Application of novel peptide UPCP in preparation of medicine for treating and / or preventing sarcopenia

Abstract

The invention discloses an application of a novel peptide UPCP in preparation of a medicine for treating and / or preventing sarcopenia. Belongs to the technical field of biological medicine. Research finds that the novel peptide UPCP can effectively relieve the muscle function, exercise ability and muscle quality of a dexamethasone-induced sarcopenia mouse model, relieve the symptoms of sarcopenia mice to a certain extent, achieve a treatment effect and provide a certain theoretical basis for clinical treatment of muscle-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more specifically to the use of a novel peptide UPCP in the preparation of a drug for treating and / or preventing sarcopenia. Background Art

[0002] Sarcopenia, or muscle loss, is a disease in which skeletal muscle mass and strength gradually decrease with age. Currently, about 50 million people worldwide suffer from sarcopenia, and it is estimated that the number of patients will reach 500 million by 2050. In Asia, the incidence of sarcopenia is 7.3%-12.0%, 5%-13% in the elderly aged 60-70, and 11%-50% in people over 80 years old. It not only affects the quality of life of the elderly, but may also lead to difficulty in movement, limited self-care ability, and even increase the risk of falls, fractures, disability and death.

[0003] At present, there are various treatments for sarcopenia, including drug therapy, exercise therapy, nutritional support, etc. Drug therapy mainly works by promoting muscle growth or reducing inflammatory response, but long-term use of certain hormone drugs may have adverse effects. Exercise therapy is widely recognized as an effective means to improve sarcopenia, especially the combination of resistance training and aerobic exercise. However, exercise therapy is not suitable for every patient, for example, it cannot be applied to patients who are bedridden or have nerve damage. In addition, some patients with severe muscle atrophy are not suitable for exercise therapy. Nutritional support is also an important component of the treatment of sarcopenia. A reasonable diet can effectively improve the patient's muscle mass and strength. However, nutritional supplementation therapy only has a good effect on patients with primary muscle atrophy, and has limited effect on muscle atrophy induced by other causes. In addition, the etiology of sarcopenia is complex, involving multiple factors and pathogenesis, and a single treatment method is difficult to fully cope with these complexities. Therefore, finding new treatments is of great significance to improving the treatment effect and quality of life of patients with sarcopenia.

[0004] Peptide drugs are widely used in medical practice because of their small molecular weight, low toxicity and strong targeting. Studies have found that MYMD-1 is mainly used to slow down the aging process and prevent sarcopenia, and has entered Phase II clinical research; Bimagrumab, as an antibody drug, is used to increase thigh muscle volume; Taldefgrobep Ala, as a protein, may help muscle and bone regeneration; Peptide of follistatin, as an inhibitor of myostatin, is used to treat sarcopenia. In addition, there are some other peptides, such as collagen peptides, glucagon-like peptide 1 receptor agonists, etc., which have certain effects on sarcopenia, suggesting that the development of peptide drugs has great application prospects for the treatment of sarcopenia. However, these peptide drugs are still in different stages of development, and their effectiveness and safety still need further clinical trials to verify.

[0005] Therefore, how to find a polypeptide that has a good therapeutic effect on sarcopenia is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0006] In view of this, the present invention provides an application of a novel peptide UPCP in the preparation of drugs for the treatment and / or prevention of sarcopenia. Through research, it was found that the novel peptide UPCP of the present invention can effectively alleviate the muscle function, motor ability and muscle mass of the dexamethasone-induced sarcopenia mouse model, alleviate the symptoms of the sarcopenia model mice to a certain extent, and achieve a therapeutic effect.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] Application of a novel peptide UPCP in the preparation of drugs for treating and / or preventing sarcopenia.

[0009] Preferably, the novel peptide UPCP is composed of 47 L-type amino acids including the transmembrane peptide HIV-TAT, and its amino acid sequence is as follows:

[0010] New UPCP: YGRKKRRQRRRYNLRSRTVLCGTCGQPADKASASGSGAQS PQNCSIM, SEQ IDNO.1.

