CN118634329A - Application of SESN1 recombinant protein in intervention of primate skeletal muscle aging - Google Patents

Abstract

The invention discloses an application of SESN1 recombinant protein in intervening primate skeletal muscle aging. The invention provides application of a substance for increasing the expression level of SESN1 of skeletal muscle cells in any one of the following: preparing a product for treating diseases caused by skeletal muscle aging or atrophy, or preparing a product for delaying skeletal muscle cell aging; or preparing a product for treating diseases caused by skeletal muscle injury; SESN1 can be used as a marker to distinguish aged skeletal muscle and young skeletal muscle of primates, can distinguish the aging degree of organisms to which serum or plasma or other body fluids belong, and can evaluate and use recombinant proteins to intervene in skeletal muscle aging-related diseases. SESN1 plays an important role in screening and identifying skeletal muscle cells, and particularly provides important molecular indicators for human skeletal muscle function assessment and disease diagnosis, and has important application value.

Description

Application of SESN1 recombinant protein in intervention of primate skeletal muscle aging

Technical Field

The invention belongs to the field of biomedicine, relates to a marker for evaluating or assisting in evaluating skeletal muscle aging and application of the marker in delaying muscle aging, and particularly relates to application of SESN1 recombinant protein in intervening primate skeletal muscle aging.

Background

Skeletal muscle is one of the largest organs of the human body, accounting for about 40% of the mass of the whole body. With age, skeletal muscle mass and strength is lost year by year, known as sarcopenia, resulting in reduced physical function and impaired quality of life for the elderly. Furthermore, since skeletal muscle is an important endocrine and metabolic organ, skeletal muscle aging may also lead to metabolic disorders through changes in muscle-derived growth factors (muscle cytokines) or metabolites, ultimately increasing the risk of age-related metabolic diseases such as diabetes and obesity. Although exercise training and diet can help maintain muscle mass, there is still a lack of effective treatments to delay or prevent skeletal muscle aging and sarcopenia in the elderly. Therefore, the deep understanding of the mechanism leading to skeletal muscle aging, the search for key factors and intervention patterns of skeletal muscle cell aging has important scientific and clinical significance.

Disclosure of Invention

The object of the present invention is to provide a primate or human skeletal muscle aging marker, and further to study skeletal muscle aging-related diseases.

In a first aspect, the invention provides the use of a substance which increases the expression level of a SESN1 protein or gene in any of the following:

1) Preparing a product for treating diseases caused by skeletal muscle aging or atrophy; of the above, the skeletal muscle aging or atrophy-induced diseases specifically such as sarcopenia and the like;

2) Preparing a product for delaying skeletal muscle cell aging; the above-mentioned delay of skeletal muscle cell aging is embodied in the decrease of diameter of human myotube cells and the decrease of SA-beta-gal activity;

3) Preparing a product for increasing the diameter of skeletal muscle cells; the skeletal muscle cell diameter is embodied by the diameter of myotube cells;

4) Preparing a product that reduces SA- β -gal activity of skeletal muscle cells; the SA-beta-gal activity of the skeletal muscle cells is embodied by the ratio of SA-beta-gal positive cells;

5) Preparing a product for treating diseases caused by skeletal muscle injury;

6) Preparing a product for promoting skeletal muscle regeneration after injury; the promotion of skeletal muscle regeneration after injury is specifically shown by the increase of the proportion of skeletal muscle PAX 7/Ki 67 positive cells and the cross-sectional area of muscle fibers;

7) The product for promoting the recovery of the motor function after the injury of the skeletal muscle of animals or humans is prepared.

The skeletal muscle or cell is derived from an animal or human, and further, the animal is primate or murine. If the animal is murine, the recovery of the exercise function after the injury is specifically reflected in the increase of the holding power; the stay time of the rotating rod is prolonged; depletion distance, time, maximum speed increase.

In an embodiment of the present invention, the skeletal muscle injury is exemplified by CTX treatment injury.

In the above application, the substance for increasing the expression level of the SESN1 protein or the gene comprises a substance for activating the endogenous SESN1 protein or the gene expression of skeletal muscle cells, SESN1 protein, any active peptide segment of the SESN1 protein or SESN1 protein with a label.

In the above, in the embodiment of the present invention, the SESN1 protein with a tag is recombinant SESN1 protein, and the catalogue number of the product from Abcam company is ab196424.

The substance for activating the endogenous SESN1 expression of skeletal muscle cells comprises a CRISPR-dCAS9 mediated endogenous activating vector.

In an embodiment of the invention, the CRISPR-dCAS9 mediated endogenous activating vector is a vector or a lentivirus,

The carrier is obtained by carrying out annealing reaction on the primer shown in the table 5 to obtain an annealed product; then connecting the annealed product with a Lenti-SAMv carrier to obtain a Lenti-SAMv2-sgSESN1 carrier;

The lentivirus is obtained by co-transfecting a Lenti-SAMv-sgSESN 1 vector, a lentivirus packaging plasmid psPAX and pMD2G into 293T cells, and packaging.

In a second aspect, the present invention provides a product comprising an agent for increasing the expression level of SESN1 as described in the first aspect;

The product has at least one of the following functions:

1) Treating diseases caused by skeletal muscle aging or atrophy;

2) Delaying skeletal muscle cell aging;

3) Increasing the diameter of skeletal muscle cells;

4) Decreasing the SA- β -gal activity of skeletal muscle cells;

5) Treating skeletal muscle injury-induced diseases;

6) Promoting skeletal muscle regeneration after injury;

7) Promoting the recovery of the motor function of animals after skeletal muscle injury.

In a third aspect, the invention provides the use of a substance that inhibits the expression of a SESN1 protein or gene in the preparation of a model of aging; the function of the aging model is to screen medicines for treating diseases caused by skeletal muscle aging or injury.

In the above application, the aging model may be an animal model or a cell model.

In the above application, the substance inhibiting expression of SESN1 protein or gene is a small interfering RNA that interferes with expression of SESN 1.

In an embodiment of the invention, the coding sequence of the small interfering RNA is SEQ ID NO.1.

In a fourth aspect, the invention provides the use of a substance for detecting expression of a SESN1 protein or gene in any of the following:

1) Preparing a product for evaluating or assisting in evaluating or identifying or assisting in identifying the degree of skeletal muscle aging;

2) Preparing a product for identifying or assisting in identifying or screening or assisting in screening skeletal muscle cells with low or high aging degree;

3) Preparing a product for identifying or assisting in identifying the degree of aging of different skeletal muscles or cells;

4) The product for identifying or assisting in identifying the aging degree of the organism to which the serum or the plasma or other body fluids belong is prepared.

