Application of NSUN2 in muscle injury repair

Abstract

The invention discloses an application of NSUN2 in muscle injury repair. Research finds that the number of activated muscle stem cells of a CTX model mouse can be remarkably increased and proliferation of the muscle stem cells can be promoted by mediating NSUN2 overexpression through AAV, and the overexpression of the NSUN2 not only improves the activation level of the muscle stem cells, but also enhances the self-renewal capacity of the muscle stem cells; in addition, overexpression of NSUN2 also significantly increases the area of muscle fibers of CTX model mice and reduces the weight of muscle atrophy. Overexpression of the NSUN2 promotes muscle fiber repair and muscle mass recovery by promoting proliferation and activation of muscle stem cells, so that healing of muscle injury is promoted, and the NSUN2 has a treatment effect on the disease. Therefore, the invention provides a new method for the treatment of muscle injury, and a new way is possibly opened up for the treatment of other muscle-related diseases.

Description

Technical Field

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

[0002] Muscle stem cells (also known as satellite cells) are a special type of adult stem cells in skeletal muscle. They have the ability to self-renew and differentiate into mature muscle cells, and are the specific executors of muscle regeneration. Under normal circumstances, muscle stem cells are in a quiescent state and do not undergo cell division. When the muscle is damaged, satellite cells are activated and proliferate and self-renew to replenish the stem cell pool. The proliferated muscle stem cells can differentiate into new muscle cells required for muscle regeneration to repair the damage. Among them, PAX7 (Paired Box 7, PAX7) is a key transcription factor for muscle stem cells. In a quiescent state, muscle stem cells express PAX7. When muscle stem cells are activated, they begin to express MyoD (Myogenic Differentiation 1, MyoD) and co-express it with PAX7. KI67 (proliferation marker) is usually used to identify cells in an active proliferative state. The good skeletal muscle regeneration ability of muscle stem cells can maintain muscle homeostasis and promote injury repair; conversely, regenerative disorders will lead to muscle atrophy and functional impairment. Therefore, muscle stem cells play a decisive role in muscle injury repair. The lack of muscle stem cells can aggravate various diseases, such as congenital muscular dystrophy, aging-related sarcopenia, permanent muscle damage and atrophy. The treatment of these diseases usually focuses on relieving symptoms, improving quality of life, and trying to restore the function of muscle stem cells. At present, the clinical strategy for acute muscle injury is mainly cryotherapy to relieve the pain of patients. Cryotherapy has a certain therapeutic effect on the body's oxidative stress, delayed muscle soreness and inflammation, but has no effect on severe muscle stem cell damage; non-steroidal anti-inflammatory drugs have a certain activation effect on muscle stem cells, but have no effect on muscle regeneration. Muscle stem cells have the ability to self-renew and can differentiate into muscle tissue, which is crucial for repairing damaged muscles and improving muscle function. Compared with traditional treatments, muscle stem cell therapy reduces complications caused by surgery and drug treatment. Muscle stem cell therapy may provide long-term effects after a single treatment, reducing the need for repeated treatment. Therefore, muscle stem cell therapy has broad application prospects in the fields of muscle diseases, sports injuries, neuromuscular diseases, and senile muscle atrophy.

[0003] The CTX (Cardiotoxin) muscle injury model is a widely used experimental model for inducing skeletal muscle injury and regeneration in animals, especially mice. CTX causes muscle fiber necrosis by destroying the muscle cell membrane. This injury activates muscle stem cells and triggers a series of regenerative processes, including stem cell proliferation, differentiation, and the formation of new muscle fibers. The CTX model can well simulate muscle inflammatory diseases and traumatic injuries and muscle atrophy, which are characterized by muscle inflammation, injury, 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 the regulation of gene expression at various 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, especially in tissue regeneration and repair, remains poorly understood. Summary of the invention

[0005] The purpose of the present invention is to explore a new strategy for effective repair of muscle tissue damage. The present invention found that AAV-mediated NSUN2 overexpression can promote the proliferation and activation of muscle stem cells, promote muscle fiber repair and muscle quality recovery, thereby promoting the healing of muscle damage and having a therapeutic effect 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 quality 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 a pharmaceutically acceptable excipient.

[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 use of NSUN2 in promoting the proliferation of muscle stem cells.

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

[0016] The present invention has found that by AAV-mediated NSUN2 overexpression, the number of activated muscle stem cells in CTX model mice can be significantly increased, and muscle stem cell proliferation can be promoted. NSUN2 overexpression not only improves the activation level of muscle stem cells, but also enhances the self-renewal ability of muscle stem cells; in addition, NSUN2 overexpression also significantly increases the area of ​​muscle fibers in CTX model mice and reduces muscle atrophy weight. NSUN2 overexpression promotes the proliferation and activation of muscle stem cells, promotes muscle fiber repair and muscle quality recovery, thereby promoting the healing of muscle damage, and 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 ways for the treatment of other muscle-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 1 is a flowchart of mouse CTX modeling and NSUN2 overexpression. A is a schematic diagram of the AAV-induced NSUN2 overexpression in the tibialis anterior muscle of mice and the CTX-induced muscle injury model; B is a Western blot detection of NSUN2 overexpression in the tibialis anterior muscle, and the n of the AAV-CTRL group and the AAV-NSUN2 group are both 3. The density quantitative data below the Western blot have been normalized according 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 PAX7 + KI67 + For the quantitative results of cells, the n values ​​for the AAV-CTRL group and the AAV-NSUN2 group 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: 100 μm; B is the immunofluorescence image of PAX7 + MyoD + Quantitative results of cells; C is PAX7 + MyoD - Quantification results of cells, n for AAV-CTRL group and AAV-NSUN2 group in B and C were 3 and 4, respectively.

