Application of NSUN2 in muscle damage repair

Abstract

The present invention discloses the application of NSUN2 in muscle injury repair. The present invention found that AAV-mediated NSUN2 overexpression can significantly increase the number of activated muscle stem cells in CTX model mice and promote muscle stem cell proliferation. NSUN2 overexpression not only increases the activation level of muscle stem cells but also enhances the self-renewal ability of muscle stem cells. Furthermore, NSUN2 overexpression also significantly increases the area of ​​muscle fibers in CTX model mice and reduces muscle atrophy weight. NSUN2 overexpression promotes the healing of muscle damage by promoting the proliferation and activation of muscle stem cells, promoting muscle fiber repair and muscle mass recovery, and thus has a therapeutic effect on this disease. Therefore, the present invention provides a new method for the treatment of muscle damage and may also open up new avenues for the treatment of other muscle-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of NSUN2 in repairing muscle damage. Background Art

[0002] Muscle stem cells (also known as satellite cells) are a specialized type of adult stem cell found in skeletal muscle. They possess the ability to self-renew and differentiate into mature muscle cells, making them the primary drivers of muscle regeneration. Normally, muscle stem cells remain quiescent and do not divide. However, upon muscle injury, satellite cells are activated and begin to proliferate and self-renew, replenishing the stem cell pool. These proliferating muscle stem cells can then differentiate into new muscle cells required for muscle regeneration and repair. PAX7 (Paired Box 7) is a key transcription factor in muscle stem cells. In a quiescent state, muscle stem cells express PAX7. Upon activation, muscle stem cells begin to express MyoD (Myogenic Differentiation 1), which co-expresses MyoD with PAX7. KI67 (a proliferation marker) is commonly used to identify cells in an actively proliferating state. The excellent skeletal muscle regeneration capacity of muscle stem cells maintains muscle homeostasis and promotes injury repair. Conversely, impaired regeneration leads to muscle atrophy and functional decline. Therefore, muscle stem cells play a crucial role in muscle injury repair. A deficiency in muscle stem cells can exacerbate various diseases, including congenital muscular dystrophy, aging-related sarcopenia, and permanent muscle damage and atrophy. Treatment for these conditions typically focuses on alleviating symptoms, improving quality of life, and attempting to restore muscle stem cell function. Current clinical strategies for acute muscle injury primarily use cryotherapy to alleviate pain. While cryotherapy has some therapeutic effects on oxidative stress, delayed onset muscle soreness, and inflammation, it is ineffective for more severe muscle stem cell damage. Nonsteroidal anti-inflammatory drugs (NSAIDs) have some activating effects on muscle stem cells but are ineffective for muscle regeneration. Muscle stem cells possess the ability to self-renew and differentiate into muscle tissue, which is crucial for repairing damaged muscle and improving muscle function. Compared to traditional treatments, muscle stem cell therapy reduces complications associated with surgery and medications. It may also provide long-term benefits after a single treatment, reducing the need for repeated treatments. Therefore, muscle stem cell therapy holds broad application prospects in areas such as muscle diseases, sports injuries, neuromuscular diseases, and age-related muscle atrophy.

[0003] The CTX (Cardiotoxin) muscle injury model is a widely used experimental model for inducing skeletal muscle injury and regeneration in animals, particularly mice. CTX damages the myocyte membrane, causing myofiber necrosis. This injury activates muscle stem cells, triggering a series of regenerative processes, including stem cell proliferation and differentiation, and the formation of new myofibers. The CTX model is an excellent model for simulating muscle inflammatory diseases, traumatic injuries, and muscle atrophy, all of which are characterized by muscle inflammation, damage, and muscle atrophy.

[0004] The maintenance of muscle stem cells relies on a complex network of regulatory mechanisms, including transcriptional and post-transcriptional control. One of the most critical mechanisms is RNA 5-methylcytosine (m5C), which plays a key role in regulating gene expression at all stages of RNA processing, including transcription, transport, stability, and translation. Aberrant m5C modification can lead to various functional defects, which are frequently observed in a range of diseases, including cancer, cardiomyopathy, and liver disease. Although m5C has been identified as a novel and abundant mRNA modification associated with energy metabolism, its regulatory function in skeletal muscle remains limited. RNA 5-methylcytosine (m5C) modification is catalyzed by the NOP2 / Sun domain methyltransferase family, which includes NSUN1-7 and DNMT2. Among them, NSUN2 is the major RNA methyltransferase responsible for mediating RNA m5C modification. Dysregulation of NSUN2 has been implicated in the pathogenesis of many diseases, such as cancer and liver injury. However, the specific role of NSUN2 in muscle stem cell maintenance, particularly in tissue regeneration and repair, remains poorly understood. Summary of the Invention

[0005] The purpose of this invention is to explore a new strategy for effective repair of muscle tissue damage. The authors discovered that AAV-mediated overexpression of NSUN2 can promote the proliferation and activation of muscle stem cells, promote myofiber repair, and restore muscle mass, thereby promoting the healing of muscle damage and having therapeutic effects on related diseases.