In the above application, the substance for detecting the expression of the SESN1 protein or gene is a substance specifically binding to the SESN1 protein or gene.

The substance binding to SESN1 protein can be monoclonal antibody or polyclonal antibody of SESN 1; the substances specifically combined with the SESN1 gene can be a specific primer pair for amplifying the SESN1 gene by PCR; in the embodiment of the invention, the specific forward direction is: TGCTTTGGGCCGTTTGGATAA; reversing: TGTAGTGCCGATAATGTAGGGGT. .

In a fifth aspect, the invention provides the use of a SESN1 gene or protein as a marker for the preparation of a product for identifying or aiding in the identification of the degree of ageing of different skeletal muscles or cells or serum or plasma or other body fluids.

In a sixth aspect, the present invention provides a kit for identifying or aiding in the identification of the degree of aging of different skeletal muscles or cells or serum or plasma or other body fluids, comprising the substance for detecting SESN1 protein or gene expression of the fourth aspect.

In the above, the skeletal muscle or cell is derived from an animal or human;

further, the skeletal muscle or cell is derived from a primate or murine, in embodiments of the invention the primate is exemplified by a cynomolgus monkey.

In the invention, when the aging degree of different skeletal muscles or cells or serum or plasma or other body fluids is identified or assisted, the aging degree of the skeletal muscles or cells or serum or plasma or other body fluids to be detected with low expression level of SESN1 is higher than that of the skeletal muscles or cells or serum or plasma or other body fluids to be detected with high expression level of SESN 1.

In the above, the skeletal muscle cell is at least one of a fast muscle type IIA, a fast muscle type IIX, a slow muscle type I, a myotube cell differentiated from a human embryonic stem cell, and the like.

The aging degree of skeletal muscle or cells (namely skeletal muscle cells) or serum or plasma or other body fluids is that of the body to which the skeletal muscle or cells (namely skeletal muscle cells) or serum or plasma or other body fluids belong.

In the invention, the product is a reagent or a kit.

Gene ID of SESN1 in the present invention 27244 (updated on 22-Sep-2022), protein ID: NP-055269.1 (updated on 28-DEC-2022).

The invention finds SESN1 as a marker, and experiments prove that the SESN1 can be used as the marker to distinguish aged skeletal muscles and young skeletal muscles of primates or humans and can also distinguish the aging degree of organisms to which serum or plasma or other body fluids belong. The markers can be used for evaluating and interfering skeletal muscle aging related diseases by using recombinant proteins. SESN1 has important functions in screening and identifying skeletal muscle cells, can evaluate skeletal muscle aging-related diseases such as sarcopenia, can detect human skeletal muscle functions, evaluate human skeletal muscle aging degree and the like, provides important molecular indications for research and intervention of skeletal muscle aging-related diseases, and has important application value particularly in human skeletal muscle function evaluation and disease diagnosis.

Drawings

FIG. 1 shows SCENIC, differential gene association analysis and gene expression correlation analysis, wherein A is common differential gene analysis between aged cynomolgus monkey myofibroblasts and FOXO3 ^-/- myotube cells, and B is common expression correlation analysis between FOXO3 and SESN 1.

FIG. 2 shows the transcriptional activation of SESN1 by FOXO3 as demonstrated by chromatin co-immunoprecipitation-fluorescent quantitative ChIP-qPCR (A) and dual luciferase reporter assay (B).

FIG. 3 shows quantitative PCR and Western blotting to identify that SESN1 mRNA (A) and protein (B) levels decreased after FOXO3 knockout and SESN1 protein levels increased after FOXO3 genetic enhancement (C).

FIG. 4 is a single cell nuclear sequencing data analysis (A), quantitative PCR (B) and Western blotting (C) to identify the reduction of mRNA and protein levels of SESN1 in aged cynomolgus skeletal muscle.

FIG. 5 is a Western blot analysis of SESN1 protein levels in human skeletal muscle.

FIG. 6 is a Western blot analysis of SESN1 protein levels in aged human myotube cells cultured over a long period of time.

FIG. 7 is a schematic representation of human myotube cells that inhibited SESN1 expression using small interfering RNA (A), identification of SESN1 protein expression from human myotube cells that inhibited SESN1 expression by protein imprinting (B), identification of diameter of human myotube cells that inhibited SESN1 expression by immunofluorescence experiments (C), identification of the degree of aging of human myotube cells that inhibited SESN1 expression by galactosidase experiments (D).

FIG. 8 is a schematic representation of CRISPR/Cas9 mediated endogenous activation of expressed human myotube cells by SESN1 (A), identification of SESN1 protein expression by endogenous activation of SESN1 expressing human myotube cells by protein imprinting (B), identification of diameter of endogenous activation of SESN1 expressing human myotube cells by immunofluorescence experiments (C), identification of the degree of senescence of endogenous activation of SESN1 expressing human myotube cells by galactosidase experiments (D).

FIG. 9 is an ELISA for identifying SESN1 content in young aged serum (A) and long-term cultured human myotube cell culture medium supernatant (B).

FIG. 10 is a schematic diagram of SESN1 recombinant protein-treated FOXO3 knockdown human myotube cells (A), immunofluorescence assay to identify SESN1 recombinant protein-treated human myotube cell diameter (B), and galactosidase assay to identify SESN1 recombinant protein-treated human myotube cell senescence (C).

FIG. 11 is a schematic diagram (A) of a model and detection of muscle injury by treatment with SESN1 recombinant protein in 16 month old mice, and the holding power (B) of the mice; stick turning residence time (C); depletion distance, time, maximum speed detection (D).

FIG. 12 shows the detection of PAX7\Ki67 positive cell proportion (A) and myofiber cross-sectional area (B, C) in skeletal muscle of SESN1 recombinant protein-treated muscle injury model by immunofluorescence staining.

FIG. 13 is a diagram of a single cell nuclear transcription component population UMAP of SESN1 recombinant protein treated muscle injury.

FIG. 14 shows functional enrichment analysis (A) and regeneration-related gene set scores (B, C) for SESN 1-induced upregulation DEGs.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The quantitative tests in the following examples were all performed in triplicate, and the results were averaged.

