Antler active peptide fragment as well as preparation method and application thereof

Abstract

The invention discloses a antler active peptide fragment as well as a preparation method and application thereof, and relates to the technical field of biological peptides. The amino acid sequence of the antler active peptide fragment is NGYDFGFDGDFYAR, and the antler active peptide fragment not only has the effect of promoting proliferation of osteoblasts, but also can inhibit reduction of cell viability and relieve osteoblast damage. Meanwhile, the antler active peptide fragment can effectively antagonize DEX-induced ferroptosis and recover the physiological function of osteoblasts to a certain extent. Specifically, the antler active peptide fragment can reduce the ROS level of osteoblasts, reduce the MDA and LPO content and iron ion accumulation, and promote the expression of osteogenic proteins Runx2 and Osterix in osteoblasts and the callback of ferroptosis proteins SLC7A11 and GPX4.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological peptides, in particular to a deer horn active peptide segment and a preparation method and application thereof. BACKGROUND

[0002] Osteoporosis (OP) is a chronic disease characterized by decreased bone mass, loss of bone integrity and increased susceptibility to fractures, which seriously affects the quality of life of patients. According to different causes, osteoporosis is divided into primary osteoporosis and secondary osteoporosis. At present, the commonly used therapeutic drugs mainly include bisphosphonates, calcitonin, bone formation promoters, vitamin D, vitamin K2, etc., but many of these drugs have adverse reactions.

[0003] Studies have shown that bioactive peptides have various biological activities, can regulate body metabolism, play multiple functions, and have characteristics such as easy digestion and absorption, and play an increasingly important role in the prevention or treatment of diseases. Polypeptides are biological macromolecules composed of amino acids, similar in structure to the body's own biological molecules, and have good biocompatibility, generally do not cause immune reactions or other serious adverse reactions. Among them, some bioactive peptides can specifically act on osteoblasts to promote bone formation, inhibit the formation and activity of osteoclasts, and reduce the absorption and destruction of bone tissue.

[0004] Deer horn is the bony appendage of deer, which is periodically regenerated by the permanent growth of frontal bone. They are the only known regenerable organs in mammals and have a very fast growth cycle. This rapid regeneration ability makes deer horn have important medical research value. The rich amino acids, polypeptides, collagen and trace elements in deer horn not only provide nutrients for osteoblasts, but also can optimize their microenvironment to promote the survival and function of osteoblasts. Therefore, it has significant display significance to extract active peptide segments from deer horn for the treatment of osteoporosis. SUMMARY

[0005] In view of the above prior art, the purpose of the present application is to provide a deer horn active peptide segment and a preparation method and application thereof. The amino acid sequence of the deer horn active peptide segment in the present application is NGYDFGFDGDFYAR, which not only has a promoting effect on osteoblast proliferation, but also can inhibit the decrease of cell activity and relieve the damage of osteoblasts. At the same time, the deer horn active peptide segment can effectively antagonize the iron death induced by DEX, and to a certain extent, restore the physiological function of osteoblasts. Specifically, the deer horn active peptide segment can reduce the ROS level of osteoblasts, reduce the content of MDA and LPO, accumulate iron ions, and at the same time promote the expression of bone formation proteins Runx2 and Osterix in osteoblasts and the adjustment of iron death proteins SLC7A11 and GPX4.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a deer horn active peptide segment, wherein the amino acid sequence of the deer horn active peptide segment is shown as SEQ ID No. 1.

[0007] Preferably, the deer horn active peptide segment has a molecular weight of 1642.6687 Da, is hydrophilic, and is stable in nature.

[0008] In a second aspect, the present application provides a preparation method of the deer horn active peptide segment, comprising the following steps: The deer horn active peptide segment is obtained by separation and purification of a deer horn gel enzymatic product, or is prepared by a solid-phase synthesis method.

[0009] Preferably, the deer horn gel enzymatic product is prepared by the following method: After adding pepsin to the deer horn gel powder, the deer horn gel enzymatic product is obtained by enzymatic hydrolysis at 40℃ and pH 4.5 for 7 hours, and collecting and drying the supernatant after enzymatic hydrolysis.

[0010] Further, the amount of pepsin used is 1.8% of the mass of the deer horn gel powder.

[0011] In a third aspect, the present application provides an application of the deer horn active peptide segment in preparing a product for preventing or treating osteoporosis.

[0012] Preferably, the deer horn active peptide segment prevents or treats osteoporosis by at least one of the following 1) to 6): 1) inhibiting lipid peroxidation of osteoblasts; 2) regulating the oxidation-reduction balance in osteoblasts; 3) promoting osteogenic differentiation of osteoblasts; 4) inhibiting ferroptosis in osteoblasts; 5) up-regulating the expression of the PI3K / AKT signaling pathway; 6) promoting the proliferation of osteoblasts.

[0013] In a fourth aspect, the present application provides an application of the deer horn active peptide segment in any one of the following 1) to 5): 1) increasing the levels of GSH and SOD in osteoblasts; 2) reducing the levels of MDA, LPO, and cellular iron content in osteoblasts; 3) reducing the ROS content in osteoblasts; 4) promoting the expression of osteogenesis proteins Runx2 and Osterix in osteoblasts; 5) promoting the regulation of ferroptosis proteins SLC7A11 and GPX4 in osteoblasts.

[0014] In a fifth aspect, the present application provides a product for preventing or treating osteoporosis, wherein the product comprises the active peptide segment of deer antler as an active ingredient.

