Polypeptides promoting osteoblast proliferation and differentiation and their applications

Abstract

The present invention discloses polypeptides for promoting osteoblast proliferation and differentiation and their applications, relating to the field of biotechnology. Using gelatin from tilapia fish scales and fish skin as raw materials, through means such as protease digestion technology, LC-MS / MS peptide identification, computer simulation screening combined with molecular docking-assisted screening, three polypeptides GM-15, AG-17, and GN-18 with the ability to promote osteoblast proliferation and improve differentiation activity are provided. These three polypeptides have relatively high cell proliferation-promoting activity, and the maximum proliferation rate can reach 124%. At the same time, these three polypeptides exhibit a strong effect on promoting osteoblast differentiation and mineralization. The three polypeptides provided by the present invention have the characteristics of good water solubility, non-toxicity, non-allergy, and strong activity in promoting osteoblast proliferation and differentiation, and have good application prospects in the fields of orthopedic drugs, health products, functional foods, etc. related to promoting osteoblast proliferation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to polypeptides that promote osteoblast proliferation and differentiation and their applications. Background Art

[0002] Osteoporosis (OP) is a chronic skeletal disease characterized by reduced bone mass and degradation of bone tissue microstructure, often leading to increased bone fragility and elevated fracture risk. Its pathogenesis is complex and involves disruption of bone metabolism balance, especially the imbalance between osteoblast and osteoclast functions. The occurrence of osteoporosis is closely related to aging. With the accelerating global aging process, it is predicted that by 2050, the proportion of the global elderly population will increase significantly, and the incidence of osteoporotic fractures will also increase accordingly, becoming one of the important issues affecting public health. Currently, although drugs such as bisphosphonates and calcitonin are widely used in the clinical treatment of osteoporosis, their long-term use may cause side effects such as gastrointestinal discomfort and osteonecrosis, and there are potential toxicity risks. Therefore, natural active substances derived from food have gradually become a research hotspot. Currently, many studies aim to find safer and more effective treatment approaches in order to provide better intervention options for osteoporosis patients.

[0003] In recent years, with the in-depth study of the mechanism of osteoporosis, bioactive peptides that promote osteoblast proliferation and differentiation have gradually attracted attention. Such peptides are usually derived from enzymatic hydrolysis products of food proteins and have the potential to promote osteoblast proliferation, differentiation, and mineralization. Research shows that certain bioactive peptides can accelerate the maturation process of osteoblasts by activating the Wnt / β-catenin signaling pathway, promoting ALP activity, and synthesizing type I collagen. In addition, active peptides can also enhance the deposition of calcium ions in the bone matrix, contributing to an increase in bone density. Compared with traditional drugs, these peptide molecules derived from food not only have good biocompatibility in vivo, but also have low toxicity and few side effects, showing broad application prospects in the prevention and treatment of osteoporosis. Future research will continue to focus on exploring the molecular mechanisms of these peptides and their potential in clinical applications, providing a scientific basis for nutritional intervention in osteoporosis.

[0004] As a widely cultured freshwater fish globally, Oreochromis niloticus has a high yield accompanied by the generation of a large number of by-products, such as fish skin, fish scales, etc. These by-products are often discarded or used for low-value-added products in traditional processing, resulting in a great waste of resources. However, studies have found that the fish skin and fish scales of Oreochromis niloticus are rich in collagen and gelatin, which not only have excellent biocompatibility but also have the potential to promote the proliferation and differentiation of osteoblasts. Through appropriate enzymatic hydrolysis treatment, high-quality collagen peptides can be extracted from these by-products, which can promote the repair and regeneration of bone tissue. Therefore, using Oreochromis niloticus by-products for the extraction of bioactive peptides not only provides potential functional components for the treatment of osteoporosis but also provides a new way for the efficient utilization of aquatic resources, meeting the concept of resource recycling and sustainable development. Summary of the Invention

[0005] The object of the present invention is to provide polypeptides that promote the proliferation and differentiation of osteoblasts and their applications to solve the problems existing in the above-mentioned prior art. The polypeptides provided by the present invention have the characteristics of good water solubility, non-toxicity, non-allergy, and strong activity in promoting the proliferation and differentiation of osteoblasts, and have good application prospects in the fields of orthopedic drugs, health products, functional foods, etc. related to promoting osteoblast proliferation.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a polypeptide GM-15 that promotes the proliferation and differentiation of osteoblasts, and its amino acid sequence is as shown in SEQ ID NO.1.

