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

Abstract

This invention discloses the application of a novel peptide UPCP in the preparation of drugs for the treatment and / or prevention of sarcopenia. It belongs to the field of biomedical technology. Research has shown that the novel peptide UPCP of this invention can effectively alleviate muscle function, motor ability, and muscle mass in a dexamethasone-induced sarcopenia mouse model, alleviating symptoms to a certain extent and achieving a therapeutic effect, thus providing a theoretical basis for the clinical treatment of muscle-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the application of a novel peptide UPCP in the preparation of drugs for the treatment and / or prevention of sarcopenia. Background Technology

[0002] Sarcopenia, or muscle loss disorder, is a condition characterized by a gradual decline in skeletal muscle mass and strength that occurs with age. It not only affects the quality of life of older adults but can also lead to difficulty in movement, limited self-care abilities, and even increase the risk of falls, fractures, disability, and death.

[0003] Currently, there are various treatment options for sarcopenia, including drug therapy, exercise therapy, and nutritional support. Drug therapy mainly works by promoting muscle growth or reducing inflammation, but long-term use of certain hormonal drugs may have adverse effects. Exercise therapy is widely recognized as an effective means of improving sarcopenia, especially the combination of resistance training and aerobic exercise. However, exercise therapy is not suitable for every patient; for example, it is not suitable for patients who are bedridden or have nerve damage. In addition, exercise therapy is not suitable for some patients with severe muscle atrophy. Nutritional support is also an important component of sarcopenia treatment; a proper diet can effectively improve muscle mass and strength. However, nutritional supplementation is only effective for patients with primary muscle atrophy, and its effectiveness is limited for muscle atrophy induced by other causes. Furthermore, the etiology of sarcopenia is complex, involving multiple factors and pathogenesis, and a single treatment method cannot comprehensively address these complexities. Therefore, finding new treatment methods is of great significance for improving the treatment outcomes and quality of life for patients with sarcopenia.

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

[0005] Therefore, finding a polypeptide that has a good therapeutic effect on sarcopenia is a technical problem that urgently needs to be solved by those skilled in the art. 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. Studies have shown that the novel peptide UPCP of the present invention can effectively alleviate muscle function, motor ability and muscle mass in dexamethasone-induced sarcopenia mouse models, and alleviate the symptoms of sarcopenia model mice to a certain extent, thus achieving a therapeutic effect.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Application of a novel peptide UPCP in the preparation of drugs for the treatment and / or prevention of 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: YGRKKRRQRRRYNLRSRTVLCGTCGQPADKASASGSGAQSPQNCSIM, SEQ ID NO.1.

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

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

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

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

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

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

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

[0018] This invention provides the application of a novel peptide UPCP in the preparation of drugs for the treatment and / or prevention of sarcopenia. Studies have shown that the novel peptide UPCP of this invention can effectively alleviate muscle function, motor ability and muscle mass in a dexamethasone-induced sarcopenia mouse model, and alleviate the symptoms of sarcopenia model mice to a certain extent, thus achieving a therapeutic effect and providing a certain theoretical basis for the clinical treatment of muscle-related diseases. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 for: Figure 1 The results of UPCP cell safety analysis are as follows: A: morphological changes in C2C12 cells; B and C: changes in Ki67 expression in C2C12 cells; D: changes in OD value of C2C12 cells; E: changes in C2C12 cell viability (ns), with no significant difference.

[0021] Figure 2 For: Construction of a C2C12 myoblast differentiation model; A: Schematic diagram of C2C12 myoblasts differentiating into myotubes after 7 days of induction with differentiation medium; B: Observation of cell morphology changes after induced differentiation under an optical microscope, scale bars = 100 μm; C: Detection of changes in MHC expression levels in C2C12 cells before and after differentiation using immunofluorescence assay, scale bars = 100 μm; D: Detection of changes in MHC expression levels during induced differentiation using Western blotting assay.

[0022] Figure 3 The objectives are as follows: A) Construction of a dexamethasone (Dex)-induced C2C12 myotube cell atrophy model; B) Schematic diagram of the C2C12 myotube cell atrophy model; C) Observation of cell morphological changes after induced atrophy under an optical microscope (Scale bars = 200 μm); D) Real-time PCR detection of changes in the mRNA expression of atrophy markers MuRF-1 and Atrogin-1 in C2C12 myotube cells after the addition of dexamethasone (Dex); E) Western blot detection of changes in MHC expression levels in C2C12 myotube cells after the addition of dexamethasone (Dex).