[0015] Preferably, the product for preventing or treating osteoporosis further comprises a pharmaceutically acceptable excipient.

[0016] Further, the pharmaceutically acceptable excipient is one or more of a coloring agent, a lubricant, a stabilizer, an emulsifier, a co-solvent, and a binder.

[0017] Preferably, the product for preventing or treating osteoporosis is in the form of granules, capsules, tablets, or oral agents.

[0018] Advantages of the present application: 1. The present application first isolates the active peptide segment of deer antler having an amino acid sequence of NGYDFGFDGDFYAR from deer antler glue, and the active peptide segment of deer antler can significantly promote the proliferation of MC3T3-E1 osteoblasts.

[0019] 2. The active peptide segment of deer antler of the present application can increase the levels of GSH and SOD in MC3T3-E1 osteoblasts, reduce the levels of MDA, LPO, cell iron content, and ROS content, promote the expression of osteogenesis proteins Runx2 and Osterix in MC3T3-E1 osteoblasts, promote the regulation of iron death proteins SLC7A11 and GPX4 in osteoblasts, and thus restore the proliferation activity and functional state of MC3T3-E1 osteoblasts.

[0020] 3. The active peptide segment of deer antler of the present application can significantly improve the activity of MC3T3-E1 osteoblasts, reduce oxidative stress indicators, and restore the expression of osteoblast-related proteins.

[0021] 4. The active peptide segment of deer antler of the present application can activate the PI3K / AKT signaling pathway, thereby promoting the osteogenic differentiation of MC3T3-E1 osteoblasts.

[0022] 5. The active peptide segment of deer antler of the present application has no cytotoxicity to MC3T3-E1 osteoblasts and exhibits extremely high biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 : Figure of peptide segment activity prediction in Example 1; Figure 2Figure A-H correspond to peptide segment NYDFGFDGDFYR, peptide segment Ac-ALGLDGRPGPLGPAG, peptide segment YNDFGFDGDFYRA, peptide segment NYDFGFDGDFYAR, peptide segment GHRGFSG, peptide segment GYDFGFDGDFYR, peptide segment NGYDFGFDGDFYAR, and peptide segment Ac-ALGGGYDFGFDGDFYR, respectively; Figure 3 Figure A-H correspond to peptide segment NYDFGFDGDFYR, peptide segment Ac-ALGLDGRPGPLGPAG, peptide segment YNDFGFDGDFYRA, peptide segment NYDFGFDGDFYAR, peptide segment GHRGFSG, peptide segment GYDFGFDGDFYR, peptide segment NGYDFGFDGDFYAR, and peptide segment Ac-ALGGGYDFGFDGDFYR, respectively; Figure 4 Figure A-E correspond to glutathione content, malondialdehyde content, superoxide dismutase content, cell iron content, and lipid peroxide content, respectively; Figure 5 Figure A-E correspond to glutathione content, malondialdehyde content, superoxide dismutase content, cell iron content, and lipid peroxide content, respectively; Figure 6 Figure A-E correspond to glutathione content, malondialdehyde content, superoxide dismutase content, cell iron content, and lipid peroxide content, respectively; Figure 7 Figure A-E correspond to glutathione content, malondialdehyde content, superoxide dismutase content, cell iron content, and lipid peroxide content, respectively; Figure 8 Figure A-E correspond to glutathione content, malondialdehyde content, superoxide dismutase content, cell iron content, and lipid peroxide content, respectively; Figure 9 Figure A-E correspond to glutathione content, malondialdehyde content, superoxide dismutase content, cell iron content, and lipid peroxide content, respectively; Figure 10: Figure of Western Blot results of PI3K and AKT signaling pathway in test example 1, wherein A is the Western Blot detection electrophoresis chart, and B is the statistical analysis of protein expression amount; Figure 11 : Figure of the influence of lipid peroxidation related indicators after adding AKT inhibitor in test example 2, wherein A-E are respectively the glutathione content chart, the malondialdehyde content chart, the superoxide dismutase content chart, the cell iron content chart and the lipid peroxide content chart; Figure 12 : Figure of ROS determination results of PI3K and AKT signaling pathway changes in transcriptome data in test example 2; Figure 13 : Figure of bone formation related protein expression level results of different treatment groups after adding AKT inhibitor in test example 2, wherein A is the Western Blot detection electrophoresis chart, and B is the statistical analysis of protein expression amount; Figure 14 : Figure of osteoblast ferroptosis related gene expression level results of different treatment groups after adding AKT inhibitor in test example 2, wherein A is the Western Blot detection electrophoresis chart, and B is the statistical analysis of protein expression amount; Figure 15 : Figure of efficacy evaluation results of different test groups on enhancing bone density in test example 3; Figure 16 : Figure of efficacy evaluation results of different test groups on zebrafish bone density in test example 3; Figure 17 : Figure of the influence of GPX4 and SLC7A11 gene expression in different test groups in test example 3; Figure 18 : Figure of the influence of PI3K and AKT gene expression in different test groups in test example 3. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0025] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific embodiments.

[0026] The experimental materials used in the embodiments of the present application are all conventional experimental materials in the art and can be purchased through commercial channels. In the present application, the cornu cervi tablets are purchased from Shandong Jianlian Shengjia Traditional Chinese Medicine Co., Ltd., the pepsin and dexamethasone (DEX) are purchased from Beijing Solabio Technology Co., Ltd., the CAS number of Fmoc-L-Arg (Pbf)-OH is 154445-77-9, and the CAS number of Fmoc-Ala-OH is 35661-39-3.

