Use of SIRT5 protein for delaying skeletal muscle aging by means of TBK1-rela signaling pathway

Abstract

The present invention belongs to the field of biotechnology. Provided is the use of an SIRT5 protein for delaying skeletal muscle aging by means of a TBK1-RelA signaling pathway. By means of in-depth exploration of the mechanism, it is found that the SIRT5 protein can regulate skeletal muscle aging / human myotube cell senescence by means of modulating the succinylation level of TBK1 proteins, thereby affecting the phosphorylation level thereof and regulating the phosphorylation activation of the downstream target gene RelA. It is also found that the inhibition of TBK1 or RelA phosphorylation activation or the restoration of SIRT5 can delay myotube cell senescence. The present invention provides rich resources for the study of tissue, cellular and molecular changes in aged and young skeletal muscles at the multi-omics level, and reveals the important role of key regulatory factor SIRT5 for regulating non-human primate skeletal muscle aging and human myotube cell senescence, and for ameliorating mouse skeletal muscle aging and muscle degeneration or atrophy by means of a TBK1-RelA signaling pathway.

Description

Application of SIRT5 protein in delaying skeletal muscle aging through the TBK1-RelA signaling pathway

[0001] This application claims priority to Chinese Patent Application No. 202510108034.0, filed January 23, 2025, entitled "Application of SIRT5 protein in delaying skeletal muscle aging through the TBK1-RelA signaling pathway" and Chinese Patent Application No. 202510308050.4, filed March 14, 2025, entitled "Application of SIRT5 protein in delaying skeletal muscle aging through the TBK1-RelA signaling pathway", the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to the application of SIRT5 protein in delaying skeletal muscle aging through the TBK1-RelA signaling pathway. Background Technology

[0003] Aging is characterized by a gradual decline in bodily function and homeostasis, ultimately leading to a variety of age-related diseases. Skeletal muscle, comprising approximately 40% of body weight and 50-75% of total body protein, is a crucial component of the body. The gradual loss of muscle mass and strength is a defining characteristic of skeletal muscle aging, which can trigger and exacerbate sarcopenia, a significant risk factor for falls and fractures in older adults. While exercise and diet can maintain muscle mass, there are currently no targeted treatments to prevent or slow the progression of skeletal muscle aging and sarcopenia. Understanding the root causes of skeletal muscle aging is key to developing innovative strategies to combat age-related muscle decline and related diseases.

[0004] Skeletal muscle, as a vital metabolic organ, controls energy metabolism and nutrient balance throughout the body. Therefore, skeletal muscle function declines with age, impacting not only physical capabilities but also increasing the risk of metabolic diseases in older adults, such as insulin resistance and type 2 diabetes. Studies have shown that metabolic disorders caused by mitochondrial dysfunction are one of the factors contributing to skeletal muscle aging. Furthermore, recent findings indicate that metabolites not only serve as energy sources and building blocks for macromolecules but also as signaling molecules in various biological processes, including protein post-translational modifications (PTMs). PTMs can affect protein folding and stability, thereby influencing their biological activity and leading to various pathophysiological conditions. However, the precise effects of metabolite- or pathway-driven PTMs on skeletal muscle aging remain unclear and require further elucidation.

[0005] Due to ethical constraints, obtaining disease-free and age-matched cardiac biopsy tissue from healthy individuals is challenging. Non-human primates (NHPs), closely resembling humans in skeletal muscle structure, electrophysiology, and pharmacokinetics, offer a viable alternative for studying primate muscle aging, sharing similarities in muscle fiber type, neuromuscular junctions, and metabolic pathways. Furthermore, human pluripotent stem cell-derived myotube models, readily genetically manipulated, have proven crucial for exploring the molecular basis of muscle aging. Utilizing these complementary models is essential for deciphering the molecular mechanisms of human skeletal muscle aging and guiding potential intervention strategies. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a signaling pathway for delaying skeletal muscle aging. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention protects novel uses of substances that regulate the SIRT5-TBK1-RelA signaling pathway.

