Milk-derived polypeptide sequence for promoting skeletal muscle cell proliferation and application thereof

Abstract

The invention discloses a milk-derived polypeptide sequence for promoting skeletal muscle cell proliferation and application of the milk-derived polypeptide sequence, and belongs to the field of bioactive peptides. The milk-derived bioactive polypeptide RHPHPHLSF provided by the invention has the effect of promoting muscle cell proliferation, and is expected to be used for preparing medicines for promoting muscle cell proliferation and preventing sarcopenia. The milk-derived bioactive polypeptide RHPHPHLSF is convenient to synthesize, can be industrially produced, and has a good application prospect in the fields of foods, medicines, cosmetics and the like.

Description

Technical Field

[0001] The invention relates to a milk-derived polypeptide sequence for promoting skeletal muscle cell proliferation and application thereof, belonging to the field of bioactive peptides. Background Art

[0002] Sarcopenia, also known as sarcopenia, is a progressive, systemic skeletal muscle disease associated with aging, characterized by decreased muscle mass, decreased muscle strength, and decreased muscle physiological function.

[0003] Sarcopenia is characterized by loss of muscle mass and impaired muscle function, including a decrease in muscle cross-sectional area and muscle strength due to aging without the body suffering from disease. At the same time, sarcopenia is often accompanied by metabolic abnormalities, including impaired insulin sensitivity, impaired oxidative defense, and decreased mitochondrial function. Various treatments have emerged to slow down sarcopenia. The usual treatment for sarcopenia is to regulate hormone levels. It has been shown that the administration of growth hormone and / or testosterone can alleviate sarcopenia by improving certain muscle functions in elderly men. However, testosterone administration is associated with a greater risk of polycythemia vera, venous thromboembolism, prostate cancer, and cardiovascular events. Therefore, the search for safer, food-derived natural alternatives that can promote muscle proliferation and alleviate muscle damage is receiving increasing attention.

[0004] In recent years, bioactive peptides and proteins are expected to make further progress in the treatment of muscle abnormalities. For example, Kitakaze et al. revealed that lactoferrin promotes C2C12 proliferation and differentiation, as well as myotube hypertrophy. And Iwasa et al. determined that peptides derived from the hydrolysis of milk casein promote glucose uptake in skeletal muscle cells and optimize human metabolism. However, compared with the rich milk-derived peptides, research reports are still relatively limited. Therefore, it is meaningful to explore milk-derived bioactive peptides that promote skeletal muscle health and prevent sarcopenia. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of a bioactive peptide in promoting skeletal muscle cell proliferation and / or preventing sarcopenia.

[0006] The present invention provides a bioactive peptide: RHPHPHLSF (9 peptides, whose amino acid sequence is Arg-His-Pro-His-Pro-His-Leu-Ser-Phe) and ARHPHPHLSF (10 peptides, whose amino acid sequence is Ala-Arg-His-Pro-His-Pro-His-Leu-Ser-Phe).

[0007] In one embodiment of the present invention, the bioactive polypeptides are milk-derived polypeptides, ARHPHPHLSF (10 peptides, SEQ ID NO. 2) and RHPHPHLSF (9 peptides, SEQ ID NO. 3) are derived from κ-casein, and are located at positions 96-105 and 97-105, respectively.

[0008] The amino acid sequence of the κ-casein is shown in SEQ ID NO.1.

[0009] κ-Casein (SEQ ID NO.1)

[0010] MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKDERFFSDKIAKYIPIQYVLSRYPSYGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTM ARHPHPHLSF MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV.

[0011] In one embodiment of the present invention, the bioactive peptide is used as food, medicine or health product; it is used to promote skeletal muscle cell proliferation and promote muscle cell growth.

[0012] Preferably, the food, medicine, health product or nutritional product may also contain a derivative of the polypeptide; the polypeptide derivative refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino terminal or carbonyl terminal of the polypeptide;

[0013] The drug also contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field;

[0014] Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition;

[0015] Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form;

[0016] Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions;

[0017] Preferably, the injection dosage form includes but is not limited to injection solution and injection powder;

[0018] Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

[0019] Preferably, the food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods;

[0020] The health care product also contains acceptable auxiliary materials.

[0021] The present invention also provides an expression vector or a recombinant microorganism, wherein the expression vector or the recombinant microorganism comprises the above-mentioned ARHPHPHLSF peptide and / or RHPHPHLSF peptide.

