CN111588854A

CN111588854A - Application of TRF2 or its up-regulating agent in preparing medicine for treating muscle disease

Info

Publication number: CN111588854A
Application number: CN201910130604.0A
Authority: CN
Inventors: 叶静; G·埃里克; 陆一鸣
Original assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Current assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Priority date: 2019-02-21
Filing date: 2019-02-21
Publication date: 2020-08-28

Abstract

The invention relates to application of TRF2 or a upregulation agent thereof in preparing a medicament for treating muscle diseases. The invention discovers that the TRF2 protein level of human skeletal muscle is negatively correlated with age; TRF2 in human striated muscle decreases with the aging of mitotic cells, and down-regulation of TRF2 in differentiated myotubes does not result in telomere deprotection, but triggers potent oxidative stress, along with increased Reactive Oxygen Species (ROS), mitochondrial dysfunction, FOXO3a activation, and autophagy, compared to mitotic cells. Therefore, the TRF2 or the upregulated agent thereof can be used for preparing a medicament for treating related muscle diseases or screening medicaments suitable for treating the related muscle diseases; the reagent for detecting the TRF2 protein or gene level can be used for preparing a reagent or a kit for detecting the physiological age of muscle of an individual.

Description

Application of TRF2 or its up-regulating agent in preparing medicine for treating muscle disease

Technical Field

The invention relates to the technical field of biological medicines, and in particular relates to application of TRF2 or a regulator thereof in preparing a medicament for treating muscle diseases.

Background

Telomere repeat binding factor 2 (TRF 2), also known as TERF2 and TRBF2, is encoded by the TERF2 gene in humans, is a protein that binds to telomeres throughout the cell cycle, and is a component of the guard telomere protein complex (shelterin) that binds to telomere DNA through the carboxy terminus, playing an important role in maintaining the structure stability of telomere DNA, preventing chromosome end-to-end fusion, and participating in DNA damage response.

Journal literature, "expression of mouse cerebral cortex telomere repeat sequence binding factor 2 and neuronal protection effect", published in "journal of Chinese aged medicine" 2014 7, observes age-increasing changes in mouse cerebral cortex TRF2 expression, and discusses the protection effect of TRF2 gene transfection on in vitro cultured cortical neurons and the effect of TRF2 in neuronal senescence. The method comprises the following steps: dividing the C57BL/6J mice into a young group (2 months old) and an old group (20 months old), and detecting the TRF2 expression of the cerebral cortex of the mice by respectively adopting a protein immunoblotting method (Western blot) and a Real-time quantitative polymerase chain reaction (Real-time PCR); pcDNA-TRF2 plasmid is transfected to 18d fetal rat cortical neuron cells, camptothecin is acted for 16h, and the survival rate of the neuron cells is detected by a tetramethyl tetrazolium blue (MTT) colorimetric method. The results show that: compared with 2-month-old mice, the expression of the TRF2 in the cerebral cortex of the 20-month-old mice is obviously reduced; after the camptothecin acted for 16h, the neuron survival rate of the transfected pcDNA-TRF2 was (75.4 +/-2.6)%, which is obviously higher than that of the transfected empty plasmid group (32.6 +/-9.3)% (t is 22.85, and P is less than 0.05). It was therefore concluded that: TFR2 expression decreased involvement in neuronal senescence, and TRF2 overexpression may be a new target for treatment of neurodegeneration.

Journal literature, "meaning of expression levels of telomere binding proteins TRF1 and TRF2 in inflammatory enteritis patients" published in the Chinese contemporary medicine, 2012, at 11 discusses the meaning of expression levels of telomere binding proteins TRF1 and TRF2 in inflammatory enteritis patients. The method comprises the following steps: expression levels of TRF1 and TRF2 were measured in 14 patients with ulcerative colitis and 13 patients with Crohn's disease and compared with normal populations. As a result, the expression levels of TRF1 and TRF2mRNA in peripheral blood of patients with inflammatory enteritis are down-regulated.