[0011] Another object of the present invention is to provide: a drug for treating and / or preventing sarcopenia, comprising the above-mentioned novel peptide UPCP or the novel peptide UPCP with a labeling group.

[0012] Preferably, the drug further comprises other pharmaceutical excipients and / or other functional active ingredients.

[0013] Preferably, the drug exerts a therapeutic effect by treating or improving the degree of muscle atrophy.

[0014] Preferably, the drug exerts a therapeutic effect by improving the body's motor ability.

[0015] Preferably, the drug exerts a therapeutic effect by improving the body's muscle mass.

[0016] Preferably, the drug exerts a therapeutic effect by improving the body's muscle function.

[0017] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides an application of a novel peptide UPCP in the preparation of a drug for treating and / or preventing sarcopenia. Through research, it is found that the novel peptide UPCP of the present invention can effectively alleviate the muscle function, motor ability and muscle mass of a dexamethasone-induced sarcopenia mouse model, alleviate the symptoms of the sarcopenia model mice to a certain extent, have a therapeutic effect, and provide a certain theoretical basis for the treatment of clinical muscle-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 for: Figure 1 Results of UPCP cell safety analysis; A: morphological changes of C2C12 cells; B and C: changes in the expression of Ki67 in C2C12 cells; D: changes in the OD value of C2C12 cells; E: changes in the survival rate of C2C12 cells, ns, no significant difference;

[0021] Figure 2 Figure 1: Construction of C2C12 myoblast differentiation model; A: Schematic diagram of C2C12 myoblasts differentiated into myotubes after 7 days of induction with differentiation medium; B: Observation of cell morphology changes after differentiation under an optical microscope, Scale bars = 100 μm; C: Immunofluorescence assay to detect changes in MHC expression levels in C2C12 cells before and after differentiation, Scale bars = 100 μm; D: Western Blot assay to detect changes in MHC expression levels during differentiation;

[0022] Figure 3A: Construction of a dexamethasone (Dex)-induced atrophy model of C2C12 myotube cells; A: Schematic diagram of the construction of atrophy model of C2C12 myotube cells; B: Observation of cell morphology changes after atrophy induction under an optical microscope, Scale bars = 200 μm; C: Real-time PCR detection of the mRNA expression changes of atrophy markers MuRF-1 and Atrogin-1 in C2C12 myotube cells after the addition of dexamethasone (Dex); D: Western blot method detection of the changes in the expression level of MHC in C2C12 myotube cells after the addition of dexamethasone (Dex);

[0023] Figure 4 A: Effect of UPCP on improving dexamethasone (Dex)-induced atrophy of C2C12 myotubes; A: UPCP alleviates Dex-induced atrophy of C2C12 myotubes; B: Optical microscopy observation of cell morphology changes after adding UPCP to Dex-induced C2C12 myotubes, Scale bars = 100 μm; C: Real-time PCR detection of the effect of adding UPCP to C2C12 myotubes on the mRNA expression of atrophy markers MuRF-1 and Atrogin-1; D: Western Blot experiment detection of MHC expression in C2C12 myotubes after adding UPCP;

[0024] Figure 5 Figure 4: Construction and verification of the sarcopenia mouse model; A: Schematic diagram of the construction of a muscle atrophy mouse model by intraperitoneal injection of dexamethasone; B: Changes in body weight of mice after daily injection of dexamethasone. C: Difference in body weight between the model group and the control group 14 days after dexamethasone injection; DF: Results of open field, rotarod fatigue, and grip strength tests 14 days after dexamethasone injection, respectively;

[0025] Figure 6 A: Diffusion of UPCP in mice after intraperitoneal injection; A: FITC-UPCP in vivo detection flow chart; B: Fluorescence microscopy observation of FITC-UPCP distribution in muscle tissue;

