Milk-derived polypeptide vsl for promoting proliferation of skeletal muscle cells and application thereof

Abstract

The application discloses a milk-derived polypeptide VSL for promoting proliferation of skeletal muscle cells and application thereof, and belongs to the field of bioactive peptides. The milk-derived bioactive polypeptide VSL has the effect of promoting proliferation of muscle cells, and is expected to be used for preparing drugs for promoting proliferation of muscle cells and preventing sarcopenia. The milk-derived bioactive polypeptide VSL is convenient to synthesize, can be produced industrially, and has a good application prospect in the fields of food, medicine and cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to a milk-derived polypeptide VSL for promoting proliferation of skeletal muscle cells and use thereof, and the field of bioactive peptides. BACKGROUND

[0002] Sarcopenia, also known as muscle loss, is a progressive, systemic skeletal muscle disease associated with aging, mainly manifested as reduced muscle mass, decreased muscle strength and decreased muscle physiological function.

[0003] Sarcopenia is characterized by loss of muscle mass and impaired muscle function, including reduced muscle cross-sectional area and muscle strength due to aging, while the body is not suffering from disease. At the same time, sarcopenia is usually accompanied by metabolic abnormalities, including impaired insulin sensitivity, impaired oxidative defense, and decreased mitochondrial function. In order to slow down muscle loss, various treatment methods have emerged. The usual treatment for sarcopenia is to regulate hormone levels. It has been proven that administration of growth hormone and / or testosterone can alleviate muscle loss by improving certain muscle functions in older men. However, testosterone administration is associated with a greater risk of polycythemia, venous thromboembolism, prostate cancer and cardiovascular events. Therefore, there is increasing interest in finding safer, food-derived natural alternatives that can promote muscle proliferation and alleviate muscle damage.

[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, and myotube hypertrophy. And Iwasa et al. determined that a milk casein hydrolysis-derived peptide promotes glucose uptake in skeletal muscle cells and optimizes human metabolism. However, compared to the rich milk-derived polypeptides, the research reports are still limited. Therefore, it is meaningful to explore milk-derived bioactive peptides that promote skeletal muscle health and prevent muscle loss. SUMMARY

[0005] The purpose of the present application is to provide a new use of bioactive peptides in promoting proliferation of skeletal muscle cells and / or preventing muscle loss.

[0006] The present application provides a bioactive peptide: VSL (3 peptides, the amino acid sequence of which is Val-Ser-Leu).

[0007] In one embodiment of the present application, the bioactive polypeptide is a milk-derived polypeptide, and VSL (3 peptides) is derived from α-lactalbumin and located at positions 5-7.

[0008] The amino acid sequences of the κ-casein and α-lactalbumin are shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0009] Alpha-lactalbumin (SEQ ID NO. 2)

[0010] MMSFVSLLLVGILFHATQAEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIMCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL

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

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

[0013] The drug also contains a pharmaceutically acceptable pharmaceutical excipient; the pharmaceutical excipient refers to a conventional drug carrier in the pharmaceutical field;

[0014] Preferably, the excipient includes one or more of the following: a binder such as a cellulose derivative, an alginate, a gelatin and a polyvinylpyrrolidone; a diluent such as starch, pre-gelatinized starch, dextrin, sucrose, lactose, mannitol; a filler such as starch, sucrose; a wetting agent such as glycerol; a disintegrant such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and dry starch; an absorption promoter such as a quaternary ammonium compound; a surfactant such as polysorbate, sorbitan fatty acid and fatty acid glyceride; a coloring agent such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red and the like; a lubricant such as hydrogenated vegetable oil, talc and polyethylene glycol; a coating material such as acrylic resin, hydroxypropyl methyl cellulose, povidone, cellulose acetate phthalate; in addition, other excipients such as a flavoring agent, a sweetener can also be added in the composition;

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

[0016] Preferably, the oral dosage form includes, but is not limited to, a tablet, a capsule, a granule, an oral liquid, an oral suspension;

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

[0018] Preferably, the inhalation dosage form includes, but is not limited to, an aerosol, a powder aerosol.