The sources of some of the reagent materials in the examples below are as follows:

PBS is available from company absin under the accession number abs962;

dNTPs are available from Fermentas under the designation R0193;

nuclease-free water was purchased from Ambion under the trade designation AM9937;

murine anti-SESN 1 was purchased from Abcam under the accession number ab67156;

Murine anti-GAPDH was purchased from Santa Cruz under the trade designation sc-365062;

Rabbit anti FOXO3 was purchased from CELL SIGNALING technology under the trade designation 2497;

Rabbit anti-IgG was purchased from CELL SIGNALING technology under the accession number 2729S;

Rabbit anti-PAX 7 was purchased from Abcam under the accession number ab34360;

Rabbit anti-Ki 67 was purchased from Abcam under the accession number ab15580;

rabbit anti-Laimin was purchased from Sigma-Aldrich under the designation L9393;

OCT ^TM compound was purchased from Sakura Finetek under the trade designation 4583;

fade resistant fixed media is available from Vector Laboratories under the designation H-1000;

protein loading buffer solution is purchased from Ding Guo, and the goods number is WB-0091;

horse serum was purchased from Gibco under the accession number 1783304;

DMEM medium was purchased from Cytiva, cat No. SH30243.01;

RNase inhibitor, superScript II reverse transcriptase, 5 XSuperScript II one-strand synthesis buffer, dithiothreitol (DTT), dynabeads MyOne streptavidin C1, alexa 488 Donkey anti-mouse IgG (H+L) secondary antibody, alexa488 Donkey anti-rabbit IgG (H+L) secondary antibody and Alexa568 Donkey anti-mouse IgG (H+L) secondary antibodies were purchased from Invitrogen under the designations AM2684, 18064-071, 65001, A21202, A21206, A21099 and A10037, respectively; rabbit antibody and murine antibody protein secondary antibodies, both purchased from Zhonghua gold bridge, were ZB-2301 and ZB-2307, respectively.

Triton X-100 was purchased from Sigma-Aldrich under the trade designation T9284;

The 2X KAPA HiFi HotStart ReadyMix and KAPA HYPER PREP library kits were purchased from KAPA company under the product numbers KK2602 and KK8054, respectively.

Cynomolgus monkey is purchased from the institute of biological resources, beijing xiaoerxin. The cynomolgus monkey experiments in the examples below were performed based on the ethical guidelines for non-human primates and have been approved by the ethical institute of animal research, academy of sciences of China.

Human embryonic stem cells were purchased from WICELL RESEARCH company under the trade designation WA09 (H9) -DL-7.

Human skeletal muscle tissue originates from the Beijing university first hospital.

Human myotube cells in the following examples were derived from human embryonic stem cells, cultured in Matrigel-coated dishes, and cultured in a low-oxygen incubator containing about 3% oxygen for 5 to 6 days using myotube cell differentiation medium (DMEM medium containing 2% horse serum by volume).

The examples below were derived from FOXO3 ^+/+ human myotube cells, FOXO3 ^-/- human myotube cells, FOXO3 ^+/+ human myotube precursor cells and FOXO3 ^-/- human myotube precursor cells, which were in the order of the names of published papers (Jing Y,Zuo Y,Yu Y,et al.Single-nucleus profiling unveils a geroprotective role of the FOXO3 in primate skeletal muscle aging.Protein&Cell.2022.), FOXO3^+/+hMyotube,FOXO3^-/-hMyotube,FOXO3^+/+hMyotube progenitor cells,FOXO3^-/-hMyotube progenitor cells.

The young group of cynomolgus monkeys is cynomolgus monkeys with the age of 4-6 years;

The aged group of cynomolgus monkeys is cynomolgus monkeys aged 18-21 years.

EXAMPLE 1 acquisition and detection of senescent skeletal muscle cell molecular markers

1. Bioinformatic analysis found that SESN1 was a downstream effector of FOXO3

Based on cynomolgus monkey skeletal muscle cell sequencing libraries of the senior group (4-6 years) and the senior group (18-21 years) in the database, SESN1 was found to have a binding motif of FOXO3 by SCENIC, differential gene association analysis and gene expression correlation analysis, and SESN1 was found to be positively correlated with gene expression of FOXO3 (FIG. 1, FIG. 1A is differential gene analysis in senior cynomolgus monkey skeletal muscle cells and FOXO3 ^-/- myotube cells, and FIG. 1B is co-expression correlation analysis of FOXO3 and SESN 1.).

2. Chromatin co-immunoprecipitation-fluorescent quantitative PCR experiments

Chromatin co-immunoprecipitation-fluorescent quantitative PCR experiments demonstrated that FOXO3 could bind to the promoter region of SESN1 (fig. 2A).

1. Protein A Dynabeads was incubated with equal amounts of FOXO3 antibody (FOXO 3 available from CELL SIGNALING technology, cat# 2497) or control IgG antibody (available from CELL SIGNALING technology, cat# 2729S) to allow magnetic beads to bind to the antibodies.

2. The digested cells were resuspended in pre-chilled PBS containing 1% formaldehyde and incubated at room temperature for 15min before crosslinking was terminated by adding glycine to a final concentration of 0.125M. The crosslinked cells were washed twice with pre-chilled PBS and centrifuged, and the supernatant was discarded and sonicated to obtain a chromatin-protein mixture of appropriate fragment size.

The cells are FOXO3 ^+/+ human myotube cells and FOXO3 ^-/- human myotube cells;

FOXO3 ^+/+ human myotube cells were FOXO3 ^+/+ human myotube cells obtained by directional differentiation of human embryonic stem cells (H9) -DL-7 from WICELL RESEARCH company, cat# WA 09;

The FOXO3 ^-/- human myotube cell is obtained by knocking out the FOXO3 Gene (Gene ID of the FOXO3 Gene is 2309 (updated on 6-Sep-2022), and Genbank Accesion is NM_001455 (updated Date 18-SEP-2022)) in 2 homologous chromosomes of the FOXO3 ^+/+ human myotube cell.

3. The antibody-bound magnetic beads were incubated with the sonicated chromatin-protein mixture and spun overnight at 4 ℃.

4. Nonspecific binding fragments were washed with RIPA buffer and TE buffer, followed by addition of proteinase K to crosslink in a metal bath at 68 ℃.

5. The precipitated DNA was extracted with phenol/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1) in two steps and dissolved with ddH ₂ O.

6. QPCR detection verifies the binding site of FOXO3 at the SESN1 promoter region.

Primers for qPCR detection described above (for amplifying the SESN1 promoter region):

SESN1-Forward GCGTTGACTTGTGGGGAATG

SESN1-Reverse GACAACCCAGCTTACAGTGC

The results are shown in fig. 2A, where IgG and αfoxo3 represent binding to Protein A Dynabeads using different antibodies, respectively, and subsequent chromatin co-immunoprecipitation-fluorescent quantitative PCR experiments were performed, and it can be seen that FOXO3 can bind to the SESN1 promoter region.