[0026] Figure 7 A: UPCP improves muscle and motor dysfunction in sarcopenic mice; A: Schematic diagram of UPCP treatment of dexamethasone-induced sarcopenic mice; B: Visualization of the movement trajectory of mice in the open field test and the statistics of the total movement distance of mice; C: Statistical results of the time mice spent on the rod in the rotating rod fatigue test; D and E: The measurement results of the grip strength of mice and the calculation results of the absolute grip strength based on body weight; F: The daily changes in body weight of the control group, model group and UPCP treatment group; G: The difference in body weight of the control group, model group and UPCP treatment group after 20 days of treatment;

[0027] Figure 8 Figure 4: UPCP improves the gastrocnemius muscle mass and function of sarcopenic mice; A: Assessment of gastrocnemius muscle mass in mice; B: Ratio of gastrocnemius muscle mass to the overall mass of mice; C: Comparison of gastrocnemius muscles in mice; D: HE staining results of gastrocnemius muscle tissue in mice; E: Statistical results of muscle fiber cross-sectional area. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The cells used in the practice of the present invention are mouse C2C12 myoblasts purchased from the American Type Culture Collection (ATCC, CCL-185).

[0030] The living animals used in the examples of the present invention are C57BL / 6 male wild-type mice purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0031] Example 1

[0032] A novel peptide UPCP is used in the preparation of a drug for treating and / or preventing sarcopenia; the novel peptide UPCP is composed of 47 L-type amino acids including the transmembrane peptide HIV-TAT, and its amino acid sequence is as follows: novel UPCP: YGRKKRRQRRRYNLRSRTVLCGTCGQPADKASASGSGAQSPQN CSIM, SEQ ID NO.1.

[0033] Example 2

[0034] Safety analysis of the new peptide UPCP

[0035] (1) Cytotoxicity test:

[0036] In order to explore whether the novel peptide UPCP is cytotoxic, experiments with different concentration gradients were set up for the novel peptide UPCP, as follows:

[0037] Experimental group 1: DMEM complete medium + 6 μM UPCP;

[0038] Experimental group 2: DMEM complete medium + 12 μM UPCP;

[0039] Control group: DMEM complete medium;

[0040] C2C12 cells (ATCC, CCL-185) were cultured in the above three culture media for 48 h, and the changes in the morphology of C2C12 cells after treatment with different concentrations of UPCP were observed under a microscope. The experimental results are as follows: Figure 1 As shown in A.

[0041] Result analysis: Figure 1 As shown in A, 6 μM and 12 μM UPCP did not affect the morphology of C2C12 cells.

[0042] (2) Cell proliferation assay:

[0043] Immunofluorescence assay was used to detect the expression of Ki67 (cell cycle-related proliferating cell nuclear protein) in C2C12 cells after 48 h of culture in three groups of experiments in step (1). The experimental results are as follows: Figure 1 B and 1C.

[0044] Result analysis: Figure 1 As shown in B and 1C, the novel peptide UPCP had no significant effect on Ki67 expression in C2C12 cells.

[0045] (3) Cell activity assay:

[0046] Cell Counting Kit-8 (CCK-8) (APExBIO k1018) was used to detect the activity of C2C12 cells after 72 hours of culture in three groups of experiments in step (1), and the effect of the new peptide UPCP on the activity of C2C12 cells was recorded. The experimental results are shown in Figure 1 D and 1E.

[0047] Result analysis: Figure 1 As shown in D and 1E, compared with the control cells, 6 μM and 12 μM of the novel peptide UPCP had no significant effect on the activity of C2C12 cells.

[0048] Therefore, the experimental results of steps (1)-(3) show that low concentrations of the new peptide UPCP have no obvious toxic effect on cells.

[0049] Example 3

[0050] Study on the effect of novel peptide UPCP on improving dexamethasone-induced C2C12 myotube atrophy

[0051] (1) Establishment of C2C12 myoblast differentiation model

[0052] Take C2C12 cells in good condition for culture. When the cell confluence is 70%-80%, use differentiation medium containing 2% horse serum to culture the cells. After 5-7 days, the C2C12 myoblasts will differentiate into myotubes. Observe the cell morphology under a microscope.