[0008] The present invention also provides a polypeptide GN-18 that promotes the proliferation and differentiation of osteoblasts, and its amino acid sequence is as shown in SEQ ID NO.2.

[0009] The present invention also provides a polypeptide AG-17 that promotes the proliferation and differentiation of osteoblasts, and its amino acid sequence is as shown in SEQ ID NO.3.

[0010] The present invention also provides the application of the above polypeptide GM-15, polypeptide GN-18, and / or polypeptide AG-17 in the preparation of drugs for preventing and / or treating osteoporosis.

[0011] The present invention also provides the application of the above polypeptide GM-15, polypeptide GN-18, and / or polypeptide AG-17 in the preparation of products for improving bone density;

[0012] The product is a health product or a functional food.

[0013] The present invention also provides a drug for preventing and / or treating osteoporosis, and the active ingredient includes at least one of the above polypeptide GM-15, polypeptide GN-18, and polypeptide AG-17.

[0014] The present invention also provides a health care product or functional food for improving bone density, and the active ingredients include at least one of the above-mentioned polypeptides GM-15, polypeptide GN-18 and polypeptide AG-17.

[0015] The present invention also provides a preparation method of the above-mentioned polypeptide GM-15, which includes the step of synthesizing the polypeptide GM-15 by solid-phase synthesis method.

[0016] The present invention also provides a preparation method of the above-mentioned polypeptide GN-18, which includes the step of synthesizing the polypeptide GN-18 by solid-phase synthesis method.

[0017] The present invention also provides a preparation method of the above-mentioned polypeptide AG-17, which includes the step of synthesizing the polypeptide AG-17 by solid-phase synthesis method.

[0018] The present invention discloses the following technical effects:

[0019] The present invention uses gelatin from tilapia fish scales and fish skin as raw materials, and through means such as protease digestion technology, LC-MS / MS peptide segment identification, computer simulation screening combined with molecular docking-assisted screening, provides three polypeptides GM-15, AG-17 and GN-18 with the ability to promote osteoblast proliferation and improve differentiation activity. These three polypeptides have relatively high cell proliferation promoting activity, and the maximum proliferation rate can reach 124%. At the same time, these three polypeptides show strong effects on promoting osteoblast differentiation and mineralization.

[0020] The three polypeptides provided by the present invention have the characteristics of good water solubility, non-toxicity, non-allergy, and strong activity in promoting osteoblast proliferation and differentiation, and have good application prospects in the fields of orthopedic drugs, health care products, functional foods, etc. related to promoting osteocyte proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is the total ion chromatogram of the alkaline protease hydrolysate of fish scale gelatin;

[0023] Figure 2 It is the total ion chromatogram of the trypsin hydrolysate of fish scale gelatin;

[0024] Figure 3 It is the total ion chromatogram of the trypsin hydrolysate of fish skin gelatin;

[0025] Figure 4 Analysis chart of the proportion of peptide segments with different molecular weights in three enzyme digestion solutions identified by LC-MS / MS;

[0026] Figure 5 2D interaction schematic diagram of the molecular docking of GM-15 and EGFR;

[0027] Figure 6 3D interaction schematic diagram of the molecular docking of GM-15 and EGFR;

[0028] Figure 7 2D interaction schematic diagram of the molecular docking of GN-18 and EGFR;

[0029] Figure 8 3D interaction schematic diagram of the molecular docking of GN-18 and EGFR;