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

[0020] The health product also contains acceptable adjuvants.

[0021] The present application also provides an expression vector or a recombinant microorganism containing the VSL 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 application also provides a food, a drug, a health product, or a nutritional product containing an effective dose of the above-mentioned polypeptide.

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

[0026] In an embodiment of the present application, the drug also contains a pharmaceutically acceptable pharmaceutical adjuvant; the pharmaceutical adjuvant refers to a conventional drug carrier in the pharmaceutical field;

[0027] Preferably, the adjuvant includes one or more of the following: a binder such as a cellulose derivative, an alginate, a gelatin, and a polyvinylpyrrolidone; a diluent such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; a filler such as starch, sucrose; a humectant such as glycerol; a disintegrant such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; an absorption promoter such as a quaternary ammonium compound; a surfactant such as polysorbate, sorbitan fatty acid, and fatty acid glyceride; a coloring agent such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red, etc.; a lubricant such as hydrogenated vegetable oil, talc, and polyethylene glycol; a coating material such as an acrylic resin, hydroxypropyl methyl cellulose, povidone, cellulose acetate phthalate; in addition, other adjuvants such as a flavoring agent, a sweetener can also be added to the composition;

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

[0029] Preferably, the oral dosage form includes, but is not limited to, a tablet, a capsule, a granule, an oral liquid, an oral suspension;

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

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

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

[0033] The health products also contain acceptable adjuvants.

[0034] The present application also provides the use of the above-mentioned VSL peptide in the preparation of food, medicine, health products or nutritional products; the food, medicine, health products or nutritional products are used to promote skeletal muscle cell proliferation and muscle cell growth.

[0035] In an embodiment of the present application, the medicine also contains a pharmaceutically acceptable carrier.

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

[0037] The medicine also contains a pharmaceutically acceptable pharmaceutical adjuvant; the pharmaceutical adjuvant refers to a conventional pharmaceutical carrier in the pharmaceutical field;

[0038] Preferably, the adjuvant includes one or more of the following: binding agents such as cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents such as starch, pre-gelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and fatty acid glyceride; colorants such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red and the like; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate phthalate; in addition, other adjuvants such as flavoring agents, sweeteners can also be added in the composition;

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

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

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

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

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

[0044] The health care product also contains acceptable adjuvants.

[0045] The present application also provides the use of bioactive peptide VSL peptide in the preparation of health care products for improving muscle mass.

[0046] In one embodiment of the present application, the health care product includes, but is not limited to, functional fermented dairy products.

[0047] The present application also provides the use of bioactive peptide VSL peptide in the preparation of drugs for promoting skeletal muscle cell proliferation and / or preventing sarcopenia.

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

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

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

[0051] Preferably, the adjuvants include one or more of the following: binding agents such as cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents such as starch, pre-gelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrating agents such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and fatty acid glyceride; colorants such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red and the like; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methyl cellulose, povidone, cellulose acetate phthalate; in addition, other adjuvants such as flavoring agents, sweeteners can also be added in the composition;

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

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

[0056] In an embodiment of the present application, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, bean products, beverages; the food also includes special dietary foods;

[0057] The health product also contains acceptable adjuvants.

[0058] The present application also provides a muscle cell proliferation promoting product, which includes the bioactive peptide VSL and a derivative of the bioactive polypeptide VSL.

[0059] In an embodiment of the present application, the muscle cell proliferation promoting product includes a health product for promoting muscle mass increase and a drug for promoting muscle cell proliferation; the derivative of the bioactive polypeptide VSL refers to a polypeptide derivative obtained by modifying the amino acid side chain group, the amino terminal or the carbonyl terminal of the bioactive polypeptide VSL by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation, etc.

