Application of yeast peptide-derived polypeptide in promoting proliferation and differentiation of myoblasts and composition

Abstract

The invention relates to application of a yeast peptide-derived polypeptide in promoting proliferation and differentiation of myoblasts and a composition, and belongs to the technical field of biological medicines and nutrition and health care. The amino acid sequence of the polypeptide comprises SEQ ID No.1 (LGGPLL), SEQ ID No.2 (PQAVTI), SEQ ID No.3 (VASGGL) or SEQ ID No.4 (LGPTGISM, and the polypeptide can be used for remarkably promoting the proliferation and the cell cycle progress of C2C12 myoblasts and up-regulating the expression level of myogenic protein. The polypeptide can be used as an active component to be applied to development of anti-sarcopenia related medicines or functional food compositions. Cell experiments prove that the polypeptide has a good muscle promoting function and is suitable for intervention of senile muscle loss such as sarcopenia and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine and nutrition and health care, and particularly relates to a yeast peptide-derived active characteristic polypeptide and application thereof in promoting muscle cell proliferation and differentiation. BACKGROUND

[0002] Skeletal muscle mass reduction, especially sarcopenia, is one of the most common and important health problems in the aging process. Studies have shown that human skeletal muscle mass begins to gradually decrease after the age of 30, with an average annual decrease of 1% to 2%, and the prevalence of sarcopenia in people over 60 years old is even more than 30%. The reduction of skeletal muscle mass not only causes the limitation of body activity function, but also is closely related to the significant increase of all-cause mortality. Studies have shown that when the muscle mass decreases by 10%, the risk of death of the individual will increase by 50%.

[0003] The fundamental reason for muscle loss caused by aging is the imbalance of myoblast regeneration, and the key mechanisms include the decrease of satellite cell activation ability, the decrease of protein synthesis ability and the damage of mitochondrial function. Specifically, the TGF-β signaling pathway of myoblast in the aging state is continuously abnormally activated, which inhibits the conversion of myoblast from the resting state (G0 phase) to the proliferative state; in addition, the sensitivity of mTORC1 signaling pathway decreases, which causes the protein synthesis rate of myosin heavy chain (MyHC) to decrease by 40%-60%; at the same time, mitochondrial dysfunction causes abnormal accumulation of reactive oxygen species (ROS), which causes the apoptosis rate of myoblast to increase by more than 3 times.

[0004] For aging muscle loss, the current conventional intervention methods include hormone replacement therapy, myostatin antibody therapy, amino acid supplements and stem cell therapy. However, these methods have certain limitations and deficiencies. For example, although hormone replacement therapy can improve muscle mass to a certain extent, it may also increase the risk of cardiovascular disease; myostatin antibody therapy can increase lean body mass, but cannot significantly enhance muscle strength; amino acid supplements (such as leucine) need to be used in a super high dose, which is easy to cause gastrointestinal discomfort; and stem cell therapy faces the challenges of strict policy supervision and high treatment cost. Therefore, it is urgent to develop a safe and effective new type of anti-aging muscle loss method with controllable cost to meet the urgent needs of the increasingly severe aging society in China for the prevention and treatment of sarcopenia.

[0005] In order to solve the problems existing in the prior art, the present inventors have found, through extensive research, that a specific active polypeptide from yeast peptide has a significant effect of promoting myoblast proliferation and differentiation, which can effectively alleviate or reverse muscle loss caused by aging, and provides an innovative solution for the field of sarcopenia prevention and treatment. SUMMARY

[0006] To overcome the problems of limited effect, significant side effects and high cost in dealing with aging muscle loss in the prior art, the inventors, based on in-depth research on the function of yeast peptide, found that a specific short peptide sequence has significant biological activity in promoting muscle cell proliferation and differentiation. Thus, the present application provides a yeast peptide-derived active feature polypeptide with a clear amino acid sequence, and a composition based on the polypeptide and its application in anti-sarcopenia. The related technical solutions are not only experimentally confirmed at the cellular level, but also have good syntheticity and industrial transformation prospects, as described below.