3. Double luciferase reporter experiments

PGL3-basic plasmids (from Promega, cat. No. E1751), pGL3-SESN 1-promter plasmids, renilla plasmids (from Promega, cat. No. 83178) were transiently transfected into human myoblast precursor cells (FOXO 3 ^+/+ or FOXO3 ^-/-) according to the procedure of the specification, respectively, and the solution was changed after 8h of transfection, using Lipofectamine 3000 kit (from Thermo FISHER SCIENTIFIC, cat. No. L3000015). After 72h transient transfection, cells were lysed using Universal Lysis Buffer in the kit, at room temperature for 5-10min. After the cleavage is completed, adding the renilla luciferase, the firefly luciferase and the firefly luciferase reaction substrates respectively, putting the substrates into a Synergy H1 full-function microplate detector, uniformly mixing the substrates, immediately measuring the mixture, and recording the luminescence value.

Construction of pGL3-SESN1-promoter plasmid.

A) The JASPAR website (http:// jaspar. Geneg. Net /) was first used to predict potential binding sites for FOXO3 at the SESN1 gene promoter region.

B) According to the position of the binding site, primers shown in the following Table 1 were designed, and the corresponding promoter region was amplified using the human genome as a template and ligated to pGL3-basic plasmid to construct pGL3-SESN1-promoter plasmid.

Table 1 shows the primer sequences for constructing pGL3-SESN 1-precursor (WT) plasmid

Construction of pGL3-SESN 1-master (Mut) mutant plasmid: based on the pGL3-SESN1-promoter (WT) plasmid which has been constructed, the base mutation at the specific position is completed. The operation was performed according to the full gold multipoint mutation kit instruction method, wherein the required primers are as follows:

Table 2 primer sequences for constructing pGL3-SESN 1-precursor mutant plasmids

The results are shown in fig. 2B, and it can be seen that the double luciferase reporter gene experiment demonstrates that FOXO3 can activate expression of SESN 1.

4. Real-time fluorescent quantitative PCR (RT-qPCR)

The mRNA expression amounts of SESN1 in human myotube FOXO3 ^+/+, human myotube FOXO3 ^-/-, eight cynomolgus monkey skeletal muscle tissues in young groups (5 years, 4 years, 5 years, 6 years) and eight cynomolgus monkey skeletal muscle tissues in old groups (18 years, 19 years, 20 years, 18 years, 19 years, 20 years, 21 years) were counted.

1. The primer sequence of the target gene was searched on PrimerBank and the primer was synthesized and dissolved in ddH ₂ O.

The real-time fluorescent quantitative PCR primer sequences are shown below (the amplified primer for SESN 1):

forward direction: TGCTTTGGGCCGTTTGGATAA; reversing: TGTAGTGCCGATAATGTAGGGGT.

2. Extracting RNA in each myotube cell or each cynomolgus monkey skeletal muscle tissue, and carrying out reverse transcription to obtain cDNA;

The cDNA was used as a template, diluted 10-fold with ddH ₂ O, amplified by RT-qPCR using the real-time fluorescent quantitative PCR primer, and placed in a fluorescent quantitative PCR instrument for reaction.

The reaction system is shown in the following Table 3;

Table 3 shows an RT-qPCR system

The reaction procedure described above is table 4;

Table 4 shows the RT-qPCR procedure

As shown in FIG. 3A, the mRNA expression levels of SESN1 in human myotube cells FOXO3 ^+/+ and human myotube cells FOXO3 ^-/- were decreased, respectively, in human myotube cells FOXO3 ^-/-.

The results of the expression level of SESN1 mRNA in skeletal muscle tissues of the young and old cynomolgus monkeys are shown in FIGS. 4A-4B, and it can be seen that the expression level of SESN1 mRNA in skeletal muscle tissues of the old cynomolgus monkeys is reduced.

5. Western blot (Western blot) detection

Statistics of human myotube cell FOXO3 ^+/+ and human myotube cell FOXO3 ^-/-, human myotube cell FOXO3 ^+/+ and human myotube cell FOXO3 ^2SA/2SA, eight cynomolgus monkey skeletal muscle tissues of young group (age 5 years, 4 years, 5 years, 6 years, respectively 6 years) and eight cynomolgus monkey skeletal muscle tissues of the aged group (ages 18 years, 19 years, 20 years, 18 years, 19 years, 20 years, 21 years), 7 individual skeletal muscle tissues of different ages (ages 54, 68, 69, 77, 78, 81 years), and the expression amounts of SESN1 protein in culture supernatants of human myotube cells.

Human embryonic stem cells derived from FOXO3 ^2SA/2SA human myotube cell differentiation are described in the following document "FOXO3-Engineered Human ESC-Derived Vascular Cells Promote Vascular Protection and Regeneration.Cell Stem Cell.2019;24(3):447-461", which is entitled FOXO3 ^2SA/2SA hESCs.

The culture supernatant of human myotube cells is obtained by culturing FOXO3 ^+/+ human myotube cells on myotube cell differentiation medium for 6-12 days, collecting culture solution, centrifuging 12000g for 10 min, and collecting supernatant.

The human myotube cells, the skeletal muscle tissues of 16 cynomolgus monkeys and the human skeletal muscle tissues are placed in a 1% SDS solution, and the human myotube cells, the skeletal muscle tissues and the human skeletal muscle tissues are fully cracked by using a tissue grinder and cell precipitation and blown for a plurality of times, and are heated to 105 ℃ for 10min, so that the protein is fully denatured. Then, western blot was performed on the same amount of protein (with reference to GAPDH expression level).

The Western blot specifically comprises the following steps:

1. An appropriate amount of concentrated protein loading buffer (Ding Guo) was added to an equal amount of protein sample. And loading the prepared protein sample into an SDS-PAGE gel hole, and carrying out a protein electrophoresis experiment.

2. Electrophoresis was stopped when bromophenol blue in the protein loading buffer reached near the bottom end of the gel.

3. After the step 2 is completed, protein transfer is performed for 2 hours by using a transfer film instrument.

4. After the step 3 is completed, the block is immediately performed with skimmed milk powder at room temperature for two hours.

5. After completion of step 4, a primary incubation was performed using SESN1 antibody (ex Abcam, cat# ab 67156) and the internal reference protein antibody GAPDH (ex Santa Cruz, cat# sc-365062), respectively, at 4℃overnight.