[0009] The present invention protects the use of substances that regulate the SIRT5-TBK1-RelA signaling pathway in any of the following A1)-A4):

[0010] A1) Delaying the aging of the body, organs, tissues, or cells;

[0011] A2) To prepare products (including drugs) that delay the aging of the body, organs, tissues or cells;

[0012] A3) Improves dysfunction of aging organisms, organs, tissues, or cells;

[0013] A4) Prepare products that improve the functional impairment of aging organisms, organs, tissues, or cells;

[0014] A5) Prepare products for studying or assisting in the study of the aging mechanisms of an organism, organ, tissue, or cell;

[0015] The substances that regulate the SIRT5-TBK1-RelA signaling pathway include substance R and / or substance S and / or substance T;

[0016] The substance R is SIRT5 protein or a substance that upregulates, enhances, or increases the activity and / or content of SIRT5 protein;

[0017] The substance S is a substance that reduces the succinylation or phosphorylation level of TBK1 protein;

[0018] The substance T is a substance that downregulates, inhibits, or reduces the activity and / or content of RelA protein.

[0019] In some embodiments of the first aspect, the substance R includes, but is not limited to, for example, nucleic acids encoding the SIRT5 protein or expression vectors containing nucleic acids encoding the SIRT5 protein, such as retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, plasmids, mRNA-lipid nanoparticles, preferably adeno-associated virus vectors, lipid nanoparticles containing mRNA encoding the SIRT5 protein, or lipid nanoparticles containing mRNA encoding enhanced SIRT5 protein expression; or small molecule activators of the SIRT5 protein;

[0020] The substance S includes, for example, siRNA or ASO targeting TBK1 mRNA or an expression vector of ASO thereof, or shRNA or sgRNA targeting TBK1, or HDAC6 activators, HDAC3 regulators, p300 / CBP acetyltransferase inhibitors such as C646 and curcumin; IKKε activators, ubiquitination regulators, natural compounds such as resveratrol; MRT67307, BX795, GS-6624, CCX872;

[0021] The substance T includes, for example, BAY 11-7082, TPCA-1, PRI-724, JSH-23, MLN4924, MZ1, and siRNA or ASO or an expression vector of ASO targeting RelA mRNA, or an expression vector of shRNA or sgRNA targeting RelA, miR-146a mimic, curcumin, resveratrol, propionic acid, acarbose, or a RelA neutralizing antibody such as AMG510.

[0022] Secondly, this invention protects a product whose function is either B1) or B2):

[0023] B1) Delaying the aging of the body, organs, tissues, or cells;

[0024] B2) Improves dysfunction of aging organisms, organs, tissues, or cells;

[0025] The active ingredient in the product is a substance that regulates the SIRT5-TBK1-RelA signaling pathway.

[0026] The substances that regulate the SIRT5-TBK1-RelA signaling pathway include substance R and / or substance S and / or substance T;

[0027] The substance R is SIRT5 protein or a substance that upregulates, enhances, or increases the activity and / or content of SIRT5 protein;

[0028] The substance S is a substance that reduces the succinylation or phosphorylation level of TBK1 protein;

[0029] The substance T is a substance that downregulates, inhibits, or reduces the activity and / or content of RelA protein.

[0030] In some embodiments of the second aspect, the substance R includes, but is not limited to, for example, nucleic acids encoding the SIRT5 protein or expression vectors containing nucleic acids encoding the SIRT5 protein, such as retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, plasmids, mRNA-lipid nanoparticles, preferably adeno-associated virus vectors, lipid nanoparticles containing mRNA encoding the SIRT5 protein, or lipid nanoparticles containing mRNA encoding enhanced SIRT5 protein expression; or small molecule activators of the SIRT5 protein;

[0031] The substance S includes, for example, siRNA or ASO targeting TBK1 mRNA or an expression vector of ASO thereof, or shRNA or sgRNA targeting TBK1, or HDAC6 activators, HDAC3 regulators, p300 / CBP acetyltransferase inhibitors such as C646 and curcumin; IKKε activators, ubiquitination regulators, natural compounds such as resveratrol; MRT67307, BX795, GS-6624, CCX872;

[0032] The substance T includes, for example, BAY 11-7082, TPCA-1, PRI-724, JSH-23, MLN4924, MZ1, and siRNA or ASO or an expression vector of ASO targeting RelA mRNA, or an expression vector of shRNA or sgRNA targeting RelA, miR-146a mimic, curcumin, resveratrol, propionic acid, acarbose, or a RelA neutralizing antibody such as AMG510.

[0033] In any of the above-described applications or products, the substance that enhances the activity and / or content of SIRT5 protein may be a nucleic acid molecule encoding SIRT5 protein or an expression cassette, recombinant vector, recombinant microorganism, or recombinant cell line containing the nucleic acid molecule.