[0023] Figure 4The objectives are as follows: A) Effect of UPCP on dexamethasone (Dex)-induced atrophy of C2C12 myotube cells; B) Schematic diagram of UPCP alleviating Dex-induced atrophy of C2C12 myotube cells; C) Effect of UPCP on the mRNA expression of atrophy markers MuRF-1 and Atrogin-1 in C2C12 myotube cells after addition, observed by optical microscopy (scale bars = 100 μm); D) Effect of UPCP on the mRNA expression of atrophy markers MuRF-1 and Atrogin-1 in C2C12 myotube cells after addition, detected by real-time PCR; E) Western blot analysis of MHC expression in C2C12 myotube cells after addition of UPCP.

[0024] Figure 5 For: Construction and validation of a mouse model of sarcopenia; A: Schematic diagram of a mouse model of muscle atrophy constructed 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;

[0025] Figure 6 The following are examples of FITC-UPCP diffusion in mice after intraperitoneal injection: A: Flowchart of FITC-UPCP in vivo detection; B: Distribution of FITC-UPCP in muscle tissue observed by fluorescence microscopy.

[0026] Figure 7 The objectives are as follows: A: UPCP improves muscle and motor dysfunction in sarcopenic mice; B: Visual image of mouse open field test trajectory and statistics of total movement distance; C: Statistical results of mouse time on the rotarod fatigue test; D and E: Measurement results of mouse grip strength and results of absolute grip strength calculated from body weight, respectively; F: Daily changes in body weight of the control group, model group, and UPCP treatment group; G: Body weight differences of the control group, model group, and UPCP treatment group after 20 days of treatment.

[0027] Figure 8 The purpose of this study was to improve the gastrocnemius muscle mass and function in sarcopenic mice using UPCP. The results included: A: Gastrocnemius muscle mass assessment in mice; B: Gastrocnemius muscle mass to overall mouse mass ratio; C: Comparison of gastrocnemius muscle mass in mice; D: HE staining results of gastrocnemius muscle tissue in mice; and E: Statistical results of muscle fiber cross-sectional area. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

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

[0031] Example 1

[0032] The application of a novel peptide UPCP in the preparation of drugs for the treatment and / or prevention of 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:

[0033] New UPCP: YGRKKRRQRRRYNLRSRTVLCGTCGQPADKASASGSGAQSPQNCSIM, SEQ ID NO.1.

[0034] Example 2

[0035] Safety analysis of novel peptide UPCP

[0036] (1) Cytotoxicity test:

[0037] To investigate whether the novel peptide UPCP has cytotoxicity, experiments were conducted with different concentration gradients of UPCP, as detailed below:

[0038] Experimental group 1: DMEM complete medium + 6 μM UPCP;

[0039] Experimental group 2: DMEM complete medium + 12 μM UPCP;

[0040] Control group: DMEM complete culture medium;

[0041] C2C12 cells (ATCC, CCL-185) were cultured in the three culture media mentioned above for 48 hours. The morphological changes 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 Figure A.

[0042] Results analysis: such as Figure 1 As shown in Figure A, 6 µM and 12 µM UPCP did not affect the morphology of C2C12 cells.

[0043] (2) Cell proliferation experiment:

[0044] Immunofluorescence assays were used to detect the expression of Ki67 (a cell cycle-related proliferating 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 As shown in B and 1C.

[0045] Results analysis: such as Figure 1 As shown in B and 1C, the novel peptide UPCP did not have a significant effect on Ki67 expression in C2C12 cells.

[0046] (3) Cell viability experiment:

[0047] The activity of C2C12 cells after 72 h of culture in step (1) was detected using Cell Counting Kit-8 (CCK-8) (APExBIO k1018). The effect of the novel peptide UPCP on the viability of C2C12 cells was recorded. The experimental results are as follows: Figure 1 As shown in D and 1E.

[0048] Results analysis: such as Figure 1 As shown in D and 1E, the novel peptide UPCP at 6 µM and 12 µM had no significant effect on the activity of C2C12 cells compared with the control group.

[0049] Therefore, the experimental results of steps (1)-(3) show that the low concentration of the novel peptide UPCP has no obvious toxic effect on cells.

[0050] Example 3

[0051] An Investigation into the Effect of Novel Peptide UPCP on Improving Dexamethasone-Induced C2C12 Myotube Atrophy

[0052] (1) Establishment of a C2C12 myoblast differentiation model

[0053] Healthy C2C12 cells were cultured. When the cell confluence reached 70%-80%, the cells were cultured in differentiation medium containing 2% horse serum. After 5-7 days, the C2C12 myoblasts differentiated into myotube cells. Cell morphological changes were observed under a microscope.

[0054] Results analysis: such as Figure 2 As shown in A and 2B, cells begin to differentiate after 3 days of culturing with 2% horse serum, produce a large number of myotubes after 5 days, and are almost completely differentiated into myotube cells after 7 days.