6. After completion of step 5, incubation was performed with the corresponding resistant protein secondary antibody at room temperature for 1 hour. Development was performed immediately after washing.

The Western blot experiment results are shown in figures 3-5,

Fig. 3B and 3C show the expression level of SESN1 protein in human myotube cells of FOXO3 ^+/+、FOXO3^-/- and FOXO3 ^2SA/2SA, and it can be seen that the expression level of SESN1 protein in human myotube cells of FOXO3 ^+/+ is higher than that in human myotube cells of FOXO3 ^-/-, and that in human myotube cells of FOXO3 ^2SA/2SA is higher than that in human myotube cells of FOXO3 ^+/+.

FIG. 4C shows SESN1 protein expression levels in skeletal muscle tissues of eight cynomolgus monkeys in young and old groups, wherein 1-8 of the young group indicates 5 years old, 4 years old, 5 years old, 6 years old, and 1-8 of the old group indicates 18 years old, 19 years old, 20 years old, 18 years old, 19 years old, 20 years old, 21 years old, respectively; it can be seen that the expression level of SESN1 protein in skeletal muscle tissue of the young group of cynomolgus monkeys is higher than that of SESN1 protein in skeletal muscle tissue of the old group of cynomolgus monkeys.

FIG. 5 shows the SESN1 protein expression level in human skeletal muscle tissue derived from different ages, and it can be seen that the SESN1 protein expression level in human skeletal muscle tissue decreases with age.

FIG. 6 shows the SESN1 protein expression level of human myotube cells cultured for different times of FOXO3 ^+/+, and it can be seen that the longer the culture time is, the lower the SESN1 protein expression level in human myotube cells is.

The results show that the protein expression quantity of SESN1 in skeletal muscle tissues and myotube cells of the aging group is obviously reduced, the protein expression quantity of SESN1 is obviously reduced after FOXO3 knockout, and the protein expression quantity of SESN1 is obviously increased after FOXO3 genetic enhancement (FOXO 3 ^2SA/2SA).

Again, the above results demonstrate that SESN1 is a novel primate or human skeletal muscle or cell senescence molecular marker and is regulated by FOXO 3. Skeletal muscle to be tested or cells thereof having a low expression level of SESN1 has a higher degree of aging than skeletal muscle to be tested or cells thereof having a high expression level of SESN 1. The degree of aging is specifically the size of skeletal muscle or its cell-derived animal or human, and the greater the age, the greater the degree of aging.

Gene ID of SESN1 Gene 27244 (updated on 22-Sep-2022), SESN1 protein ID: NP-055269.1 (updated on 28-DEC-2022).

Example 2 functional verification of molecular marker SESN1 for skeletal muscle cell aging degree

1. Verification of SESN1 function by Small interfering RNA (siRNA)

First), human myotube cells FOXO3 ^+/+ are divided into two groups, i.e., si-SESN1 group and si-NC group, and the expression of inhibition SESN1 in human myotube cells is detected by small interfering RNAs (sirnas).

1. Transfection

Human myotube cells of si-SESN1 and si-NC groups were transfected with Lipofectamine RNAiMAX kit (available from Thermo FISHER SCIENTIFIC, cat. No. 13778150) as follows:

si-SESN1 group: human myotube cell FOXO3 ^+/+ transfected SESN1 small interfering RNA (si-SESN 1: ACAGGAATGTCGAGATGAA, SEQ ID NO. 1);

si-NC group: human myotube cell FOXO3 ^+/+ transfected negative control small interfering RNA (si-NC);

after transfection, each group was cultured for 48 hours, and cells were harvested to obtain si-SESN1 group cells and si-NC group cells.

2. Detection of

The expression level of SESN1 in the cells of the si-SESN1 group and the cells of the si-NC group was detected by using Western blot (SESN 1 antibody is purchased from Abcam, cat. No. ab 67156).

The results are shown in FIGS. 7A-7B, wherein A is a schematic diagram for inhibiting SESN1 expression in human myotube cells by small interfering RNA (siRNA), and B is a Western blot method for detecting SESN1 expression in siI-SESN 1 group cells and siI-NC group cells, and the obvious reduction of SESN1 expression of the siI-SESN 1 group relative to the siI-NC group cells indicates that SESN1 gene expression is successfully inhibited.

Two) diameter of human myotube cell after SESN1 expression inhibition by experimental detection

The above-mentioned si-SESN1 group cells and si-NC group cells were fixed with 4% paraformaldehyde for 30 minutes, and washed with PBS 3 times for 5 minutes each. Then permeabilized with 0.4% Triton X-100 solution for 15min. The cells were washed 3 times with PBS for 5min each, and then blocked with 10% donkey serum for 1 hour at room temperature. Incubation resistance: the blocking solution was discarded, and the cells were covered with an anti-dilution solution and incubated overnight at 4 ℃. Secondary antibody incubation: the next day was washed 3 times with PBS for 5min each, followed by addition of the corresponding fluorescent secondary antibody and Hoechest 33342 and incubation at room temperature for 1 hour. Wash 3 times with PBS for 10min each on a shaker.

As shown in FIG. 7C, the immunofluorescence experiment shows that the diameter of human myotube cells inhibiting SESN1 expression is obviously lower than that of human myotube cells in si-SESN1 group, and the result shows that after the SESN1 expression is inhibited in the diameter of human myotube cells, the diameter of the cells is obviously reduced.

Third) SA-beta-gal Activity of human myotube cells after inhibition of SESN1 expression by experimental detection

Human myotube cells 48 hours after transfection of the si-SESN1 group and the si-NC group were taken out, the medium was discarded, washed 2 times with PBS, and fixed in a fixing solution at room temperature for 5min. Then, the wells of each 6-well plate were washed 3 times with PBS, 2mL of staining solution was added, and incubated overnight at 37℃in the absence of light. The next day, the staining solution was discarded, washed 2 times with PBS, and subsequently stained with Hoechest33342 at a 1:1000 ratio with PBS (5 min). After washing twice with PBS, photographs were taken with a microscope.

The formula of the fixing solution comprises the following steps: PBS containing 0.2% glutaraldehyde and 2% formaldehyde.

The formula of the dyeing liquid comprises the following steps: 40mM citrate-sodium phosphate buffer (pH＝6.0),100mM K₄[Fe(CN)₆]·3H₂O,100mM K₃[Fe(CN)6],2mM MgCl₂·6H₂O,150mM NaCl and X-gal at a final concentration of 1 mg/mL.