[0027] In the present application, the primers and primer sequences used are as follows.

[0028] Example 1 1. The cornu cervi gum powder was prepared according to the method in the Chinese Pharmacopoeia (2020 edition). Specifically as follows: After the cornu cervi tablets 500 g were crushed, 5 times the amount of distilled water was added for soaking for 1 h, and the decoction liquid was taken out every 3 h, and after adding water, it was decocted again, a total of two times, the decoction liquid was combined, concentrated to a thick paste, and then cooled to room temperature to obtain the cornu cervi gum; the cornu cervi gum was pre-cooled and freeze-dried for 48 h, dehydrated, and passed through a 120 mesh sieve to obtain the cornu cervi gum powder, which was stored at -20℃ for standby.

[0029] 2. Preparation of cornu cervi polypeptide: Pepsin was added to the cornu cervi gum powder, the amount of pepsin was 1.8% of the mass of the cornu cervi gum powder, the cornu cervi gum powder concentration was 0.08 mg / mL, and the enzyme was hydrolyzed at 40℃ and pH 4.5 for 7 h, the supernatant after enzyme hydrolysis was collected, -20℃ overnight, freeze-dried for 48 h, and passed through a 120 mesh sieve to obtain the enzyme hydrolysis product, which was stored at -20℃ for standby; the enzyme hydrolysis product was subjected to reduction alkylation treatment to stabilize the polypeptide structure to obtain the cornu cervi polypeptide. The specific steps are as follows: the enzyme hydrolysis product was dissolved in 50 nM NH4HCO3 solution, then DTT solution was added to a final concentration of 10 mmol / L in the system, and reduction was carried out at 56℃ water bath for 1 h; IAM solution was added to a final concentration of 55 mmol / L in the system, and the reaction was carried out in the dark for 40 min; DTT solution was added to a final concentration of 20 mmol / L in the system to neutralize the unreacted IAM; desalting was performed using a self-filled desalting column, and the solvent was dried in a vacuum centrifugal concentrator at 45℃ to obtain the cornu cervi polypeptide.

[0030] 3. HPLC-MS / MS detection of cornu cervi polypeptide: The detection was performed using a liquid chromatograph Easy-nLC 1200, and the liquid chromatograph conditions were as follows: the analysis column was 100 μm i.d. x 180 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm; the mobile phase was A: 0.1% formic acid aqueous solution; the mobile phase B: 80% ACN / 0.1% formic acid aqueous solution; the flow rate was 600 nL / min; and the analysis time of each component was 66 min. The mass spectrometry conditions were as follows: the primary mass spectrometry parameters: Resolution: 70,000, AGCtarget: 3e6, Maximum IT: 100 ms, Scanrange: 100 to 1500 m / z. The secondary mass spectrometry parameters: Resolution: 17500, AGCtarget: 1e5, Maximum IT: 50 ms, TopN: 20, NCE / stepped NCE: 28.

[0031] 4. Polypeptide sequence identification and screening: The PEAKS Studio 10.6 Denovo was used to analyze the mass spectrometry data, and it was predicted that 42% of the peptide segments had biological activity, and the results are shown in Table 1. Figure 1 According to previous research reports, low-molecular-weight proteins after enzymolysis are considered to be a good source for screening bioactive peptides, and the bioactivity is higher than that of high-molecular-weight protein hydrolysates. Peptide molecules with a peptide sequence higher than 20 amino acids can be cut into smaller peptide segments by digestive enzymes in the human gastrointestinal digestion, so that the activity is changed. Therefore, the present application focuses on analyzing the peptide segments with a length less than 20 in the results.

[0032] Then, based on the calculation tools such as PeptideRanker, CPPpred, PepDraw and ProtParam, a total of 9 high-confidence peptide segments were identified, as shown in Table 1. It is confirmed by the UniProt protein sequence database that the above-mentioned 9 peptide segments are all new peptide segments.

[0033] Table 1: Identified active polypeptides and physicochemical properties 5. Synthesis and structure identification of active peptide segments: Taking the peptide segment NGYDFGFDGDFYAR as an example, the solid-phase synthesis method was used for preparation, and the preparation methods of the remaining peptide segments were the same. The specific steps are as follows: Take 2-chlorotrityl chloride resin in a reaction vessel, add DCM (dichloromethane) to swell for 15 min, remove DCM, wash with DMF (dimethylformamide) for 4 times, and remove DMF; add DIEA (N,N-diisopropylethylamine), DMF and DMC to Fmoc-L-Arg(Pbf)-OH, stir and aerate for 6 h, remove the liquid after the reaction is completed, add MeOH:DIEA (1:1) solution to stir for 30 min to close the unreacted active site, remove the solution, wash with DMF for 3 times, obtain Fmoc-L-Arg(Pbf) resin, then add DP (20% piperidine+80% DMF) to react for 20 min to remove the Fmoc protecting group, remove DP and wash with DMF for 3 times, repeat the above deprotection group steps in turn, and then couple the next amino acid; weigh Fmoc-Ala-OH, HOBt (1-hydroxybenzotriazole) and DIC (N,N-diisopropylcarbodiimide) in turn and add them to the reaction vessel, dissolve in DMF and stir, aerate for 2 h, take the resin and use ninhydrin test, confirm the successful coupling, remove the liquid, wash with DMF for 3 times, remove the Fmoc protecting group by DP and wash; after coupling Tyr(tBu), Phe, Asp(OtBu), Gly, Asp(OtBu), Phe, Gly, Phe, Asp(OtBu), Tyr(tBu), Gly, Asn(Trt) in turn according to the above, NGYDFGFDGDFYAR resin is obtained; add 3 times the volume of cleavage solution (TFA / water / triisopropylsilane, 95:2.5:2.5, v / v / v) to NGYDFGFDGDFYAR resin and mix, stir at room temperature for 3 h, filter, collect the filtrate, add the filtrate to 8 times the volume of cold diethyl ether, stir, filter, collect the white precipitate, wash with cold diethyl ether for 4 times, and obtain the crude peptide; vacuum dry the crude peptide, dissolve in pure water, filter, purify by HPLC, collect the fraction of the target peak, remove the solvent, and freeze-dry for 48-72 h to obtain the purified NGYDFGFDGDFYAR peptide.

