinew collagen at the cellular and molecular level.The synergistic effects of ginseng and deer sinew were studied by cell proliferation and transcriptome sequencing.Results:1.Through single factor analysis and orthogonal test,the extraction and preparat ion process of deer sinew collagen was established.The specific process was to soa k deer’s tendon granules in 5% acetic acid solution for 48 h,then add pepsin in acco rdance with the enzyme base ratio of 1:1000 after boiling for 1.5h,and continue en zymatic hydrolysis for 6h,with the yield up to 58.56% and purity up to 48.4%.2.The CCK-8 method was used to elucidate the proliferation promoting effect of deer’s fascia collagen on MC-3T3 cells in the concentration range of 4mg/ml to 10mg/ml,and the dose-effect relationship was certain,and the proliferation promoting effect was most obvious at the concentration of 8mg/ml.3.Through the RNA-seq technologies and means,and the establishment of a deer sinew collagen transcriptome database,through bioinformatics analysis method,were identified in 852 differentially expressed genes,compared with the blank group,deer sinew collagen treatment group was significantly raised 502 differentially expressed genes,these genes are mainly involved in the regulation of osteogenesis cells metabolism,proliferation regulation;There were 350 significantly down-regulated differentially expressed genes thatare involved in the regulation of osteogenic processes.The results of GO functional enrichment analysis showed that these differentially expressed genes were mainly involved in the regulation of cell metabolism,signaling pathways,immune system processes,stress response,biological processes,positive regulation of cell tissue components,and reproductive proliferation regulation.The enrichment analysis of the KEGG metabolic pathway further indicated that these differentially expressed genes were mainly involved in the regulation of the pi3k-akt signaling pathway,the htlv-i infection signaling pathway,the MicroRNAs in cancer signaling pathway,and the Focal cytokines signaling pathway.4.Through cck-8 method and transcriptomic sequencing technology,the synergistic effect of ginseng water extract and deer sinew collagen was studied.The results showed that ginseng water extract could significantly improve the effect of deer sinew collagen,and the effect of deer sinew on proliferation was significantly enhanced when the two were mixed in equal proportion.A Mc-3t3 transcriptome database was constructed after the proportional mixing of deer’s tendon collagen and ginseng extract.The results of GO functional enrichment analysis showed that the regulatory effects of deer’s tendon and ginseng extract on the regulation of cell metabolism,immune stress response,cell positive regulation,reproductive proliferation and other aspects to achieve the regulatory effects on MC-3T3 cells.The enrichment analysis results of KEGG metabolic pathway indicated that the major signaling pathways involved included Prolactin signaling pathway,PPAR signaling pathway,cytokine-cytokine receptor interaction,mRNA surveillance signaling pathway,RNA transport,and nod-like receptor pathway.The gene expression that can regulate cell proliferation after the synergistic action of deer tendon collagen and ginseng extract includes Tmem254,Vps35,MALAT1,Smc2,KIF5 B,Dlk2,Atp5g2,and Apoo-ps,which can regulate the gene expression level of cell proliferation to maintain cell proliferation.Conclusion: Deer sinew collagen can promote the proliferation of MC-3T3 osteoblasts and significantly improve the proliferation-promoting activity of MC-3T3 osteoblasts.Meanwhile,deer sinew collagen can regulate the gene expression level involved in the regulation of cell proliferation,thus achieving the effect of bone strengthening.In addition,ginseng water extract can significantly improve the effect of deer sinew collagen,and the effect of deer sinew on promoting proliferation is significantly enhanced after the mixture of the two in equal proportion...