TWI767672B - Use of short peptides and compositions thereof for the treatment or/and prevention of skeletal muscular dystrophy or its related diseases - Google Patents

Abstract

The embodiments of the present invention disclose the use of a short peptide or a mixture containing the same for a composition for preventing and/or skeletal muscle atrophy-related diseases, wherein the amino acid code of the short peptide is SEQ ID No.: The sequence shown in 1 or the homologous amino acid sequence derived from the substitution, deletion or addition of one or more amino acids.

Description

Use of short peptides and compositions thereof for the treatment or/and prevention of skeletal muscular dystrophy or related diseases

The present invention relates to the use of a short peptide, especially the use of a short peptide and its composition for treating or/and preventing skeletal muscular dystrophy or its related diseases.

Press, skeletal muscle plays an important role in the elderly, helping the elderly to maintain better physical activity function. Generally speaking, the lean muscle mass of young adults accounts for about 50% of the total body weight. After the age of 40, the amount of muscle tissue will gradually decline, about 8% every 10 years to about 70 years old, and then continue to decline at a rate of 15% every 10 years. Sarcopenia, also known as sarcopenia or skeletal muscle atrophy, occurs mostly in the elderly. The main feature of the disease is the progressive and general loss of skeletal muscle mass and function. The incidence of sarcopenia is related to falls, fractures, inconvenience of movement, disability, infection, and physiological and metabolic disorders. Therefore, sarcopenia will reduce the quality of life of the elderly and increase the mortality rate.

At present, sarcopenia is considered to be a new geriatric syndrome. With the increase in human lifespan and the increase in the number of people with metabolic syndrome, research estimates that by 2050, about 200 million people worldwide will suffer from sarcopenia. and, according to research, the annual direct health care costs for sarcopenia exceed $18.5 billion, accounting for approximately 1.5% of total health care expenditures, therefore, the treatment or/or prevention of sarcopenia and its related Disease has become a major research target in care and health promotion this year. If the morbidity rate of sarcopenia can be reduced or the age of onset of sarcopenia can be delayed, medical expenses and national public health burden will be greatly saved.

The main purpose of the present invention is to provide a second use of a short peptide, which can be used to protect muscle cells, promote protein synthesis in muscles, and improve the efficiency of mitochondrial synthesis of energy, so as to achieve prevention or/and treatment and bone Efficacy of muscular dystrophy-related diseases and their complications.

Another object of the present invention is to provide a second use of a short peptide, which can protect muscle cells from damage caused by high-glucose environment or the oxidative stress induced by them, and can maintain the activity of muscle cells and continue to grow, so that Achieve the effect of preventing or/and treating skeletal muscle atrophy-related diseases and their complications.

The reason is that, in order to achieve the above purpose, the embodiments of the present invention disclose the use of a short peptide or a composition containing the same for preventing and/or skeletal muscle atrophy-related diseases, wherein the short peptide The amino acid coding is the sequence shown in SEQ ID No.: 1 or the homologous amino acid sequence derived from one or more amino acid substitutions, deletions, and additions.

Among them, the disease related to skeletal muscle atrophy is muscular dystrophy or sarcopenia.

Among them, the composition can regulate the expression of MAFbx and MuRF1.

Another embodiment of the present invention discloses the use of the short peptide or the mixture containing it for preparing a muscle protein synthesis factor promoter.

Among them, the muscle protein synthesis factor promoter can increase the expression levels of P-ERK/ERK, P-Akt/Akt, P-mTOR/ mTOR and P-FOXO3a/FOXO3a.

In another embodiment of the present invention, the use of short peptides or mixtures containing them for preparing myotube differentiation promoting agents is disclosed.

Among them, the myotube differentiation promoter can inhibit the expression of GSK3β.

In the embodiment of the present invention, the amino acid encoding of the short peptide is the sequence shown in SEQ ID No.: 1.

In the embodiment of the present invention, the mixture containing the short peptide is a potato hydrolyzate.