[0020] Figure 4 Overexpression of NSUN2 repairs muscle fibers and reduces muscle atrophy. A is the HE staining of muscle tissue in CTX injury model mice, scale: 100μm; B is the muscle overexpressing NSUN2 in the CTX injury model formed a larger muscle fiber area; C is the muscle overexpressing NSUN2 in the CTX injury model reduced atrophy, in B, the n of AAV-CTRL group and AAV-NSUN2 group were 8 and 7, respectively, in C, the n of AAV-CTRL group and AAV-NSUN2 group were 5 and 7, respectively. DETAILED DESCRIPTION

[0021] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are all 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 antibody information 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] Eight-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). Muscle injury was performed two weeks later. To induce muscle injury, mice were anesthetized with sodium pentobarbital and 50 μL of cardiotoxin (50 μM) was injected into the tibialis anterior muscle of the right leg. An equal amount of saline was injected into the left leg. Seven days after injury, the tibialis anterior muscle was collected for weighing, Western Blot, HE staining, and immunofluorescence staining.

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

[0034] 1) Add 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 gradient dilution of the standard sample with 1×PBS according to the BCA kit; for protein samples, dilute the supernatant prepared in step 2) 10-fold with 1×PBS to prepare the test samples, wherein the final volumes of the test sample and reference sample solutions are both 20 μL.

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

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

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

[0039] 6) After incubation, use an ELISA 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, and dilute to 1L to prepare the storage solution. Dilute to 1×Running Buffer to prepare the electrophoresis working solution for use.

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

[0044] ③10×Transfer Buffer: Add 30.3g Tris powder, 144g glycine, and ddH2O as solvent, and dilute to 1L to prepare the storage solution. Dilute to 1×Transfer Buffer according to the formula of 10×Transfer Buffer 100mL+methanol 200mL+ddH2O 700mL to prepare the transfer working solution for use.

[0045] 2) Prepare separation gel and stacking gel

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

[0047] Table 2 Preparation of 10% separation gel

[0048]

[0049] ②Prepare 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 solution into its tank, check for leakage, pull out the comb, add the marker and sample into the gel wells in sequence, add the electrophoresis solution into the electrophoresis tank, set the program to perform constant voltage electrophoresis at 80V for 30min and then switch to 120V for constant voltage electrophoresis for 1h.

[0053] 4) Transfer: First, use methanol to activate the PVDF membrane, and take out the gel in step 3), assemble the transfer clip in the order of the "sandwich" structure, and be careful not to leave bubbles during the assembly. Assemble the transfer device, add the transfer working solution, set the program to 250mA constant current for 2h, and place it on ice for transfer.

[0054] 5) Blocking: After the transfer, take out the membrane, add 1×TBST and place it on a shaker at room temperature for washing for 5 minutes, then discard it, and add 5% skim milk as blocking solution and place it on a shaker at room temperature for blocking. The blocking time is 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 clean, discard the liquid, 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, add secondary antibody prepared with 5% skim milk at a ratio of 1:5000, and incubate on a shaker at room temperature for 1 hour.

[0058] 9) Same as step 7).

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

[0060] 4. Immunofluorescence Staining

[0061] Mouse muscles were fixed with 4% polyformaldehyde (PFA, Sigma) for 2 hours at room temperature and then embedded with 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 prepared 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 slices. 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 one-sample t-test. *p<0.05, ***p<0.001.

[0066] 1. In order to explore the repair effect of NSUN2 on muscle damage, we used C57BL / 6J mouse background and constructed an acute muscle damage model by CTX (cardiotoxin) injection. Flowchart ( Figure 1 A) in Figure 1 shows the modeling process. That is, we used AAV-NSUN2 and control AAV virus (AAV-CTRL) to overexpress NSUN2 in the tibialis anterior muscle of mice. After 14 days of AAV overexpression, CTX was injected into the tibialis anterior muscle of mice for modeling. After 7 days of CTX modeling, the tibialis anterior muscle of mice was collected for related marker detection. Western blot detection results showed that NSUN2 was significantly overexpressed in the tibialis anterior muscle ( Figure 1 B).

[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 the overexpression of NSUN2 significantly enhances the regeneration 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 C). Therefore, these data fully demonstrate that mice overexpressing NSUN2 show better effects on myofiber repair and muscle mass recovery than the control group.

[0070] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of NSUN2 in the preparation of drugs for promoting muscle damage 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 muscle injury includes: traumatic muscle injury or non-traumatic muscle injury.

4. The use according to claim 3, characterized in that: 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.

5. A drug for promoting muscle damage repair, characterized in that: The invention comprises a vector for over-expressing the NSUN2 gene and pharmaceutically acceptable auxiliary materials.

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

7. The drug according to claim 5, characterized in that The dosage form of the drug is injection.

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

9. Application of NSUN2 in promoting the proliferation of muscle stem cells.

10. The use according to claim 9, characterized in that: The method comprises the steps of increasing the expression level of NSUN2 gene in cells.

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