| Purpose:The antler polypeptides(A～E)with different molecular weight were isolated and purified.To study the effect of antler polypeptides(A～E)on the proliferation and osteogenic differentiation of MC3T3-E1 osteoblasts,the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells,the effect of A～E on osteoporosis of kidney deficiency type in rats,and to explore the mechanism of A～E on the prevention and treatment of osteoporosis.The composition of pilose antler polypeptide A was analyzed by liquid chromatography-mass spectrometry(HPLC-MS).The surface structure of pilose antler polypeptide a was analyzed and characterized by atomic force microscopy(AFM).The target gene,target protein and related signal pathway of pilose antler polypeptide A in the prevention and treatment of osteoporosis were explored by using the methods of network pharmacology and bioinformatics.Guidance.Materials and methods:1 MaterialThe raw material of pilose antler was purchased from xifeng medicinal materials market in liaoning province.Professor Li feng from the department of TCM identification of liaoning university of traditional Chinese medicine identified it as the unossifying and densely hairy young horns of red deer Cervus elaphus Linnaeus.Pilose antler polypeptide,were made by the laboratory.2 Methods2.1 The purification and molecular weight distribution of Cervus antler polypeptide(A～E)5 kinds of pilose antler polypeptide(A～E)with different molecular weights were isolated and purified by multistage membrane separation technology.The molecular weight of pilose antler polypeptide(A～E)was determined by high performance gel filtration chromatography and SDS-PAGE electrophoresis.2.2 The proliferation and differentiation of MC3T3-E1 osteoblasts by Cervus antler polypeptide(A～E)MTT method was used to study the proliferation of MC3T3-E1 osteoblasts,and the effect of antler polypeptide(A～E)on the differentiation of MC3T3-E1 osteoblasts was studied by ELISA,with ALP as the indicator.2.3 The effect of Cervus antler polypeptide(A～E)on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)MTT method was used to study the effect of pilose antler polypeptide(A～E)on the proliferation of bone marrow mesenchymal stem cells;ALP,BGP and BMP-2were used as indicators to study the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)by pilose antler polypeptide(A～E).The expression of BMP-2,Smad1,Smad5,Runx2 genes and proteins in BMSCs were detected by RT-PCR and Western blot respectively.2.4 The effect of pilose antler polypeptide A on osteoporosis of kidney deficiency type in ratsThe osteoporosis model of kidney deficiency type was established by ovariectomy in female rats.Progynova and XLGB were used as positive control drugs,respectively,and pilose antler polypeptide a was given for 12 weeks.Bone mineral density(BMD)of rats was measured by bone densitometer,uterus index was determined,estradiol(E2),alkaline phosphatase(ALP),osteocalcin(BGP),bone morphogenetic protein-2(BMP-2),tartrate resistant acid phosphatase(TRAP),peroxisome proliferator activated receptor γ 2(PPAR γ 2)and BALP,B in bone tissue were detected by enzyme-linked immunosorbent assay(ELISA).MP-2,Smad1,Smad5,Runx2,TGF-β1,and TIEG1,TGF-β1 in hypothalamus and kidney.The relative expression of BMP-2,Smad1,Smad5,Runx2 gene and protein were detected by RT-PCR and Western blot respectively.He staining was used to observe the morphological changes of femur and vertebrae,and immunohistochemistry was used to observe the morphological changes and positive expression of bone tissue.2.5 Composition and characteristics of pilose antler polypeptide AIn this paper,the method of precolumn derivatization and automatic amino acid analyzer were used to determine the kinds and contents of amino acids after hydrolysis of pilose antler polypeptide A.The component analysis of polypeptide A in pilose antler was determined by LTQ Orbitrap HPLC-MS.The surface morphology of pilose antler polypeptide A was measured by AFM with the parameters of roughness,surface height analysis and power spectral density.Using the methods of GO analysis,KEGG analysis and string protein interaction network,we analyzed the target gene and protein related to osteoporosis and the related signal transduction pathway in 72 polypeptide fragments of pilose antler polypeptide A.Results:1 Five kinds of polypeptides with different molecular weight were obtained,i.e.peptide A(200-3000 Da),peptide B(3000-5000 Da),peptide C(5000-10000 Da),peptide D(10000-50000 Da),and peptide E(> 50000 Da).2 Pilose antler polypeptide(A～E)can promote the proliferation and osteogenic differentiation of MC3T3-E1 osteoblasts,among which polypeptide A with the molecular weight of 200-3000 Da,at a concentration of 200 μg· m