[0030] Figure 9 2D interaction schematic diagram of the molecular docking of AG-17 and EGFR;

[0031] Figure 10 3D interaction schematic diagram of the molecular docking of AG-17 and EGFR;

[0032] Figure 11 Secondary mass spectrometry diagram of GM-15;

[0033] Figure 12 Secondary mass spectrometry diagram of AG-17;

[0034] Figure 13 Secondary mass spectrometry diagram of GN-18;

[0035] Figure 14 High performance liquid chromatography diagram of GM-15;

[0036] Figure 15 High performance liquid chromatography diagram of AG-17;

[0037] Figure 16 High performance liquid chromatography diagram of GN-18;

[0038] Figure 17 Statistical chart of the proliferation viability of Saos-2 cells under the action of GM-15, GN-18 and AG-17 at different concentrations;

[0039] Figure 18ALP staining images and ALP activity statistical graphs of Saos-2 cells under the action of GM-15, GN-18, and AG-17 at different concentrations; among them, A and B are the ALP staining images and ALP activity statistical graphs of Saos-2 cells under the action of GM-15 at different concentrations; C and D are the ALP staining images and ALP activity statistical graphs of Saos-2 cells under the action of GN-18 at different concentrations; E and F are the ALP staining images and ALP activity statistical graphs of Saos-2 cells under the action of AG-17 at different concentrations; in A, C, and E, before is the cell morphology before microscopic staining, and after is the cell morphology after microscopic staining;

[0040] Figure 19 Mineralization staining images and mineralization degree statistical graphs of Saos-2 cells under the action of GM-15, GN-18, and AG-17 at different concentrations; among them, A and B are the mineralization staining images and mineralization degree statistical graphs of Saos-2 cells under the action of GM-15 at different concentrations; C and D are the mineralization staining images and mineralization degree statistical graphs of Saos-2 cells under the action of GN-18 at different concentrations; E and F are the mineralization staining images and mineralization degree statistical graphs of Saos-2 cells under the action of AG-17 at different concentrations;

[0041] Figure 20 Statistical graphs of the expression levels of osteogenesis-related genes and Wnt / β-catenin-related genes under the influence of GM-15, GN-18, and AG-17; among them, A-I are ALP, IBSP, OCN, OSX, Runx2, Coll-α, β-catenin, LRP5, and GSK3β respectively. Detailed implementation manners

[0042] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0044] Unless otherwise noted, 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 invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0045] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0046] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0047] Example 1

[0048] 1. Extraction of fish skin gelatin and fish scale gelatin

[0049] (1) Experimental materials

[0050] Tilapia fish skin and scales are sourced from the local aquatic product market and obtained after processing fresh fish.

[0051] (2) Experimental methods

[0052] Fish skin gelatin extraction process: Wash tilapia fish skin three times with distilled water to remove impurities, and then air-dry naturally. Cut the dried fish skin into pieces, and soak it in 0.05 mol / L NaOH solution for 45 minutes according to a solid-liquid ratio of 1:10 (m:v) to remove the non-collagen part. Wash the residual NaOH on the fish skin with distilled water, and then stir and soak it in 0.05 mol / L HCl solution for 15 minutes according to a solid-liquid ratio of 1:10 to make the fish skin expand. Then wash it 7 to 8 times with distilled water until the pH value is close to neutral. Mix the wet fish skin with distilled water at a ratio of 1:10 (m / v), and heat it in a water bath at 60 °C for 6 hours. Filter out larger impurities with non-woven fabric, and then centrifuge at 10,000 g for 15 minutes at room temperature. Collect the supernatant and perform freeze-drying treatment with a freeze dryer for further research.

[0053] In addition to the same steps as the above fish skin gelatin extraction process, the extraction process of tilapia fish scale gelatin also requires the following steps after soaking in NaOH: Soak it in 0.4 mol / L HCl solution for 3 hours according to a solid-liquid ratio of 1:10 for decalcification treatment.