[0034] 6. Screening of the peptide by in vitro test: (1) The effect of different peptide segments on the activity of MC3T3-E1 cells was detected by CCK-8 method. Specifically as follows: After the MC3T3-E1 cells were recovered, they were inoculated in a T-25 culture bottle and cultured in a CO2 incubator. When the cell coverage reached 85%, the old culture medium was discarded and the cells were passaged. The MC3T3-E1 cells in the logarithmic growth phase were taken, the complete culture medium was discarded, washed with PBS for 2 times, trypsinized, and the cell concentration was adjusted to 5×10 7cfu / L in a 96-well culture plate, 100 μL of cell suspension was added to each well, the supernatant was discarded after the cells adhered to the wall and the cells were divided into a blank control group and different concentrations of peptide groups (the concentration gradient of each peptide group was 0, 10, 25, 50, 100, 200 μg / mL); after culturing for 24 hours, the serum-free medium was replaced, 10 mL of CCK-8 reagent was added to each well, and the reaction was carried out at 37°C for 55 minutes. Then, the effect of different peptides on the proliferation of MC3T3-E1 cells was detected and calculated using a microplate reader at a wavelength of 450 nm. The results are shown in Figure 2. Figure 2 shown.

[0035] Depend on Figure 2 It can be seen that compared with the control group, the peptides Ac-ALGLDGRPGPLGPAG, NYDFGFDGDFYAR, and GHRGFSG have toxic effects on cells, but the peptide NGYDFGFDGDFYAR shows a certain pro-proliferation effect on cells.

[0036] (2) Explore the effect of peptides on the decreased activity of MC3T3-E1 cells induced by DEX, and screen out peptides with therapeutic effects, as follows: Take MC3T3-E1 cells in the logarithmic growth phase, discard the complete medium, wash twice with PBS, digest with trypsin, and adjust the cell density to 5×107 7 cfu / L in a 96-well culture plate, 100 μL of cell suspension was added to each well. After the cells adhered, the supernatant was discarded and the cells were divided into blank control group, DEX group (800 μM), and DEX+DAP group; among them, the concentration gradient of DEX+DAP group was: 0, 10, 25, 50, 100 and 200 μg / mL, and DAP was the 8 peptides synthesized above. After 24 hours, the serum-free medium was replaced, 10 μL of CCK-8 reagent was added to each well, and the reaction was incubated at 37°C for 55 minutes. The results were detected at a wavelength of 450 nm on a microplate reader to obtain the effects of different peptides on the proliferation of MC3T3-E1 cells treated with DEX. The results are shown in Figure 2. Figure 3 shown.

[0037] Depend on Figure 3 As can be seen, compared with the model group, only the peptide NGYDFGFDGDFYAR (100 μg / mL) intervention treatment significantly inhibited the DEX-induced decrease in MC3T3-E1 cell viability. It is speculated that NGYDFGFDGDFYAR is an effective active peptide for alleviating osteoblast damage and improving osteoporosis. Therefore, this peptide was selected for subsequent experiments.

[0038] Test Example 1: Cell test After MC3T3-E1 cells were revived, they were inoculated into T-25 culture flasks and cultured in a CO2 incubator. When the cell confluence reached 85%, the old culture medium was discarded and the cells were passaged. When the cells grew to the logarithmic phase, 1.5×10 5 Cells were seeded in 6-well plates and divided into 4 groups, with 2 replicates in each group.

[0039] There are 4 treatment groups in this experiment, as follows: Blank control group: normal culture without treatment; DEX group: treated with 800 μM DEX; DEX+low-concentration NGY group: A mixture of DEX and the complete culture medium of the antler active peptide NGY in Example 1 was used as the treatment solution, wherein the DEX concentration was 800 μM and the antler active peptide NGY concentration was 50 μg / mL; DEX+high-concentration NGY group: A mixture of DEX, the antler active peptide NGY described in Example 1, and complete culture medium was used as the treatment solution, wherein the DEX concentration was 800 μM and the antler active peptide NGY concentration was 100 μg / mL; After culturing each treatment group for 24 h, the lipid peroxidation index, intracellular ROS content and other parameters of each treatment group were detected.