L-1,after 48 hours of action,has the most significant effect on the proliferation and osteogenic differentiation.3 Effect of Pilose antler polypeptide(A～E)on proliferation and differentiation of BMSCs cells3.1 Pilose antler polypeptide(A～E)can promote the proliferation and osteogenic differentiation of BMSCs,among which polypeptide A with the molecular weight of200-3000 Da has the most significant effect on the proliferation and osteogenic differentiation of BMSCs at a concentration of 200 μg·m L– 1.3.2 The expression of BMP-2,Smad1,Smad5,Runx2 genes and proteins was enhanced by Pilose antler polypeptide(A～E);the expression of BMP-2,Smad1,Smad5,Runx2 genes and proteins was up-regulated by polypeptide A,which wasclose to the inducible solution group,indicating that it might be related to the activation of BMP-2 / Smad1,Smad5 / Runx2 signal transduction pathway.4 Effect of pilose antler polypeptide A on osteoporosis of kidney deficiency type in rats4.1 The detection results of bone metabolism related indexes in serumPilose antler polypeptide A increased the level of E2,ALP,BGP,BMP-2,and decreased the level of trap and PPAR γ 2.4.2 Uterine indexCompared with the normal group,the uterus of the model group was obviously atrophied,and the uterus index of each group was significantly lower than that of the normal group;compared with the model group,the uterus index of the polypeptide A high and low dose group,western medicie postive control group and traditional Chinese medicine postive control group had no statistical significance.4.3 The results of bone mineral density testThe bone mineral density of rats was increased in each group,and the effect of high dose group of pilose antler polypeptide A was the most significant.4.4 The detection results of related indexes in bone tissueCompared with the normal group,BALP,BGP,BMP-2,Smad1,Smad5,Runx2,TGF-β1 and TIEG1 in the bone tissue of the model group were significantly reduced;compared with the model group,the high and low dose group,western medicine postive control group and traditional Chinese medicine postive control group of pilose antler polypeptide A could significantly improve the levels of BALP,BGP,BMP-2,Smad1,Smad5,Runx2,TGF-β1 and TIEG1 in the bone tissue of the model group,and the high dose group of polypeptide A could significantly increase the levels of BALP,BGP,BMP-2,Smad1,Runx2,TGF-β1 and TIEG1 in the bone tissue of the model group.The effect is the most significant.4.5 The expression of TGF-β 1 and TIEG 1 in hypothalamusThe expression of TGF-β 1 and TIEG 1 in hypothalamus was enhanced in each group,and the effect of high dose of polypeptide A was the most significant.4.6 The expression of TGF-β 1 and TIEG 1 in kidneyThe expression levels of TGF-β1 and TIEG1 were enhanced in each group,and the effect of high dose of peptide A was the most significant.4.7 The expression of related genes in bone tissueThe relative expression of BMP-2,Smad1,Smad5 and Runx2 gene in bone tissue of rats in each group was significantly increased;the relative expression of BMP-2,Smad1,Smad5 and Runx2 gene in high dose group of polypeptide A was better than that in other groups.4.8 The expression of related proteins in bone tissueCompared with the normal group,the gray value of BMP-2,Smad1,Smad5 and Runx2 protein in the bone tissue of the model group was significantly reduced;the protein expression of BMP-2,Smad1,Smad5 and Runx2 was increased in each group,and the effect of high-dose peptide A group was the most significant.4.9 The results of bone histomorphology4.9.1 The results of HE staining testIn the high and low dose group of pilose antler polypeptide A,the trabecula was thickened,the structure was close to the model group,the marrow cavity was smaller,and osteogenic cells were seen in the periosteum.4.9.2 The immunohistochemical results of bone tissueCompared with the normal group,the average optical density of BMP-2,Smad1,Smad5 and Runx2 in the model group decreased significantly;compared with the model group,the average optical density of BMP-2,Smad1,Smad5 and Runx2 in the model group was significantly higher,the average optical density of Smad1 and Runx2 in the high and low dose group of pilose antler polypeptide A was significantly higher than that in the western medicine postive control group;BMP-2,Smad1,Smad5 and Runx2 in the high and low dose group of polypeptide A.The average optical density was close to or even higher than that of positive group.5 Component analysis of polypeptide A from pilose antler5.1 The types and contents of amino acids in pilose antler polypeptide AAfter hydrolysis,there are 18 kinds of amino acids,accounting for 26.4%;histidine content is the highest,Aspartic acid content is the lowest,and the contentof 