[0007] In one possible implementation, the present application provides a yeast peptide-derived active feature polypeptide, whose amino acid sequence comprises the sequence shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

[0008] In one possible implementation, the amino acid sequence of SEQ ID No. 1 is LGGPLL, the amino acid sequence of SEQ ID No. 2 is PQAVTI, the amino acid sequence of SEQ ID No. 3 is VASGGL, and the amino acid sequence of SEQ ID No. 4 is LGPTGISM.

[0009] In one possible implementation, the above-mentioned polypeptide can effectively promote myoblast proliferation and differentiation, improve the activity and regenerative capacity of muscle cells by increasing the proportion of G2 / M phase cells in the cell cycle and up-regulating the expression of muscle-derived proteins such as Myh1.

[0010] In one possible implementation, the present application further relates to the use of the above-mentioned polypeptide in the preparation of medicines or health foods, particularly for resisting aging muscle loss. The polypeptide of the present application can effectively slow down the development of sarcopenia by improving the function of myoblasts, improve the muscle function of the elderly population, and significantly improve their quality of life.

[0011] In one possible implementation, the present application also provides an anti-aging composition, which comprises any one or more of the above-mentioned polypeptides, and can further be combined with pharmaceutically or dietetically acceptable adjuvants or carriers to prepare various dosage forms, including but not limited to oral preparations, injections, transdermal preparations, functional foods, drinks or nutritional supplements.

[0012] Based on the technical scheme, the yeast peptide-derived active feature polypeptide is aimed at the problems of more side effects, poor curative effect and high economic cost of the existing anti-sarcopenia scheme, realizes muscle regeneration and sarcopenia intervention through the clear amino acid sequence and the significant promotion of muscle cell proliferation and differentiation. The polypeptide can effectively improve the cell proliferation activity, wherein the proliferation rate of C2C12 myoblast cells can be increased by 53.05%, and the G2 / M phase cell ratio is significantly improved, indicating that it has a clear advantage in promoting cell cycle progression and cell proliferation. In addition, the polypeptide can effectively up-regulate the expression of key muscle-derived proteins such as myosin heavy chain (Myh1), which is beneficial to improve the problem of insufficient protein synthesis capacity in the aging environment, and further enhances the muscle regeneration effect.

[0013] The polypeptide and composition provided by the application can be widely used in the development of therapeutic and preventive preparations for aging muscle loss, including but not limited to medicine, functional food and nutritional health products. Specifically, the dosage form of the polypeptide in the pharmaceutical preparation can be prepared into oral dosage form, injection dosage form, transdermal preparation, etc. according to specific needs, to adapt to different clinical or daily use needs; and in the field of functional food, suitable food-grade adjuvants such as protein carriers, carbohydrates, dietary fibers, etc. can be further prepared into health foods convenient for ingestion, which can meet the daily health care needs of the elderly population.

[0014] Further, the preparation process of the polypeptide is clear, stable and controllable, which is synthesized by Fmoc solid-phase synthesis method, and strict quality control measures are adopted, including accurate mass spectrometry verification (m / z value deviation controlled within 0.05%), high-performance liquid chromatography purification (main peak area ratio ≥ 95%), which ensures the high purity, high activity and good safety of the polypeptide product, so that the technical scheme has obvious competitive advantage in large-scale production process.

[0015] The application further clearly defines the specific mechanism of the yeast peptide-derived polypeptide against sarcopenia, that is, it can down-regulate the expression of TGF-β signaling pathway related proteins, improve the cell microenvironment, and significantly improve the proliferation and differentiation activity of myoblasts, thereby fundamentally realizing muscle mass recovery and function improvement. The technical scheme of the application is not only suitable for the prevention or treatment of sarcopenia in people over 60 years old, but also can effectively promote muscle regeneration after sports injury, which is helpful to speed up the rehabilitation speed of athletes or ordinary people after sports injury.