[0053] Result analysis: Figure 2 As shown in A and 2B, the cells began to differentiate after 3 days of culture with 2% horse serum, and a large number of myotubes were produced after 5 days of differentiation. After 7 days, the cells were almost completely differentiated into myotubes.

[0054] At the same time, immunofluorescence assay and Western blot were used to measure the differentiation marker MHC in C2C12 cells after 7 days of culture with horse serum. The experimental results were as follows: Figure 2 C and Figure 2 As shown in D, the results showed that after 7 days, the expression of differentiation marker MHC in cells increased significantly, and its expression showed an increasing trend with the increase of differentiation days.

[0055] In conclusion, culturing C2C12 cells with 2% horse serum for 7 days can induce C2C12 cell differentiation, and a C2C12 myoblast differentiation model was successfully established.

[0056] (2) Establishment of C2C12 myotube atrophy model

[0057] Based on the C2C12 cell differentiation model, a dexamethasone-induced C2C12 myotube atrophy model was used. Well-differentiated C2C12 myotube cells were taken and treated with 50 μM dexamethasone (Dex) for 24 h ( Figure 3 A), Observe the morphological changes of C2C12 myotube cells under a microscope, such as Figure 3 As shown in B, Dex induced morphological changes in myotube cells.

[0058] After 24 hours of Dex treatment, the genes of atrophy markers MuRF-1 and Atrogin-1 in the cells were detected by qPCR. The primer sequences for detecting atrophy markers MuRF-1 and Atrogin-1 are as follows:

[0059] β-actin(mouse)-sense: 5'-CCTCTATGCCAACACAGTGC-3', SEQ ID NO.2;

[0060] β-actin (mouse)-antisense: 5'-ACATCTGCTGGAAGGTGGAC-3', SEQ ID NO.3; this gene was used as an internal reference gene for local control;

[0061] Atrogin-1(mouse)-sense: 5'-CAGCTTCGTGAGCGACCTC-3', SEQ ID NO.4;

[0062] Atrogin-1(mouse)-antisense: 5'-GGCAGTCGAGAAGTCCAGTC-3', SEQ ID NO.5;

[0063] MuRF-1(mouse)-sense: 5'-CCAGGCTGCGAATCCCTAC-3', SEQ ID NO.6;

[0064] MuRF-1(mouse)-antisense: 5'-ATTTTCTCGTCTTCGTGTTCCTT-3', SEQ ID NO.7.

[0065] The changes in the expression of atrophy marker genes were detected. Figure 3 As shown in C, the expression levels of atrophy markers MuRF-1 and Atrogin-1 genes in C2C12 myotube cells treated with Dex were significantly upregulated compared with those in the control group.

[0066] Western blot was used to detect the expression of MHC after dexamethasone-induced myotube atrophy. The experimental results are as follows Figure 3 As shown in D, the results showed that the expression of MHC was significantly decreased after dexamethasone induced myotube atrophy.

[0067] Based on the above results, myotube atrophy cell model was successfully induced after myotube cells were treated with 50μM dexamethasone for 24h.

[0068] (3) The novel peptide UPCP alleviates Dex-induced atrophy of C2C12 myotube cells

[0069] Experimental group: C2C12 myotube cells in good condition were cultured. When the cell confluence was 70%-80%, 50 μM dexamethasone (Dex) was added for 24 h. New DMEM + 2% horse serum medium was replaced, and 6 μM of the novel peptide UPCP was added for 48 h. The experimental process is as follows: Figure 4 As shown in A Figure 4 A).

[0070] Control group: Different from the experimental group, Dex was not added as the control group.