(I) The DI-10 peptide disclosed in the present invention has an amino acid sequence of SEQ ID No.: 1, a chemical formula of C ₅₅ H ₉₁ N ₁₃ O ₁₅ , and a structure as shown in the following formula (I).

(I)

The DI-10 peptide disclosed in the present invention has the ability to enhance muscle cell proliferation, differentiation and protein synthesis, as well as enhance mitochondrial biosynthesis, reduce intracellular oxidative stress and inhibit Glycogen synthase kinase 3β (GSK3β) gene. Protein expression, which in turn promotes myogenic differentiation and improves myotube and myosin heavy chain (MyHC) protein synthesis. And it can be used as an active ingredient of the composition to achieve the effect of preventing or/and treating diseases related to muscle atrophy.

Furthermore, the DI-10 peptides disclosed in the present invention are obtained by artificial synthesis, recombinant organism production platform or isolated from plants according to the common knowledge in the art and current technology. In previous studies, it was also revealed that the DI-10 peptide can be separated from potato or Solanaceae by enzymatic hydrolysis, which means that the hydrolyzed product of potato or Solanaceae contains the DI-10 disclosed in the present invention. Peptides, such as potatoes, are hydrolyzed with Alcalase to obtain hydrolyzed products containing DI-10 peptides, and the hydrolyzed products have the effect of promoting lipolysis.

The so-called "artificial synthesis method" in the present invention refers to a method in which amino acids are sequentially connected to form a polypeptide by artificial methods such as chemical synthesis method or peptide synthesizer. Chemical synthesis methods include solid-phase peptide synthesis method and liquid-phase peptide synthesis method; solid-phase synthesis method is to carry out the bonding reaction of peptides on solid polymer particles (or polymer supports) in a solvent.

The so-called "recombinant organism production platform" in the present invention refers to constructing a nucleic acid for expressing a specific protein on an expression vector through biological recombination technology to obtain a recombinant vector, and the recombinant expression vector is expressed in a host cell. The specific protein can be obtained by further steps such as separation and purification of the nucleic acid, wherein the host cell can be Bacillus subtilis, yeast, lactic acid bacteria and the like.

The so-called "composition" in the present invention refers to comprising an effective amount of the desired compound or active ingredient to produce a specific effect, and at least one pharmaceutically or a food acceptable carrier. As would be understood by those of ordinary skill in the art to which the present invention pertains, the form of the composition may vary depending on the administration method to induce specific effects, such as lozenges, powders, injections, etc., and the carrier also varies with Depending on the form of the composition, it can be solid, semi-solid or liquid. For example, carriers include, but are not limited to, gelatin, emulsifiers, hydrocarbon mixtures, water, glycerol, saline, buffered saline, lanolin, paraffin, beeswax, simethicone, ethanol.

Hereinafter, in order to describe the technical features and effects of the present invention in detail, some examples and drawings are given for further explanation as follows.

The cell lines used in the following examples are all available for purchase, and are only used to verify the efficacy of the DI-10 peptide disclosed in the present invention, and are not intended to limit the description of the present invention and the scope of the patent application.

In the following examples, unless otherwise specified, the basal medium used for culturing cells is DMEM medium.

Example 1: Preparation of short peptides

The DI-10 peptide with the amino acid sequence of SEQ ID No.: 1 was prepared by chemical synthesis, as shown in Figure 1, and the purity of the DI-10 peptide was confirmed by HPLC and Mass instrument, and the results were shown in Figure 2 and As shown in Figure 3, it is shown that the peptides obtained by chemical synthesis have high purity (about 98%).

Example 2: Preparation of Potato Hydrolyzate

The yield and hydrolysis rate of potato hydrolyzed protein under the same hydrolysis time with 6% hydrolyzing enzyme (Alcalase), the results are shown in Figure 4 and Figure 5, showing that the potato was hydrolyzed with 6% hydrolyzing enzyme (Alcalase) for 22 hours. The hydrolysis rate and yield are the highest.

According to this result, in the following examples of the present invention, the potato hydrolyzate (hereinafter referred to as PP622 hydrolyzate) produced under the conditions of 6% hydrolysis enzyme (Alcalase) and the hydrolysis time of 22 hours is used in the following examples. .