[0016] In summary, the yeast peptide-derived active feature polypeptide and the composition thereof of the present application have the technical advantages of high efficiency, safety and economy, make up for many deficiencies of the prior art, have great clinical and industrial application potential, can effectively improve the life quality of the elderly population, have broad market prospects and important social value. The technical path of the present application can be realized in batches through commercial synthesis means, has good production economy and application expansion BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The concentration-dependent curve of the promoting proliferation effect of the yeast peptide-derived active feature polypeptide of the present application on C2C12 myoblast cells shows the change of the cell proliferation rate under the treatment of polypeptides with different concentrations.

[0018] Figure 2 The influence diagram of the polypeptide of the present application on the cell cycle distribution of C2C12 myoblast cells after the polypeptide acts on the cells reflects the difference in the proportion of cells in each stage of the cell cycle between the polypeptide treatment group and the control group.

[0019] Figure 3 The differential protein heat map of the proteomics analysis result shows the difference in the protein expression level of C2C12 myoblast cells treated by the polypeptide of the present application compared with the control group, and the color change represents the increase or decrease of the protein expression level.

[0020] Figure 4 The primary mass spectrum of the yeast-derived active feature polypeptide of the present application confirms the accuracy of the amino acid sequence and the purity of the polypeptide sample. DETAILED DESCRIPTION

[0021] The technical solutions of the present application are described in detail through specific embodiments, so that those skilled in the art can more clearly and completely understand the features, implementation methods and beneficial effects of the present application. It should be pointed out that the present embodiment is only used for illustration and is not used to limit the protection scope of the present application. Those skilled in the art can make appropriate adjustments or improvements to the following embodiments according to specific needs without departing from the concept and technical essence of the present application.

[0022] Example 1: verification of the promoting proliferation effect

[0023] 1. Experimental materials and instruments

[0024] 1.1 Experimental reagents

[0025] Yeast peptide sample: used for evaluating its effect on cell proliferation.

[0026] Trypsin: purchased from Thermo Fisher Scientific, USA, used for cell digestion.

[0027] Penicillin-streptomycin mixed antibiotic: also purchased from Thermo Fisher Scientific, USA, for sterility control during cell culture process.

[0028] PBS buffer: purchased from Dattexil Biotech Co., Ltd., Shanghai, China, for cell washing and other operations.

[0029] BCA protein quantification kit: purchased from Lambright Biotech Co., Ltd., for protein quantification analysis.

[0030] CCK-8 kit: purchased from Lambright Biotech Co., Ltd., for cell viability detection.

[0031] DMEM medium: purchased from Thermo Fisher Scientific, USA, as the basic medium for cell culture.

[0032] Fetal bovine serum (FBS): purchased from Thermo Fisher Scientific, USA, to provide essential growth factors and nutrients for cells.

[0033] Cell cycle and apoptosis detection kit: purchased from Thermo Fisher Scientific, USA, for possible subsequent cell cycle and apoptosis analysis.

[0034] 1.2 Experimental instruments

[0035] Analytical balance (AL104): produced by Mettler Toledo, for accurate weighing of reagents.

[0036] Centrifuge (PK165): produced by Hunan Pingke Scientific Instrument Co., Ltd., for centrifugal separation of cell suspension.

[0037] Liquid chromatograph (U 3000) and triple quadrupole liquid chromatograph-mass spectrometer (Quantum ACCESS MAX): both purchased from Thermo Fisher Scientific, USA, for qualitative and quantitative analysis of yeast peptide samples (although the analysis results are not directly shown in this example, technical support is provided to ensure sample quality).

[0038] Data processing software (Xcalibur 3.1): for processing and analyzing liquid chromatograph and mass spectrometer data.

[0039] 2. Experimental methods

[0040] C2C12 myoblast cells in logarithmic growth phase were seeded at 1 x 10 4The cells were inoculated in a concentration of 1.5×105 / mL in sterilized culture bottles and incubated in a cell incubator at 37°C and 5% CO2. When the cells grew to 80% confluence and were in good condition, they were subcultured.