[0040] 1. Lipid peroxidation index Lipid peroxidation indicators include GSH concentration, MDA concentration, SOD concentration, cellular iron concentration and LPO concentration. The detection method is: The GSH, MDA, SOD, iron and LPO content in the MC3T3-E1 osteoblasts were detected using a GSH kit, MDA kit, SOD kit, iron content kit and LPO kit (all purchased from Beijing Solebow Biotechnology Co., Ltd.), respectively. Then, the protein concentration of MC3T3-E1 cells was detected using a BCA kit (purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.). The GSH, MDA, SOD, iron and LPO concentrations were obtained. The results are shown in Table 1. Figure 4 shown.

[0041] Depend on Figure 4 It can be seen that treatment with the deer antler active peptide fragment of the present invention significantly inhibited the DEX-induced decrease in GSH and SOD levels and the increase in MDA, LPO, and cellular iron concentrations, increasing GSH and SOD levels and reducing MDA, LPO, and cellular iron levels in a dose-dependent manner. This suggests that the deer antler active peptide fragment of the present invention can inhibit lipid peroxidation in osteoblasts and exert a bone-protective effect.

[0042] 2. ROS content The ROS content in cultured MC3T3-E1 osteoblasts was detected using the fluorescent probe DCFH-DA reactive oxygen species detection kit (purchased from Biyuntian Biotechnology Co., Ltd.). Figure 5 shown.

[0043] Depend on Figure 5 As can be seen, compared with the blank control group, the green fluorescence intensity of cells in the DEX group increased, indicating a significant increase in ROS levels. After intervention with the deer antler active peptide of the present invention, intracellular ROS levels were reduced compared to the model group. This shows that the deer antler active peptide of the present invention can regulate the intracellular redox balance and reduce ROS accumulation.

[0044] 3. Expression of bone formation-related proteins Western blotting was used to investigate the effect of the active peptide fragments of deer antlers on the expression of bone formation-related proteins. Figure 6 shown.

[0045] Depend on Figure 6 It can be seen that intervention with the deer antler active peptide fragment of the present invention can effectively inhibit the decrease in Runx2 and Osterix expression caused by DEX, and significantly increase the expression levels of Runx2 and Osterix. This shows that the deer antler active peptide fragment of the present invention can alleviate the inhibitory effect of DEX on bone formation-related proteins.

[0046] 4. Expression of genes related to bone cell death Western blotting was used to investigate the effects of the active peptide fragments of deer antlers on the expression of genes related to bone cell death. Figure 7 shown.

[0047] Depend on Figure 7 As can be seen, there are differences in protein expression levels between the different treatment groups. Compared with the blank control group, the levels of protein SLC7A11 and GPX4 in the DEX group were significantly reduced. After intervention with the deer antler active peptide of the present invention, the protein expression levels recovered or further changed compared with the DEX group, indicating that the deer antler active peptide of the present invention can inhibit the decline of SLC7A11 and GPX4, and there may be a concentration-dependent effect, indicating that the deer antler active peptide of the present invention can activate SLC7A11 / GPX4 to exert a therapeutic effect on cell ferroptosis.

[0048] 5. Differential expression of cellular genes mRNA sequencing and analysis were used to explore the effects of the active peptide fragments of deer antlers on the genes of DEX-treated cells. The specific steps were: (1) Sample processing and RNA extraction: MC3T3-E1 cells stored at -80℃ were used as the research object, and the whole process was operated in an RNase-free environment. Total RNA was extracted using the TaKaRa RNAiso Plus kit, and protein and genomic DNA contamination was removed by chloroform phase separation and magnetic bead purification technology. The quality of the RNA was evaluated by the Agilent Bioanalyzer 2100 system, and only samples with an RNA integrity value (RIN≥8.0) and a 28S / 18S ratio≥1.8 were retained to ensure the reliability of subsequent analysis.

[0049] (2) Library construction and high-throughput sequencing: Based on the Illumina platform, a strand-specific cDNA library was constructed: mRNA was enriched by oligo(dT) magnetic beads, double-stranded cDNA was synthesized using a high-fidelity reverse transcriptase (Superscript IV), and 200-500 bp inserts were selected using AMPure XP magnetic beads. After the library quality was verified by Agilent 2100 Bioanalyzer and Qubit 2.0 fluorescence quantification, the libraries were mixed at equal molar concentrations, and double-end 150 bp sequencing was completed on a NovaSeq 6000 sequencer (target data volume≥40M reads / sample).

[0050] (3) Data analysis and functional annotation: The raw data were quality controlled by FastQC and Trimmomatic software, and low-quality sequences (Phredscore<30) and adapter contamination were removed to obtain high-quality clean reads. The sequences were accurately mapped to the mouse reference genome (GRCm39) using the STAR alignment tool, and transcript quantification was performed by StringTie. DESeq2 algorithm was used to screen differential expression genes (DEGs), and Benjamini-Hochberg method was used to correct multiple hypothesis testing errors. Key gene sets were annotated by GO function and KEGG pathway enrichment analysis by DAVID and clusterProfiler.

[0051] To explore the effect of the active peptide segment of deer antler on the genes of DEX-treated cells, the expression profile of the transcriptome mRNA of the DEX group and the DEX+NGY group was detected, and the DEGs were screened with a condition of padj <0.05. The results are shown in Figure 8 As can be seen from Figure 8 , compared with the DEX group, 1737 genes were up-regulated and 1587 genes were down-regulated after treatment with the active peptide segment of deer antler, indicating that the active peptide segment of deer antler can change the mRNA expression profile of osteoblasts caused by DEX treatment.