Example 3: Cell Viability Assay

The C2C12 cells were taken and cultured from day 0 to day 2 in DMEM medium supplemented with 0.25μM DEX (dexamethasone), 0.5μM IBMX (3-isobutyl-1-methylxanthanine) and 1 1μg/ml insulin for differentiation culture. , from the 2nd day to the 8th day of culture, the culture medium was replaced with DMEM medium supplemented with insulin for culture, and different concentrations of DI-10 peptides (the amino acid sequence of SEQ ID) were added from the 8th day of culture. No.: 1) and PP622 hydrolyzate were cultured for 3 days. After the culture was completed, the survival rates of C2C12 cells under different culture conditions were detected respectively. The results are shown in Figure 6A and Figure 6B.

It can be seen from the results in Fig. 6A and Fig. 6B that the DI-10 peptide and the PP622 hydrolyzate disclosed in the present invention are not toxic to cells and will not affect cell growth.

Example 4: Detection of muscle-stimulating protein performance

As shown in Example 3, on the 8th day of cell culture, C2C12 cells were treated with different concentrations of DI-10 peptide or PP622 hydrolyzate. Peptide and PP622 hydrolyzate were cultured in C2C12 cells for the expression of proteins related to muscle synthesis, including ERK, Akt, mTOR and FOXO3a, and were quantified to analyze different concentrations of DI-10 peptide and PP622 hydrolyzate, respectively. The results are shown in Figures 7 and 8 for the effects of Akt/mTOR signaling pathways in cells related to myogenesis.

From the results in Figure 7 and Figure 8, it can be seen that DI-10 peptide and PP622 hydrolyzate can induce Akt and mTOR protein phosphorylation, respectively, and can increase the expression of phosphorylated myogenic transcription factor Foxo3a, which shows that this The DI-10 peptide and PP622 hydrolyzate disclosed in the present invention can activate the Akt/mTOR signaling pathway, and with the increase of the additive dose, it means that the DI-10 peptide and PP622 hydrolyzate disclosed in the present invention can promote muscle synthesis, respectively. It can achieve the effect of improving or treating sarcopenia, muscular dystrophy or related diseases caused by it.

Example 5: Detection of intracellular microtubule formation

As shown in the procedure of Example 3, starting from the 8th day of culture, C2C12 cells were cultured in DMEM medium supplemented with different concentrations of DI-10 peptide or PP622 hydrolyzate for 48 hours, and then observed under the microscope after different concentrations of DI-10 Whether the peptide or PP622 hydrolyzate was cultured in C2C12 cells had Microtubule formation, the results are shown in Figure 9A and Figure 9B.

9A and 9B, it can be seen that after treating C2C12 cells with DI-10 peptide or PP622 hydrolyzate at a concentration of 10 μg/ml or more for a predetermined time, microtubules can be formed in the cells. This result shows that the DI-10 peptide or PP622 hydrolyzate disclosed in the present invention can promote the formation of microtubules in C2C12 cells, which means that the DI-10 peptide or PP622 hydrolyzate disclosed in the present invention can promote the structural integrity of muscle fiber cells, Then, it can promote the generation of muscle cells, so as to achieve the effect of improving or treating sarcopenia, muscular dystrophy or related diseases caused by it.

Example 6: Cell viability test under high glucose treatment

The C2C12 cells were taken and cultured in the medium supplemented with different concentrations (0, 5, 15, 30 mM) of glucose, and the cell viability was observed. The results are shown in Figure 10. The results in Figure 10 show that when cells are treated with glucose at a concentration of 30 mM, the cells will encounter high oxidative stress caused by high glucose, resulting in a significant decrease in cell viability.

C2C12 cells were taken, cultured in a medium with a concentration of 30 mM glucose as a high-glucose induction environment, and treated with different concentrations of DI-10 peptide or PP622 hydrolyzate, respectively, and then the survival rate of each C2C12 cell was detected. The results are as follows 11 and 12.