[0006] According to one aspect of the present invention, there is provided use of NSUN2 in the preparation of a medicament for promoting muscle damage repair.

[0007] Preferably, in the above application, the NSUN2 can promote the activation and proliferation of muscle stem cells, promote muscle fiber repair and muscle mass recovery.

[0008] Preferably, the muscle injury includes traumatic muscle injury or non-traumatic muscle injury.

[0009] Preferably, the non-traumatic muscle injury includes any one or more of the following: myogenic muscle atrophy, disuse muscle atrophy, muscular dystrophy, sarcopenia, muscle disease and muscle weakness.

[0010] According to a second aspect of the present invention, a drug for promoting muscle damage repair is provided, comprising a vector for overexpressing the NSUN2 gene and pharmaceutically acceptable excipients.

[0011] Preferably, the vector overexpressing the NSUN2 gene is an adeno-associated virus vector overexpressing the NSUN2 gene.

[0012] Preferably, the dosage form of the drug is an injection.

[0013] Preferably, the injection includes injection solution and lyophilized powder injection.

[0014] According to a third aspect of the present invention, there is provided a use of NSUN2 in promoting the proliferation of muscle stem cells.

[0015] Preferably, the above application includes a step of increasing the expression level of the NSUN2 gene in cells.

[0016] The present study found that AAV-mediated overexpression of NSUN2 significantly increased the number of activated muscle stem cells and promoted muscle stem cell proliferation in CTX model mice. NSUN2 overexpression not only enhanced muscle stem cell activation but also enhanced the self-renewal capacity of muscle stem cells. Furthermore, NSUN2 overexpression significantly increased myofiber area and reduced muscle atrophy in CTX model mice. NSUN2 overexpression promotes muscle stem cell proliferation and activation, promoting myofiber repair and muscle mass recovery, thereby promoting the healing of muscle damage and possessing a therapeutic effect on this disease. Therefore, this invention provides a new approach for treating muscle damage and may also open new avenues for the treatment of other muscle-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Flowchart for establishing the CTX model in mice and overexpressing NSUN2. A is a schematic diagram of the AAV-induced NSUN2 overexpression in the tibialis anterior muscle and the CTX-induced muscle injury model in mice. B is a Western blot analysis of NSUN2 overexpression in the tibialis anterior muscle. The n values ​​for both the AAV-CTRL and AAV-NSUN2 groups were 3. Density quantification data below the Western blots were normalized to GAPDH and expressed relative to the AAV-CTRL group.

[0018] Figure 2Overexpression of NSUN2 significantly activated quiescent muscle stem cells. A is the immunofluorescence image of KI67 (green), PAX7 (red) and DAPI (blue) of NSUN2 overexpressing muscle in CTX injury model, scale bar: 100μm; B is the immunofluorescence image of PAX7 + KI67 + For the quantitative results of cells, the n values ​​for the AAV-CTRL and AAV-NSUN2 groups were 3 and 4, respectively.

[0019] Figure 3 Overexpression of NSUN2 improves the activation level and self-renewal ability of muscle stem cells. A is the immunofluorescence image of MyoD (green), PAX7 (red) and DAPI (blue) in NSUN2-overexpressing muscles in the CTX injury model, scale bar: 100μm; B is the immunofluorescence image of PAX7 + MyoD + Quantitative results of cells; C is PAX7 + MyoD - Quantification results of cells. The n values ​​for the AAV-CTRL and AAV-NSUN2 groups in B and C are 3 and 4, respectively.

[0020] Figure 4 Overexpression of NSUN2 repairs myofibers and reduces muscle atrophy. A shows HE staining of muscle tissue from a CTX-injured mouse model (scale bar: 100 μm). B shows that muscles overexpressing NSUN2 in the CTX-injured model developed larger myofiber areas. C shows that overexpressing NSUN2 reduced muscle atrophy in the CTX-injured model. In B, the n values ​​for the AAV-CTRL and AAV-NSUN2 groups were 8 and 7, respectively; in C, the n values ​​for the AAV-CTRL and AAV-NSUN2 groups were 5 and 7, respectively. DETAILED DESCRIPTION

[0021] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents and materials used are all commercially available unless otherwise specified.

[0023] Example 1

[0024] 1. Materials

[0025] AAV2 / 9-NSUN2 was purchased from Heyuan Biotechnology, and AAV2 / 9-CONTROL was purchased from Heyuan Biotechnology.