[0060] Beneficial effects

[0061] The milk-derived bioactive polypeptide VSL of the present application has a significant effect of promoting skeletal muscle cell proliferation, and is expected to be used for preparing a drug or functional food for promoting skeletal muscle cell proliferation and preventing sarcopenia. The VSL of the present application is convenient to synthesize and can be produced industrially, and has a good application prospect in the fields of food, medicine and cosmetics, etc. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 : Schematic diagram of screening whey peptide and FGFR1 receptor molecule docking. Pymol 1.5 and LigPlot are used to show the specific binding site of the polypeptide and FGFR1. The interaction force includes hydrogen bond combination and hydrophobic interaction force.

[0063] Figure 2 : Proliferation of polypeptide on skeletal muscle cells. The proliferation of skeletal muscle cells in the presence of bioactive peptide VSL at a concentration of 0.01 μM was tested; the absorbance at 450 nm was measured by a microplate reader to quantify the CCK-8 assay. 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 3EdU proliferation staining results of polypeptides. (a) Cells were treated with modified DMEM medium and bioactive peptide VSL at a concentration of 0.01 μΜ. Then, the 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 were the average values ± SEM from 9 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the control group.

[0065] Figure 4 Effect of polypeptides on muscle cell cycle. The effect of bioactive peptide VSL at a concentration of 0.01 μΜ on skeletal muscle cell cycle was tested; the number of cells at each stage was collected and analyzed by flow cytometry, and Modefit5 was used for plotting and data analysis. n = 3, data were expressed as mean ± SEM, and analyzed by one-way ANOVA followed by Tukey's multiple comparison test (p < 0.05).

[0066] Figure 5 Effect of polypeptides on muscle cell reactive oxygen species content. The effect of bioactive peptide VSL at a concentration of 0.01 μΜ on skeletal muscle cell reactive oxygen species damage was tested; the relative content of reactive oxygen species in the cells was quantified by measuring the absorbance at 532 nm with a microplate reader. n = 3, data were 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.

[0067] Figure 6 Effect of polypeptides on muscle cell MDA content. The effect of bioactive peptide VSL at a concentration of 0.01 μΜ on skeletal muscle cell oxidative damage was tested; the MDA content in the cells was quantified by measuring the absorbance at 532 nm with a microplate reader, and normalized by BCA protein content. n = 3, data were 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

[0068] The EdU working solution and cell cycle detection kit used in the following examples were purchased from Wuhan Savi Biological Technology Co., Ltd. and Shanghai Biyun Tian Biological Technology Co., Ltd., respectively.

[0069] The LC-MS / MS analysis method used in the following examples is as follows:

[0070] LC-MS / MS analysis was performed using an EASY nLC 1200 system equipped with an Acclaim PepMap™ RSLC column (50 pm x 15 cm, 2 pm, 100 pm). Mobile phase A consisted of water containing 0.1% (v / v) formic acid and 2% (v / v) acetonitrile, while mobile phase B was water containing 0.1% (v / v) formic acid and 90% (v / v) acetonitrile. The gradient elution program was: 6-20% B, 0-40 min; 20-32% B, 40-52 min; 32-80% B, 52-56 min, at a flow rate of 0.2 pL / min. The MS spectrum acquisition range was 150-2000 m / z with a resolution of 60000. The MS / MS data collection range was 180-2000 m / z with a scan time of 0.1 s.

[0071] The active peptide sequence identification method involved in the following examples is:

[0072] Peptide identification was performed using bovine proteins from the UniProt database (https: / / www.uniprot.org / ) as a reference database. Raw data were searched using Maxquant (v.1.5.2.8) in the non-specific enzyme cleavage 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 protein and peptide segment matching false discovery rate (FDR) threshold was set to 1%. In order to find bioactive peptides, three repeated analyses were performed for each sample, and if a peptide appeared at least twice, it was considered to be significant.