[0034] The recombinant vector is a recombinant expression vector obtained by ligating FLAG-SIRT5 cDNA into the pLE4 vector, which expresses SIRT5 protein with a FLAG tag.

[0035] The substance that reduces TBK1 protein activity can be a protein, polypeptide, or small molecule compound that inhibits TBK1 protein function.

[0036] The substance that reduces RelA protein content may be a substance that inhibits RelA protein synthesis activation, promotes RelA protein degradation, or knocks down or eliminates the RelA protein encoding gene. Alternatively, the substance that downregulates, inhibits, or reduces RelA protein activity or content may be an RNA molecule that inhibits, reduces, or downregulates the expression of the RelA protein encoding gene, or an RNA molecule that inhibits, reduces, or downregulates the activity or content of the protein, or the encoding gene of the RNA molecule, or an expression cassette containing the gene, or a recombinant vector, recombinant microorganism, or recombinant cell line containing the gene or expression cassette.

[0037] Furthermore, the recombinant microorganism may be a recombinant virus, which may be a recombinant lentivirus, a recombinant retrovirus, a recombinant adenovirus, a recombinant adeno-associated virus, or a recombinant herpesvirus.

[0038] The substance that knocks down the TBK1 protein encoding gene can be siRNA or shRNA that inhibits TBK1 gene expression.

[0039] The substance that knocks out the RelA protein-coding gene can be a RelA gene editing system; the RelA gene editing system includes a Cas9 endonuclease and sgRNA targeting the RelA gene.

[0040] Furthermore, the substance that enhances the activity and / or content of SIRT5 protein is a recombinant lentiviral vector containing a nucleic acid molecule encoding the SIRT5 protein or a lentivirus expressing SIRT5. Specifically, the recombinant lentiviral vector is a vector obtained by ligating mouse SIRT5 cDNA into a pLE4 vector. The lentivirus expressing SIRT5 is obtained by transfecting the recombinant lentiviral vector into lentiviral packaging cells and then culturing the cells.

[0041] The substance used to knock out the RelA protein-encoding gene can specifically be the sgRelA vector.

[0042] Thirdly, the present invention protects novel uses of substances for detecting SIRT5 expression levels and / or substances for detecting TBK1 protein succinylation / phosphorylation levels and / or substances for detecting RelA expression levels.

[0043] This invention protects the use of substances for detecting SIRT5 expression levels and / or substances for detecting TBK1 protein succinylation / phosphorylation levels and / or substances for detecting RelA protein expression or activation levels in any of the following C1) or C2):

[0044] C1) To identify or assist in identifying the level of aging in an organism, organ, tissue, or cell;

[0045] C2) Prepare products for identifying or assisting in the identification of the aging level of an organism, organ, tissue, or cell.

[0046] Fourthly, this invention protects a product for identifying or assisting in the identification of the level of aging in an organism, organ, tissue, or cell;

[0047] The product includes substances for detecting SIRT5 expression levels and / or substances for detecting TBK1 succinylation or phosphorylation levels and / or substances for detecting RelA protein expression or protein activity levels.

[0048] Fifthly, this invention protects novel uses of substances that regulate the SIRT5-TBK1-RelA signaling pathway;

[0049] The present invention protects the use of substances that regulate the SIRT5-TBK1-RelA signaling pathway in any of the following D1)-D6):

[0050] D1) Constructing senescent cells;

[0051] D2) Preparation of products for constructing senescent cells;

[0052] D3) Screening or assisting in the screening of drugs that delay aging;

[0053] D4) Prepare products (including cells, such as senescent cells prepared by the method described in F4 below) for screening or assisting in the screening of drugs that delay aging.

[0054] D5) To study or assist in the study of the aging mechanisms of an organism, organ, tissue, or cell;

[0055] D6) Prepare products for researching or assisting in the research of aging mechanisms in organisms, organs, tissues, or cells;

[0056] The substances that regulate the SIRT5-TBK1-RelA signaling pathway include substance X and / or substance Y and / or substance Z;

[0057] The substance X is a substance that downregulates, inhibits, or reduces the activity and / or content of SIRT5 protein;

[0058] The substance Y is a substance that increases the succinylation and / or phosphorylation level of TBK1 protein;

[0059] The substance Z is RelA protein or a substance that upregulates, enhances, or increases the activity and / or content of RelA protein.