[0054] 2. Preparation of Enzymatic Hydrolysate

[0055] (1) Experimental Materials

[0056] Trypsin and Alkaline Protease

[0057] (2) Experimental Methods

[0058] For fish skin and fish scale gelatin, alkaline protease and trypsin hydrolysis were carried out respectively under the conditions of enzyme addition amount (mass ratio: 2%), optimal temperatures of 37°C (trypsin) and 50°C (alkaline protease), optimal pH values of 7.5 (trypsin) and 8.0 (alkaline protease), and enzymatic hydrolysis time of 6 h. The most suitable pH and temperature were selected for both proteases. During the hydrolysis process, 0.05 mol / L NaOH and 0.05 mol / L HCl were used to adjust and maintain the required pH value through a pH meter. After the hydrolysis was completed, the hydrolyzate was heated in a boiling water bath for 10 minutes to inactivate the protease. Then, the hydrolysis product was centrifuged at 10000 g for 10 minutes at room temperature, and the supernatant was collected and freeze-dried for further use.

[0059] 3. LC-MS / MS Identification and Computer-Aided Screening of Enzymatic Hydrolysate

[0060] Experimental Method: First, the peptides identified by LC-MS / MS were de-duplicated and then summed. Peptides with phosphorylation or dephosphorylation and other modifications at different amino acid positions were regarded as the same peptide, and their relative contents were calculated according to their intensity. To study the main active part in the enzymatic hydrolysate, machine learning screening was carried out on peptides with a content percentage of more than 0.01% (fish skin trypsin enzymatic hydrolysate: 578, accounting for about 55.52% of the total; fish scale alkaline protease enzymatic hydrolysate: 486, accounting for about 49.79% of the total; fish scale trypsin enzymatic hydrolysate: 594, accounting for about 61.74% of the total). The LC-MS / MS identification result diagram is shown in Figure 1-4 .

[0061] Use the PeptideRanker (http: / / bioware.ucd.ie / ~compass / biowareweb / ) tool to calculate the scores of peptides in the three enzymatic hydrolysates. The peptide scores given by PeptideRanker are in the range of 0 - 1. The highest score (1) indicates the peptide segment most likely to have biological activity, and the higher the value, the greater the possibility of having biological activity.

[0062] Toxicity of peptides is one of the important concerns in the development of gelatin functional food ingredients. Therefore, computer prediction of toxicity of gelatin-derived peptides with PeptideRanker scores greater than 0.5 was investigated using ToxinPred (http: / / www.imtech.res.in / raghava / toxinpred / index.html). A SVM (support vector machine) based prediction method with a threshold of 0.0 was selected to separate toxic and non-toxic peptides.

[0063] GRAVY Calculator (https: / / www.gravy-calculator.de / ) can be used to calculate the hydrophilicity of proteins or peptides based on the amino acid sequence. A value less than zero is considered hydrophilic, and a larger absolute value represents a stronger hydrophilicity (hydrophobicity). The hydrophilicity of peptides identified and screened by LC-MS / MS can be obtained based on computer prediction, which can reflect the difficulty of their synthesis.

[0064] One of the key issues in developing gelatin-derived functional food ingredients is the allergenicity of peptides. Therefore, AllerTop1.0 (https: / / ddg-pharmfac.net / AllerTOP / index.html) is used to predict the allergenicity of the screened peptides, and "NON-ALLERGEN" means non-allergenic, indicating that the peptide can be used for subsequent experiments.