[0041] According to the results of the previous cell viability pre-experiment, the digested myoblast cell suspension was inoculated into a 96-well plate at a density of about 1×10 4 cells per well, with 4-5 parallel holes in each group. After the cells adhered, yeast peptide solutions with concentrations of 25, 50, 100, 250, and 500 μg / mL were added to the complete culture medium, and the control group was added with the same volume of complete culture medium. The culture plates were incubated at 37°C and 5% CO2. After 24, 48, and 72 hours of incubation, 10 μL of CCK-8 reagent solution was added to each well, and after 2 hours of incubation, the absorbance value of each well was measured at 450 nm using a microplate reader. The proliferation rate was calculated according to the cell viability formula to evaluate the effect of yeast peptide on the proliferation of C2C12 myoblast cells.

[0042] 3. Experimental results

[0043] The specific results are shown in Figure 1 and Figure 2 . As can be seen from Figure 1 , within the concentration range of 25-500 μg / mL, yeast peptide significantly promotes the proliferation of mouse C2C12 myoblast cells within 24-72 hours. When the yeast peptide concentration is 100 μg / mL, the proliferation of myoblast cells is most significant after 72 hours of incubation (P<0.01), which is 53.05% higher than the control group. This result shows that yeast peptide at a specific concentration can effectively promote the proliferation of myoblast cells, providing important experimental basis for subsequent related research.

[0044] Example 2: Cell cycle regulation

[0045] 1. Experimental method

[0046] Myoblast cells C2C12 were inoculated in a 6-well plate at a density of 4.5-5×10 5 cells per well, using standard growth medium and incubated at 37°C and 5% CO2 for 3 hours to ensure that the cells were fully adherent. Then, the original growth medium was discarded and replaced with serum-free DMEM medium to starve the cells for 16 hours to synchronize the cell cycle to the G0 phase.

[0047] After cell synchronization, the serum-free DMEM was discarded and replaced with growth medium containing different concentrations of yeast peptide (25 μg / mL, 100 μg / mL, 250 μg / mL). The cells were incubated at 37°C, 5% CO2 for another 24 hours.

[0048] After incubation, the cells were collected and fixed with 70% ethanol at 4°C overnight to maintain cell morphology and fix DNA. The next day, the cells were washed with PBS buffer to remove residual ethanol. Then, the cells were stained using a cell cycle and apoptosis detection kit (according to the instructions) at room temperature and in the dark for 30 minutes.

[0049] After staining, the cells were analyzed by flow cytometry (excited with a 488 nm laser line). The data obtained by flow cytometry were used for subsequent analysis of cell cycle distribution.

[0050] 2. Experimental results

[0051] The experimental results showed that different concentrations of yeast peptide (50 μg / mL, 100 μg / mL, 250 μg / mL) could effectively stimulate DNA synthesis in C2C12 myoblast cells, promote the transition from diploid to tetraploid state, accelerate cell cycle progression, and affect the proportion of cell proliferation compared with the blank control group.

[0052] Specific data showed that when the concentration of yeast peptide was 50 μg / mL, the proportion of cell tetraploid was 15.8% higher than the control group. When the concentration of yeast peptide increased to 100 μg / mL, the proportion of cell tetraploid significantly increased, which was 29.2% higher than the control group. When the concentration of yeast peptide was 250 μg / mL, although the proportion of cell tetraploid increased, it was only 16.7% higher than the control group, showing a concentration-dependent response characteristic.

[0053] Further analysis of the proportion of each phase of the cell cycle found that in the 100 μg / mL yeast peptide treatment group, the proportion of G2 / M phase cells reached 92.3%, while the control group was only 63.1%, indicating that yeast peptide at this concentration significantly promoted cells to enter and arrest in the G2 / M phase, thereby accelerating cell cycle progression.

[0054] In summary, yeast peptide can significantly regulate the cell cycle progression of C2C12 myoblast cells within a certain concentration range, promote cell proliferation, and provide important evidence for subsequent research on the mechanism of yeast peptide in cell proliferation and differentiation.