[0073] The method of screening the docking ability of whey peptides with FGFR1 receptor molecules involved in the following examples is:

[0074] For virtual screening and molecular docking, the molecular structure of FGFR1 receptor (PDB ID: 1EVT) was downloaded from RCSB Protein Data Bank (https: / / www.rcsb.org / ). Water molecules and irrelevant atoms were removed before docking. Peptide structure was built and energy minimized using Discovery Studio 2019. Virtual screening was performed using AutoDock Vina 1.2. The docking method was referred to, the center position of the box was set to -10.083, 2.000, -12.444, the size was 120x120x120, and other parameters were kept as default. The conformation with the highest affinity was selected as the result. Under the same conditions, the binding mode and direction of FGFR1 receptor were analyzed and selected. The first three conformations of each peptide docking 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.

[0075] The culture media involved in the following examples are as follows:

[0076] DMEM complete medium: purchased from Thermo Fisher Scientific (China) Co., Ltd. Shanghai, China.

[0077] ARHPHPHLSF (10 peptides) involved in the following examples is derived from kappa-casein, located at positions 96-105.

[0078] Kappa-casein (SEQ ID NO. 1)

[0079] MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKDERFFSDKIAKYIPIQYVLSRYPSYGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV.

[0080] Example 1: Preparation of biologically active polypeptides

[0081] 1. Isolation and preparation of polypeptides

[0082] (1) Preparation of fermented whey

[0083] The purified strain CCFM1263 (described in the Chinese invention patent application document with publication number CN117305158A) was subcultured on a first generation MRS solid plate and two generations of MRS liquid tube, centrifuged at 6000 x g for 5 min, resuspended with 11% (w / w) skim milk, inoculated into 11% (w / w) skim milk at an inoculum of 2% (v / v), shaken uniformly, and then cultured in an anaerobic incubator at 37°C for 18 h. Then, the same method was used for the skim milk subculture, and after being cultured in an anaerobic incubator at 37°C for 18 h, the protein was washed with Tris acid, and the viable cell count was adjusted to 1 x 10 6 CFU / mL, inoculated into 11% (w / w) skim milk at an inoculum of 2% (v / v), and fermented in a constant temperature incubator at 37°C for 48 h.

[0084] Different fermented milk samples were taken, 1 mol / L sodium hydroxide solution was added to adjust the sample pH to 4.6, and then pasteurized in a high-pressure steam sterilization pot at 95°C for 10 min. The sterilized fermented milk sample was centrifuged at 10000 x g at 4°C for 10 min, and the supernatant was taken and filtered with a 0.45 μm organic filter membrane to obtain the Lactobacillus helveticus fermented milk whey sample. The collected Lactobacillus helveticus fermented milk whey was freeze-dried into powder and stored in a freeze-dried bottle at 4°C for standby use.

[0085] (2) The C18 solid phase extraction column was used for desalting and purification of the fermented whey. The C18 solid phase extraction desalting column was used for desalting and purification of the sample, and the loading amount was 3 mg. It was divided into five steps of wetting, leveling, loading, desalting, and washing, and the flow rate was controlled by a constant pressure pump.

[0086] (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, and then subjected to LC-MS / MS analysis.

[0087] The sequence of the peptide in the mixed solution was analyzed.

[0088] 2. Identification and screening of active peptide sequences

[0089] The sequence of the peptide obtained in step 1 was identified, and the docking ability of the whey peptide with the FGFR1 receptor molecule was screened.

[0090] The results are shown in Table 1

[0091] Table 1: Molecular docking results

[0092]

[0093]

[0094] Results show that: the lower the binding energy, the better the effect of binding with the receptor, and ultimately a group of polypeptides with better virtual binding effect with the receptor are obtained, ARHPHPHLSF and VSL. The schematic diagram of the docking of the two groups of polypeptides with FGFR1 molecules is shown in Figure 1 .

[0095] Example 2: Effect of bioactive polypeptides on muscle cells

[0096] 1. Effect on muscle cell proliferation

[0097] The specific method is as follows:

[0098] (1) Chemically synthesized by Shanghai Gil Peptide Co., Ltd. to prepare ARHPHPHLSF polypeptide powder and VSL polypeptide powder, respectively.

