Use of mrpl41 gene / protein in preparation of medicine for treating senile myopathy

Abstract

This invention belongs to the field of biomedical technology, specifically involving the application of the MRPL41 gene / protein in the preparation of drugs for treating age-related sarcopenia. This invention is the first to demonstrate that MRPL41 is a key rate-limiting factor in the assembly of mitochondrial ribosomes in aging muscle satellite cells, establishing it as a core target for the treatment of age-related sarcopenia. Overexpression of MRPL41 can promote the assembly of mitochondrial 55S holoenzymes, improve the translation efficiency of mitochondrial-encoded proteins, repair mitochondrial dysfunction at its source, thereby promoting muscle repair, improving muscle atrophy, and achieving the treatment of age-related sarcopenia. This invention provides a precise microscopic target for the treatment of age-related sarcopenia, overcoming the limitations of existing clinical therapies (such as hormone replacement and simple nutritional supplementation) due to significant side effects and "anabolism resistance," providing a new direction for the development of innovative drugs specifically for the treatment of age-related sarcopenia, and has excellent application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of the MRPL41 gene / protein in the preparation of drugs for treating age-related sarcopenia. Background Technology

[0002] Sarcopenia, an age-related degenerative skeletal muscle disease, is characterized by decreased muscle mass, strength, and function, severely impacting the quality of life and increasing the risk of death in older adults. Current clinical interventions for sarcopenia primarily rely on exercise therapy, nutritional supplementation, and hormone replacement therapy. However, these existing therapies have significant limitations: exercise therapy suffers from poor adherence due to physical limitations in older adults; nutritional supplementation often fails to overcome age-related anabolic resistance; and hormone therapy carries potential systemic side effects (such as carcinogenic risks). Therefore, in-depth exploration of the core pathological mechanisms leading to age-related muscle regeneration disorders and the identification of specific new targets have become crucial issues that urgently need to be addressed.

[0003] Muscle satellite cells (MuSCs) are crucial for maintaining muscle regeneration, and their activity is highly dependent on mitochondrial metabolic homeostasis. The biosynthesis of the mitochondrial respiratory chain complex is controlled by mitochondrial ribosomes, and the assembly efficiency of ribosomes is the decisive factor limiting mitochondrial translation function. Mitochondria possess an independent genetic information translation system, and its core machinery—the mitochondrial ribosome—is responsible for the synthesis of key subunits encoding the respiratory chain complex. This process strictly depends on the precise assembly of the large (39S) and small (28S) subunits of the mitochondrial ribosome to form the catalytically active 55S monozyme.

[0004] Although current research recognizes a significant decline in mitochondrial translation efficiency associated with aging, the exact molecular structural defects leading to the dysfunction of this translational machine remain unclear. Existing technologies have not yet revealed the specific assembly obstacles at the microscopic level that cause the decline in mitochondrial function, particularly lacking definitive evidence regarding changes in the stability of mitochondrial ribosomal proteins during aging and their impact on subunit assembly. Due to the lack of clear microscopic targets, there are currently no drugs in clinical practice that can specifically reverse mitochondrial translation defects and restore muscle regeneration potential at its source.

[0005] In summary, current technologies lack intervention strategies targeting key rate-limiting factors in mitochondrial ribosome assembly, thus failing to address the mitochondrial translational dyscompensation problem in age-related sarcopenia. Therefore, developing a therapeutic strategy and drug screening method capable of precisely repairing the mitochondrial translational machinery and improving muscle satellite cell function is of significant scientific and clinical value. Summary of the Invention

[0006] This invention aims to overcome the shortcomings and deficiencies of existing drugs for treating age-related sarcopenia, such as poor compliance, difficulty in overcoming "anabolism resistance", and accompanying systemic side effects, and provides the application of reagents that promote the expression of MRPL41 gene / protein in the preparation of drugs for the prevention or treatment of age-related sarcopenia.

[0007] The first objective of this invention is to provide the use of the MRPL41 gene / protein in the preparation of drugs for the prevention or treatment of age-related sarcopenia.

[0008] A second objective of this invention is to provide the use of reagents that promote the expression of the MRPL41 gene / protein in the preparation of drugs for the prevention or treatment of age-related sarcopenia.

[0009] A third objective of this invention is to provide a medicament for the prevention or treatment of age-related sarcopenia.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention demonstrates that injecting a reagent that promotes MRPL41 gene / protein expression (AAV9-MRPL41 vector) can reverse the pathological characteristics of aging skeletal muscle, significantly improve muscle regeneration quality and metabolic function, and significantly improve mitochondrial dysfunction in aging muscle satellite cells. Based on the broad-spectrum mechanism by which the MRPL41 gene / protein promotes the regenerative potential of muscle satellite cells by repairing mitochondrial ribosome assembly, overexpression of the MRPL41 gene / protein is also applicable to improving or treating skeletal muscle injury or degenerative muscle atrophy.