[0055] Meanwhile, the differentiation marker MHC in C2C12 cells cultured in horse serum for 7 days was measured using immunofluorescence and Western blot methods. The experimental results are as follows: Figure 2 C and Figure 2 As shown in Figure D, the results indicate that after 7 days, the expression of the differentiation marker MHC in cells increased significantly, and its expression showed an increasing trend with the increase of differentiation days.

[0056] In summary, culturing C2C12 cells in 2% horse serum for 7 days can induce C2C12 cell differentiation, successfully constructing a C2C12 myoblast differentiation model.

[0057] (2) Establishment of the C2C12 myotube atrophy model

[0058] Based on the C2C12 cell differentiation model, a C2C12 myotube atrophy model was induced using dexamethasone.

[0059] Well-differentiated C2C12 myotube cells were 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 induces morphological changes in myotube cells.

[0060] After 24 h of Dex treatment, qPCR was performed to detect the atrophy markers MuRF-1 and Atrogin-1 in the cells. The primer sequences for detecting the atrophy markers MuRF-1 and Atrogin-1 are as follows:

[0061] β-actin(mouse)-sense: 5'-CCTCTATGCCAACACAGTGC-3', SEQ ID NO.2;

[0062] β-actin(mouse)-antisense: 5'-ACATCTGCTGGAAGGTGGAC-3', SEQ ID NO.3; This gene is used as an internal reference gene for local control.

[0063] Atrogin-1(mouse)-sense: 5'-CAGCTTCGTGAGCGACCTC-3', SEQ ID NO.4;

[0064] Atrogin-1(mouse)-antisense: 5'-GGCAGTCGAGAAGTCCAGTC-3', SEQ ID NO.5;

[0065] MuRF-1(mouse)-sense: 5'-CCAGGCTGCGAATCCCTAC-3', SEQ ID NO.6;

[0066] MuRF-1(mouse)-antisense: 5'-ATTTTCTCGTCTTCGTGTTCCTT-3', SEQ ID NO.7.

[0067] The changes in the expression levels of atrophy marker genes were detected, and the results were as follows: Figure 3 As shown in Figure C, the expression levels of the atrophy markers MuRF-1 and Atrogin-1 genes in C2C12 myotube cells treated with Dex were significantly upregulated compared to the control group.

[0068] Western blot analysis was used to detect MHC expression levels after dexamethasone-induced myotube atrophy. The results are as follows: Figure 3 As shown in Figure D, the results indicate that MHC expression levels were significantly reduced after dexamethasone-induced myotube atrophy.

[0069] Based on the above results, treatment of myotube cells with 50 µM dexamethasone for 24 h successfully induced a myotube atrophy cell model.

[0070] (3) The novel peptide UPCP alleviates Dex-induced atrophy of C2C12 myotube cells

[0071] Experimental group: Healthy C2C12 myotube cells were cultured. When the cell confluence reached 70%-80%, 50 µM dexamethasone (Dex) was added for 24 h. The medium was then replaced with fresh DMEM + 2% horse serum medium, and 6 µM of the novel peptide UPCP was added for 48 h of culture. The experimental procedure is as follows: Figure 4 As shown in A ( Figure 4 A).

[0072] Control group: Unlike the experimental group, no Dex was added as the control group.

[0073] Morphological experiments showed that, compared with the control group, the diameter of C2C12 myotube cells treated with Dex was significantly reduced, indicating that Dex induced atrophy of C2C12 myotube cells. When UPCP was added to the atrophied C2C12 myotube cells, the diameter of the myotubes increased to some extent. Figure 4 B).

[0074] After culturing the cells with the novel peptide UPCP for 48 h, qPCR was performed on each group of cells. The primer sequences for detecting the atrophy markers MuRF-1 and Atrogin-1 are shown above. The experiment showed that the expression levels of the atrophy markers MuRF-1 and Atrogin-1 genes in C2C12 myotube cells treated with Dex were significantly upregulated compared with the control group, while the expression levels of the corresponding atrophy genes in C2C12 myotube cells treated with the novel peptide UPCP were significantly downregulated. Figure 4 C).

[0075] Western blot analysis was used to detect the MHC expression levels of the novel peptide UPCP after treatment. The results showed that MHC expression was downregulated after Dex treatment, but recovered after the addition of the novel peptide UPCP. Figure 4 D).

[0076] In summary, the above experimental results indicate that UPCP can alleviate Dex-induced atrophy of C2C12 myotube cells.

[0077] Example 4

[0078] Investigation into the effect of novel peptide UPCP on dexamethasone-induced sarcopenia phenotype in mice

[0079] (1) Construction of Dex-induced sarcopenia mouse model

[0080] Sarcopenia model group: Eight-week-old male wild-type mice of strain C57BL / 6 were intraperitoneally injected daily for two weeks after one week of feeding. Figure 5 A);

[0081] Control group: Eight-week-old male wild-type mice of the C57BL / 6 strain were injected intraperitoneally with an equal volume of physiological saline every day for two weeks after being fed for one week.