A paper "Telomelength and regulatory proteins in human clinical muscles with and with a telomeric generating cycles" published in periodical literature Experimental Physiology 2012, 6, evaluates telomere length and specific proteins involved in telomere regulation in patients with dermatomyositis and polymyositis and in healthy persons. In particular, mean and minimum telomere lengths and the expression of telomerase, tankyrase 1, TRF2 and POT1 were studied in skeletal muscle samples of 12 patients and 13 healthy subjects. As a result, it was found that the expression levels of telomerase, tankyrase 1, TRF2, and POT1 were six-fold, seven-fold, three-fold, and five-fold higher, respectively, (P < 0.05) in the skeletal muscle nucleus portion of the patient, as compared to the healthy control. This suggests that there may be endogenous mechanisms to allow for a sustained cycle of degeneration and regeneration of telomere regulation in skeletal muscle, and that there is a pattern in which regulatory factors may be involved in telomere protection in skeletal muscle.

Although the structure, location and function of the TRF2 protein are known to some extent, many problems still need to be further discussed and studied. The role of TRF2 in post-mitotic differentiated tissues and cells, for example, is not clear.

Skeletal muscle makes up 40% of body weight, fluctuating with age and health. Skeletal muscle is composed primarily of postmitotic, differentiated multinucleated cells and geostationary satellite cells containing renewal potential. Depletion of proliferating cells during aging limits muscle regeneration or repair, leading to sarcopenia or age-related muscle atrophy, the first cause of the onset of anomalcule age. However, little is still known about the cascade of injury events associated with changes in function and structure of aging muscles.

There is no report on the relationship between TRF2 and sarcopenia or age related muscle wasting disease.

Disclosure of Invention

The invention aims to provide a new application of TRF2 aiming at the defects in the prior art.

In the first invention of the present invention, there is provided the use of TRF2 or a upregulation thereof in the manufacture of a medicament for the treatment of a muscle disease.

As a preferred example, the muscle disease is sarcopenia.

As another preferred example, the muscle disease is selected from sarcopenia, muscular dystrophy, amyotrophic lateral sclerosis, Charcot Marie Tooth disease, Dejin stocks disease, muscle pine disease and galangal disease caused by aging.

Preferably, the muscular dystrophy is selected from becker muscular dystrophy, congenital muscular dystrophy, duchenne muscular dystrophy, distal muscular dystrophy, emerley-delbruffs muscular dystrophy, limb and facet shoulder brachial muscular dystrophy, girdle muscular dystrophy, myotonic muscular dystrophy, oculopharyngeal muscular dystrophy, spinal muscular atrophy, brown vialato VanLaelic syndrome, and Fazio Londe syndrome.

Preferably, the muscle atrophy is selected from the group consisting of cancer-induced muscle atrophy, aids-induced muscle atrophy, congestive heart failure-induced muscle atrophy, chronic obstructive pulmonary disease with muscle atrophy, renal failure-accompanied muscle atrophy, severe burns with muscle atrophy and long-term bed-ridden muscle atrophy.

As another preferred example, the upregulating agent is selected from the group consisting of peptides, peptidomimetics, small organic molecules, and aptamers.

In a second aspect of the present invention, there is provided a method of screening for a drug suitable for treating a muscle disease, comprising:

I) providing a test compound; and

II) determining the ability of the test compound to activate TRF2 expression or activity.

In a third aspect of the invention, there is provided a method of treating a muscle disorder comprising sequentially administering to a subject an effective amount of a compound treatment to activate TRF2 expression or activity.

As a preferred example, the subject is selected from mammals.

Preferably, the mammal is selected from the group consisting of rodents, felines, canines and primates.

More preferably, the subject is a human, especially an elderly person over the age of 70.

In a fourth aspect of the invention, there is provided a use of a reagent for detecting the level of TRF2 protein or gene in the preparation of a reagent or kit for detecting the physiological age of muscle of an individual.

The invention has the advantages that: the invention discovers that the TRF2 protein level of human skeletal muscle is negatively correlated with age; TRF2 in human striated muscle decreases with the aging of mitotic cells, and down-regulation of TRF2 in differentiated myotubes does not result in telomere deprotection, but triggers potent oxidative stress, while increasing Reactive Oxygen Species (ROS), mitochondrial dysfunction, FOXO3a activation and autophagy compared to mitotic cells; the TRF2 protein injection can obviously prevent rat sarcopenia. Therefore, the TRF2 or the upregulated agent thereof can be used for preparing related medicines for treating muscle diseases; the reagent for detecting the TRF2 protein or gene level can be used for preparing a reagent or a kit for detecting the physiological age of muscle of an individual.

