Study On The Structural Characterization Of Deer Antler Protein And Its Mechanism Of Improving Osteoporosis In Rats With Kidney-yang Deficiency

Cervi Cornu Pantotrichum is a valuable traditional Chinese medicine in China,among which protein is the most abundant.In view of the traditional effects of deer antler on tonifying kidney and strengthening bones and muscles,the material basis and the biological mechanism of its effect is not clear enough.Therefore,it is particularly important to clarify the material basis and mechanism of the traditional efficacy of velvet antler.Objective: To screen and clarify the bone protective effect of deer antler protein(DAP),to clarify the structural and functional characteristics of DAP,and to determine its protective effect on osteoporosis caused by kidney-yang deficiency in rats.Based on non-targeted metabonomics,to further clarify the metabolic regulation mechanism and biological pathway of DAP in improving osteoporosis caused by kidney-yang deficiency,and to reveal its traditional mechanism of action.Methods:1.Extraction and activity identification of DAP: Deer antler extracts with different concentrations of ethanol and water were extracted by solvent method,and the cell viability of MC3T3-E1 cell injury model induced by dexamethasone was used as the evaluation index to screen the effective extracts of deer antler.The main difference components in the extracts were compared to determine the active component of deer antler,namely DAP.The protein in the effective deer antler extract was extracted by alkali extraction and acid precipitation,and the optimum extraction process of DAP was determined by single factor test and orthogonal test with the protein extraction rate as the evaluation index.The total components of DAP were analyzed by kjeldahl method,HPLC,GC and LC-MS.The bone protective effect was verified by MC3T3-E1 cells injury model induced by dexamethasone,and the effect of DAP on the mineralization ability of MC3T3-E1 cells was clarified by alizarin red staining.2.Structural characterization and property analysis of DAP: the molecular weight distribution of DAP was detected by SDS-PAGE gel electrophoresis combined with Coomassie brilliant blue staining,the protein composition of DAP was analyzed by Label-free technology,and the amino acid composition of DAP was detected by HPLC.The structural characteristics and stability of DAP were evaluated by UV spectroscopy,CD,FTIR,endogenous fluorescence spectroscopy and the detection of thermal stability and disulfide bonds.The molecular morphology of DAP was confirmed by SEM and TEM.3.Evaluation of therapeutic effect of DAP on OP rats with kidney-yang deficiency: 50 male SD rats were randomly divided into 5 groups: blank control,model,DAP low-dose(100mg/kg/d),DAP high-dose(200mg/kg/d)and positive control(Jinkui Shenqi Pills200mg/kg/d).Rats in each group were given intramuscular hydrogenation for 7 days,except the blank control group.Observe the symptoms such as "exhaustion" of rats in each group and record the changes of body mass;The changes of hormone levels of T,ACTH,CORT and TSH in rats in each group were analyzed by ELISA,and the changes of serum CRE,BUN and UA were detected.The contents of Calcitriol,OPN,PINP and CTX,bone histomorphology and bone analysis parameters were detected and analyzed by micro-CT,and the histological morphology of kidney and femur in rats was analyzed by HE staining.4.Based on metabonomics,the therapeutic mechanism of DAP on osteoporosis rats with kidney-yang deficiency was discussed.Non-targeted metabonomics was detected by UPLC-MS,and the differential metabolites among kidney,femur and serum of rats in blank group,model group and DAP group were screened,and the metabolic pathways and regulatory networks of these differential metabolites were analyzed.5.The mechanism of DAP in the intervention of osteoporosis with kidney-yang deficiency was discussed based on PI3K/AKT signaling pathway: Based on the damage model of MC3T3-E1 cells induced by dexamethasone and the model of kidney-yang deficiency rats induced by hydrocortisone,the levels of inflammatory factors IL-1β,IL-6and TNF-α in the in vitro and in vivo models were assayed by ELISA.The cell apoptosis rates were quantified by flow cytometry.The gene expression levels of Runx2,Spp1,Col1a1 and Alpl were analyzed by q RT-PCR,and the protein expression levels of apoptosis-related proteins and PI3K/AKT signaling pathway were analyzed by Western blot.The kidney of rats was analyzed by