[0099] (2) Add ARHPHPHLSF and VSL polypeptide powder obtained in step (1) to DMEM complete culture medium to make the final concentration of polypeptides in the culture medium 0.001 μmol / L, 0.01 μmol / L and 0.1 μmol / L, respectively. Finally, DMEM complete culture medium containing 0.001 μmol / L, 0.01 μmol / L and 0.1 μmol / L bioactive peptide ARHPHPHLSF is prepared.

[0100] DMEM complete culture medium containing 0.001 μmol / L, 0.01 μmol / L and 0.1 μmol / L bioactive peptide VSL.

[0101] (3) Mouse myoblast C2C12 cells were inoculated in a 96-well plate at a seeding amount of 2×10 4 cells / well, and cultured at 37°C for 24h. Then, DMEM complete culture medium containing 0 μmol / L, 0.001 μmol / L, 0.01 μmol / L and 0.1 μmol / L bioactive peptide ARHPHPHLSF (named ARH10) and VSL was added, respectively, and cultured at 37°C for 24h. Then, 10 μL CCK-8 solution was added to each well under sterile conditions, and incubated in an incubator (37°C) for 30min. Shake well, and measure the absorbance at 450nm using a microplate reader.

[0102] The results are shown in Table 2 and Figure 2 .

[0103] Table 2: Effect of polypeptides with different concentrations on muscle cell proliferation

[0104]

[0105] The results show that the milk-derived peptide VSL can promote the proliferation of myoblasts, and the effect is optimal when the concentration reaches 0.01 μmol / L, and the proliferation rate reaches 128% when the concentration of the milk-derived peptide VSL is 0.01 μmol / L. ARHPHPHLSF can also promote the proliferation of myoblasts, and the effect is improved with the increase of the dose.

[0106] 2, Effect on EdU proliferation staining of muscle cells

[0107] The specific method is as follows:

[0108] (1) Chemically synthesized by Shanghai Gilmore Peptide Co., Ltd. to prepare ARHPHPHLSF polypeptide powder and VSL polypeptide powder respectively.

[0109] (2) Add ARHPHPHLSF polypeptide and VSL polypeptide powder obtained in step (1) to DMEM complete medium respectively, so that the final concentration of ARHPHPHLSF polypeptide in the medium is 0.1 μmol / L, and the final concentration of VSL polypeptide is 0.01 μmol / L. Prepare DMEM complete medium containing ARHPHPHLSF polypeptide with a final concentration of 0.1 μmol / L and DMEM complete medium containing VSL polypeptide with a final concentration of 0.01 μmol / L.

[0110] (3) Seed mouse myoblast C2C12 cells in a 96-well plate at a seeding amount of 2×10 4 cells / well, and culture at 37°C for 24 h. Then add DMEM complete medium containing 0 μmol / L and 0.1 μmol / L of bioactive peptide ARHPHPHLSF (named ARH10) and 0.01 μmol / L of bioactive peptide VSL respectively, and culture at 37°C for 24 h.

[0111] (4) Add 500 μL of 37°C preheated EdU working solution to the intervened cells and incubate for 2 h. Discard the EdU working solution, add 1 mL of fixing solution, and fix at room temperature for 15 min. Discard the fixing solution and wash with PBS containing 5% BSA for 3 times. 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 with washing solution for 3 times. Add 500 μL of Click reaction solution and incubate at room temperature for 30 min in the dark. Discard the Click reaction solution and wash with washing solution for 3 times. Add 1 mL of Hoechsty solution and incubate at room temperature for 10 min in the dark. Discard the Hoechsty solution and wash with washing solution for 3 times. Collect images under fluorescence microscope.