8 kinds of essential amino acids is 2071.24 mg.g-1,accounting for 53.04%.Free amino acids contain 18 kinds of amino acids,accounting for 23.6%;threonine content is the highest,proline content is the lowest;8 kinds of essential amino acids are 553.81 mg.g-1,accounting for 60.18%.The content of essential amino acid in the binding amino acid was 1517.43 mg.g-1,up to 50.84%.5.2 The determination of the composition of polypeptide a from pilose antler by HPLC-MS72 proteins with molecular weight of 700-2000 Da and isoelectric point of4.33-11.45 were obtained.5.3 The surface morphology of pilose antler polypeptide A5.3.1 The overview of surface morphologyThe surface of pilose antler polypeptide A showed a sharp centroid and irregular distribution on the surface.When the concentration increased from 0.1μg·m L-1 to5μg·m L-1,the roughness Ra / RQ decreased,but the change was not obvious.5.3.2 The surface height distributionWhen the concentration of 0.1 μg·m L-1 is 6.13969 nm,the concentration of 0.25μg·m L-1 is 39.4588 nm,the concentration of 0.5 μg·m L-1 is 14.5105 nm,the concentration of 1μg·m L-1 is 16.6944 nm,the concentration of 5 μg·m L-1 is 28.6132 nm.When the concentration of 0.1 μg·m L-1 is 6.13969 nm,the concentration of 0.25μg·m L-1 is 39.4588 nm,the concentration of 0.5 μg·m L-1 is 14.5105 nm,the concentration of 1 μg·m L-1 is 16.6944 nm,the concentration of 5 μg·m L-1 is28.6132 nm.5.3.3 Power spectral density(PSD)When the wavelength is 0.208 μ m and the frequency is 4.80/μ m,the PSD value of pilose antler peptide A is 40.1 nm3 in horizontal axis,44.7 nm3 in vertical axis and 75055 nm4 in two dimensions respectively;when the wavelength is 0.0971 μ m and the frequency is 10.3/μ m,the PSD value of pilose antler peptide is 3.29 nm3 in horizontal axis,6.3 nm3 in vertical axis and 2931 nm4 in two dimensions respectively.5.4 The network pharmacology of pilose antler polypeptide AThe main target protein of polypeptide A is collagen α-1(I)chain(COL1A1),mitogen activated protein kinase 14(MAPK14),fibrin 2(FBN2),etc.it can be concluded that PI3K-Akt signaling pathway,Ras signaling pathway,MAPK signaling pathway,FOX signaling pathway may activate the mechanism of action of pilose antler in the prevention and treatment of osteoporosis.The discovery and speculation of these signaling pathways is a supplement to the mechanism of antler peptides in the prevention and treatment of osteoporosis of kidney deficiency type.It is speculated that the role of pilose antler peptides to "nourish the kidney and strengthen bones" may be the result of the synergy of these several signaling pathways.Conclusion:1 Pilose antler polypeptide was separated and purified into five different molecular weight peptides.The molecular weight of pilose antler polypeptide was determined by high performance gel filtration chromatography and SDS-PAGE electrophoresis.2 Pilose antler polypeptide(A～E)can promote the proliferation and differentiation of MC3T3-E1 osteoblasts,and the effect of polypeptide a with molecular weight of200-3000 Da is better than that of other polypeptides.3 Pilose antler polypeptide(A～E)can promote the proliferation and osteogenic differentiation of BMSCs,and the effect of polypeptide A with a molecular weight of 200-3000 Da is better than that of other polypeptides.The mechanism may be related to its up regulation of BMP-2,Smad1,Smad5 and Runx2 signal transduction pathway.4 Pilose antler polypeptide A can prevent and treat osteoporosis of kidney deficiency type in rats.The mechanism of action may be the activation of BMP-2,Smad1,Smad5 and Runx2 signaling pathways.5 Based on the analysis of 72 polypeptide fragments of pilose antler polypeptide A by liquid chromatography-mass spectrometry(HPLC-MS),combined with the methods of network pharmacology and bioinformatics,the direct and indirect target proteins and target genes related to osteoporosis,as well as other signal pathways,target gene target proteins and multiple signal pathways coordination were found,which can prevent and treat osteoporosis.6 AFM was used to analyze the surface structure of pilose antler polypeptide A from microcosmic point of view.The surface of pilose antler polypeptide A has a concave and convex body shape,which is distributed irregularly on the molecular surface.It is speculated that the irregular cone structure of polypeptide A may make polypeptide A enter cells more easily and play a role in drug efficacy.The lower the concentration of polypeptide A solution,the clearer the surface morphology can be observed.AFM’s in-depth analysis of the structure of peptide A lays the foundation for studying the structure-activity relationship of pilose antler peptides. |
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