[0065] 4. Molecular docking

[0066] Experimental methods: The crystal structure of EGFR was obtained from RCSB PDBk (PDB ID: 1IVO, ), first remove the same structure of the homodimer, retain only one of the chains, then eliminate the ligands and water molecules that come with the protein, check the selected protein, fix its end, and add hydrogen, and finally minimize the energy to complete the preparation of the protein receptor. For the structural preparation of the polypeptide ligand, ChemDraw 20.0 is used to draw the polypeptide sequence, and the saved cdxml file is directly imported into the ChemDraw 3D software and the PDB file of the polypeptide is exported. The PDB file of the polypeptide is hydrogenated and the energy is minimized in the Discovery Studio 2019 software to complete the structural preparation of the polypeptide ligand. The position of the EGF factor in the EGFR crystal complex is used as the center to confirm the docking box, and its size is adjusted to completely wrap the position of EGF binding, that is, to complete the confirmation of the docking box. In order to study the interaction between the peptide identified by LC-MS / MS and EGFR, the present invention defines EGFR as a receptor and the polypeptide as a ligand. Molecular docking adopts the CDOCKER semi-flexible docking method of Discovery Studio 2019 software.

[0067] Analysis results: According to the above screening and docking results, the peptides with CDOCKER less than -90 were finally selected as those with better binding efficacy for further screening. Among them, peptides with a content greater than 0.1% were considered to be abundant in the enzymatic hydrolysate. Therefore, the last six peptides were selected for analysis, considering the binding sites, the number of hydrogen bonds, and other scores comprehensively. The results showed that the interacting amino acids between GMPGERGAAGLPGLR (SEQ ID NO.1) and EGFR included Lys13, Thr15, Gln16, and Tyr45. They were also involved in the interaction sites between EGFR and EGF. GNPGAAGSAGPQGPIGPR (SEQ ID NO.2) included Lys13, Thr15, and Gln16. The two peptides mainly relied on hydrogen bonds and hydrophobic forces to maintain the structure of the complex. In terms of the number of hydrogen bonds, the hydrogen numbers of these two peptides were 8 and 6 respectively. The results indicated that the peptides GMPGERGAAGLPGLR and GNPGAAGSAGPQGPIGPR binding to EGFR might play a role in promoting the proliferation activity of osteoblasts at the active site. Therefore, these two peptides were selected for subsequent synthesis experiments. At the same time, considering that AGPPGPPGLGGNFAAQY (SEQ ID NO.3) was abundant in the enzymatic hydrolysate of fish skin trypsin and had a good molecular docking score, approximately 90.7113, three peptides were selected from the enzymatic hydrolysates of scale alkaline protease, scale trypsin, and fish skin trypsin, namely GMPGERGAAGLPGLR (GM-15), GNPGAAGSAGPQGPIGPR (GN-18), and AGPPGPPGLGGNFAAQY (AG-17). At the same time, the secondary mass spectrometry diagrams of the three peptides were obtained (see Figures 11-13 ). The 2D and 3D interaction schematic diagrams of GM-15, GN-18, and AG-17 docking with the EGFR molecule are shown in Figures 5-10 .

[0068] 5. Artificial synthesis of peptides (solid-phase synthesis method)

[0069] Experimental method: Sort the sequences of the three peptides according to "Sequence: N-terminal - A1A2A3... An - C-terminal". First, activate the resin. Weigh the resin Fmoc - An - Resin according to the required amount starting from An at the C-terminal and place it in the reaction column for soaking for 30 minutes. After soaking the resin for 30 minutes, draw off the solution, and then perform deprotection. Carry out the deprotection reaction with piperidine for 30 minutes. Within 30 minutes of deprotection, calculate the amount of amino acid required for each step and the reaction amount of the condensing agent NMM according to the amount prepared, and then weigh the next amino acid Fmoc - An - 1. After 30 minutes of deprotection, draw off the piperidine and then wash with DMF 6 times. After washing 6 times, detect the deprotection color and record it. After washing the deprotection and detecting, add the weighed materials in sequence according to the order of each peptide, then add a little reaction solution, then add base, NMM, adjust the gas evenly, wash off the resin adhered to the inner wall of the reaction column with DCM, and then record the reaction time. React for 30 minutes. After reacting for 30 minutes, draw off the reaction solution, wash with DMF 3 times and then detect to check whether the reaction is complete. After detecting that it has been connected, repeat the above steps to connect the next amino acid. If the detection fails, soak it in DCM and then, without deprotection, continue to connect the amino acid in this step until it is connected.