It can be seen from the results in Figure 11 and Figure 12 that the DI-10 peptide and PP622 hydrolyzate disclosed in the present invention can resist the oxidative stress caused by high sugar regardless of the concentration, so that the cells continue to grow, and the cell viability will increase as the concentration increases.

In other words, the results in Figure 11 and Figure 12 show that the DI-10 peptide or PP622 hydrolysate disclosed in the present invention indeed has the effect of promoting the growth of muscle fiber cells, and can still protect the muscle fiber cells even under high oxidative stress induced by high glucose. Muscle cells are not damaged by high oxidative stress. Therefore, the DI-10 peptide or PP622 hydrolyzate disclosed in the present invention has the effect of improving or treating sarcopenia, muscular dystrophy or related diseases caused by it.

Example 7: Detecting the ability of muscle cells to differentiate under high glucose environment

C2C12 cells were taken, cultured in a medium supplemented with 30 mM glucose as a high-glucose induction environment, and then treated with different concentrations of DI-10 peptide or PP622 hydrolyzate, and differentiated and cultured. After the culture was completed, the expression of MyHC (myosin heavy chain) protein in each C2C12 cell was analyzed by Western blotting method and quantified. The results are shown in Figure 13A and Figure 13B .

From the results of Figure 13A and Figure 13B, it can be seen that the DI-10 peptide or PP622 hydrolyzate disclosed in the present invention can promote the differentiation of myotubes at high concentrations (above 10 μg/ml), which means that when the muscle cells are in a high-glucose environment Or when encountering high oxidative stress, an effective dose of the disclosed DI-10 peptide or PP622 hydrolyzate of the present invention can not only protect muscle cells from being destroyed by oxidative stress and die, but also promote muscle protein synthesis and accelerate muscle growth. cell growth. It can be seen that the DI-10 peptide or PP622 hydrolyzate disclosed in the present invention can effectively achieve the effect of improving or treating sarcopenia, muscular dystrophy or related diseases caused by it.

Example 8: Detecting the expression of proteins related to muscle synthesis and mitochondrial biosynthesis in muscle cells under high glucose environment

The process of this example is basically the same as that of Example 7, the difference is that after the culture is completed, the expression of protein synthesis-related proteins and mitochondrial synthesis-related proteins in each C2C12 cell or myotube is analyzed by Western blotting method: pAkt/ Akt, pmTOR/mTOR, pAMPK/AMPK, pFC1, pGSK3β/GSK3β, MAFbx, Murf1, Nrf1, TFam, CoxIV were quantified, and the results are shown in Figure 14 to Figure 27 . In addition, the protein synthesis activity of each C2C12 cell subjected to different treatments under a high glucose environment was observed under a microscope, and the results are shown in FIG. 28 .

From the results in Figure 14 to Figure 28, it can be seen that in a high glucose environment or under high glucose-induced oxidative stress, after the muscle cells are treated with the DI-10 peptide or PP622 hydrolyzate disclosed in the present invention, the levels of pAkt, pmTOR, pAMPK in muscle cells are increased. , Nrf1, TFam, CoxIV, pFC1 increased, and MAFbx, Murf1, pGSK3β/GSK3β and other downstream regulatory factors decreased, showing that the DI-10 peptide or PP622 hydrolyzate disclosed in the present invention can not only protect muscle cells from high glucose or The damage caused by oxidative stress can also promote protein synthesis in muscle cells and improve the efficiency of mitochondrial synthesis of energy.

From the above results, it can be seen that under the high glucose environment or the high oxidative stress induced by it, the DI-10 peptide or PP622 hydrolyzate disclosed in the present invention can effectively enhance the activity of muscle cells, so as to effectively improve or treat muscle cells. Efficacy of hypothyroidism, muscular dystrophy or related diseases caused by it.