[0060] In some embodiments of the fifth aspect, the substance X includes, for example, a CRISPR / Cas9 gene editing system that targets and knocks out SIRT5 (e.g., which includes a Cas9 endonuclease and an sgRNA targeting the SIRT5 gene, the target sequence of which is, for example, GCGAGTCGTGGTC), small molecule inhibitors targeting SIRT5 enzyme activity, such as AK-7, SirReal2, SIRT5-IN-1, C-176, and EX-527 derivatives; substances that reduce SIRT5 protein content, such as SIRT5-PROTAC-1, and molecular glue degraders, such as ML265 analogs; gene silencing tools, such as siRNA and shRNA lentiviral vectors targeting the SIRT5 mRNA 3'UTR; siRNA targeting SIRT5 mRNA; and natural compounds and endogenous regulatory molecules, such as quercetin, berberine, α-ketoglutarate, estrogen, and SIRT5 neutralizing antibodies;

[0061] The substance Y includes, for example, succinic acid, IKKε agonists, MRT67307, berberine, and N-acetylcysteine;

[0062] The substance Z includes, for example, classical NF-κB pathway activators such as TNF-α, IL-1β, lipopolysaccharide, EBV-LMP1; IKK kinase agonists such as IKKε selective activators such as PS1145; NEMO binding domain peptides; TAK1 agonists such as 5Z-7-oxozeanol; RelA dephosphorylation inhibitors such as okadaic acid and Calyculin A; RelA protein stability enhancers such as MI-63, MG132, emetine, and miR-146a inhibitors; HDAC inhibitors such as trichostatin A and C646; chromatin remodeling agents such as RG108; curcumin, triptolide, and lenalidomide.

[0063] Sixthly, this invention protects a product whose function is any one of the following E1)-E3):

[0064] E1) Constructing senescent cells;

[0065] E2) Preparation of drugs for screening or assisting in the screening of drugs that delay aging;

[0066] E3) Research or assist in the research of aging mechanisms of the body, organs, tissues or cells;

[0067] The active ingredient in the product is a substance that regulates the SIRT5-TBK1-RelA signaling pathway;

[0068] The substances that regulate the SIRT5-TBK1-RelA signaling pathway include substance X and / or substance Y and / or substance Z;

[0069] The substance X is a substance that downregulates, inhibits, or reduces the activity and / or content of SIRT5 protein;

[0070] The substance Y is a substance that increases the succinylation and / or phosphorylation level of TBK1 protein;

[0071] The substance Z is RelA protein or a substance that upregulates, enhances, or increases the activity and / or content of RelA protein.

[0072] In some embodiments of the sixth aspect, the substance X includes, for example, a CRISPR / Cas9 gene editing system that targets and knocks out SIRT5 (e.g., comprising a Cas9 endonuclease and an sgRNA targeting the SIRT5 gene, the target sequence of which is, for example, GCGAGTCGTGGTC); small molecule inhibitors targeting SIRT5 enzyme activity, such as AK-7, SirReal2, SIRT5-IN-1, C-176, and EX-527 derivatives; substances that reduce SIRT5 protein content, such as SIRT5-PROTAC-1, and molecular glue degraders, such as ML265 analogs; gene silencing tools, such as siRNA and shRNA lentiviral vectors targeting the SIRT5 mRNA 3'UTR; siRNA targeting SIRT5 mRNA; and natural compounds and endogenous regulatory molecules, such as quercetin, berberine, α-ketoglutarate, estrogen, and SIRT5 neutralizing antibodies;

[0073] The substance Y includes, for example, succinic acid, IKKε agonists, MRT67307, berberine, and N-acetylcysteine;

[0074] The substance Z includes, for example, classical NF-κB pathway activators such as TNF-α, IL-1β, lipopolysaccharide, EBV-LMP1; IKK kinase agonists such as IKKε selective activators such as PS1145; NEMO binding domain peptides; TAK1 agonists such as 5Z-7-oxozeanol; RelA dephosphorylation inhibitors such as okadaic acid and Calyculin A; RelA protein stability enhancers such as MI-63, MG132, emetine, and miR-146a inhibitors; HDAC inhibitors such as trichostatin A and C646; chromatin remodeling agents such as RG108; curcumin, triptolide, and lenalidomide.