[0055] Example 3: Proteomics analysis

[0056] 1. Experimental method

[0057] 1.1 Cell culture and treatment

[0058] C2C12 myoblast cells were inoculated into a T25 culture flask and cultured in a cell incubator at 37°C with 5% CO2. When the cells were adherent and the confluence reached 80%, the original culture medium was discarded and replaced with complete culture medium containing 100 μg / mL yeast peptide for 24 hours of continuous culture.

[0059] 1.2 Cell lysis and protein extraction

[0060] After the culture was completed, the culture medium was discarded and the cells were washed three times with PBS buffer to remove residual culture medium and impurities. Then, an appropriate amount of RIPA lysis solution was added to the T25 culture flask and reacted on ice for about 15 minutes, during which the cells were repeatedly blown with a pipette to ensure complete lysis. After lysis was complete, the lysate was transferred to a centrifuge tube and centrifuged at 12000 r / min (4°C) for 10 minutes. The supernatant was then collected and stored at low temperature for use as the cell protein extract.

[0061] 1.3 Protein concentration determination

[0062] According to the instructions of the BCA protein quantification kit, the reaction solution was prepared and the protein concentration in the extract was calculated. First, a standard curve was prepared by adding known concentrations of protein standard solution to a 96-well plate and diluting with buffer to a certain concentration. Then, the extracted protein samples were diluted to an appropriate concentration, and a certain amount of reaction solution was accurately pipetted into a 96-well plate. After incubation at 37°C for 30 minutes, the absorbance was measured at 562 nm using an enzyme-labeled instrument. The protein concentration of each group of supernatant was calculated according to the standard curve.

[0063] 1.4 Protein digestion and mass spectrometry analysis

[0064] The cell protein extract was diluted to the same concentration, and trypsin was added according to the conditions of trypsin to protein mass ratio of 1:20, and was fully digested at 37°C for 17 hours. After digestion was complete, the trypsin was inactivated by high temperature treatment, and was cooled to room temperature. Then, 8 μL of 1 mol / L DTT solution was added and reacted at 37°C for 1 hour to reduce disulfide bonds. Next, 1 mol / L iodoacetamide was added and stored at room temperature in the dark for 30 minutes to alkylate cysteine residues. After the reaction was complete, the sample was centrifuged at 10000 r / min for 10 minutes, and the supernatant was collected for subsequent mass spectrometry analysis. The Orbitrap high-resolution mass spectrometer was used to analyze the sample to obtain protein identification and quantification information.

[0065] 2. Experimental results

[0066] As Figure 3As shown, the differential protein heat map shows that the expression of Myh1 and other up-regulated proteins is significantly enhanced between the yeast peptide treatment group and the control group, and the color change reflects the trend of expression level change.

[0067] Through proteomic analysis, we identified a total of 1706 proteins between the yeast peptide treatment group and the control group. After further screening and analysis, a total of 177 differentially expressed reported proteins were found, of which 11 proteins were up-regulated and 58 proteins were down-regulated.

[0068] In particular, we identified 10 up-regulated proteins including Myh1 (myosin heavy chain), the expression of which was significantly increased by 2-5 times in the yeast peptide treatment group. These up-regulated proteins are mainly involved in biological processes such as muscle contraction, cytoskeletal structure and energy metabolism, indicating that yeast peptides may affect the proliferation and differentiation of myoblasts by regulating the expression of these proteins.

[0069] In summary, the proteomic analysis results reveal the significant impact of yeast peptides on the protein expression profile of myoblasts, providing an important molecular basis for further studying the mechanism of action of yeast peptides.