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Figure 1 shows the results of confirming the amino acid sequence of the DI-10 peptide disclosed in the present invention. Figure 2 shows the results of confirming the purity of the DI-10 peptide by HPLC, wherein the analysis conditions are as follows: the gradient is 21-46% B buffer for 25 minutes; the buffer A is 0.1% TFA (Trifluoroacetic acid) and 100% Water; Buffer B is 0.1% TFA and 100% acetonitrile. Fig. 3 is the result of confirming the purity of DI-10 peptide by Mass apparatus. Figure 4 shows the hydrolysis rate of potato hydrolyzed protein by 6% hydrolyzing enzyme (Alcalase) at different hydrolysis time. Figure 5 shows the yield of potato hydrolyzed protein with 6% hydrolysis enzyme (Alcalase) at different hydrolysis times. Figure 6A shows the cell viability obtained after adding different concentrations of DI-10 peptide for culture. Figure 6B shows the cell viability obtained after adding different concentrations of PP622 hydrolyzate for culture. Figure 7A shows the expression of proteins related to muscle synthesis in C2C12 cells treated with different concentrations of DI-10 peptide. Figure 7B is the result of quantifying the expression of P-ERK/ERK protein in Figure 7A. Figure 7C is the result of quantifying the expression of P-Akt/Akt protein in Figure 7A. Figure 7D is the result of quantifying the expression of P-mTOR/mTOR protein in Figure 7A. Figure 7E is the result of quantifying the expression of P-FOXO3a/FOXO3a protein in Figure 7A. Figure 8A shows the expression of proteins related to muscle synthesis in C2C12 cells treated with different concentrations of PP622 hydrolyzate. Figure 8B is the result of quantifying the expression of P-ERK/ERK protein in Figure 8A. Figure 8C is the result of quantifying the expression of P-Akt/Akt protein in Figure 8A. Figure 8D is the result of quantifying the expression of P-mTOR/mTOR protein in Figure 8A. Figure 8E is the result of quantifying the expression of P-FOXO3a/FOXO3a protein in Figure 8A. Figure 9A shows the results of microtubule formation in C2C12 cells treated with different concentrations of DI-10 peptide by microscopy. Figure 9B shows the results of microtubule formation in C2C12 cells treated with different concentrations of PP622 hydrolyzate by microscopy. FIG. 10 is the result of examining the cell viability after culturing C2C12 cells with different concentrations of glucose. Figure 11 shows the results of detecting cell viability after treating C2C12 cells with different concentrations of DI-10 peptide under high glucose environment (30mM glucose). Figure 12 shows the results of detecting cell viability after treating C2C12 cells with different concentrations of PP622 hydrolyzate under high glucose environment (30 mM glucose). Figure 13A shows the results of detecting the expression of MyHC protein in each C2C12 cell after treating C2C12 cells with different concentrations of DI-10 peptide under a high glucose environment (30 mM glucose). Figure 13B shows the results of detecting the expression of MyHC protein in each C2C12 cell after treating C2C12 cells with different concentrations of PP622 hydrolyzate under a high glucose environment (30 mM glucose). Figure 14A shows the results of detecting the expression of pAkt/Akt protein in each C2C12 cell after treating C2C12 cells with different concentrations of DI-10 peptide under a high glucose environment (30 mM glucose). Figure 14B is the quantification result of Figure 14A.

Figure 15A shows the results of detecting the expression of pmTOR/mTOR protein in each C2C12 cell after treating C2C12 cells with different concentrations of DI-10 peptide under a high glucose environment (30 mM glucose).

Figure 15B shows the quantitative results of Figure 15A.

Figure 16A shows the results of detecting the expression of pAMPK/AMPK protein in each C2C12 cell after treating C2C12 cells with different concentrations of DI-10 peptide under a high glucose environment (30 mM glucose).

Figure 16B is the quantification result of Figure 16A.

Figure 17A shows the results of detecting the expression of pFC1 protein in each C2C12 cell after treating C2C12 cells with different concentrations of DI-10 peptide under a high glucose environment (30 mM glucose).

Figure 17B is the quantification result of Figure 17A.

Figure 18A shows the results of detecting the expression of pAkt/Akt protein in each C2C12 cell after treating C2C12 cells with different concentrations of PP622 hydrolyzed protein under a high glucose environment (30 mM glucose).