[0075] In any of the above-described applications or products, the substance that reduces SIRT5 protein activity may be a protein, polypeptide, or small molecule compound that inhibits SIRT5 protein function.

[0076] The substance that reduces SIRT5 protein content may be a substance that inhibits SIRT5 protein synthesis, promotes SIRT5 protein degradation, or knocks down or eliminates the SIRT5 gene.

[0077] The substance that downregulates, inhibits, or reduces the content and / or activity of SIRT5 protein is an RNA molecule that inhibits, reduces, or downregulates the expression of the gene encoding SIRT5 protein, or an RNA molecule that inhibits, reduces, or downregulates the activity or content of the protein, or the gene encoding the RNA molecule, or an expression cassette containing the gene, or a recombinant vector, recombinant microorganism, or recombinant cell line containing the gene or expression cassette.

[0078] The substance that upregulates, enhances, or increases the activity and / or content of TBK1 / RelA protein may be a nucleic acid molecule encoding TBK1 / RelA protein or an expression cassette, recombinant vector, recombinant microorganism, or recombinant cell line containing the nucleic acid molecule.

[0079] Furthermore, the substance that knocks down the SIRT5 protein-coding gene can be siRNA or shRNA that inhibits SIRT5 gene expression.

[0080] The substance that knocks out the SIRT5 protein-coding gene can be a SIRT5 gene editing system; the SIRT5 gene editing system includes a Cas9 endonuclease and sgRNA that targets the SIRT5 gene.

[0081] The target sequence of the sgRNA targeting the SIRT5 gene is specifically: GCGAGTCGTGGTC.

[0082] Seventhly, the present invention protects any one of the following methods (F1)-F7):

[0083] F1) A method for delaying aging of an organism, organ, tissue, or cell, comprising adjusting the methods described in the "first aspect" above.

[0084] The steps of contacting the substance controlling the SIRT5-TBK1-RelA signaling pathway with the body, organ, tissue, or cell;

[0085] F2) A method for improving dysfunction of an aging organism or organ or tissue or cell, comprising the step of contacting the substance that regulates the SIRT5-TBK1-RelA signaling pathway as described in the "First Aspect" above with the aging organism or organ or tissue or cell.

[0086] F3) A method for identifying or assisting in identifying the level of aging of an organism or organ or tissue or cell, comprising detecting the expression level of SIRT5 and / or the succinylation or phosphorylation level of TBK1 and / or the phosphorylation or expression level of RelA in a sample from the subject;

[0087] F4) A method for constructing senescent cells, comprising introducing the substance that regulates the SIRT5-TBK1-RelA signaling pathway as described in the "Fifth Aspect" above into recipient cells to obtain recombinant cells, wherein the recombinant cells are senescent cells;

[0088] Compared with the recipient animal cells, the recombinant cells have at least one of the following characteristics:

[0089] K1) The expression level of the gene encoding the SIRT5 protein is decreased or reduced; and / or the content of the SIRT5 protein is decreased or reduced; and / or the activity of the SIRT5 protein is reduced;

[0090] Increased succinylation and / or phosphorylation levels of K2)TBK1 protein;

[0091] K3) The expression level of the gene encoding the RelA protein is upregulated or increased; and / or the content of the RelA protein is upregulated or increased; and / or the activity of the RelA protein is enhanced.

[0092] The recipient animal cells can be isolated cells.

[0093] The animal cells may exclude animal germ cells, animal fertilized eggs, and animal embryonic stem cells, and may be somatic cells or cell lines. The recipient animal cells may be human embryonic stem cells.

[0094] F5) A method for screening or assisting in the screening of drugs for delaying aging, comprising the step of screening senescent cells constructed using the method described in F4) for drugs for delaying aging.

[0095] F6) A method for studying or assisting in the study of the aging mechanisms of an organism, organ, tissue or cell, including the steps of studying the aging mechanisms of an organism, organ or tissue or cell using senescent cells constructed by the method described in F4);

[0096] F7) A method for altering the senescent state of isolated cells, comprising the step of introducing a substance that regulates the SIRT5-TBK1-RelA signaling pathway as described in the "First Aspect" or "Fifth Aspect" above into recipient cells.