[0026] The information of antibodies used in the experiment is shown in Table 1:

[0027] Table 1 Antibodies

[0028]

[0029] 2. Methods

[0030] 1. Cardiotoxin-induced muscle injury model

[0031] 8-week-old C57 / BL6J mice were injected into the tibialis anterior muscle of both legs with 50 μL of AAV2 / 9-NSUN2 or

[0032] AAV2 / 9-CONTROL (5×10 10 vg / side). Two weeks later, muscle injury was performed. To induce muscle injury, mice were anesthetized with sodium pentobarbital and injected with 50 μL of cardiotoxin (50 μM) into the tibialis anterior muscle of the right leg. An equal amount of normal saline was injected into the left leg. Seven days after injury, the tibialis anterior muscle was harvested for weighing, Western blot, H&E staining, and immunofluorescence staining.

[0033] 2. Total protein extraction and BCA protein quantification

[0034] 1) Add an appropriate amount of RIPA to the tissue to be tested and grind it in a grinder with steel balls.

[0035] 2) Prepare reference samples of known concentrations by serial dilution of the standard sample using 1× PBS according to the BCA kit. For protein samples, dilute the supernatant prepared in step 2) 10-fold using 1× PBS to prepare the test samples. The final volume of both the test sample and reference sample solutions was 20 μL.

[0036] 3) Prepare the working solution: Prepare the working solution in a ratio of solution A: solution B = 50:1.

[0037] 4) Add the working solution prepared in step 3) to the test sample and reference sample solutions, with 200 μL of working solution per tube, and mix thoroughly.

[0038] 5) 200 μL of each sample prepared in step 4) was pipetted into a 96-well plate and incubated at 37° C. for 30 min.

[0039] 6) After incubation, use a microplate reader to measure the absorbance of the sample at 562 nm, draw a standard curve (where the R value must be greater than 0.99), and calculate the protein concentration of each sample to be tested.

[0040] 3. Western blot experiment

[0041] 1) Prepare liquid:

[0042] ①10× Running Buffer: Add 144g glycine, 10g SDS powder, 30.3g Tris powder, and ddH2O as solvent to 1L to prepare the stock solution. Dilute to 1× Running Buffer to prepare the electrophoresis working solution.

[0043] ②5×SDS Loading Buffer: Add 4 g of SDS powder, 20 mg of bromophenol blue, 3.085 g of DTT, 10 mL of Tris-HCl (1 M pH 6.8), and 20 mL of glycerol in sequence, using ddH2O as the solvent, and adjust the volume to 40 mL.

[0044] ③10× Transfer Buffer: Add 30.3g Tris powder, 144g glycine, and ddH2O as solvent to 1L to prepare the stock solution. Dilute the solution to 1× Transfer Buffer by adding 100mL of 10× Transfer Buffer, 200mL of methanol, and 700mL of ddH2O to prepare the working transfer solution.

[0045] 2) Prepare separation gel and stacking gel

[0046] ①Prepare 10% separation gel (10 mL):

[0047] Table 2 Preparation of 10% separation gel

[0048]

[0049] ② Prepare 5% stacking gel (5 mL):

[0050] Table 3 Preparation of 5% stacking gel

[0051]

[0052] 3) Protein Gel Electrophoresis: Assemble the prepared gel and add the newly prepared electrophoresis fluid to the tank. Check for leakage. Remove the comb and add the marker and sample to the gel wells in sequence. Add electrophoresis fluid to the electrophoresis tank and set the program to perform constant voltage electrophoresis at 80V for 30 minutes and then switch to 120V for 1 hour.

[0053] 4) Transfer: First, activate the PVDF membrane with methanol. Remove the gel from step 3) and assemble the transfer cassettes in a "sandwich" configuration, taking care not to trap any air bubbles. Assemble the transfer apparatus, add the transfer working solution, set the program to a constant current of 250 mA for 2 hours, and place on ice for transfer.

[0054] 5) Blocking: After transfer, remove the membrane, add 1×TBST and place on a shaker for washing at room temperature for 5 minutes, then discard. Add 5% skim milk as blocking solution and place on a shaker for blocking at room temperature for 1 hour.

[0055] 6) Primary Antibody Incubation: Discard the blocking solution, add 1×TBST and wash on a shaker at room temperature until the blocking solution is completely washed out. Discard the solution, add the primary antibody prepared with 5% BSA as the antibody diluent, and incubate on a shaker at 4°C overnight.

[0056] 7) Wash the membrane: Wash the membrane three times with 1×TBST, 10 min each time.

[0057] 8) Secondary antibody incubation: Discard the solution and add secondary antibody prepared with 5% skim milk at a ratio of 1:5000. Incubate on a shaker at room temperature for 1 hour.

[0058] 9) Same as step 7).