[0052] The differential genes of up-regulation and down-regulation were subjected to KEGG enrichment analysis, and the results are shown in Table 1. Figure 9 Figure 9 As can be seen, after the treatment of the active peptide segment of deer antler according to the present application, the differential genes of up-regulation are significantly enriched in PI3K / AKT, Human papillomavirus infection and other signal pathways; and the differential genes of down-regulation are significantly enriched in Amyotrophic lateral sclerosis, Huntington disease and other signal pathways. After excluding the signal pathways related to diseases, the differential genes of PI3K / AKT signal pathway account for the most, and thus it can be seen that the active peptide segment of deer antler according to the present application can up-regulate the expression of PI3K / AKT signal pathway.

[0053] The results of PI3K / AKT signal pathway involved in KEGG enrichment analysis in the transcriptome data were verified by Western Blot, and the results are shown in Table 2. Figure 10 Figure 10 As can be seen, compared with the blank control group, the phosphorylation levels of PI3K and AKT proteins in the DEX treatment group are significantly reduced, indicating that the activity of PI3K / AKT signal pathway is inhibited; and in the DEX+NGY treatment group, the phosphorylation levels of PI3K and AKT are increased compared with the DEX treatment group, indicating that the active peptide segment of deer antler according to the present application can partially restore the activity of PI3K / AKT signal pathway. This further verifies that the active peptide segment of deer antler according to the present application plays a protective role by regulating PI3K / AKT signal pathway in the process of DEX-induced cell oxidative stress.

[0054] Test Example 2 After the MC3T3-E1 cells were recovered, they were inoculated in a T-25 culture flask and placed in a CO2 incubator for culture. When the cell coverage reached 85%, the old culture medium was discarded for subculture. The cultured MC3T3-E1 cells were inoculated in a six-well plate, and four treatment groups were set up, respectively as follows: Blank control group: normal culture without treatment; DEX group: treated with 800 μM DEX; DEX+NGY group: a mixed solution of DEX, the active peptide segment of deer antler NGY in Example 1 and complete culture medium was used as the treatment solution; wherein the concentration of DEX was 800 μM, and the concentration of the active peptide segment of deer antler NGY was 100 μg / mL; ​​DEX+NGY+inhibitor group: A mixture of DEX, the antler active peptide NGY described in Example 1, the AKT inhibitor MK2206, and complete culture medium was used as the treatment solution; wherein the concentration of DEX was 800 μM, the concentration of the antler active peptide NGY was 100 μg / mL, and the concentration of the AKT inhibitor MK2206 was 5 μM; After culturing each treatment group for 24 h, the lipid peroxidation index, intracellular ROS content and ferroptosis-related proteins of each treatment group were detected. The results are as follows Figures 11-14 shown.

[0055] Studies have found that DEX treatment can significantly reduce the antioxidant capacity of cells (decreased GSH content and activity), and significantly increase cell oxidative damage (increased MDA and LPO content) and iron accumulation levels. Figure 11 It can be seen that intervention with the deer antler active peptides of the present invention partially reversed these DEX-induced changes, specifically manifested by increases in GSH content and SOD activity, and decreases in MDA, LPO, and cellular iron levels. Adding an AKT inhibitor reversed these changes. This suggests that the inhibitory effect of the deer antler active peptides of the present invention on lipid peroxidation is antagonized by the AKT inhibitor, suggesting that the deer antler active peptides inhibit lipid peroxidation via the PI3K-AKT pathway.

[0056] Depend on Figure 12 As can be seen, compared with the blank control group, the green fluorescence intensity of cells in the model group increased, indicating a significant increase in ROS levels. After intervention with the antler active peptide NGY of the present invention, intracellular ROS levels were reduced compared to the model group. The addition of an AKT inhibitor reversed the ROS-inhibiting effect of the antler active peptide NGY. These results demonstrate that AKT inhibitors can antagonize the ROS-inhibiting effect of NGY, suggesting that NGY inhibits excessive ROS production through the PI3K-AKT pathway.

[0057] Depend on Figure 13 Significant differences in RUNX2 and OSX protein expression levels were observed between the different treatment groups. The DEX group showed significantly lower RUNX2 and OSX protein expression levels compared to the blank group (P < 0.01). Treatment with the antler active peptide NGY significantly increased these levels compared to the DEX group, indicating that the antler active peptide NGY increased RUNX2 and OSX protein expression. However, this effect was reversed and weakened by the addition of an AKT inhibitor. These results indicate that AKT inhibition can antagonize the effect of the antler active peptide NGY on increasing bone-related protein expression, suggesting that the antler active peptide NGY increases bone-related protein expression through the PI3K / AKT pathway.

[0058] Figure 14It can be seen that there are significant differences in the expression levels of SLC7A11 and GPX4 proteins among different treatment groups. The expression levels of SLC7A11 and GPX4 proteins in the DEX group are significantly lower than those in the blank group (P<0.01), and the expression levels of SLC7A11 and GPX4 proteins in the NGY treatment group are significantly higher than those in the DEX group, indicating that the active peptide segment NGY of antler can increase the expression of SLC7A11 and GPX4 proteins; but this effect is reversed and weakened after the addition of AKT inhibitor. These results show that AKT inhibition can antagonize the effect of active peptide segment NGY of antler on increasing the expression of ferroptosis-related proteins, suggesting that NGY regulates the expression of SLC7A11 and GPX4 through the PI3K / AKT pathway to inhibit ferroptosis.