Drawings

FIG. 1: a: the biopsy chart used in the study, including patient sex, biopsy age and puncture site, was healthy with no muscle related disease in all patients. B: westernblot assays for TRF2 protein levels in muscle biopsy specimens. C: TRF2 protein levels correlated with age of skeletal muscle biopsies, with 95% confidence intervals showing discontinuous lines and equations and associated goodness of fit (R)²)。

FIGS. 2 to 3: telomere-Induced DNA damage response signal analysis (TIF, Telomeric Induced Foci) of virus-transduced myoblasts (FIG. 2) and myotubes (FIG. 3) using immunofluorescence coupled with Deltavision microscopy imaging. Fixed cell staining of telomeric probes (PNA, green) and 53BP1 (red) antibody indicated DNA damage. Stacking after 100Z 331 images are acquired is deconvoluted (Deltavision)

GE). Transduction of shtrf2 induced an increase in TIF in myoblasts (shtrf2 compared to shscarmble, P0.0005; one-way anova, Kruskal Wallis multiple comparison test, P0.05), indicating that modulated mitotic cell telomere damage increased myotube expression at TRF2 with only in-focus multinucleated cell counts (minimum 40 nuclei per condition). Note: middle school of researchTRF2 knock-out plasmids were used commercially, SIGMA corporation, SHCLNG-NM-005652 (human), SHCLNG-NM-009353 (murine).

FIG. 4: a: ROS detection application

detection kit (GE) combined with Deltavision ultra high resolution microscopy imaging. Hydrogen peroxide treatment resulted in an increase in the signal number of ROS (no-load and no-load + H)₂O₂For comparison, P ═ 0.0274; empty and TRF2+ H₂O₂For comparison, P-0.0291; Holm-Sidak's multiple comparison test; p ═ 0.05). TRF2 downregulation significantly increased ROS (shscarmble vs. shtrf2, P < 0.0001; Holm-Sidak's multiple comparison test; P ═ 0.05). EGCG (epigallocatechin gallate), epigallocatechin gallate, antioxidant contrast agent. B: by using

The kit detects autophagy foci of the transduced shtrf2 myotube cells. 100Z-axis change pictures (deltavision) are acquired on average under each condition

GE). The image is then acquired and processed using imaris. Monocytes were excluded from the analysis.

FIGS. 5 to 7: FIG. 5: in myotube cells, TRF2 was down-regulated and Foxo3a and PGC1 gene expression was increased as confirmed by RT-PCR. FIGS. 6 to 7: immunoblotting and immunofluorescence confirmed that TRF 2-damaged cells had elevated FOXO3a levels and nuclear localization at telomeres.

Detailed Description

The inventor analyzes the protein level of TRF2 from skeletal muscle biopsy specimens of donors of different ages through extensive and intensive research, and firstly discovers that the protein level of TRF2 is reduced along with the increase of the age; TRF2 expression was down-regulated in a post-mitotic cell model, and it was found that post-mitotic cell down-regulation of TRF2 did not trigger telomere deprotection, but rather mitochondrial dysfunction and ROS production. The TRF2 is suggested to have protective effect on human striated muscle cells and avoid triggering a series of oxidative damage pathways. Therefore, TRF2 or a upregulation thereof can be used to treat muscle disorders; and a substance useful for treating a muscle disease can be screened based on the above-mentioned function of TRF 2.

TRF2 and application thereof

In the present invention, the TRF2 protein may be naturally occurring, for example, it may be isolated or purified from a mammal. In addition, the TRF2 protein can also be artificially prepared, for example, the recombinant TRF2 protein can be produced according to the conventional genetic engineering recombination technology. Preferably, the present invention may employ a recombinant TRF2 protein.

Any suitable TRF2 protein may be used in the present invention. The TRF2 protein comprises full-length TRF2 protein or a bioactive fragment thereof. Preferably, the amino acid sequence of the TRF2 protein may be substantially identical to the sequence shown in GenBank accession No. np _ 005643.2.

The amino acid sequence of the TRF2 protein formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. The TRF2 protein or biologically active fragment thereof includes a partial substitution of conserved amino acids in the sequence that does not affect its activity or retains some of its activity. Appropriate substitutions of amino acids are well known in the art and can be readily made and ensure that the biological activity of the resulting molecule is not altered. These techniques allow one of skill in the art to recognize that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al, Molecular Biology of The Gene, fourth edition, 1987, The Benjamin/Cummings Pub. Co. P224.