[0022] Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector;

[0023] Preferably, the recombinant microorganism is a bacterium or a fungus.

[0024] The present invention also provides a food, medicine, health product or nutritional product, wherein the food, medicine, health product or nutritional product comprises an effective dose of at least one of the polypeptides;

[0025] Preferably, the food, medicine, health product or nutritional product may also contain a derivative of the polypeptide; the polypeptide derivative refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino terminus or carbonyl terminus of the polypeptide.

[0026] In one embodiment of the present invention, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field;

[0027] Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition;

[0028] Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form;

[0029] Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions;

[0030] Preferably, the injection dosage form includes but is not limited to injection solution and injection powder;

[0031] Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

[0032] In one embodiment of the present invention, the food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, bean products, and beverages; the food also includes special dietary foods;

[0033] The health care product also contains acceptable auxiliary materials.

[0034] The present invention also provides the use of the above-mentioned ARHPHPHLSF peptide and / or RHPHPHLSF peptide in the preparation of food, medicine, health care product or nutritional product; the food, medicine, health care product or nutritional product is used to promote skeletal muscle cell proliferation and promote muscle cell growth.

[0035] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable carrier.

[0036] In one embodiment of the present invention, the application includes but is not limited to promoting muscle cell proliferation.

[0037] The drug also contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field;

[0038] Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition;

[0039] Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form;

[0040] Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions;

[0041] Preferably, the injection dosage form includes but is not limited to injection solution and injection powder;

[0042] Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

[0043] In one embodiment of the present invention, the food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods;

[0044] The health care product also contains acceptable auxiliary materials.

[0045] The present invention also provides the use of the bioactive peptide RHPHPHLSF and / or ARHPHPHLSF peptide in the preparation of health products that help improve muscle quality.

[0046] In one embodiment of the present invention, the health care products include but are not limited to functional fermented dairy products.

[0047] The present invention also provides the use of the bioactive peptide RHPHPHLSF and / or ARHPHPHLSF peptide in the preparation of a drug for promoting skeletal muscle cell proliferation and / or preventing sarcopenia.

[0048] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable carrier.

[0049] In one embodiment of the present invention, the application includes but is not limited to promoting muscle cell proliferation.

[0050] The drug also contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field;

[0051] Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition;

[0052] Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form;

[0053] Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions;

[0054] Preferably, the injection dosage form includes but is not limited to injection solution and injection powder;

[0055] Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

[0056] The present invention also provides a product for promoting muscle cell proliferation, wherein the product comprises a bioactive peptide RHPHPHLSF or ARHPHPHLSF peptide or a derivative of the bioactive peptide RHPHPHLSF or ARHPHPHLSF peptide;

[0057] In one embodiment of the present invention, the product for promoting muscle cell proliferation includes health products for promoting muscle quality improvement and drugs for promoting muscle cell proliferation; the derivative of the biologically active polypeptide RHPHPHLSF or ARHPHPHLSF peptide refers to a polypeptide derivative obtained by modifying the amino acid side chain group, amino terminus or carbonyl terminus of the biologically active polypeptide RHPHPHLSF or ARHPHPHLSF peptide by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation.

[0058] Beneficial Effects

[0059] The milk-derived bioactive polypeptide RHPHPHLSF of the present invention has a significant effect of promoting skeletal muscle cell proliferation, and is expected to be used to prepare drugs or functional foods for promoting skeletal muscle cell proliferation and preventing sarcopenia. The RHPHPHLSF of the present invention is easy to synthesize, can be industrially produced, and has good application prospects in the fields of food, medicine, and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 :Schematic diagram of docking of whey peptides with FGFR1 receptor molecules. Pymol 1.5 and LigPlot are used to show the specific binding sites between peptides and FGFR1. The interaction forces include hydrogen bonding and hydrophobic interaction.

[0061] Figure 2 : Effect of peptides on the proliferation of skeletal muscle cells. The bioactive peptide RHPHPHLSF at a concentration of 0.1 μM was tested for its effect on the proliferation of skeletal muscle cells; the CCK-8 assay was quantified by measuring the absorbance at 450 nm with a microplate reader. n = 3, data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey's multiple comparison test (p < 0.05).