[0059] 10) Exposure and Development: Prepare developer (A solution: B solution = 1:1 ratio, prepare and use immediately), place the film on the plate, add developer dropwise onto the film, shake evenly, perform exposure and development on the machine, and save the results.

[0060] 4. Immunofluorescence Staining

[0061] Mouse muscles were fixed with 4% paraformaldehyde (PFA, Sigma) for 2 hours at room temperature and then embedded in 30% sucrose (Sigma) at 4°C overnight. The tissue was then frozen with OCT (SAKURA) and cut into 10 μm sections. The sections were blocked with 5% BSA and 0.5% Triton X-100 blocking solution in PBS solution for 1 hour at room temperature and then incubated with primary antibodies at 4°C overnight. Secondary antibodies (Life Technologies, Waltham, MA, USA) were incubated at room temperature for 1 hour. The sections were imaged using a fluorescence microscope (Zeiss, Oberkochen, Germany). Primary antibodies included Ki67 (AF7649-SP, R&D System), P63 (ET1610-44, HUABIO), PAX7 (AB-528428, DSHB) and MyoD (18943-1-AP, Proteintech). Secondary antibodies included donkey anti-rabbit IgG (Alexa Fluor TM 555, Life Technologies, A-31572), goat anti-mouse IgG (Alexa Fluor TM 488, Life Technologies, A-10680) and goat anti-mouse IgG (Alexa Fluor TM488, Life Technologies, A-11006).

[0062] 5. Hematoxylin and Eosin Staining

[0063] All mice were anesthetized with isoflurane and sacrificed. Isolated muscles were fixed with 4% paraformaldehyde. Fixed tissues were dehydrated, embedded in paraffin, and sectioned. Frozen tissue blocks for hematoxylin and eosin (H&E) staining were cut into 10 μm sections. H&E staining was performed by Wuhan Saiwei Biotechnology Co., Ltd. according to standard protocols.

[0064] 3. Results

[0065] All data are presented as mean ± standard deviation (SD), and statistical significance was assessed using a one-sample t-test. *p < 0.05, ***p < 0.001.

[0066] 1. To explore the role of NSUN2 in repairing muscle damage, we used C57BL / 6J mice and established an acute muscle injury model by injecting CTX (cardiotoxin). Flowchart ( Figure 1 Figure A) shows the modeling process. We used AAV-NSUN2 and control AAV virus (AAV-CTRL) to overexpress NSUN2 in the tibialis anterior muscle of mice. 14 days after AAV overexpression, CTX was injected into the tibialis anterior muscle of mice to establish the model. 7 days after CTX establishment, the tibialis anterior muscle of mice was collected for related marker detection. Western blot analysis showed that NSUN2 was significantly overexpressed in the tibialis anterior muscle ( Figure 1 B) in.

[0067] 2. We performed immunofluorescence staining on the tibialis anterior muscles of model mice and found that NSUN2 overexpression significantly increased the number of activated muscle stem cells, namely PAX7 + Ki67 + The proportion of double-positive muscle stem cells increased, indicating that NSUN2 overexpression significantly activated quiescent muscle stem cells ( Figure 2 ).

[0068] 3. NSUN2 overexpression also increased activation (PAX7 + MyoD + , Figure 3 B) and self-renewing muscle stem cells (PAX7 + MyoD - , Figure 3 C), indicating that overexpression of NSUN2 not only increased the activation level of muscle stem cells, but also enhanced the self-renewal ability of muscle stem cells.

[0069] 4. We found that overexpression of NSUN2 produced significant effects in model mice. Specifically, overexpression of the NSUN2 gene significantly increased the area of ​​muscle fibers ( Figure 4 This result indicates that overexpression of NSUN2 significantly enhances the regenerative and repair capacity of muscle fibers. In addition, compared with the control group, the degree of muscle atrophy in NSUN2-overexpressing mice was significantly reduced, and the weight of muscle atrophy was reduced ( Figure 4 Therefore, these data clearly demonstrate that NSUN2-overexpressing mice exhibit better myofiber repair and muscle mass recovery than the control group.

[0070] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of NSUN2 in the preparation of drugs for promoting skeletal muscle damage and repair.

2. The use according to claim 1, characterized in that The NSUN2 can promote the activation and proliferation of muscle stem cells, promote muscle fiber repair and muscle quality recovery.

3. The use according to claim 1, characterized in that The skeletal muscle injury includes: traumatic skeletal muscle injury or non-traumatic skeletal muscle injury.

4. The use according to claim 1, characterized in that The medicine comprises a vector for overexpressing the NSUN2 gene and pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that The vector for overexpressing the NSUN2 gene is an adeno-associated virus vector for overexpressing the NSUN2 gene.

6. The use according to claim 1, characterized in that The dosage form of the medicine is injection.

7. The use according to claim 6, characterized in that The injection includes injection solution and freeze-dried powder injection.

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