Any biologically active fragment of TRF2 protein can be used in the present invention. Herein, a biologically active fragment of a TRF2 protein is meant to be a polypeptide that still retains all or part of the function of the full-length TRF2 protein. Typically, the biologically active fragment retains at least 50% of the activity of the full-length TRF2 protein. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99% or 100% of the activity of the full-length TRF2 protein.

The present invention may also employ modified or improved TRF2 proteins, e.g., TRF2 proteins modified or improved to promote their half-life, effectiveness, metabolism, and/or potency of the protein. The modified or improved TRF2 protein may be a conjugate of the TRF2 protein, or it may comprise substituted or artificial amino acids. The modified or improved TRF2 protein may have little commonality with the naturally occurring TRF2 protein, but also avoid triggering oxidative damage pathways in human striated muscle cells without additional adverse effects or toxicity. That is, any of the variations which do not affect the biological activity of the TRF2 protein can be used in the present invention.

The corresponding nucleotide coding sequence can be conveniently derived from the amino acid sequence of the TRF2 protein.

Preferably, the nucleotide sequence of the TRF2 protein may be substantially identical to the sequence shown in GenBank accession No. nm _ 005652.4.

In addition, the amino acid sequence and nucleotide sequence of the mouse TRF2 protein have also been disclosed in GenBank accession nos. np _001076587.1 and nm _ 001083118.2.

Based on the description of the invention, TRF2 is a novel drug target related to muscle diseases. Various therapeutic means against TRF2 can be a novel and effective means for preventing and treating muscle diseases in animals (particularly humans). Also, TRF2 can be used as a target for drug screening to screen drugs that protect striated muscle cells from oxidative damage by increasing the expression or activity of TRF 2.

The term "muscle disease" as used herein refers to disorders of the muscular system that affect muscle function. The muscular system refers to an organ system composed of bones, smooth muscles and cardiac muscles. Muscle diseases are selected from, but not limited to, muscular dystrophy (e.g., becker muscular dystrophy, congenital muscular dystrophy, duchenne muscular dystrophy, distal muscular dystrophy, emerley-delbruffs muscular dystrophy, limb and facet shoulder brachial muscular dystrophy, girdle muscular dystrophy, myotonic muscular dystrophy, oculopharyngeal muscular dystrophy, spinal muscular atrophy, brown vialato Van laidic syndrome, Fazio Londe syndrome); muscle atrophy (e.g., muscle atrophy caused by cancer, muscle atrophy caused by AIDS, muscle atrophy caused by congestive heart failure, chronic obstructive pulmonary disease with muscle atrophy, muscle atrophy with renal failure, severe burns with muscle atrophy, and muscle atrophy with prolonged bed rest); amyotrophic lateral sclerosis; charcot Marie Tooth disease; dejin Stotas disease; myotonia or ganleydi disease. In a particular example, the muscle disease is sarcopenia. The term "muscle relaxation disease" refers to a syndrome characterized by progressive and systemic loss of skeletal muscle mass and strength, with the risk of physical disability, poor quality of life, and mortality, as adverse consequences.

In a specific embodiment, the muscle disease is Sarcopenia (Sarcopenia). Sarcopenia refers to a decrease in skeletal muscle mass, strength decline, and physical decline of any cause. The disease can be divided into two main categories of primary and secondary sarcopenia according to the etiology. Sarcopenia which occurs with the aging process of a human body is called primary sarcopenia. The prevalence rate of the population above 65 years is 5% -10%, and the prevalence rate of the population above 70 years is over 40%. Sarcopenia is a pathological result of a variety of factors. It has been found that absence of innervation, mitochondrial dysfunction, inflammation and altered hormone levels may all contribute to decreased skeletal muscle quality and function. Oxygen radical clearance in sarcopenia patients is also impaired.

TRF2 up-regulating agent and application thereof

Based on the above new findings of the present inventors, the present invention provides the use of an up-regulator of TRF2 for the preparation of a composition for preventing striated muscle cells from triggering oxidative damage pathways, thereby treating muscle diseases.

As used herein, the up-regulation of TRF2 includes promoters, agonists, and the like. Any substance that can increase the activity of TRF2 protein, maintain the stability of TRF2 protein, promote the expression of TRF2 protein, promote the secretion of TRF2 protein, prolong the effective duration of TRF2 protein, or promote the transcription and translation of TRF2 can be used in the present invention.