[0011] Therefore, this invention claims protection for the following applications: Application of MRPL41 gene / protein in the preparation of drugs for the prevention or treatment of age-related sarcopenia.

[0012] Application of reagents that promote MRPL41 gene / protein expression in the preparation of drugs for the prevention or treatment of age-related sarcopenia.

[0013] Specifically, in the above applications, the drug treats age-related sarcopenia by repairing mitochondrial 55S holoenzyme assembly disorders.

[0014] Application of reagents that promote MRPL41 gene / protein expression in the preparation of drugs for the prevention or treatment of skeletal muscle injury.

[0015] Application of reagents that promote MRPL41 gene / protein expression in the preparation of drugs for promoting muscle repair.

[0016] Application of reagents that promote MRPL41 gene / protein expression in the preparation of drugs for the prevention or treatment of degenerative muscle atrophy.

[0017] Application of MRPL41 gene / protein as a target in the preparation of drugs for the prevention or treatment of mitochondrial dysfunction.

[0018] Application of reagents that promote MRPL41 gene / protein expression in the preparation of drugs for the prevention or treatment of mitochondrial dysfunction.

[0019] Specifically, in the above application, the mitochondrial dysfunction refers to mitochondrial dysfunction caused by age-related sarcopenia.

[0020] Application of MRPL41 gene / protein as a target in screening drugs for the prevention or treatment of age-related sarcopenia.

[0021] In the above application, the screening method is as follows: the compound to be tested is contacted with a senescent muscle satellite cell model, and the expression level of MRPL41 is detected. If the expression level of MRPL41 is significantly higher than that of the control group, the compound is determined to be a candidate drug for the prevention or treatment of senile sarcopenia.

[0022] This invention provides a drug for the prevention or treatment of age-related sarcopenia, containing a reagent that promotes the expression of the MRPL41 gene / protein.

[0023] Preferably, in the above-mentioned drug, the reagent for promoting MRPL41 gene / protein expression is an overexpression vector of the MRPL41 gene.

[0024] Preferably, in the above-mentioned drug, the overexpression vector is an adeno-associated virus vector.

[0025] Preferably, in the above-mentioned drug, the adeno-associated virus vector is an AAV9 serotype vector.

[0026] Preferably, the above-mentioned drug further contains pharmaceutically acceptable pharmaceutical excipients.

[0027] Optionally, the pharmaceutical excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, emulsifier, cosolvent, surfactant, or buffer.

[0028] Optionally, the pharmaceutical excipients may further include any one or a combination of at least two of the following: colorants, pH adjusters, antioxidants, and antibacterial agents.

[0029] Optionally, the carrier includes liposomes, micelles, dendritic macromolecules, microspheres, or microcapsules.

[0030] The present invention has the following beneficial effects: This invention is the first to demonstrate that MRPL41 is a key rate-limiting factor in the assembly of mitochondrial ribosomes in senescent muscle satellite cells. By promoting MRPL41 gene / protein expression (AAV9-MRPL41 vector), this invention significantly reverses muscle function decline in age-related sarcopenia mice. Specifically, it significantly restores limb grip strength and exercise endurance, increases wet weight and muscle mass index in the tibialis anterior and gastrocnemius muscles; histopathological examination shows a significant increase in muscle fiber cross-sectional area, improved atrophy, and restoration of glycogen reserves within muscle fibers; simultaneously, it significantly upregulates the expression levels of key transcription factors for muscle regeneration, proving that this invention can effectively overcome "anabolism resistance" and promote muscle regeneration.

[0031] Furthermore, experiments showed that overexpression of MRPL41 effectively restored intracellular mitochondrial membrane potential (TMRE signaling), significantly reduced reactive oxygen species (ROS) accumulation, and inhibited oxidative stress damage; simultaneously, it significantly restarted the protein expression of PGC-1α, a core regulator of mitochondrial biogenesis. This indicates that the present invention can reverse aging-induced mitochondrial dysfunction at the cellular metabolic level.