[0059] Test Example 3: Animal Experiment AB wild-type zebrafish and transgenic hard bone green fluorescent zebrafish were used as objects for animal experiments. The AB wild-type zebrafish and transgenic hard bone green fluorescent zebrafish were provided by Hangzhou Huan Te Biological Technology Fish Center, and the feeding conditions were as follows: circulating water constant temperature 28℃, light cycle 14h light and 10h dark, feeding at 9am and 3pm, and live brine shrimp larvae incubated for 24h were used as feed.

[0060] The day before the experiment, adult fish were placed in a breeding box at a ratio of 2:1 for males and females, with male and female fish separated by a transparent barrier and placed at 28℃ overnight. On the day of the experiment, the transparent barrier was removed at 9am, and the zebrafish began natural mating and spawning, with the time after fertilization recorded as dpf. Zebrafish eggs were selected according to the zebrafish embryo development chart, and the selected eggs were treated in a special way and fed until 3dpf for subsequent experimental research.

[0061] 1. Maximum detection concentration (MTC) Select 3dpf (days old) transgenic hard bone green fluorescent zebrafish, place 30 randomly in each well, and distribute them into 6-well plates. Different experimental groups were given different concentrations of test samples, specifically: positive control group, normal control group, model control group, and test group. The positive control group used 5.00μg / mL ALN solution (alendronate sodium tablets, batch number X020681, solvent: ultrapure water), the normal control group was not treated, and the test group used different concentrations of active peptide segments of antler horn. Except for the normal control group, 10.00μM DEX was added to the rest of the groups to construct a zebrafish osteogenesis damage model. After treating each treatment group of zebrafish at 28℃ for 4 days, the MTC of the sample on the model zebrafish was detected, as shown in Table 2.

[0062] Table 2: Sample Osteoporosis Improvement Efficacy Concentration Exploration Experiment Results (n=30) As can be seen from Table 2, the active peptide segment of antler of the present application does not cause death of zebrafish and no obvious abnormality is observed at a concentration range of 31.2-125 μg / mL. When the concentration is increased to 250 and 500 μg / mL, the mortality rates reach 97% and 100% respectively, which is significantly higher than that of the model control group. Therefore, the maximum detection concentration (MTC) of the active peptide segment of antler prepared in the present application for improving the efficacy of osteoporosis is 125 μg / mL.

[0063] 2. Evaluation of efficacy of enhancing bone density 3dpf wild-type AB strain zebrafish are selected, 30 zebrafish are placed in each well and randomly distributed into 6-well plates, and 12.5 μg / mL of the active peptide segment of antler of Example 1 and 125 μg / mL of the active peptide segment of antler of Example 1 are respectively given in water solution, 5.00 μg / mL of ALN is given to the positive control group, and a normal control group and a model control group are simultaneously set.

[0064] Except for the normal control group, the rest of the groups are given 2.00 μM of DEX in water solution to establish the osteoporosis model of zebrafish. After being treated at 28°C for 4 days, 50.0 μg / mL of alizarin red is used for staining for 24 h, 10 zebrafish randomly selected from each group are placed under a fluorescence microscope for photographing, and the results are shown in Figure 15

[0065] As shown in Figure 15 Compared with the blank group, the fluorescence intensity of the head bone of the zebrafish in the model group is significantly decreased (P<0.01), which indicates that the osteoporosis model is successfully established. Compared with the model group, the fluorescence intensity of the head bone of the zebrafish in the positive control group is obviously increased (P<0.01), which shows a good anti-osteoporosis effect. In the active peptide segment of antler group of the present application, the fluorescence intensity is increased to a certain extent in the low concentration (12.5 μg / mL) group compared with the model group, but the difference is not significant (P>0.05). When the concentration is high (125 μg / mL), the fluorescence intensity of the head bone of the zebrafish is not much different from that of the blank group, which indicates that the bone density has basically returned to the normal level. Therefore, the high-concentration active peptide segment of antler can improve the problem of decreased bone density of zebrafish with osteoporosis.

[0066] 3. Efficacy of promoting bone formation 3dpf (day old) transgenic hard bone green fluorescent zebrafish are selected, 30 zebrafish are placed in each well and randomly distributed into 6-well plates, and 12.5 μg / mL of the active peptide segment of antler of Example 1 and 125 μg / mL of the active peptide segment of antler of Example 1 are respectively given in water solution, 5.00 μg / mL of ALN is given to the positive control group, and a normal control group and a model control group are simultaneously set. Except for the normal control group, the rest of the groups are given 10.00 μM of DEX in water solution to establish the osteogenesis injury model of zebrafish. After being treated at 28°C for 4 days, 10 zebrafish randomly selected from each group are placed under a fluorescence microscope for photographing, and the results are shown in Figure 16 .​

[0067] By Figure 16 It can be seen that compared with the blank group, the bone fluorescence intensity of zebrafish in the model group was significantly reduced after DEX treatment (P<0.01), indicating that the osteoporosis model was successfully established. Compared with the model group, the bone fluorescence intensity of the positive group was significantly increased (P<0.01), indicating that alendronate sodium had good anti-osteoporosis effect in the model. In terms of intervention of the active peptide segment NGY of antler, at a low concentration (12.5 μg / mL), the bone fluorescence intensity of zebrafish was slightly improved compared with the model group, but the difference did not reach a significant level (P>0.05); at a high concentration (125 μg / mL), the bone fluorescence intensity was close to that of the blank group, indicating that the bone density had basically returned to the normal level. In summary, the active peptide segment of antler at a high concentration can increase the bone formation function and improve the osteoporosis of zebrafish.