[0070] GM - 15, AG - 17, and GN - 18 were purified by HPLC technology to obtain the chromatograms of GM - 15, AG - 17, and GN - 18 after high - performance liquid chromatography analysis ( Figures 14-16 ). When analyzed by high - performance liquid chromatography, GM - 15, AG - 17, and GN - 18 were separated according to the separation conditions respectively and the main peaks were collected. After lyophilization, the purity of the samples could be above 95%.

[0071] 6. Osteoblast proliferation activity promoted by GM - 15, AG - 17, and GN - 18

[0072] Experimental method: Saos - 2 cells were grown in McCoy's 5A medium containing 1% antibiotic and 10% fetal bovine serum (antibiotic: 100 U / mL penicillin and 100 μg / mL streptomycin). The incubator environment was a humid environment at 37 °C with 5% CO2. Digest the cells with 0.25% trypsin every 2 - 3 days for sub - culture. If the sub - culture density is not reached, perform a medium change.

[0073] Use the CCK8 assay method to detect the proliferation effects of GM - 15, AG - 17, and GN - 18 on Saos - 2 cells respectively. Seed the cells at 5×10 per well 3Inoculate the cells at a density into a 96-well plate and culture them for 24 hours at 37°C and 5% CO₂. Then, replace the complete medium with a basal medium (McCoy's 5A basal medium supplemented with 1% penicillin-streptomycin and no serum) for 4 hours to keep the cells in a uniform state. Then, replace the basal medium with complete media containing GM-15, AG-17, or GN-18 at concentrations of 0 μg / mL (blank control), 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL, respectively, and incubate for 24 hours. Subsequently, discard the medium and gently rinse with PBS. Add CCK8 solution (v:v = 10% in complete medium) to each well and incubate in the dark in the cell culture incubator for 1 hour until the corresponding absorbance is reached. Finally, measure the absorbance at 450 nm using a microplate reader.

[0074] Analysis results: As Figure 17 shown, compared with the blank control group, both GM-15 and GN-18 showed better osteoblast proliferation activity with increasing concentration. Among them, GM-15 showed the best proliferation activity at a concentration of 10 μg / mL, and the proliferation effect of GN-18 continuously increased with increasing concentration. The proliferation effect of AG-17 was not obvious, and only at a concentration of 50 μg / mL showed good proliferation activity.

[0075] 7. Effects of GM-15, AG-17, and GN-18 on ALP activity and staining

[0076] Experimental method:

[0077] ALP activity: Inoculate the cells into a 6-well plate at a density of 1×10 6 cells / well. When the cell density grows to about 80%, treat with differentiation media containing different concentrations of osteogenic peptides GM-15, AG-17, or GN-18 (0, 1, 10, and 50 μg / mL) for 7 days. The differentiation medium is changed every two days. After 7 days, the protein concentration and ALP activity of the cells are measured using a BCA protein assay and an ALP assay kit, respectively.

[0078] ALP staining: Inoculate the cells into a 12-well plate at a density of 1×10 5Cells / well. When the cell density reaches about 80%, the cells are treated with differentiation media containing different concentrations of osteogenic peptides GM-15, AG-17, or GN-18 (0, 1, 10, and 50 μg / mL) for 7 days. The differentiation media is changed every two days. An ALP staining kit is used to stain alkaline phosphatase (ALP) in Saos-2 cells after 7 days of differentiation. The cells are fixed with 4% paraformaldehyde at 37°C for 15 minutes, and the ALP staining procedure is started. The samples are incubated with a solution containing nitroblue tetrazolium chloride and 5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt for 30 minutes until the desired color intensity is achieved, and micrographs are captured using an inverted optical microscope.