[0070] Example 4: Method for synthesizing polypeptides

[0071] The yeast peptide-derived active characteristic polypeptides (SEQ ID No. 1: LGGPLL; SEQ ID No. 2: PQAVTI; SEQ ID No. 3: VASGGL; SEQ ID No. 4: LGPTGISM) described in the present application are prepared by Fmoc solid-phase synthesis. The specific steps are as follows:

[0072] (e) The amino acid sequence is gradually connected on the resin according to the Fmoc solid-phase synthesis method, and the resin is Wang resin;

[0073] (f) After the completion of polypeptide synthesis, a TFA: EDT: TIPS: H2O mixed solvent (volume ratio 92.5:2.5:2.5:2.5) is used to cleave the resin and release the polypeptide;

[0074] (g) Mass spectrometry is used to verify the mass of the obtained polypeptide, and the deviation of the polypeptide molecular m / z value is required to be controlled within ±0.05%;

[0075] (h) The obtained polypeptide sample is purified by reverse phase high performance liquid chromatography (HPLC), using a C18 chromatographic column (4.6x250mm, 5μm), and the mobile phase is acetonitrile-water system containing 0.1% TFA. The main peak area of the purified polypeptide is not less than 95%, and the purified target polypeptide is obtained.

[0076] As Figure 4As shown, the main peak m / z value of the MALDI-TOF mass spectrum of the polypeptide sample is highly consistent with the theoretical molecular weight,

[0077] The error is less than ±0.05%, verifying the accuracy of the molecular structure of the synthesized polypeptide.

[0078] The SEQ ID sequences in the application are specifically defined as follows:

[0079] SEQ ID No. 1: LGGPLL;

[0080] SEQ ID No. 2: PQAVTI;

[0081] SEQ ID No. 3: VASGGL;

[0082] SEQ ID No. 4: LGPTGISM.

[0083] The SEQ ID numbers are identification numbers of amino acid sequences given in sequence, for facilitating subsequent clear search and accurate reference of the sequences. All sequences are expressed by standard single-letter amino acid code.

[0084] In summary, through the above specific embodiments, the application determines the preparation method of the active characteristic polypeptide from yeast peptides and its significant promoting effect on myoblast proliferation, cell cycle regulation and protein synthesis, which can be effectively used for developing anti-aging muscle loss medical and health food preparations, and has clear technical advantages and good application prospect.

[0085] The above embodiments are only used to specifically illustrate the technical solutions and advantages of the application, and are not used to limit the scope of the application. Based on the understanding of the application, those skilled in the art can make appropriate adjustments, improvements or equivalent replacements to the specific embodiments of the application, and these adjustments, improvements or replacements still belong to the protection scope defined by the claims of the application.

Claims

1. A yeast peptide-derived active polypeptide, characterized in that, The amino acid sequence of the polypeptide includes the sequence shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No.

4.

2. The polypeptide according to claim 1, characterized in that, The amino acid sequence of SEQ ID No. 1 is LGGPLL, the amino acid sequence of SEQ ID No. 2 is PQAVTI, the amino acid sequence of SEQ ID No. 3 is VASGGL, and the amino acid sequence of SEQ ID No. 4 is LGPTGISM.

3. The application of the polypeptide according to claim 1 or 2 in promoting myocyte proliferation and differentiation.

4. The application according to claim 3, characterized in that, The application is in the preparation of pharmaceuticals or health foods that combat age-related muscle loss.

5. An anti-aging composition, characterized in that, The composition comprises the polypeptide of claim 1 or 2.

6. The method for preparing the polypeptide according to claims 1-2, characterized in that... Including the following steps: (a) Amino acid sequences were sequentially linked onto the resin using the Fmoc solid-phase synthesis method. The resin was Wang resin. (b) After the peptide synthesis was completed, the resin was lysed using a mixed solvent of TFA:EDT:TIPS:H2O (volume ratio 92.5:2.5:2.5:2.5) to release the peptide; (c) The obtained peptides were verified by mass spectrometry using a triple quadrupole mass spectrometer, and the deviation of the peptide molecules' m / z values ​​was required to be controlled within ±0.05%. (d) The obtained peptide sample was purified by reversed-phase high-performance liquid chromatography (HPLC) using a C18 column (4.6×250mm, 5μm) and a mobile phase of acetonitrile-water system containing 0.1% TFA. The purified peptide had a main peak area of ​​not less than 95%, thus obtaining the purified target peptide.

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