[0097] In the above method, the substance regulating the SIRT5-TBK1-RelA signaling pathway includes at least one of N1, N2, and N3, wherein N1 is any one of the following:

[0098] N1), an RNA molecule that inhibits, reduces or downregulates the expression of the gene encoding the SIRT5 protein, or an RNA molecule that inhibits, reduces or downregulates the activity or content of the protein, or a gene encoding the RNA molecule, or an expression cassette containing the gene, or a recombinant vector, recombinant microorganism or recombinant cell line containing the gene or expression cassette.

[0099] N2) Substances that increase the succinylation and / or phosphorylation levels of TBK1 protein;

[0100] N3) Substances that upregulate, enhance, or increase the activity and / or content of RelA protein are nucleic acid molecules encoding RelA protein or expression cassettes, recombinant vectors, recombinant microorganisms, or recombinant cell lines containing said nucleic acid molecules.

[0101] In F3 above, the SIRT5 expression level can be the SIRT5 protein expression level or the SIRT5 mRNA expression level.

[0102] The substance used to detect SIRT5 expression levels can be a substance that detects SIRT5 expression levels by immunofluorescence tissue staining, plasma ELISA, (single-cell) transcriptomics, plasma proteomics, or a combination thereof.

[0103] The T expression level can be the RelA protein activation or expression level or the RelAm RNA expression level. The substance used to detect the RelA expression level can be a substance that detects the RelA protein activation or expression level by immunofluorescence tissue staining, plasma ELISA, (single-cell) transcriptomics, plasma proteomics, or a combination thereof.

[0104] The succinylation or phosphorylation level of the TBK1 protein can be the succinylation level of lysine residue 137 and the phosphorylation level of serine residue 172 in the TBK1 protein. The substance used to detect the succinylation or phosphorylation level of the TBK1 protein can be a substance that detects the succinylation level of lysine residue 137 and the phosphorylation level of serine residue 172 in the TBK1 protein using Western blotting technology.

[0105] The method may specifically include the following steps: measuring the SIRT5 expression level and / or TBK1 succinylation / phosphorylation level and / or RelA protein expression or activation level in the subject sample; comparing the SIRT5 expression level and / or TBK1 succinylation / phosphorylation level and / or RelA protein expression or activation level with reference values; and identifying or assisting in identifying the aging level of the subject.

[0106] Preferably, the sample is selected from tissue or blood samples, such as whole blood, serum, or plasma.

[0107] Preferably, the reference values ​​are the SIRT5 expression level (or average expression level) or TBK1 succinylation / phosphorylation level (or average succinylation / phosphorylation level) or RelA protein expression or activation level (or average expression or activation level) in the same type of samples from young, healthy individuals (or groups) with normal skeletal muscle function.

[0108] Preferably, the aging level includes normal aging, accelerated aging, and / or delayed aging.

[0109] Preferably, when the SIRT5 expression level in the sample from the subject is lower than the reference value and / or the TBK1 succinylation / phosphorylation level is higher than the reference value and / or the RelA protein expression or activation level is higher than the reference value, it indicates that the subject is in an aging state. The aging state of the subject is specifically manifested at the tissue level as follows: decreased muscle fiber area in skeletal muscle tissue; a shift from fast to slow muscle fiber types, i.e., a decrease in type IIA fibers and an increase in type I fibers; increased fibrotic areas and lipid deposition in skeletal muscle tissue; a decrease in the number of neurofilament-labeled axonal regions and terminal clasps, muscle stem cells, and capillaries; decreased expression of nuclear membrane and related proteins; and a decrease in proteins associated with heterochromatin. There is also an increase in immune cells, macrophages, and pro-inflammatory M1 macrophages, as well as an increased level of inflammation.

[0110] Wherein, the cross-sectional area of ​​muscle fibers in the skeletal muscle tissue is the area of ​​skeletal muscle fibers assessed by MYH1 / MYH2 / MYH7 staining and / or WGA staining, and the MYH2-positive type IIA muscle fibers and MYH7-positive type I muscle fibers.

[0111] Fibrosis in the skeletal muscle tissue is reflected by the positive area in Masson staining.

[0112] Increased lipid deposition in the skeletal muscle tissue is observed via the BODIPY chromosome.

[0113] The number of muscle stem cells in the skeletal muscle tissue was determined by the number of PAX7-positive cells in IF staining.

[0114] The number of capillaries in the skeletal muscle tissue was reflected by the number of CD31-positive cells in IF staining.