[0082] a: Mouse weight monitoring:

[0083] To evaluate the modeling effect of Dex, the weight of the mice was monitored throughout the modeling process.

[0084] The results are as follows Figure 5 As shown in B and 5C, compared with the control group, the body weight of mice in the sarcopenia model group showed a significant decreasing trend. Two weeks after modeling, the body weight of mice in the sarcopenia model group was significantly lower than that of mice in the control group.

[0085] b: Mouse motor ability test:

[0086] Two weeks after modeling, the sarcopenia model group mice showed muscle atrophy, with decreased muscle mass and function, and correspondingly weakened motor ability. To further evaluate the effect of dexamethasone on inducing muscle atrophy in mice, open field tests, rotarod tests, and grip strength tests were performed on the mice two weeks after modeling. The experimental results are as follows: Figure 5 As shown in D-5F.

[0087] The results showed that, in terms of motor ability, the mice in the sarcopenia model group had significantly reduced motor ability compared with the control group.

[0088] In summary, the above results demonstrate that a dexamethasone-induced muscular atrophy mouse model has been successfully established.

[0089] (2) Investigation on the diffusion of the novel peptide UPCP in mouse muscle tissue

[0090] To detect the diffusion of UPCP in mouse muscle tissue, FITC-UPCP (synthesized by Shanghai Jier Company with FITC group labeled at the N end of UPCP) was injected into mice intraperitoneally at a dose of 20 mg / kg. 24 hours after injection, muscle tissue was collected by perfusion and prepared into frozen sections. FITC fluorescence signals were collected under a fluorescence microscope.

[0091] The results showed that FITC-UPCP diffused into mouse muscle tissue 24 hours after injection, and a strong FITC signal could be clearly detected in mouse muscle tissue. Figure 6 This indicates that FITC-UPCP has strong penetrability and can diffuse from the abdominal cavity into muscle tissue within a certain period of time.

[0092] (3) Investigation on the improvement of dexamethasone-induced sarcopenia phenotype in mice by the novel peptide UPCP

[0093] To investigate the therapeutic effect of UPCP, the above-mentioned 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 it (model group); the above-mentioned control group mice were used as controls, and the above three groups of mice were subjected to the following operations ( Figure 7 A):

[0094] UPCP treatment group: UPCP (20 mg / kg) was injected intraperitoneally every other day;

[0095] Control group: Injected with an equal volume of normal saline, once every other day;

[0096] Model group: Injected with an equal volume of physiological saline, once every other day;

[0097] a: Mouse weight monitoring

[0098] To evaluate the therapeutic effect of the novel peptide UPCP, the body weight of mice was monitored throughout the treatment process.

[0099] The results are as follows Figure 7 As shown in Figure FG, compared with the control group, the body weight of mice in the model group (treated with dexamethasone) showed a significant decreasing trend, while the body weight of sarcopenic mice treated with UPCP did not significantly improve compared with the untreated group (model group). Previous studies have shown that dexamethasone treatment increases fat in mice, which is likely the reason why the body weight of untreated sarcopenic mice tends to increase in the later stages. However, the body weight of mice in the UPCP treatment group did not continue to increase compared with the untreated group, suggesting that the novel peptide UPCP may inhibit the increase of fat in sarcopenic mice to some extent.

[0100] b: Mouse muscle function and motor ability testing

[0101] To evaluate the therapeutic effect of UPCP on muscular atrophy mice, the open field test was used to assess the therapeutic effect of UPCP on day 19 after intraperitoneal injection. Figure 7 B); Rotor test was performed on day 20 after intraperitoneal injection of UPCP ( Figure 7 C), the time mice spent on the stick significantly increased after UPCP treatment. Simultaneously, the grip strength of the treated mice also increased. Figure 7 D-7E).

[0102] The experimental results show that UPCP can improve muscle function and motor ability in mice with sarcopenia.

[0103] c: Measurement of gastrocnemius muscle mass and function in mice

[0104] In addition, after UPCP treatment, all mice were euthanized by cervical dislocation, and the gastrocnemius muscle was separated, weighed, and fixed in paraformaldehyde for HE staining. The experimental results are as follows: Figure 8 As shown in A-8E.

[0105] Experimental results showed that UPCP could improve the mass of the gastrocnemius muscle in sarcopenic mice and increase the cross-sectional area of ​​muscle fibers.

[0106] The above experiments suggest that UPCP can improve muscle function and motor ability in mice with sarcopenia.

[0107] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0108] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of a novel peptide UPCP in the preparation of a drug for treating sarcopenia, wherein 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 shown in SEQ ID NO.1; The novel peptide UPCP exerts its therapeutic effect by improving the degree of muscle atrophy, the body's exercise capacity, the body's muscle mass, and the body's muscle function.

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