immunohistochemistry.Results:1.Extraction and activity identification of DAP: Through screening,it was found that the water-soluble component of deer antler had better activity on osteoblasts than the alcohol-soluble component,and the active component was protein.The optimum extraction process of DAP was determined as follows: the ratio of material to liquid was 1:20,the protein extraction p H value was 10.0,the time of extraction was 2 h,and the p H value of protein precipitation was 4.0.Under these conditions,the extraction rate of DAP was the highest,reaching 20.97%.The total components of DAP include 80.77% protein,5.76%fatty acids,and trace amounts of nucleotides and steroid hormones.DAP can inhibit the loss of osteoblasts and promote the proliferation and mineralization of osteoblasts.2.Structural characterization and property analysis of DAP: The molecular composition of DAP was analyzed,and it was found that the main molecular weight distribution of DAP was 80-43 k Da and less than 14.4 k Da,and the protein components with high abundance were albumin,adult β-globin,actin,vimentin and serum transferrin.DAP is rich in glutamic acid,lysine,aspartic acid and leucine,as well as rich in essential amino acids.The structural analysis of DAP shows that DAP has obvious protein characteristic structure and good stability.3.Evaluation of the therapeutic effect of DAP on osteoporosis rats with kidney-yang deficiency: DAP significantly increased the body weight of rats with kidney-yang deficiency,improved the symptoms such as "exhaustion",restored the hormone level of rats with kidney-yang deficiency,improved the renal function,restored the bone density and trabecular structure of rats with kidney-yang deficiency,and improved the phenotype of rats with kidney-yang deficiency and osteoporosis induced by hydrocortisone.4.To explore the therapeutic mechanism of DAP on osteoporosis rats with kidney-yang deficiency based on metabonomics: In the model group and DAP group of osteoporosis rats with kidney-yang deficiency,the differential metabolites in kidney,bone and blood mainly include steroid hormones and their precursors,amino acids,adenosine,glycerides and phospholipids.The metabolic pathways involved in these differential metabolites mainly include steroid hormone biosynthesis pathway,aldosterone synthesis and secretion pathway,etc.The metabolic network formed by the interaction of metabolic pathways shows that the regulation of some differential metabolites is related to PI3K/AKT signaling pathway.5.Based on PI3K/AKT signaling pathway,the mechanism of DAP in the intervention of osteoporosis with kidney-yang deficiency was discussed: DAP can significantly inhibit the release of inflammatory factors IL-1β,IL-6 and TNF-α in vivo and in vitro,improve the apoptosis level of MC3T3-E1 cells induced by dexamethasone,up-regulate the gene expression levels of Runx2,Spp1,Col1a1 and Alpl in vivo and in vitro models,and up-regulate the bcl-2/in vivo models.In addition,DAP significantly increased the phosphorylation levels of AKT,PI3 K and m TOR proteins in in vitro and in vivo models.Conclusion: DAP is an active component with bone protection effect in deer antler,which has stable molecular structure characteristics.It is rich in amino acids such as glutamic acid,lysine,aspartic acid and leucine,which may be the basis of DAP’s good bone protection effect.DAP can regulate hypothalamus-pituitary-target gland axis,restore hormone level and renal function in rats with kidney-yang deficiency,and improve the phenotype of osteoporosis in rats with kidney-yang deficiency.DAP can improve OP in rats with kidney-yang deficiency mainly by influencing the metabolism of steroid hormones and their precursors,amino acids,etc.,and participating in steroid hormone biosynthesis pathway,aldosterone synthesis and secretion pathway,etc.Further study on its biological mechanism found that DAP inhibited the secretion of inflammatory factors and the expression of apoptosis-related proteins through the activation of PI3K/AKT phosphorylation,thus inhibiting the inflammatory reaction and apoptosis of cells and rats with kidney-yang deficiency.Moreover,the activation of AKT stimulated the phosphorylation of m TOR,thus promoting the proliferation of osteoblasts.That is,DAP inhibits inflammatory reaction and apoptosis caused by kidney-yang deficiency through PI3K/AKT signaling pathway,and promotes the proliferation of osteoblasts,thereby reducing bone loss,increasing bone formation and improving the symptoms of osteoporosis.