As shown in the detection result in FIG. 7D, the degree of aging of the human myotube cells inhibiting SESN1 expression is identified by a galactosidase experiment, and the proportion of the galactosidase positive cells of the human myotube cells in the si-SESN1 group is obviously higher than that of the human myotube cells in the si-NC group, so that the degree of aging is increased after the expression of SESN1 is inhibited in the human myotube cells.

The above results indicate that inhibiting the expression of SESN1 promotes senescence or reduces the diameter of human myotube cells.

2. Verification of SESN1 function by CRISPR/Cas9 mediated endogenous activation of SESN1

First), FOXO3 ^-/- human myotube cells were divided into two groups, namely CRISPRa-sgNTC control group and CRISPRa-sgSESN1 activation group.

Construction of endogenous activating vector mediated by CRISPR-dCas9 fig. 8A is a schematic diagram of human myotube cells expressed by endogenous activation of SESN1 mediated by CRISPR/Cas 9.

Acquiring a SESN1 transcription activated sgRNA sequence by referring to related data and documents, specifically, carrying out an annealing reaction on a synthesized primer shown in a table 5 to obtain an annealed product; and then carrying out enzyme digestion on the Leni-SAMv 2 carrier (purchased from Addgene, product # 75112), and carrying out a ligation reaction on the digested carrier and an annealed product to obtain the Leni-SAMv 2-sgSESN carrier. The Lenti-SAMv2-sgSESN1 vector was then co-transfected into 293T cells with lentiviral packaging plasmid psPAX (available from Addgene, cat# 12260) and pMD2G (available from Addgene, cat# 12259), the culture supernatants were harvested 48 and 72 hours later, and pellet was resuspended in DMEM medium after centrifugation at 19,400G for 2.5 hours at 4℃to give Lenti-SAMv2-sgSESN1 lentivirus.

The same procedure was used to prepare the Lenti-SAMv-sgNTC lentivirus and the Lenti-MPHv lentivirus (plasmid available from Addgene, cat# 167934, prepared as above).

The sgRNA primer sequences described above are shown in table 5 below:

Table 5 shows primer sequences

CRISPRa-sgNTC control group: after co-infecting the human myotube precursor cells FOXO3 ^-/- with Lenti-SAMv-sgNTC lentivirus and Lenti-MPHv lentivirus (MOI value 0.3-0.4) for 48 hours, 5ug/mL Blasticidin (available from Invivogen, cat# ant-bl-1) and 25ug/mL Hygromycin B (available from Invivogen, cat# ant-hg-1) were added to the infection system, and cultured for 6 days to obtain the selected cells, which were further differentiated and cultured in myotube cell differentiation medium for 5 days to obtain differentiated and cultured myotube cells.

CRISPRa-sgSESN1 active group: after co-infecting the human myotube precursor cells FOXO3 ^-/- for 48 hours with the Lenti-SAMv-sgNTC lentivirus and the Lenti-MPHv lentivirus (MOI value 0.3-0.4), 5ug/mL Blasticidin (available from Invivogen, cat. No. ant-bl-1) and 25ug/mL Hygromycin B (available from Invivogen, cat. No. ant-hg-1) were added to the infected system, and cultured for 6 days to obtain infected cells, and the infected cells were further differentiated in a differentiation medium for 5 days to obtain differentiated myotube cells.

And detecting the expression quantity of SESN1 in the cells after the differentiation culture by using a Western blot method.

As shown in FIG. 8B, the expression of SESN1 in CRISPRa-sgSESN1 activation group is increased, and the result shows that SESN1 is successfully activated.

Two) detection of the diameter of human myotube cells after endogenous activation of SESN1 by experiments

The method is similar to the method for inhibiting the diameter of human myotube cells after SESN1 expression.

The CRISPRa-sgNTC control cells obtained in one) above and CRISPRa-sgSESN1 activation components were subjected to differentiation culture to examine the diameters of human myotube cells after endogenous activation of SESN 1.

As shown in FIG. 8C, the diameter of human myotube cells after endogenous SESN1 activation is obviously higher than that of the control group.

Third) detection of SA-beta-gal Activity of human myotube cells after endogenous activation of SESN1 by experiments

The control CRISPRa-sgNTC obtained in one) above was used to control the SA-. Beta. -gal activity detected by cells after constitutive culture and cells after constitutive culture of CRISPRa-sgSESN1 (methods were as before).

The method can inhibit SA-beta-gal activity of human myotube cells after SESN1 expression.

As a result, as shown in FIG. 8D, after activating SESN1 expression in human myotube cells, the SA-. Beta. -gal activity of the cells was significantly decreased.

The above results indicate that activating expression of endogenous SESN1 reduces human myotube cell senescence or increases human myotube cell diameter.

Example 3 functional verification of SESN1 recombinant protein

1. The SESN1 content in the serum of the aged and in the myotube cell culture medium supernatant was identified by means of an Enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA, kit purchased from Finetest company, cat# EH 0909).

The aged serum is ex-vivo serum from a person 18-25 years of age.

The young human serum is ex vivo serum from humans aged 65-80 years.

Myotube cell culture medium supernatants were prepared as follows: human myotube cells FOXO3 ^+/+ were cultured in myotube cell differentiation medium for 6, 10 or 12 days, centrifuged at 1000rpm/min for 5min, and the supernatant was collected.

The following samples were aged serum, young serum, human myotube cell culture supernatant of 6 days, human myotube cell culture supernatant of 10 days, and human myotube cell culture supernatant of 12 days.

1. All reagents were left at room temperature prior to the experiment. The standard was diluted in concentration gradients.

2. The 96-well plate was taken out, 100. Mu.L of standard substance or sample was sequentially added to the well plate, and the well plate was sealed with a sealing film and incubated at 37℃for 90min.

3. The eluate was washed 4 times, 100. Mu.L of biotin-labeled antibody detection solution was added, and incubated at 37℃for 60 minutes.

4. After washing 4 times with eluent, 100. Mu.L of avidin-HRP solution was added to each well and incubated at 37℃for 30min.

5. After washing 5 times with eluent, 90. Mu.L of freshly prepared reaction substrate was added to each well and incubated for 15min at 37℃in the absence of light.

6. To each well, 50. Mu.L of stop solution was added, respectively, and gently mixed, and the color in the well changed to yellow.

7. And detecting the absorbance at 450nm and 570nm by using an enzyme-labeled instrument respectively, subtracting the 570nm readout value from the 450nm readout value to obtain the actual absorbance, drawing a corresponding standard curve according to the standard substance, and calculating to obtain the target protein concentration in the sample.