[0062] Figure 3 :EdU proliferation staining results of peptides. (a) Cells were treated with modified DMEM medium and different concentrations of 0.1μM bioactive peptide RHPHPHLSF. Then, cells were stained with EdU staining solution and images were taken with an inverted fluorescence microscope; (b) The number of positive cells was counted using image software, and the data are the mean ± SEM from 9 independent experiments. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 compared with the control group.

[0063] Figure 4 :Effects of peptides on muscle cell cycle. The effects of the bioactive peptide RHPHPHLSF at a concentration of 0.1 μM on the skeletal muscle cell cycle were tested; the number of cells in each period was collected and analyzed by flow cytometry, and the data were plotted and analyzed using Modefit5. n=3, data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey's multiple comparison test (p<0.05).

[0064] Figure 5:Effect of peptides on the content of reactive oxygen species in muscle cells. The bioactive peptide RHPHPHLSF at a concentration of 0.1 μM was tested for its effect on reactive oxygen species damage in skeletal muscle cells; the relative content of reactive oxygen species in cells was quantified by measuring the absorbance at 532 nm using a microplate reader. n=3, data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey's multiple comparison test (p<0.05). ****p<0.0001, compared with the control group.

[0065] Figure 6 : Effect of peptides on MDA content in muscle cells. The bioactive peptide RHPHPHLSF at a concentration of 0.1 μM was tested for its effect on oxidative damage in skeletal muscle cells; the MDA content in cells was quantified by measuring the absorbance at 532 nm with a microplate reader and normalized with BCA protein content. n = 3, data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey's multiple comparison test (p < 0.05). *p < 0.05, **p < 0.01, compared with the control group. DETAILED DESCRIPTION

[0066] The EdU working solution and cell cycle detection kit involved in the following examples were purchased from Wuhan Saiweier Biotechnology Co., Ltd. and Shanghai Biyuntian Biotechnology Co., Ltd., respectively.

[0067] The LC-MS / MS analysis method involved in the following embodiments is:

[0068] LC-MS / MS analysis was performed using an EASY nLC1200 system equipped with an Acclaim PepMapTM RSLC column (50μm×15cm, 2μm, 100μm). The mobile phase A consisted of an aqueous solution containing 0.1% (v / v) formic acid and 2% (v / v) acetonitrile, while the mobile phase B was an aqueous solution containing 0.1% (v / v) formic acid and 90% (v / v) acetonitrile. The gradient elution program was: 6% to 20% B, 0-40min; 20% to 32% B, 40-52min; 32% to 80% B, 52-56min, with a flow rate of 0.2μL / min. The MS spectrum acquisition range was 150-2000m / z, with a resolution of 60000. The MS / MS data collection range was 180-2000m / z, with a scan time of 0.1s.

[0069] The active peptide sequence identification method involved in the following embodiments is:

[0070] Peptide identification was performed using bovine proteins from the UniProt database (https: / / www.uniprot.org / ) as a reference database. Raw data were retrieved using Maxquant (v.1.5.2.8) in nonspecific enzyme fragmentation mode with a mass tolerance of 0.02 Da, a tolerance of 10 ppm for de novo synthesis, and a tolerance of 7 ppm for isotope removal in MS / MS. The false discovery rate (FDR) threshold for protein and peptide matching was set to 1%. To find bioactive peptides, each sample was analyzed in triplicate, and if a peptide appeared at least twice, its presence was considered significant.

[0071] The method for screening the docking ability of whey peptides with FGFR1 receptor molecules involved in the following embodiments:

[0072] For virtual screening and molecular docking, the molecular structure of FGFR1 receptor (PDB ID: 1EVT) was downloaded from the RCSB Protein Data Bank (https: / / www.rcsb.org / ). Before docking, water molecules and irrelevant atoms were removed. Peptide structures were built and energy minimized using Discovery Studio 2019. Virtual screening was performed using AutoDock Wiener 1.2. For the docking method reference, the center position of the box was set to -10.083, 2.000, -12.444, the size was 120 × 120 × 120, and other parameters remained default. The conformation with the highest affinity was selected as the result. Under the same conditions, the binding mode and orientation of the FGFR1 receptor were analyzed and selected. The top three conformations of each peptide docked with FGFR1 were generated, and the best conformation was selected for further analysis based on the docking results. Discovery Studio (DS) 2019, Pymol 1.5 and LigPlot were used.