[0112] The results are shown in Table 3 and Figure 3

[0113] ​Table 3: Effect of different concentrations of polypeptides on the proportion of positive cells in muscle cells

[0114]

[0115] The results are shown in Table 3. The milk-derived peptide VSL can significantly increase the proportion of positive cells in muscle cells, and the proliferation effect is significantly higher than that of ARHPHPHLSF on C2C12. When the concentration of milk-derived peptide VSL is 0.01 μmol / L, the proportion of positive cells increases by 20.11% compared with the normal group. Figure 3

[0116] 3. Effect on the growth cycle of muscle cells

[0117] The specific method is as follows:

[0118] (1) Chemically synthesized by Shanghai Gilmore Peptide Co., Ltd. to prepare ARHPHPHLSF polypeptide powder and VSL polypeptide powder, respectively.

[0119] (2) Add ARHPHPHLSF polypeptide and VSL polypeptide powder obtained in step (1) to DMEM complete medium to make the final concentration of ARHPHPHLSF polypeptide in the medium 0.1 μmol / L and the final concentration of VSL polypeptide 0.01 μmol / L. Prepare DMEM complete medium containing ARHPHPHLSF polypeptide with a final concentration of 0.1 μmol / L and DMEM complete medium containing VSL polypeptide with a final concentration of 0.01 μmol / L.

[0120] (3) Seed mouse myoblast C2C12 cells in a 12-well plate at a seeding amount of 1 x 10 5

[0121] ​​(4) Discard the culture medium in the culture plate, and after lysing the cells with a lysis solution, collect the cells in a 1.5 mL centrifuge tube, discard the supernatant after centrifugation, and then add pre-cooled 0.5 mL PBS buffer solution to the precipitate to dissolve it, discard the supernatant after centrifugation, and repeat the washing with the PBS solution 2-3 times. Then add propidium iodide (PI) staining solution to disperse the cells, and then stain overnight at 4°C in the dark (more than 24 h). Finally, analyze the fluorescence of the cells using a flow cytometer. The DNA signal represented by the red fluorescence staining intensity after PI staining is collected by computer, and the forward scatter (FSC) and side scatter (SSC) data are real-time collected.

[0122] The results are shown in Table 4 and Figure 4

[0123] Table 4: Effect of different polypeptides on muscle cell cycle

[0124]

[0125] The results are shown in Table 4 and Figure 4 As shown in Table 4, the milk-derived VSL can accelerate the progress of the muscle cell cycle. When the concentration of the milk-derived peptide VSL is 0.01 μmol / L, the number of G0 / G1 phase cells is significantly reduced by 10.9% compared to the normal group, and the number of S phase cells is significantly increased by 9.15%, indicating that VSL treatment accelerates the progress of the cell cycle and promotes cell growth.

[0126] 4. Determination of ROS content in muscle cells

[0127] The specific method is as follows:

[0128] (1) Chemically synthesized by Shanghai Gilmore Peptide Co., Ltd. to prepare ARHPHPHLSF polypeptide powder and VSL polypeptide powder, respectively.

[0129] (2) Add the ARHPHPHLSF and VSL polypeptide powders obtained in step (1) to the DMEM complete culture medium to make the final concentration of the polypeptides in the culture medium 0.1 μmol / L and 0.01 μmol / L, respectively. At the same time, dissolve the TNF-α powder synthesized by Shanghai Genechem in the DMEM culture medium to make the final concentration of TNF-α in the culture medium 20 ng / mL. Finally, prepare DMEM complete culture medium containing 0.1 μmol / L bioactive peptide ARHPHPHLSF, DMEM complete culture medium containing 0.01 μmol / L bioactive peptide VSL, and DMEM complete culture medium containing 20 ng / mL TNF-α.

[0130] (3) Seed mouse myoblast C2C12 cells into a six-well plate at a density of 1 x 10​5 After 24h of culture, the cells were intervened in groups. DMEM complete medium containing 0 μmol / L, 0.1 μmol / L bioactive peptide ARHPHPHLSF (named ARH10), 0.01 μmol / L bioactive peptide VSL and DMEM complete medium containing 20 ng / mL TNF-α were added respectively, and the cells were cultured at 37°C for another 24h.