Typically, up-regulation of TRF2 expression has a biological effect on one or more of the following events: (1) generating an RNA template from the DNA sequence (e.g., by transcription); (2) processing of RNA transcripts (e.g., by splicing, editing, 5 'cap formation, and/or 3' end formation); (3) translating the RNA into a polypeptide or protein; and/or (4) post-translational modification of the polypeptide or protein.

The up-regulator is a compound which activates the expression or activity of TRF2, and specifically can be a peptide, a peptide mimetic, a small organic molecule or an aptamer. The term "peptidomimetic" refers to a short, proteinaceous chain that resembles a peptide. The term "small organic molecule" refers to a molecule of comparable size to the organic molecules commonly used in pharmaceuticals. The term does not include biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5KDa, more preferably up to 2KDa, and most preferably up to about 1 KDa.

Composition comprising a metal oxide and a metal oxide

The invention also provides a composition comprising an effective amount (e.g., 0.000001-50 wt%, preferably 0.00001-20 wt%, more preferably 0.0001-10 wt%) of the TRF2 protein or a upregulating agent thereof, and a pharmaceutically acceptable carrier.

The composition of the present invention can be directly used for muscle diseases, particularly sarcopenia. In addition, it may be used in combination with other therapeutic agents or adjuvants.

Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8.

As used herein, the term "comprising" means that the various ingredients can be applied together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising. As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The composition of the invention contains a safe and effective amount of TRF2 protein and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation is usually adapted to the administration mode, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the invention can also be prepared into a sustained release preparation.

The effective amount of TRF2 protein or a upregulation thereof according to the present invention may vary with the mode of administration and the severity of the disease to be treated, among other things. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: the TRF2 protein or the pharmacokinetic parameters of the TRF2 protein up-regulated, such as bioavailability, metabolism, half-life and the like; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. Generally, satisfactory results are obtained when the TRF2 protein or a upregulated dose thereof of the present invention is administered daily at a dosage of about 0.00001mg to about 50mg per kg of animal body weight, preferably about 0.0001mg to about 10mg per kg of animal body weight. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The invention also provides a method of treating a muscle disorder comprising sequentially administering to a subject an effective amount of a compound treatment to activate TRF2 expression or activity.

The TRF2 protein or its up-regulator of the present invention is not particularly limited in its administration mode, and may be systemic or local. For example, it can be administered by intraperitoneal injection, intravenous injection, oral administration, subcutaneous injection, intradermal injection, etc. The mode of administration of the up-regulator of TRF2 protein depends primarily on the type and nature of the up-regulator, as can be assessed by one skilled in the art.

Screening method

Knowing the utility of the TRF2 protein in protecting striated muscle from triggering oxidative damage pathways, substances that promote the expression or activity of TRF2 can be screened based on this feature.

Accordingly, the present invention provides a method of screening for a drug suitable for the treatment of a muscle disease, comprising: I) providing a test compound; and II) determining the ability of the test compound to activate TRF2 expression or activity.

In a preferred embodiment of the present invention, a control group may be provided in order to more easily observe the change in the expression or activity of TRF2 in the screening, and the control group may be a system expressing TRF2 without adding the candidate substance.

The system for expressing TRF2 can be, for example, a cell (or cell culture) system, and the cell can be a cell endogenously expressing TRF 2; or may be a cell recombinantly expressing TRF 2. The TRF2 expression system can be (but is not limited to) a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model) and the like.

As a preferred embodiment of the present invention, the method further comprises: the potential substances obtained are subjected to further cell experiments and/or animal experiments to further select and identify substances that are truly useful for the treatment of muscle diseases.

The method for detecting the expression, activity, amount of TRF2 protein or secretion of TRF2 protein according to the present invention is not particularly limited. Conventional protein quantitative or semi-quantitative detection techniques may be employed, such as (but not limited to): SDS-PAGE, Western-Blot, ELISA, etc.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1

We evaluated skeletal muscle biopsy specimens TRF2 protein levels from donors of different ages. We found that TRF2 protein levels were inversely correlated with age (correlation, R)²0.6967) that dropped sharply after the third decade of life, consistent with shortening of telomere length (fig. 1).