As shown in fig. 9, a is the detection of the average content of SESN1 in serum of young and old people, B is the detection of the content of SESN1 in supernatant of human myotube cell culture medium at different culture times, the content of SESN1 in serum of young people is higher than that of old serum, and the content of SESN1 in supernatant is reduced when human myotube cell culture time is long; the results indicated that the SESN1 content in the supernatant of aged human serum and myoblast cell culture medium was reduced for a long period of time.

2. And the function of SESN1 is verified by intervening myotube cells with the SESN1 recombinant protein.

First), FOXO 3-deleted human myotube cells FOXO3 ^-/- were divided into two groups, namely a control group and a SESN1 recombinant protein-treated group.

Control group: human myotube cell FOXO3 ^-/- was cultured in myotube cell differentiation medium for 8 days;

SESN1 recombinant protein treatment group: human myotube cells FOXO3 ^-/- were cultured in myotube cell differentiation medium for 4 days, cells were continuously cultured in myotube cell differentiation medium containing 0.5 μg/mL SESN1 recombinant protein (0.5 μg/mL SESN1 recombinant protein was added to myotube cell differentiation medium) for 4 days on day 5 of myotube cell differentiation, and medium was changed 1 time for 2 days during the continuous culture.

Two) detecting the diameter of human myotube cells after SESN1 recombinant protein treatment through experiments

The diameters of human myotube cells in the cells of the control group and the SESN1 recombinant protein treatment group were detected.

The method is similar to the method for inhibiting the diameter of human myotube cells after SESN1 expression.

The results of the measurements are shown in FIGS. 10A-10B, wherein A is a schematic diagram of the control group and SESN1 recombinant protein treated human myotube cells, and B is a diameter measurement of the control group (denoted as Vehicle in the figure) and SESN1 recombinant protein (denoted as rSESN1 in the figure) treated human myotube cells, and it can be seen that the diameter of the SESN1 recombinant protein treated human myotube cells is significantly higher than that of the control group.

Third) detection of SA-beta-gal Activity of human myotube cells after SESN1 recombinant protein treatment by experiment

The SA- β -gal activity of cells of the control group and SESN1 recombinant protein-treated group was examined.

The method can inhibit SA-beta-gal activity of human myotube cells after SESN1 expression.

As a result, as shown in FIG. 10C, the SA-. Beta. -gal activity of the cells after treatment with SESN1 recombinant protein (rSESN. Sup. Th 1. Sup. In the human myotube cells was significantly reduced as compared with the control group (referred to as Vehicle).

The results show that the SESN1 recombinant protein reduces the aging of human myotube cells or increases the diameter of human myotube cells.

3. Verification of SESN1 function by intervention of SESN1 recombinant protein in mouse skeletal muscle injury model

First) establishing a model of skeletal muscle injury in mice

Cardiotoxin (CTX, latoxan) powder was dissolved in PBS (ph=7.2-7.4) to 10 μm and mice of the C57BL/6J variety (body weight 30-40 g) were injected separately into tibialis anterior (25 μl, two injection points) and quadriceps femoris (50 μl, two injection points) of both lower limbs. The intact control group was injected with an equal amount of PBS in the same manner. The next day of injection, the behavioral index or skeletal muscle tissue morphology is detected, and whether the model establishment is successful or not is judged, and the damaged mice treated with CTX are the skeletal muscle injury model of the mice.

Two) intervention of SESN1 recombinant protein on damaged skeletal muscle

The continuous 7-day in situ injection of SESN1 recombinant protein into tibialis anterior and quadriceps of mice treated with CTX and C57BL/6J strain mice is specifically as follows:

post-injury control group: the injured mice treated with CTX were injected with 30 μl of PBS for tibialis anterior, and 60 μl of PBS for quadriceps;

post-injury r SESN1 treatment group: the concentration of SESN1 recombinant protein solution was 0.5. Mu.g/mL (solvent PBS), 30. Mu.L of SESN1 recombinant protein solution was injected into the tibialis anterior muscle of the injured mice treated with CTX, and 60. Mu.L of SESN1 recombinant protein solution was injected into the quadriceps femoris.

Undamaged group: c57BL/6J mice were injected with PBS 30. Mu.L for tibialis anterior, and PBS 60. Mu.L for quadriceps;

Third), behavioural detection:

each of the above groups was tested on day 9 post injection for each of the groups obtained in the two) above:

1. Grip test

The grip of the mouse limb was measured using a grip detector (Panlab GRID STRENGTH METER, LE902,902). The maximum grip of the mice during this procedure was recorded, each mouse was tested 10 times per time, and the average value was recorded as the grip of that mouse.

2. Rotating rod test

Mouse motor coordination was assessed using a stick-turning instrument (YIYAN TECH, YLS-4C). The rotating rod was started at a speed of 4rpm and accelerated at an acceleration of 8rpm/min ² until the speed reached 44rpm, after which it was constant. The time the mice remained on the rotor was recorded, and each mouse was recorded three times and averaged as the average rotor residence time. Before the formal experiment, animals are trained for 3 times a day for 3 consecutive days, so that the mice adapt to the training environment, and errors caused by factors such as environment and the like are reduced.

3. Running machine performance test

Mice were trained on a treadmill (SANS bioassay, SA 101) 3 days prior to formal experiments. On the day of testing, the treadmill was set to an acceleration of 2rpm/min ². When the mouse stays on the electrode for more than 10 seconds and cannot return to the running machine to continue running, the maximum speed, time and running distance at the moment are recorded, and the distance is the depletion distance.

The results are shown in fig. 11, wherein A is a schematic diagram of muscle injury model and detection by SESN1 recombinant protein treatment of 16-month-old mice, and B is grasping force detection of the mice; c is detection of the stay time of the rotating rod of the mouse; d is the running distance, time and maximum speed detection of the depletion of the mice, and the improvement of the grabbing force of the mice after the SESN1 recombinant protein treatment can be seen compared with the control group after injury; the stay time of the rotating rod is prolonged; depletion distance, time, maximum speed increase.

Fourth), tissue immunofluorescent staining:

Detection of the mice in the two groups obtained above were subjected to detection of ice-cold section and immunofluorescence staining of quadriceps at day 10 after injection.

Frozen sections of quadriceps femoris were removed from the refrigerator at minus 80℃and left at room temperature for 15min, after one wash with PBS, fixed with 4% PFA solution, 0.4% Triton X-100 solution permeabilized for 15min, and blocked with 10% donkey serum at room temperature. The primary antibody was incubated for 4 degrees overnight and the secondary antibody was incubated at room temperature for 1 hour. Each muscle fiber was labeled with a Laminin antibody (available from Sigma-Aldrich under the designation L9393).