[0131] (4) Discard the old culture medium, add 500 μL DCFH-DA solution with a final concentration of 10 μmol / L, and incubate at 37°C for 20-40 min. Wash with serum-free medium (purchased from Thermo Fisher Scientific) for 3 times to remove the DCFH-DA that does not enter the cells, and finally measure the absorbance value at 532 nm.

[0132] The results are shown in Table 5 and Figure 5

[0133] Table 5: Effect of polypeptides with different concentrations on the content of ROS in muscle cells

[0134]

[0135] The results are shown in Table 5 and Figure 5 The content of ROS in the milk peptide VSL treatment group was reduced by 1.90 times compared with the modeling group.

[0136] 5. Determination of MDA content in muscle cells

[0137] The specific method is as follows:

[0138] (1) ARHPHPHLSF polypeptide powder and VSL polypeptide powder were prepared by chemical synthesis from Shanghai Gilmore Peptide Co., Ltd.

[0139] (2) ARHPHPHLSF and VSL polypeptide powder obtained in step (1) were added to DMEM complete medium to make the final concentration of polypeptide in the medium 0.1 μmol / L and 0.01 μmol / L respectively, and TNF-α powder synthesized by Shanghai Genechem was dissolved in DMEM medium to make the final concentration of TNF-α in the medium 20 ng / mL. Finally, DMEM complete medium containing 0.1 μmol / L bioactive peptide ARHPHPHLSF, DMEM complete medium containing 0.01 μmol / L bioactive peptide VSL and DMEM complete medium containing 20 ng / mL TNF-α were prepared.

[0140] (3) Mouse myoblast C2C12 cells were inoculated into a six-well plate at 1 × 10 5 ​After 24h, the cells were intervened in groups. DMEM complete medium containing 0 μmol / L, 0.1 μmol / L bioactive peptide ARHPHPHLSF (named ARH10), 0.01 μmol / L bioactive peptide VSL and 20 ng / mL TNF-α were added respectively, and the cells were cultured at 37℃ for another 24h.

[0141] (4) The treated cells were lysed using lysis solution, and the supernatant was collected in a 1.5 mL centrifuge tube after centrifugation at 12000g for 10 min. The supernatant was used for subsequent determination. 0.1 ml of homogenate, lysis solution as a blank control, 0.1 ml of different concentrations of standard for making standard curve, and 0.1 ml of sample for determination were added in a new centrifuge tube; then 0.2 ml of MDA detection working solution was added. After mixing, 100℃ or boiling water bath heating was performed for 15 min. The water bath was cooled to room temperature, and centrifugation was performed at 1000g for 10 min at room temperature. 200 μL of supernatant was taken and added to a 96-well plate, and then the absorbance was determined at 532 nm by using an enzyme-labeled instrument.

[0142] Results are shown in Table 6 and Figure 6

[0143] Table 6: Effect of different concentrations of polypeptides on the content of MDA in muscle cells

[0144]

[0145] Results are shown in Table 6 and Figure 6 As shown in the results, compared with the modeling group, the content of MDA in the milk peptide VSL treatment group was reduced by 2.05 times compared with the TNF-α group.

[0146] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.​

Claims

1. Application of peptides with the amino acid sequence VSL in the preparation of drugs for the prevention of sarcopenia.

2. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

3. The application according to claim 2, characterized in that, The excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents: starch, dextrin, sucrose, lactose, mannitol; Fillers: starch, sucrose; wetting agents: glycerin; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers: quaternary ammonium compounds; surfactants: polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red. Lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; Coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; Excipients: flavoring agents and sweeteners.

4. The application according to claim 3, characterized in that, The drug is available in oral, injectable, or inhaled dosage forms.

5. The application according to claim 4, characterized in that, The oral dosage forms are tablets, capsules, granules, and oral liquids.

6. The application according to claim 4, characterized in that, The oral dosage form is an oral suspension.

7. The application according to claim 4, characterized in that, The injectable dosage forms are injection solutions and injection powders for injection.

8. The application according to claim 4, characterized in that, The inhalation dosage form is an aerosol or powder inhaler.

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