[0071] The results of morphological experiments showed that the diameter of C2C12 myotubes treated with Dex was significantly reduced compared with the control group, indicating that Dex induced atrophy of C2C12 myotubes. When UPCP was added to the atrophied C2C12 myotubes, the diameter of the myotubes increased to a certain extent ( Figure 4 B).

[0072] After culturing with the novel peptide UPCP for 48 hours, the cells in each group were subjected to qPCR detection. The primer sequences for detecting atrophy markers MuRF-1 and Atrogin-1 were as above. The experiment showed that the expression levels of atrophy markers MuRF-1 and Atrogin-1 genes in C2C12 myotubes treated with Dex were significantly upregulated compared with those in the control group, while the expression levels of the corresponding atrophy genes in C2C12 myotubes treated with the novel peptide UPCP were significantly downregulated ( Figure 4 C).

[0073] Western blot was used to detect the expression of MHC after treatment with the novel peptide UPCP. The experimental results showed that the expression of MHC was downregulated after Dex treatment, while the expression of MHC was restored after adding the novel peptide UPCP ( Figure 4 D).

[0074] In summary, the above experimental results show that UPCP can alleviate Dex-induced atrophy of C2C12 myotube cells

[0075] Example 3

[0076] Study on the effect of novel peptide UPCP on improving the sarcopenia phenotype induced by dexamethasone in mice

[0077] (1) Construction of Dex-induced sarcopenia mouse model

[0078] Sarcopenia model group: 8-week-old C57BL / 6 male wild-type mice were intraperitoneally injected with dexamethasone (20 mg / kg, diluted in saline) every day after feeding for one week for two weeks ( Figure 5 A);

[0079] Control group: 8-week-old male wild-type mice of the C57BL / 6 strain were intraperitoneally injected with an equal amount of normal saline every day after being raised for one week for two weeks.

[0080] a: Mouse weight monitoring:

[0081] In order to evaluate the modeling effect of Dex, the body weight of mice was monitored throughout the modeling process.

[0082] The results are as follows Figure 5As shown in B and 5C, the body weight of the mice in the sarcopenia model group showed a significant downward trend compared with the control group. Two weeks after modeling, the body weight of the mice in the sarcopenia model group was significantly lower than that of the mice in the control group.

[0083] b: Mouse exercise ability test:

[0084] Two weeks after modeling, the mice in the sarcopenia model group had muscle atrophy, and the muscle mass and function of the mice decreased, and the corresponding exercise ability was also weakened. In order to further evaluate the effect of dexamethasone on inducing muscle atrophy in mice, the mice were subjected to open field test, rotating rod test and grip strength test two weeks after modeling. The experimental results are as follows: Figure 5 As shown in D-5F.

[0085] The results showed that in terms of athletic ability, the athletic ability of mice in the sarcopenia model group was significantly weakened compared with that in the control group.

[0086] In summary, the above results indicate that the dexamethasone-induced muscle atrophy mouse model was successfully established.

[0087] (2) Study on the diffusion of the novel peptide UPCP in mouse muscle tissue

[0088] In order to detect the diffusion of UPCP in the muscle tissue of mice, FITC-UPCP (FITC group was labeled at the N-terminus of UPCP, synthesized by Shanghai Jier Company) was injected into mice via intraperitoneal injection at a dose of 20 mg / kg. After 24 hours of injection, the muscle tissue was collected by perfusion and made into frozen sections, and the FITC fluorescence signal was collected under a fluorescence microscope.

[0089] The results showed that FITC-UPCP could diffuse into the mouse muscle tissue 24 hours after injection, and a strong FITC signal could be clearly detected in the mouse muscle tissue ( Figure 6 ), indicating that FITC-UPCP has strong penetrability and can diffuse from the abdominal cavity to the muscle tissue within a certain period of time.