[0068] 4. Real-time fluorescent quantitative RT-PCR detection (1) Zebrafish sample processing Select 3 dpf (days of age) transgenic hard bone green fluorescent zebrafish, place 30 randomly in each well, and allocate them into 6-well plates. Respectively, give 12.5 μg / mL of the active peptide segment of antler in Example 1 and 125 μg / mL of the active peptide segment of antler in Example 1, and give 5.00 μg / mL of ALN to the positive control group (ALN), and set up a normal control group (CON) and a model control group (DEX). Except for the normal control group, 10.00 μM of DEX is added to the rest of the groups to construct the zebrafish osteogenesis injury model. The zebrafish in each treatment group is treated at 28°C for 4 days.

[0069] (2) RNA extraction Grind the zebrafish into fine powder, and then transfer the fine powder into a centrifuge tube containing 1 mL SparkZol for lysis. Add 0.2 mL of chloroform, shake for 15 s, and then place at room temperature for 2-3 min. Centrifuge at 4°C, 12000 rpm for 12 min. Collect the water sample layer after centrifugation, mix with an equal volume of isopropanol, and place at room temperature for 10 min. Centrifuge at 4°C, 12000 rpm for 10 min, discard the supernatant, and then centrifuge the precipitate at 4°C, 12000 rpm for 3 min. Discard the supernatant, and then place at room temperature for 2-3 min. Dry. Place at room temperature for 2-3 min, dry, and then add 30 μL of RNase Free H2O to fully dissolve the RNA. Obtain the total RNA, and store at -80°C. Take the total RNA extracted above, perform 1.0% agarose gel electrophoresis at 135 V for 20 min, and then perform statistical analysis after reverse transcription and real-time PCR detection. The results are shown in Figure 17 and Figure 18 .

[0070] By Figure 17 It can be seen that the expression of SLC7A11 gene did not change in each group, while GPX4 showed a certain trend. Compared with the blank group, the expression of GPX4 mRNA in the model group decreased; in the NGY low and high dose treatment groups and the positive drug treatment group, the expression level of GPX4 increased, but the differences were not statistically significant. The results showed that the active peptide segment NGY of antler could inhibit the ferroptosis of the in vivo model of osteoporosis to a certain extent.

[0071] By Figure 18 It can be seen that compared with the blank group, the expression of PI3K and AKT mRNA in the model group decreased; the high dose of active peptide segment NGY of antler and the positive drug treatment could increase the expression level of PI3K and AKT, but the increase effect of AKT was not as significant as that of PI3K. The results showed that the PI3K / AKT signal pathway was inhibited in the zebrafish osteoporosis model, while the active peptide segment NGY of antler treatment could activate the PI3K / AKT signal pathway.

[0072] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An active peptide fragment of deer antler, characterized in that, The amino acid sequence of the active peptide segment of deer antler is shown as SEQ ID No.

1.

2. The method of claim 1, wherein the active peptide fragment of deer antler is prepared by the steps of: The method comprises the following steps: The active peptide segment of deer antler is obtained after separation and purification of enzymatic products of deer antler glue, or is prepared by solid-phase synthesis.

3. The method for preparing the active peptide fragment of deer antler according to claim 2, characterized in that: The enzymatic products of deer antler glue are prepared by the following method: After adding pepsin to the deer antler glue powder, the enzymatic products of deer antler glue are obtained by enzymolysis at 40℃ and pH 4.5 for 7h, and collecting the supernatant after drying.

4. The method for preparing the active peptide fragments of deer antlers according to claim 3, characterized in that: The amount of pepsin is 1.8% of the mass of the deer antler glue powder.

5. The active peptide segment of deer antler of claim 1 is used for preparing a product for preventing or treating osteoporosis.

6. The use according to claim 5, wherein the compound is ###0002### The active peptide segment of deer antler prevents or treats osteoporosis by at least one of the following 1) to 6): 1) inhibiting lipid peroxidation of osteoblasts; 2) regulating the redox balance in osteoblasts; 3) promoting osteogenic differentiation of osteoblasts; 4) inhibiting ferroptosis in osteoblasts; 5) up-regulating the expression of PI3K / AKT signaling pathway; 6) promoting the proliferation of osteoblasts.

7. The active peptide segment of deer antler of claim 1 is used for any one of the following 1) to 5): 1) increasing the levels of GSH and SOD in osteoblasts; 2) reducing the levels of MDA, LPO and cellular iron content in osteoblasts; 3) reducing the content of ROS in osteoblasts; 4) promoting the expression of osteogenesis proteins Runx2 and Osterix in osteoblasts; 5) promoting the regulation of ferroptosis proteins SLC7A11 and GPX4 in osteoblasts.

8. A product for preventing or treating osteoporosis, characterized by, The active peptide segment of deer antler of claim 1 is used as an active ingredient.

9. The product for preventing or treating osteoporosis according to claim 8, wherein The product for preventing or treating osteoporosis further comprises a pharmaceutically acceptable excipient.

10. The product for preventing or treating osteoporosis according to claim 8, wherein the product is a food product. The dosage form of the product for preventing or treating osteoporosis is granules, capsules, tablets or oral preparations.

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