The results are shown in FIG. 12, and are the detection of PAX 7/Ki 67 positive cell proportion (A) and myofiber cross-sectional area (B, C) in the skeletal muscle of the SESN1 recombinant protein treated muscle injury model by immunofluorescence staining; the results show that the skeletal muscle PAX 7/Ki 67 positive cell proportion and the muscle fiber cross-sectional area of the SESN1 recombinant protein treated group (rSESN 1) after injury are increased compared with the control group after injury and the non-injury group, which indicates that the SESN1 recombinant protein can promote regeneration of skeletal muscle after injury.

Fifth), sequencing and analysis of mouse skeletal muscle tissue single cell nuclear transcriptome:

Preparation of frozen tissue single cell nucleus samples: the frozen tissue is taken out from the liquid nitrogen, the tissue is respectively ground into powder in a mortar precooled by the liquid nitrogen, the powder is transferred into a grinding tube, homogenate and steel balls are added, and the mixture is ground in a tissue grinder.

After tissue milling was completed, the supernatant was removed by centrifugation at 4℃after collection of the liquid and the double positive nuclei of Hoechst 33342 and PI were screened by flow cytometry after resuspension with PBS containing BSA.

Single cell nuclear transcriptome library preparation: nuclei were captured in the droplet emulsion and library construction was performed according to the instructions associated with 10 x Genomics. Taking 7000 nuclei per sample as a standard, all reactions were performed in a Bio-RadC1000 thermal cycler equipped with 96-well deep well plates, reverse transcribed into cDNA, and amplified. The cDNA fragments and the average fragment length of the library were evaluated using a fragment analyzer, library construction was performed using a Chromium SINGLE CELL 3'GEM Library and Gel Bead kit v3, and sequencing was performed using NovaSeq 6000Sequencing System after library preparation was completed.

For the sequencing library, the differentiation genes of skeletal muscle of mice after the SESN1 recombinant protein stem prognosis are obtained through Seurat software package find_all_ markers function, and the screening threshold is |LogFC| >0.5 and the corrected P value is <0.05. The single nuclei obtained were clustered using Uniform Manifold Approximation and Projection (UMAP) based on cell markers, and the results are shown in fig. 13. Gene functional enrichment analysis of the differential gene was performed using a gene annotation tool METASCAPE (http:// metacape. Org /), the results of which are shown in FIG. 14A. A common gene set is obtained from Regeneration Roadmap (https:// ngdc. Cncb. Ac. Cn/regeneration /) databases. Each cell within the dataset was scored for the corresponding gene set using the AddModuleScore function of the Seurat software package, the results are shown in fig. 14B-14C.

The result shows that the SESN1 recombinant protein can accelerate the recovery of the motor function after the skeletal muscle of the mice is damaged and enhance the regeneration capability.

The results prove that SESN1 is an effector of FOXO3, and inhibiting SESN1 expression can cause cell senescence phenotypes such as smaller diameter of human myotube cells, increased SA-beta-gal activity and the like, and endogenous activation of SESN1 can delay the senescence of myotube cells. Meanwhile, SESN1 is down-regulated in serum of the old, and the SESN1 recombinant protein can delay skeletal muscle cell aging and promote injured muscle regeneration, so that the SESN1 is proved to be a potential biomarker for predicting human skeletal muscle aging, and the recombinant SESN1 treatment can provide a feasible treatment strategy for delaying skeletal muscle aging.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

1. Use of a substance that increases the expression level of a SESN1 protein or gene in any of the following:

1) Preparing a product for treating diseases caused by skeletal muscle aging or atrophy;

2) Preparing a product for delaying skeletal muscle cell aging;

3) Preparing a product for increasing the diameter of skeletal muscle cells;

4) Preparing a product that reduces SA- β -gal activity of skeletal muscle cells;

5) Preparing a product for treating diseases caused by skeletal muscle injury;

6) Preparing a product for promoting skeletal muscle regeneration after injury;

7) The product for promoting the recovery of the motor function after the injury of the skeletal muscle of animals or humans is prepared.

2. The use according to claim 1, characterized in that:

the substances for improving the expression level of the SESN1 protein or the gene comprise substances for activating the endogenous SESN1 protein or the gene expression of skeletal muscle cells, SESN1 proteins, any active peptide fragments of the SESN1 proteins or SESN1 proteins with labels.

3. A product comprising the agent for increasing the expression level of SESN1 protein or gene according to claim 1 or 2;

The product has at least one of the following functions:

1) Treating diseases caused by skeletal muscle aging or atrophy;

2) Delaying skeletal muscle cell aging;

3) Increasing the diameter of skeletal muscle cells;

4) Decreasing the SA- β -gal activity of skeletal muscle cells;

5) Treating skeletal muscle injury-induced diseases;

6) Promoting skeletal muscle regeneration after injury;

7) Promoting the recovery of the motor function of animals after skeletal muscle injury.

4. Application of substances for inhibiting SESN1 protein or gene expression in preparing aging models;

the function of the aging model is to screen medicines for treating diseases caused by skeletal muscle aging or injury.

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

The substances for inhibiting SESN1 protein or gene expression are small interfering RNA for interfering SESN1 expression.

6. Use of a substance for detecting expression of a SESN1 protein or gene in any of the following:

1) Preparing a product for evaluating or assisting in evaluating or identifying or assisting in identifying the degree of skeletal muscle aging;

2) Preparing a product for identifying or assisting in identifying or screening or assisting in screening skeletal muscle cells with low or high aging degree;

3) Preparing a product for identifying or assisting in identifying the degree of aging of different skeletal muscles or cells;

4) The product for identifying or assisting in identifying the aging degree of the organism to which the serum or the plasma or other body fluids belong is prepared.

7. The use according to claim 6, characterized in that:

The substances for detecting the SESN1 protein or gene expression are substances specifically combined with the SESN1 protein or gene.

Use of a sesn1 gene or protein as a marker for the preparation of a product for the identification or assisted identification of the degree of senescence of different skeletal muscles or cells or serum or plasma or other body fluids.

9. A kit for or aiding in the identification of the degree of aging of different skeletal muscles or cells or serum or plasma or other body fluids comprising a substance for detecting the expression of a SESN1 protein or gene as claimed in claim 6 or 7.

10. The use according to any one of claims 1 or 2 or 4-8 or the kit according to claim 9, characterized in that: the skeletal muscle or cell is derived from an animal or human;

Further, the skeletal muscle or cell is derived from primate or murine.