[0090] (3) Study on the effect of the novel peptide UPCP on improving the sarcopenia phenotype induced by dexamethasone in mice

[0091] In order to explore the therapeutic effect of UPCP, the above successfully established sarcopenia model mice were divided into two groups, one group was given the novel peptide UPCP (UPCP treatment group), and the other group was not given (model group); the above control group mice were taken as controls, and the above three groups of mice were subjected to the following operations ( Figure 7 A):

[0092] UPCP treatment group: UPCP (20 mg / kg) was injected intraperitoneally once every other day;

[0093] Control group: injected with an equal amount of normal saline every other day;

[0094] Model group: injected with an equal amount of normal saline every other day;

[0095] a: Mouse weight monitoring

[0096] To evaluate the therapeutic effect of the novel peptide UPCP, the body weight of the mice was monitored throughout the treatment.

[0097] The results are as follows Figure 7 As shown in FG, compared with the control group, the body weight of the model group mice (treated with dexamethasone) showed a significant downward trend, while the body weight of the sarcopenic mice after UPCP treatment did not significantly improve compared with the untreated group (model group). Previous studies have shown that dexamethasone treatment will increase the fat of mice to a certain extent, which is probably the reason why the weight of untreated sarcopenic mice will show an upward trend in the later period, while the weight of the mice in the UPCP treatment group did not continue to increase compared with the untreated group, suggesting that the new peptide UPCP is likely to inhibit the increase of fat in sarcopenic mice to a certain extent.

[0098] b: Mouse muscle function and motor ability detection

[0099] In order to evaluate the therapeutic effect of UPCP on muscular atrophy mice, the open field test was used to evaluate the therapeutic effect of UPCP on the 19th day after intraperitoneal injection of UPCP ( Figure 7 B); Rotarod test was performed on the 20th day after intraperitoneal injection of UPCP ( Figure 7 C), the time mice stayed on the rod increased significantly after UPCP treatment. At the same time, the grip strength of the treated mice also increased ( Figure 7 D-7E).

[0100] The experimental results show that UPCP can improve the muscle function and motor ability of sarcopenic mice.

[0101] c: Gastrocnemius muscle mass and function testing in mice

[0102] In addition, after UPCP treatment, all mice were killed by cervical dislocation, and the gastrocnemius muscles were isolated, weighed, and fixed in paraformaldehyde for HE staining. The experimental results are shown in Figure 8 As shown in A-8E.

[0103] The experimental results showed that UPCP could improve the gastrocnemius muscle mass and increase the cross-sectional area of ​​muscle fibers in sarcopenic mice.

[0104] The above experiments suggest that UPCP can improve the muscle function and motor ability of sarcopenic mice.

[0105] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0106] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of a novel peptide UPCP in the preparation of drugs for the treatment and / or prevention of sarcopenia.

2. The use according to claim 1, characterized in that: The novel peptide UPCP is composed of 47 L-type amino acids including the transmembrane peptide HIV-TAT, and its amino acid sequence is as follows: New UPCP: YGRKKRRQRRRYNLRSRTVLCGTCGQPADKASASGSGAQS PQNCSIM, SEQ ID NO.

1.

3. A drug for treating and / or preventing sarcopenia, characterized in that: It comprises the novel peptide UPCP as described in claim 1 or 2 or the novel peptide UPCP with a labeling group.

4. The drug for treating and / or preventing sarcopenia according to claim 3, characterized in that It also includes other pharmaceutical excipients and / or other functional active ingredients.

5. The drug for treating and / or preventing sarcopenia according to claim 3 or 4, characterized in that: The drug exerts a therapeutic effect by treating or improving the degree of muscle atrophy.

6. The drug for treating and / or preventing sarcopenia according to claim 3 or 4, characterized in that: The drug exerts its therapeutic effect by improving the body's motor ability.

7. The drug for treating and / or preventing sarcopenia according to claim 3 or 4, characterized in that: The drug exerts its therapeutic effect by improving the body's muscle mass.

8. The drug for treating and / or preventing sarcopenia according to claim 3 or 4, characterized in that: The drug exerts its therapeutic effect by improving the body's muscle function.

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