[0073] The culture medium involved in the following examples is as follows:

[0074] DMEM complete medium: purchased from Thermo Fisher Scientific (China) Co., Ltd., Shanghai, China.

[0075] Example 1: Preparation of biologically active polypeptides

[0076] 1. Separation and preparation of peptides

[0077] (1) Preparation of fermented whey

[0078] The purified strain CCFM1263 (described in the Chinese invention patent application document with publication number CN117305158A) was subcultured on the first-generation MRS solid plate and the second-generation MRS liquid tube, centrifuged at 6000×g for 5 min, resuspended in 11% (w / w) skim milk, inoculated into 11% (w / w) skim milk at a 2% (v / v) inoculum, shaken evenly, and cultured in an anaerobic incubator at 37°C for 18 h. Then, the skim milk was subcultured in the same way, and after culturing in an anaerobic incubator at 37°C for 18 h, the protein was washed with Tris acid, and the viable count was adjusted to 1×10 6 CFU / mL, inoculated with 11% (w / w) skim milk at an inoculum size of 2% (v / v), and fermented in a constant temperature incubator at 37°C for 48 h.

[0079] Take different fermented milk samples, add 1 mol / L sodium hydroxide solution to adjust the sample pH to 4.6, and pasteurize in a high pressure steam sterilizer at 95°C for 10 minutes. Centrifuge the sterilized fermented milk sample at 10000×g, 4°C for 10 minutes, take the supernatant, and filter it with a 0.45μm organic filter membrane to obtain the Lactobacillus helveticus fermented milk whey sample. The collected Lactobacillus helveticus fermented milk whey is freeze-dried into powder and placed in a freeze-drying bottle at 4°C for standby use.

[0080] (2) Desalting and purifying the fermented whey using a C18 solid phase extraction column. The sample was desalted and purified using a C18 solid phase extraction desalting column with a sample loading of 3 mg. The process was divided into five steps: moistening, leveling, loading, desalting, and washing, and the flow rate was controlled by a constant pressure pump.

[0081] (3) After purification, the peptide solution was dried using a cold centrifugal vacuum concentrator and reconstituted in an aqueous solution consisting of 0.1% (v / v) formic acid and 2% (v / v) acetonitrile to achieve a peptide concentration of 0.5 mg / mL. After stirring the solution at 12000g and 4°C for 15 min, the whey was collected and stored in a sample vial before LC-MS / MS analysis.

[0082] The sequences of the peptides in the mixed solution were analyzed.

[0083] 2. Identification of active peptide sequences and screening of whey peptides for their ability to dock with FGFR1 receptor molecules

[0084] The peptide sequence obtained in step 1 was identified, and the whey peptide was screened for its docking ability with the FGFR1 receptor molecule.

[0085] The results are shown in Table 1

[0086] Table 1: Molecular docking results

[0087]

[0088]

[0089] The results showed that the lower the binding energy, the better the binding effect with the receptor. Finally, two groups of peptides with good virtual binding effect with the receptor were obtained, ARHPHPHLSF and RHPHPHLSF. The schematic diagram of the docking of the two groups of peptides with FGFR1 molecules is shown in Figure 1 shown.

[0090] Example 2: Effects of bioactive peptides on muscle cells

[0091] 1. Effect on muscle cell proliferation

[0092] The specific method is as follows:

[0093] (1) ARHPHPHLSF polypeptide powder and RHPHPHLSF polypeptide powder were prepared by chemical synthesis by Shanghai Jier Peptide Co., Ltd.

[0094] (2) Adding the ARHPHPHLSF and RHPHPHLSF polypeptide powders obtained in step (1) to DMEM complete medium respectively, so that the final concentrations of the polypeptides in the medium are 0.001 μmol / L, 0.01 μmol / L, and 0.1 μmol / L, respectively. Finally, DMEM complete medium containing 0.001 μmol / L, 0.01 μmol / L, and 0.1 μmol / L of the bioactive peptide ARHPHPHLSF is prepared;

[0095] DMEM complete medium containing 0.001 μmol / L, 0.01 μmol / L, and 0.1 μmol / L bioactive peptide RHPHPHLSF.