[0079] Result analysis: As Figure 18 shown, after induction with differentiation media supplemented with GM-15, AG-17, and GN-18, the staining intensity was positively correlated with the concentration, and the best promotion effect on differentiation was shown at the highest concentration of 50 μg / mL. Moreover, all three osteogenic peptides could significantly improve the differentiation degree of Saos-2. At the same time, the results of the induction and differentiation of Saos-2 cells by the three enzymatic hydrolysates were compared. The promotion effect of the enzymatic hydrolysates at the same concentration (400 μg / mL) on Saos-2 cells was not as obvious as that of the osteogenic peptides. However, according to the measurement results of the ALP alkaline phosphatase detection kit, the enzymatic hydrolysates at this concentration also played a positive role in the differentiation of Saos-2 cells.

[0080] 8. Effects of GM-15, AG-17, and GN-18 on Mineralization Degree and Alizarin Red Staining

[0081] Experimental method:

[0082] Mineralization degree: Cells are seeded in 12-well plates at a density of 3×10 6 Cells / well. When the cell density reaches about 80%, the cells are treated with differentiation media containing different concentrations of osteogenic peptides GM-15, AG-17, or GN-18 (0, 1, 10, and 50 μg / mL) for 21 days. The differentiation media is changed every two days, and in the middle and late stages of differentiation, the media is changed in a semi-replacement manner to prevent the calcium nodules from being washed away.

[0083] Mineralization staining: An alizarin red staining kit is used to stain the calcium nodules in Saos-2 cells after 21 days of differentiation to compare their mineralization degree. The cells are fixed with 4% paraformaldehyde at 37°C for 30 minutes, and the alizarin red staining procedure is started. Then, the stained calcium nodules are dissolved with a 10% (w / v) cetylpyridinium chloride solution for 30 minutes, and subsequently, the extracted stained calcium nodules are measured with an enzyme-linked immunosorbent assay (ELISA) reader at 562 nm.

[0084] Result analysis: Mineralization is the last step of osteoblast differentiation and can intuitively reflect the degree of bone formation. Alizarin red chelates with calcium ions to produce a dark red or purple complex, which is commonly used to observe calcium nodules during the mineralization process of osteoblasts. As Figure 19 shown, the number of mineralized nodules in the blank control group was the lowest. After treatment with GM-15, AG-17, GN-18, and the three enzymatic hydrolysates, the number of mineralized nodules increased significantly, and the staining intensity of alizarin red also increased significantly. However, the results of the mineralization degree and staining performance did not show a concentration-dependent relationship, indicating that mineralization is a long-term and very complex system and process. However, as long as GM-15, AG-17, or GN-18 collagen peptides are added, a significant increase in the mineralization degree and a deepening of the staining degree can be significantly observed.

[0085] 9. qPCR Analysis of GM-15, AG-17, and GN-18

[0086] Experimental method: Saos-2 cells were seeded in 6-well plates at a density of 1×10 6 cells / well. When the cells grew to 80% confluence, they were treated with differentiation medium containing 50 μg / mL osteogenic peptide GM15, AG17, or GN18 for 7 days, and the differentiation medium was changed every two days. The total RNA samples obtained were reverse transcribed into cDNA using the oligo(dT) primer of the PrimeScript cDNA Synthesis Kit. Using GADPH as the internal reference gene, the relative expression levels of ALP, IBSP, OCN, RUNX2, COL1A1, OSX, β-catenin, GSK-3Beta, and LRP5 were measured. The fold change was calculated using the 2 -ΔΔCt method.

[0087] Result analysis:

[0088] Osteogenesis-related genes (6): To confirm the above speculation, the present invention detected the expression of osteogenic activity-related genes. Since the incubation time was 7 days for all, the concentration with the best ALP activity (50 μg / mL) was selected for qPCR analysis. ALP is an early marker of differentiation, while IBSP appears in the middle stage of osteogenic differentiation. OCN is mainly synthesized and secreted by mature osteoblasts and is the main non-collagen protein in the bone matrix. It promotes mineral deposition and calcification in normal bone tissue and its regulation of bone, and its production time is in the late stage of differentiation. The Coll-α gene (i.e., the collagen alpha chain gene) is mainly responsible for encoding collagen, which is a structural protein widely present in connective tissues. Runx2 has been described as a key osteogenic transcription factor involved in the differentiation and maturation of osteoblasts, which is crucial for bone tissue formation and mineralization. OSX is a direct downstream transcriptional regulator of Runx2 and is also a marker of bone formation. As Figure 20As shown in A - F, compared with the blank control group, both osteogenic peptides and enzymatic hydrolysates showed enhanced expression of osteogenesis - related genes. Among them, GN - 18 showed good significance in all six genes, proving that it regulates the entire process of osteoblast proliferation and differentiation. AG - 17 significantly up - regulated the RUNX - 2 gene by more than 600%, indicating that it may promote osteogenesis by participating in regulatory factors and pathways related to RUNX - 2. GM - 15 played an obvious role in up - regulating the ALP gene, which proved that it regulated osteogenesis by regulating the ALP gene in the early stage of osteogenic differentiation. From the above, it can be concluded that all collagen peptides can promote the expression of related genes in osteoblasts, but the regulatory methods for their expression still need to be explored through subsequent experiments.

[0089] Genes related to the Wnt / β - catenin pathway (3): There are some signaling pathways that regulate the proliferation and differentiation of osteoblasts, including the OPG / RANKL / RANK signaling pathway, BMPs signaling pathway, MAPK signaling pathway, and Wnt / β - catenin signaling pathway. Among these pathways, the Wnt / β - catenin signaling pathway is the most commonly used way to study osteogenesis. It is speculated in this invention that these three collagen peptides may promote the differentiation of Saos - 2 cells by docking with EGFR and screening, and may activate the Wnt / β - catenin signaling pathway. The mRNA and protein expressions of Wnt / β - catenin - related factors were determined, including signal pathways involving β - catenin, LRP5, and GSK3β, to verify the hypothesis of this invention. All three collagen peptides significantly down - regulated the expression of the GSK3β gene. The over - expression of GSK3β inhibits Wnt / β - catenin, indicating that GM - 15, AG - 17, and GN - 18 can all express the Wnt / β - catenin pathway by inhibiting GSK3β ( Figure 20 as shown in G - I). β - catenin is an important factor in this pathway. AG - 17 showed an expression level of more than 200% of β - catenin, and the expressions of the other two peptides were not significant, indicating that AG - 17 is most likely to regulate the proliferation and differentiation of osteoblasts through the Wnt / β - catenin pathway.

[0090] The above - mentioned embodiments only describe the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A polypeptide GM-15 that promotes the proliferation and differentiation of osteoblasts, characterized in that, The amino acid sequence is as shown in SEQ ID NO.

1.

2. A polypeptide AG-17 that promotes osteoblast proliferation and differentiation, characterized in that, The amino acid sequence is as shown in SEQ ID NO.

3.

3. Use of a polypeptide in the preparation of a drug for preventing and / or treating osteoporosis, characterized in that, The amino acid sequence of the polypeptide is as shown in any one of SEQ ID NOs. 1-3.

4. Use of a polypeptide in the preparation of a product for improving bone density, characterized in that, The amino acid sequence of the polypeptide is as shown in any one of SEQ ID NOs. 1-3; The product is a health care product or a functional food.

5. A drug for preventing and / or treating osteoporosis, characterized in that, The active ingredient includes a polypeptide whose amino acid sequence is as shown in at least one of SEQ ID NOs. 1-3.

6. A health product or functional food for improving bone density, characterized in that, The active ingredient includes a polypeptide whose amino acid sequence is as shown in at least one of SEQ ID NOs. 1-3.

7. A method for preparing the polypeptide GM-15 as described in claim 1, characterized in that, It includes the step of synthesizing the polypeptide GM-15 by solid-phase synthesis method.

8. A method for preparing polypeptide AG-17 as described in claim 2, characterized in that, It includes the step of synthesizing the polypeptide AG-17 by solid-phase synthesis method.

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