[0032] This invention promotes mitochondrial ribosomal subunit binding by overexpressing MRPL41 protein, restoring 55S holoenzyme assembly efficiency and specifically improving the translation efficiency of mitochondrial-encoded proteins, thereby restoring muscle regeneration potential from its source. This invention provides a precise microscopic target for the treatment of age-related sarcopenia, overcoming the limitations of existing clinical therapies (such as hormone replacement and simple nutritional supplementation) due to significant side effects and "anabolism resistance." It offers a new direction for developing innovative drugs specifically for the treatment of age-related sarcopenia and has excellent application prospects. Attached Figure Description

[0033] Figure 1 The results of targeted intervention of MRPL41 on skeletal muscle regeneration impairment in aging sarcopenia mice are shown in Figure A. The results of skeletal muscle function testing in each group of mice are shown in Figure B. The H&E staining results of tibialis anterior muscle tissue in each group of mice are shown in Figure B. The scale bar in the figure is 50 µm. The results of PAS staining results of tibialis anterior muscle tissue in each group of mice are shown in Figure C. The scale bar in the figure is 50 µm. The results of expression analysis of key transcription factors for muscle regeneration in each group of mice are shown in Figure D.

[0034] Figure 2The results of targeted intervention on MRPL41 on mitochondrial dysfunction in muscle satellite cells of aging mice are shown in Figure A, which is a representative fluorescence image and quantitative statistical graph of TMRE staining; Figure B is a representative fluorescence image and quantitative statistical graph of ROS staining; Figure C shows the in situ expression of PGC-1α, a core regulator of mitochondrial biogenesis in each group of mice. Scale bar: 10µm.

[0035] Figure 3 To investigate the effects of targeted intervention on MRPL41 on the correct assembly and translation of the mitochondrial 55S holoenzyme; Figure A shows the continuous absorption peak patterns of polyribosomal sucrose density gradient centrifugation in each group of mice; Figure B shows the quantitative analysis results of the peak area ratio of the mitochondrial 55S holoenzyme to the free subunit [55S / (28S + 39S)]; Figure C shows the relative expression level of the mitochondrial coding gene COX1 mRNA in muscle satellite cells of each group detected by real-time PCR; Figure D shows the expression level of the mitochondrial translation core target protein COX1 in muscle satellite cells of each group detected by Western blotting. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0037] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0038] Cardiotoxin (CTX), CAS No.: 2918768-05-3, structural formula:

[0039] Example 1: Construction and Grouping Intervention of an Age-Related Sarcopenia Mouse Model I. Laboratory Animals Healthy male C57BL / 6 mice were selected as experimental subjects. Two-four-month-old mice served as the natural aging sarcopenia model group (referred to as the "aging group"), simulating the muscle atrophy state of human aging; two-month-old mice served as the young normal control group (referred to as the "young group"). All mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in a standard SPF-grade environment.

[0040] II. Construction of a Muscle Injury Regeneration Model To assess muscle regeneration capacity, acute muscle injury models were established in aging and young mice before intervention. Specifically, the acute muscle injury model was established by injecting 50 μl of 10 μM cardiotoxin (CTX) at multiple sites into the tibialis anterior (TA) muscle of C57BL / 6 mice.

[0041] III. Viral Vector Information Adeno-associated virus serum type 9 (AAV9-MRPL41) overexpressing MRPL41 under the CAG promoter was purchased from Shandong Weizhen Biotechnology Co., Ltd. (Shandong, China).

[0042] The full-length cDNA sequence of the mouse MRPL41 gene (GenBank Accession No. NM_001031808.2) is shown in SEQ ID NO.1: SEQ ID NO.1: ATGGGTTTCCTGACTGCCGTGACTCAAGGCCTGGTGCGGGGAGCTGACAGGATGAGCAAGTGGACAAGCAAGCGGGGACCACGCACCTTCACTAAGAGTCGGGGTGCCAAGAAAACAGGCATCTATACTTCTGATAGGAAGTTTGTGCAAATAAAAGAAATGGTTCCAGAATTTGTCGTCCCGGACTTGACGGGCTTCAAGCTC AAGCCCTACGTTAATTACCGAGCTCCTGCAGGCATAGACACACCTCTGACCGCCAAAGCGCTCTTCCAGGAAACAGTTGCACCCGCTATCGAAAAAGACTTTAAAGAAGGGACATTTGATGCTAACAACCTGGAGAAATACGGCTTCGAGCCCACACAGGAAGGCAAGCTGTTCCAATTATATCCTAAGAATTTCCCACGCTAG.

[0043] The full-length amino acid sequence of mouse MRPL41 protein (GenBank Accession No. NP_001026978.2) is shown in SEQ ID NO.2: SEQ ID NO.2: MGFLTAVTQGLVRGADRMSKWTSKRGPRTFTKSRGAKKTGIYTSDRKFVQIKEMVPEFVVPDLTGFKLKPYVNYRAPAGIDTPLTAKALFQETVAPAIEKDFKEGTFDANNLEKYGFEPTQEGKLFQLYPKNFPR.