[0096] (3) Mouse myoblast C2C12 cells were inoculated in a 96-well plate at a seeding volume of 2×10 4 cells / well, cultured at 37°C for 24h, then added DMEM complete medium containing 0μmol / L, 0.001μmol / L, 0.01μmol / L, and 0.1μmol / L bioactive peptides ARHPHPHLSF (named: ARH10) and RHPHPHLSF (named: RH9), respectively, and cultured at 37°C for 24h. Then, 10μL of CCK-8 solution was added to each well under sterile conditions, and the cells were cultured in an incubator (constant temperature of 37°C) for 30min. The cells were shaken and the absorbance was measured at 450nm with an enzyme reader.

[0097] The results are shown in Table 2 and Figure 2 As shown:

[0098] Table 2: Effects of different concentrations of peptides on muscle cell proliferation

[0099]

[0100]

[0101] The results showed that the milk peptide RHPHPHLSF can promote the proliferation of myoblasts, and the rate of increase increases with the increase in dosage. When the concentration of the milk peptide RHPHPHLSF is 0.1 μmol / L, the rate of increase reaches 127%. ARHPHPHLSF can also promote the proliferation of myoblasts, and the rate of increase increases with the increase in dosage.

[0102] 2. Effects on EdU proliferation staining of muscle cells

[0103] The specific method is as follows:

[0104] (1) ARHPHPHLSF polypeptide powder and RHPHPHLSF polypeptide powder were prepared by chemical synthesis by Shanghai Jier Peptide Co., Ltd.

[0105] (2) Add the ARHPHPHLSF polypeptide and RHPHPHLSF polypeptide powders obtained in step (1) to DMEM complete medium respectively, so that the final concentration of the polypeptide in the medium is 0.1 μmol / L. DMEM complete medium containing ARHPHPHLSF polypeptide at a final concentration of 0.1 μmol / L and DMEM complete medium containing RHPHPHLSF polypeptide at a final concentration of 0.1 μmol / L are prepared.

[0106] (3) Mouse myoblast C2C12 cells were inoculated in a 96-well plate at a seeding volume of 2×10 4 cells / well, cultured at 37°C for 24 h, and then added with DMEM complete culture medium containing 0 μmol / L and 0.1 μmol / L bioactive peptides ARHPHPHLSF (named: ARH10) and RHPHPHLSF (named: RH9), respectively, and cultured at 37°C for 24 h.

[0107] (4) Add 500 μL of EdU working solution preheated at 37°C to the treated cells and incubate for 2 h. Discard the EdU working solution, add 1 mL of fixative solution, and fix at room temperature for 15 min. Discard the fixative solution and wash three times with PBS containing 5% BSA. Add 1 mL of 0.3% Triton X-100 to each well and incubate at room temperature for 15 min. Discard Triton X-100 and wash three times with the wash solution. Add 500 μL of Click reaction solution and incubate at room temperature in the dark for 30 min. Discard the Click reaction solution and wash three times with the wash solution. Add 1 mL of Hoechsty solution and incubate at room temperature in the dark for 10 min. Discard the Hoechsty solution and wash three times with the wash solution. Collect images using a fluorescence microscope.

[0108] The results are shown in Table 3 and Figure 3 As shown:

[0109] Table 3: Effects of different concentrations of peptides on the proportion of positive cells in muscle cells

[0110]

[0111] The results are as follows Figure 3 As shown, the milk peptide RHPHPHLSF can significantly increase the proportion of positive cells in myoblasts, and the proliferation effect is significantly higher than the proliferation effect of ARHPHPHLSF on C2C12. When the concentration of the milk peptide RHPHPHLSF is 0.1 μmol / L, the proportion of positive cells increases by 20.11% compared with the normal group.

[0112] 3. Impact on the growth cycle of muscle cells

[0113] The specific method is as follows:

[0114] (1) ARHPHPHLSF polypeptide powder and RHPHPHLSF polypeptide powder were prepared by chemical synthesis by Shanghai Jier Peptide Co., Ltd.

[0115] (2) Add the ARHPHPHLSF polypeptide and RHPHPHLSF polypeptide powders obtained in step (1) to DMEM complete medium respectively, so that the final concentration of the polypeptide in the medium is 0.1 μmol / L. DMEM complete medium containing ARHPHPHLSF polypeptide at a final concentration of 0.1 μmol / L and DMEM complete medium containing RHPHPHLSF polypeptide at a final concentration of 0.1 μmol / L are prepared.