Figure 18B is the quantification result of Figure 18A.

Figure 19A shows the results of detecting the expression of pmTOR/mTOR protein in each C2C12 cell after treating C2C12 cells with different concentrations of PP622 hydrolyzed protein under a high glucose environment (30 mM glucose).

Fig. 19B is the quantitative result of Fig. 19A.

Figure 20A shows the results of detecting the expression of pAMPK/AMPK protein in each C2C12 cell after treating C2C12 cells with different concentrations of PP622 hydrolyzed protein under a high glucose environment (30 mM glucose).

Figure 20B shows the quantitative results of Figure 20A.

Figure 21A shows the results of detecting the expression of pFC1 protein in each C2C12 cell after treating C2C12 cells with different concentrations of PP622 hydrolyzed protein under a high glucose environment (30 mM glucose).

Figure 21B is the quantitative result of Figure 21A.

Figure 22A shows the results of detecting the expression of Nrf1, TFam and CoxIV proteins in C2C12 cells after treating C2C12 cells with different concentrations of DI-10 peptide under high glucose environment (30 mM glucose).

Figure 22B is the quantification result of Figure 22A.

Figure 23A shows the results of detecting the expression of Nrf1, TFam and CoxIV proteins in each C2C12 cell after treating C2C12 cells with different concentrations of PP622 hydrolyzed protein under a high glucose environment (30 mM glucose). Figure 23B is the quantification result of Figure 22A. Figure 24A shows the results of detecting the expression of pGSK3β/GSK3β protein in each C2C12 cell after treating C2C12 cells with different concentrations of DI-10 peptide under a high glucose environment (30 mM glucose). Figure 24B is the quantification result of Figure 24A. Figure 25A shows the results of detecting the expression of pGSK3β/GSK3β protein in each C2C12 cell after treating C2C12 cells with different concentrations of PP622 hydrolyzed protein under a high glucose environment (30 mM glucose). Figure 25B is the quantification result of Figure 25A. Figure 26A shows the results of detecting the expression of MAFbx and Murf1 proteins in each C2C12 cell after treating C2C12 cells with different concentrations of DI-10 peptide under a high glucose environment (30 mM glucose). Figure 26B is the result of the quantification of Figure 26A. Figure 27A shows the results of detecting the expression of MAFbx and Murf1 proteins in each C2C12 cell after treating C2C12 cells with different concentrations of PP622 hydrolyzed protein under a high glucose environment (30 mM glucose). Figure 27B is the quantification result of Figure 27A. Figure 28 shows the results of microscopic observation of protein synthesis in C2C12 cells treated with different treatments in a high-glucose culture environment.

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Claims (9)

Use of a short peptide or a mixture containing the same for preparing a composition for treating or/and preventing skeletal muscle atrophy-related diseases, wherein the amino acid encoding of the short peptide is the sequence shown in SEQ ID No.: 1 . The use according to claim 1, wherein the disease related to skeletal muscle atrophy is muscular dystrophy or sarcopenia. The use according to claim 1, wherein the mixture containing the short peptide is a potato hydrolyzate. Use of a short peptide or a mixture containing it for preparing a muscle protein synthesis factor promoter, wherein the amino acid encoding of the short peptide is the sequence shown in SEQ ID No.: 1. The use according to claim 4, wherein the muscle protein synthesis factor promoter can increase the expression levels of P-ERK/ERK, P-Akt/Akt, P-mTOR/mTOR and P-FOXO3a/FOXO3a. The use according to claim 4, wherein the mixture containing the short peptide is a potato hydrolysate. Use of a short peptide or a mixture containing the same for preparing a myotube differentiation promoting agent, wherein the amino acid encoding of the short peptide is the sequence shown in SEQ ID No.: 1. The use according to claim 7, wherein the myotube differentiation promoter is capable of inhibiting the expression of GSK3β. The use according to claim 7, wherein the mixture containing the short peptide is a potato hydrolyzate.

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