[0044] The AAV9-MRPL41 target sequence sequencing primers used for overexpression of MRPL41 are as follows: 5' sequencing primer: CCTCTGCTAACCATGTTCAT (SEQ ID NO.3); 3' sequencing primer: AGGATGTCCCAGGCGAAGG (SEQ ID NO.4).

[0045] IV. Virus Intervention and Grouping On day 5 after CTX injection, adeno-associated virus 9 (AAV9) intervention was initiated. Twenty-four-month-old senescent mice were randomly divided into three groups, along with a two-month-old young control group, resulting in a total of four experimental groups (n=6 per group): Young control group: 2-month-old mice injected with physiological saline; Aging model group: 24-month-old mice injected with physiological saline; Aging + AAV9-MRPL41 treatment group: 24-month-old mice were injected with AAV9 virus overexpressing MRPL41 (AAV9-MRPL41). Senescent + AAV9-Vector empty vector group: 24-month-old mice were injected with empty AAV9 virus (AAV9-Vector).

[0046] Treatment: C57BL / 6 mice were deeply anesthetized and administered a titer of 8×10⁻⁶. 10 The viral vector at a concentration of vg / mL was injected locally at multiple points in the muscle, and samples were collected for experiments on the 28th day of intervention.

[0047] V. Sample Collection On day 28 of intervention, mice were euthanized, and tibialis anterior muscle, gastrocnemius muscle tissue, and primary muscle satellite cells were collected for subsequent experiments.

[0048] Example 2: Overexpression of MRPL41 improves skeletal muscle regeneration impairment in age-related sarcopenia mice. This embodiment aims to verify the effect of overexpression of MRPL41 on improving muscle function and morphology in aging mice. Experiments were conducted on mice in each group of Example 1.

[0049] I. Detection of Key Indicators of Skeletal Muscle Function in Mice The grip strength of mice was tested using a mouse grip strength tester, and the exercise endurance of mice was assessed using an animal treadmill.

[0050] The results are as follows Figure 1 As shown in Figure A, the results indicate that, compared with the aging group, the treated mice showed a significant recovery in muscle grip strength and muscle mass.

[0051] II. Pathological evaluation of mouse skeletal muscle regeneration tissue After euthanasia, the tibialis anterior muscle tissue was fixed with 4% paraformaldehyde. After preparing tissue sections, the muscle tissue structure and muscle fiber size were observed by H&E staining, and the glycogen reserves and metabolic status within the muscle fibers were detected by PAS staining.

[0052] The results are as follows Figure 1 As shown in Figures B and C, the results indicate that, compared with the aging group, the treated mice exhibited significant morphological and metabolic improvements: H&E staining showed a significant increase in the cross-sectional area of ​​muscle fibers and improved atrophy; PAS staining showed a significant recovery in glycogen content within muscle fibers. These histological results demonstrate that MRPL41 overexpression effectively reverses the pathological characteristics of aging skeletal muscle and significantly improves muscle regeneration quality and metabolic function.

[0053] III. Detection of the effect of MRPL41 overexpression on muscle regeneration-related genes by quantitative real-time PCR After homogenizing and extracting mRNA from mouse muscle tissue, cDNA was obtained by reverse transcription. The expression of key muscle regeneration transcription factors Pax7, MyoD, and MyoG was detected by quantitative real-time PCR using a Roche LC480 system. GAPDH was used as an internal control, and the data were analyzed using the ΔΔCt method. The primers used for quantitative real-time PCR are shown in Table 1.

[0054] The results of quantitative real-time PCR are as follows Figure 1 As shown in Figure D, the results indicate that, compared with the aging group, the expression levels of muscle regeneration-related genes Pax7, MyoD, and MyoG in the treatment group mice were significantly restored.

[0055] Table 1 Primers used in real-time PCR

[0056] The above experimental results indicate that in vivo administration of AAV9-MRPL41 significantly improved skeletal muscle regeneration impairment in aging mice.

[0057] Example 3: Overexpression of MRPL41 improves mitochondrial dysfunction in senescent muscle satellite cells The establishment and grouping of the age-related sarcopenia mouse model were the same as in Example 1. After 28 days of intervention, the mice were sorted using FACS flow cytometry (biochemical marker: CD45). - CD31 - Sca1 - Integrin α7 + Primary myosatellite cells were sorted and extracted for experiments.

[0058] I. Detection of mitochondrial membrane potential and oxidative stress level in muscle satellite cells Cells were stained and sorted using TMRE fluorescent probes, and mitochondrial membrane potential was quantitatively analyzed by fluorescence microscopy and flow cytometry. The intensity of red fluorescence represents the membrane potential. Simultaneously, ROS fluorescent probes were used to detect intracellular reactive oxygen species accumulation to assess the degree of mitochondrial oxidative stress damage.