[0116] (3) Mouse myoblast C2C12 cells were inoculated in a 12-well plate at a seeding volume of 1×10 5 cells / well, cultured at 37°C for 24 h, and then added with DMEM complete culture medium containing 0 μmol / L and 0.1 μmol / L bioactive peptides ARHPHPHLSF (named: ARH10) and RHPHPHLSF (named: RH9), respectively, and cultured at 37°C for 24 h.

[0117] (4) Discard the culture medium in the culture plate, use the lysis solution to lyse the cells, collect the cells into a 1.5 mL centrifuge tube, discard the supernatant after centrifugation, add pre-cooled 0.5 mL PBS buffer solution to dissolve the precipitate, discard the supernatant after centrifugation, and repeat washing with PBS solution 2-3 times. Add propidium iodide (PI) staining solution to blow away the cells, and then stain them overnight at 4°C in the dark (more than 24 hours). Finally, use a flow cytometer to analyze the fluorescence of the cells. The DNA signal represented by the red fluorescence staining intensity after PI staining is collected by a computer, while the forward scatter (FSC) and side scatter (SSC) data are collected in real time. All data are analyzed using Modefit5.

[0118] The results are shown in Table 4 and Figure 4 As shown:

[0119] Table 4: Effects of different peptides on the cell cycle of muscle cells

[0120]

[0121] The results are as follows Figure 4 As shown, milk-derived RHPHPHLSF can accelerate the process of muscle cell cycle. When the concentration of milk-derived peptide RHPHPHLSF was 0.1 μmol / L, compared with the normal group, RHPHPHLSF significantly reduced the number of G0 / G1 phase cells by 5.6%, and significantly increased the number of S phase cells by 5.71%, indicating that RHPHPHLSF treatment accelerated the cell cycle process and promoted cell growth.

[0122] 4. Determination of ROS content in muscle cells

[0123] The specific method is as follows:

[0124] (1) ARHPHPHLSF polypeptide powder and RHPHPHLSF polypeptide powder were prepared by chemical synthesis by Shanghai Jier Peptide Co., Ltd.

[0125] (2) The ARHPHPHLSF and RHPHPHLSF polypeptide powders obtained in step (1) are added to the DMEM complete medium respectively, so that the final concentration of the polypeptide in the medium is 0.1 μmol / L. At the same time, the TNF-α powder synthesized by Shanghai Biochemical is dissolved in the DMEM medium, so that the final concentration of TNF-α in the medium is 20 ng / mL. Finally, the following are prepared: DMEM complete medium containing 0.1 μmol / L bioactive peptide ARHPHPHLSF, DMEM complete medium containing 0.1 μmol / L bioactive peptide RHPHPHLSF, and DMEM complete medium containing 20 ng / mL TNF-α.

[0126] (3) Mouse myoblast C2C12 cells were seeded into six-well plates, with 1×10 cells per well. 5 After culturing for 24 hours, the cells were divided into groups for intervention. DMEM complete medium containing 0 μmol / L and 0.1 μmol / L bioactive peptides ARHPHPHLSF (named: ARH10) and RHPHPHLSF (named: RH9) and DMEM complete medium containing 20 ng / mL TNF-α were added respectively and cultured at 37°C for another 24 hours.

[0127] (4) Discard the old culture medium, add 500 μL of DCFH-DA solution with a final concentration of 10 μmol / L, and incubate at 37°C for 20-40 min. Wash three times with serum-free culture medium (purchased from Thermo Fisher Scientific) to remove DCFH-DA that has not entered the cells, and finally measure the absorbance at a wavelength of 532 nm.

[0128] The results are shown in Table 5 and Figure 5 As shown:

[0129] Table 5: Effects of different concentrations of peptides on the content of ROS in muscle cells

[0130]

[0131] The results are as follows Figure 5 As shown, compared with the modeling group, the milk-derived peptide RHPHPHLSF treatment group reduced the content of reactive oxygen species (ROS) by 1.79 times.

[0132] 5. Determination of MDA content in muscle cells

[0133] The specific method is as follows:

[0134] (1) ARHPHPHLSF polypeptide powder and RHPHPHLSF polypeptide powder were prepared by chemical synthesis by Shanghai Jier Peptide Co., Ltd.