We simulated a model of TRF2 regulating postmitotic cells and in vivo observations confirmed that TRF2 regulates skeletal muscle postmitotic cells. Myoblasts were obtained from the deltoid biopsy and cultured in differentiation medium (day 0; 2% horse serum) to confluency on day 5 to form multinucleated myotubes, which were transduced with lentiviral particles on day 7. On day 17, myotubes were collected. Meanwhile, the virus was transduced and proliferated into myoblasts for 5 days, and the cells were collected. As observed in other mitotic cells, decreased expression of TRF2 in myoblasts triggers telomere deprotection, i.e., recruitment of DNA Damage Response (DDR) factors to telomeres, forming signals resulting from telomere dysfunction (fig. 2). Importantly, post-mitotic cell TRF2 down-regulation failed to trigger similar TIF and ATM activation (fig. 3), suggesting the existence of a myotube-specific telomere protection mechanism. Importantly, down-regulation of TRF2 did not affect morphological differential changes such as myogenesis, apoptosis, etc. We investigated whether inhibition of TRF2 affects other markers affecting aging. We first analyzed whether modulating TRF2 expression would affect the mitochondrial network, mitochondrial content and quantitate Reactive Oxygen Species (ROS). In myotubes with loss of TRF2 protein expression, the mitochondrial network appeared to be interrupted and disconnected compared to controls. In addition, quantitative analysis of mitochondrial DNA when TRF2 was consumed showed higher levels of mitochondrial DNA (8-fold, P ═ 0.0003, PCR detection of mitochondrial DNA), suggesting a possible compensatory mechanism for mitochondrial damage. At the same time, reducing TRF2 expression produces high levels of ROS, reaching H₂O₂ROS levels after treatment, indicating the presence of mitochondrial dysfunction. In addition, antioxidant treatment simultaneously reversed ROS production and improved mitochondrial network connectivity (fig. 4). Mitochondria can regulate autophagy through ROS production, we assessed autophagy activity and found that TRF2 is down-regulated and significantly increased in autophagic lesion number (fig. 4)). Next, we evaluated TRF2 transcription of genes (anabolism and catabolism) that regulate muscle cell homeostasis in muscle cells. We observed that TRF2 was down-regulated, with increased expression of Foxo3a and PGC1 genes, which may directly trigger increased ROS production due to TRF2 inhibition (fig. 5). Immunoblotting and immunofluorescence confirmed that TRF 2-damaged cells had elevated FOXO3a levels and nuclear localization at telomeres (fig. 6 and 7). These results indicate that extra-telomeric function of TRF2 is essential for myotube redox balance.

Taken together, these findings indicate that high levels of TRF2 protect postmitotic cells from triggering a series of oxidative damage pathways, such as mitochondrial dysfunction and ROS production.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

Use of TRF2 or a upregulation thereof in the manufacture of a medicament for the treatment of a muscle disease.
2. The use according to claim 1, wherein the muscle disease is sarcopenia.
3. The use of claim 2, wherein the sarcopenia is primary sarcopenia.
4. The use according to claim 1, wherein the muscle disease is selected from the group consisting of sarcopenia, muscular dystrophy, amyotrophic lateral sclerosis, Charcot Marie Tooth disease, DejinStotas disease, muscle pine disease and Ganmadex disease caused by aging.
5. The use according to claim 4, wherein the muscular dystrophy is selected from Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emmery-Delivers muscular dystrophy, limb and facet humeral muscular dystrophy, girdle muscular dystrophy, myotonic muscular dystrophy, oculopharyngeal muscular dystrophy, spinal muscular atrophy, brown Vialeto Van Laelic syndrome, and Fazio Londe syndrome.
6. The use of claim 4, wherein the muscle atrophy is selected from the group consisting of cancer-induced muscle atrophy, AIDS-induced muscle atrophy, congestive heart failure-induced muscle atrophy, chronic obstructive pulmonary disease with muscle atrophy, muscle atrophy with renal failure, severe burns with muscle atrophy and muscle atrophy with prolonged bed rest.
7. The use of claim 1, wherein the upregulating agent is selected from the group consisting of peptides, peptidomimetics, small organic molecules, and aptamers.
8. A composition for treating a muscle disorder, said composition comprising TRF2 or a upregulation thereof.
9. A method of screening for a drug suitable for treating a muscle disease, comprising:

I) providing a test compound;

II) determining the ability of the test compound to activate TRF2 expression or activity.
10. Use of a reagent for detecting the level of TRF2 protein or gene in the preparation of a reagent or a kit for detecting the physiological age of muscle of an individual.