[0059] Experimental results are as follows Figure 2 As shown in Figures A and B, compared with the aging group, representative fluorescence images and quantitative statistical plots of TMRE staining show that the intensity of TMRE red fluorescence signal in the muscle satellite cells of the treatment group is significantly increased, indicating that the damaged mitochondrial membrane potential is effectively restored. Simultaneously, representative fluorescence images and quantitative statistical plots of ROS staining show that the intensity of ROS fluorescence signal in the treatment group is significantly reduced, indicating that excessive intracellular oxidative stress is significantly inhibited. These results confirm that overexpression of MRPL41 can significantly improve mitochondrial functional homeostasis in aging muscle satellite cells.

[0060] II. Immunofluorescence staining assessment of mitochondrial biogenesis core protein expression Immunofluorescence staining was performed on myosatellite cells in each group, and the in situ expression of PGC-1α, a core regulatory factor in mitochondrial biogenesis, was observed and quantitatively analyzed using confocal microscopy.

[0061] The results are as follows Figure 2 As shown in Figure C, compared with the aging group, quantitative analysis of mean fluorescence intensity (MFI) revealed that the fluorescence signal of PGC-1α in the cells of the aging group was extremely faint; however, after treatment with AAV9-MRPL41, the fluorescence signal intensity of PGC-1α in muscle satellite cells showed a highly significant increase. These morphological and quantitative results conclusively demonstrate that overexpression of MRPL41 successfully restarted the mitochondrial biogenesis program in aging muscle satellite cells at the protein level.

[0062] Example 4: Overexpression of MRPL41 promotes mitochondrial 55S holoenzyme assembly and core subunit translation. The establishment and grouping intervention of the age-related sarcopenia mouse model were the same as in Example 1. Skeletal muscle tissue and sorted muscle satellite cells were collected on day 28 of the intervention treatment for experiments.

[0063] I. Sucrose density gradient centrifugation to detect mitochondrial ribosome assembly Skeletal muscle tissue and sorted myosal cell lysates were gently spread on the top layer of centrifuge tubes containing a 10%–50% sucrose density gradient. Polyribosome sedimentation analysis was performed using an ultracentrifuge. After centrifugation, a UV detection and collection system was used for continuous monitoring at 260 nm, collecting a total of 15 fractions. A continuous absorption peak spectrum was plotted by measuring the absorbance at 260 nm, and the peak area ratio of the 55S holoenzyme to the free subunit of mitochondrial ribosomes [55S / (28S + 39S)] and the distribution characteristics of the polymeric region were quantitatively analyzed.

[0064] Combining the absorption peak spectrum and its quantitative analysis results, respectively as follows: Figure 3 As shown in Figures A and B, the results indicate that the 55S / (28S + 39S) ratio in the treatment group significantly increased compared to the aging group. This result clearly demonstrates that overexpression of MRPL41 effectively reversed the defect of abnormal subunit release in the aging state, promoted the binding of the 39S large subunit to the 28S small subunit, and thus significantly restored the correct assembly of the mitochondrial ribosomal 55S holoenzyme.

[0065] II. Detection of Mitochondrial Genome (mtDNA) Encoding Genes by Quantitative Real-Time PCR mRNA was extracted, and quantitative real-time PCR was used to detect COX1 (cytochrome c oxidase subunit I), a representative target gene for mitochondrial ribosome translation.

[0066] The results are as follows Figure 3 As shown in Figure C, there was no significant difference in COX1 transcription levels among the groups.

[0067] III. Western Blot Detection of Mitochondrial Translation Core Target Proteins Proteins were extracted from mouse myosatellite cells, subjected to SDS-PAGE electrophoresis, transferred to a membrane, and incubated overnight at 4°C with COX1 antibody. After washing, the cells were incubated with secondary antibody for imaging.

[0068] Experimental results are as follows Figure 3 As shown in Figure D, compared with the aging group, the expression level of COX1 protein translated by mitochondrial ribosomes in the muscle satellite cells of the treated mice was significantly restored. These experimental results indicate that in vivo administration of AAV9-MRPL41 effectively repaired the aging-induced mitochondrial 55S holoenzyme assembly disorder and restored mitochondrial translation quality control.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of an adeno-associated virus vector overexpressing the MRPL41 gene in the preparation of drugs for treating age-related sarcopenia, characterized in that, The nucleotide sequence of the MRPL41 gene is shown in SEQ ID NO.1.

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