[0135] (2) The ARHPHPHLSF and RHPHPHLSF polypeptide powders obtained in step (1) are added to the DMEM complete medium respectively, so that the final concentration of the polypeptide in the medium is 0.1 μmol / L, and the TNF-α powder synthesized by Shanghai Biochemical is dissolved in the DMEM medium, so that the final concentration of TNF-α in the medium is 20 ng / mL. Finally, the following are prepared: DMEM complete medium containing 0.1 μmol / L bioactive peptide ARHPHPHLSF, DMEM complete medium containing 0.1 μmol / L bioactive peptide RHPHPHLSF, and DMEM complete medium containing 20 ng / mL TNF-α.

[0136] (3) Mouse myoblast C2C12 cells were seeded into six-well plates, with 1×10 cells per well. 5 After culturing for 24 hours, the cells were divided into groups for intervention. DMEM complete medium containing 0 μmol / L and 0.1 μmol / L bioactive peptides ARHPHPHLSF (named: ARH10) and RHPHPHLSF (named: RH9) and DMEM complete medium containing 20 ng / mL TNF-α were added respectively and cultured at 37°C for another 24 hours.

[0137] (4) The treated cells were lysed with lysis buffer, collected in a 1.5 mL centrifuge tube, and centrifuged at 12000 g for 10 minutes to obtain the supernatant for subsequent determination. Add 0.1 ml of homogenate and lysis buffer to a new centrifuge tube as a blank control, add 0.1 ml of standard of different concentrations to prepare a standard curve, add 0.1 ml of sample for determination; then add 0.2 ml of MDA detection working solution. After mixing, heat at 100 ° C or in a boiling water bath for 15 minutes. Cool to room temperature in a water bath and centrifuge at 1000 g for 10 minutes at room temperature. Take 200 μL of supernatant and add it to a 96-well plate, and then measure the absorbance at 532 nm using an enzyme reader.

[0138] The results are shown in Table 6 and Figure 6 As shown:

[0139] Table 6: Effects of different concentrations of peptides on the content of MDA in muscle cells

[0140]

[0141] The results are as follows Figure 6 As shown, compared with the modeling group, the milk-derived peptide RHPHPHLSF-treated group reduced the MDA content by 1.15 times.

[0142] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A polypeptide, characterized in that The polypeptide is RHPHPHLSF.

2. The polypeptide according to claim 1, characterized in that It is used as food, medicine or health product; it is used to promote skeletal muscle cell proliferation and promote muscle cell growth.

3. An expression vector or a recombinant microorganism, characterized in that: The expression vector or recombinant microorganism comprises the polypeptide according to claim 1; Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector; Preferably, the recombinant microorganism is a bacterium or a fungus.

4. A food, medicine, health product or nutritional product, characterized in that: The food, medicine, health product or nutritional product contains an effective dose of the polypeptide according to claim 1 or 2; preferably, the food, medicine, health product or nutritional product may also contain the polypeptide according to claim 1 or 2; the polypeptide derivative refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino terminal or carbonyl terminal of the polypeptide; Preferably, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition; Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form; Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions; Preferably, the injection dosage form includes but is not limited to injection solution and injection powder; Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

5. The food, medicine, health product or nutritional product according to claim 4, characterized in that: The food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods; The health care product also contains acceptable auxiliary materials.

6. Use of ARHPHPHLSF peptide and / or RHPHPHLSF peptide in the preparation of food, medicine, health care product or nutritional product; the food, medicine, health care product or nutritional product is used to promote skeletal muscle cell proliferation and promote muscle cell growth.

7. The use according to claim 6, characterized in that: The drug also contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition; Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form; Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions; Preferably, the injection dosage form includes but is not limited to injection solution and injection powder; Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

8. The use according to claim 6, characterized in that: The food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods; The health care product also contains acceptable auxiliary materials.

9. Use of ARHPHPHLSF peptide and / or RHPHPHLSF peptide in the preparation of health products that help improve muscle quality or in the preparation of drugs that promote skeletal muscle cell proliferation and / or prevent sarcopenia.

10. A method for preparing the food, medicine, health product or nutritional product according to claim 4 or 5, characterized in that: The method comprises mixing the polypeptide or the derivative of the polypeptide as claimed in claim 1 or 2 with at least one acceptable excipient; preferably, the polypeptide derivative refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino terminus or carbonyl terminus of the polypeptide.

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