[0115] The nuclear membrane and its related proteins in the skeletal muscle tissue were reflected by the number of LaminB1 and LAP2 positive cells in IF staining.

[0116] The heterochromatin-associated proteins in the skeletal muscle tissue were identified by the fluorescence intensity of H3K9me3 and the number of HP1γ-positive cells in IF staining.

[0117] The number of immune cells in the skeletal muscle tissue was determined by the number of CD45-positive cells in IF staining.

[0118] The number of macrophages in the skeletal muscle tissue was determined by the number of CD68-positive cells in IF staining.

[0119] The number of pro-inflammatory M1 macrophages in the skeletal muscle tissue was determined by the number of CD86-positive cells in IF staining.

[0120] The level of inflammation in the skeletal muscle tissue was reflected by the number of S100A8 and S100A9 positive cells in IHC staining and by detecting the transcriptional expression levels of IFNb and IL1 using RT-qPCR.

[0121] In F4 above, the recipient cell is a skeletal muscle cell derived from human embryonic stem cells and / or human induced pluripotent stem cells.

[0122] The phenotype of the senescent cells is characterized by at least one of the following: decreased skeletal muscle cell diameter; increased proportion of SA-β-gal positive cells; increased levels of muscle atrophy-related markers Fbx32 and MurF1; increased levels of inflammatory factors IL-6, IL-8, and MCP1; increased mitochondrial superoxide anion; decreased mitochondrial membrane potential; decreased respiratory function; and increased oxidative damage.

[0123] The levels of the inflammatory factors IL-6, IL-8, and MCP1 are the transcriptional expression levels detected by RT-qPCR and / or the secretion levels of IL-6 and TNFα proteins in cell culture supernatant detected by enzyme-linked immunosorbent assay (ELISA).

[0124] The changes in mitochondrial superoxide anion were assessed using Mitosox staining to evaluate the changes in fluorescence intensity.

[0125] The changes in mitochondrial membrane potential were assessed using the fluorescence intensity of TMRM in the TMRM staining method.

[0126] The mitochondrial respiratory function was measured using Western blotting to detect changes in the oxidative respiratory chain complex.

[0127] The oxidative damage was detected using Western blotting, which was used to detect changes in the expression levels of 4-HNE and SOD1 proteins.

[0128] In F6 above, the aging mechanism may be the knockout or knockdown of SIRT5 that promotes aging, or the overexpression of SIRT5.

[0129] Mechanisms for delaying aging, or mechanisms by which TBK1 / RelA overexpression promotes aging, or mechanisms by which TBK1 / RelA knockout or knockdown delays aging.

[0130] In any of the above-described applications, products, or methods, the improvement of dysfunction in aging organisms, organs, tissues, or cells refers to the improvement of skeletal muscle dysfunction (or functional decline) in aging.

[0131] The improvement in aging skeletal muscle dysfunction (or functional decline) is manifested in improved motor ability in aged mice. Tissue-level staining shows a decrease in the total muscle fiber area of ​​skeletal muscle tissue, a decrease in the proportion of fast-twitch muscle fibers, and a decrease in immune cell infiltration.

[0132] Specifically, intramuscular injection of a lentivirus overexpressing SIRT5 into the skeletal muscle of aged mice significantly increased net-grabbing time, rotarod dwell time, maximum exhaustion time, and distance compared to the control group. It also reduced the proportion of CD45 and CD68 positive cells, decreased the level of the inflammatory cytokine IL6, and decreased the levels of the inflammatory cytokines Tnfa and MCP1.

[0133] In one embodiment of the present invention, the SIRT5 protein interacts with the TBK1 protein, specifically verified by an exogenous Co-IP experiment in HEK293T cells overexpressing SIRT5 protein or overexpressing TBK1 protein, or by an exogenous Co-IP experiment in wild-type cardiomyocytes overexpressing SIRT5 protein.

[0134] In one embodiment of the present invention, the SIRT5 protein can desuccinylate TBK1 protein at the K137 site. Specifically, in HEK293T cells, overexpression of TBK1, SIRT5, and the SIRT5-H158Y mutant protein shows that the succinylation level of TBK1 protein decreases when overexpressing wild-type SIRT5 protein and significantly increases when overexpressing the SIRT5-H158Y mutant protein. Immunoprecipitation experiments were performed in SIRT5+ / + and SIRT5- / - human myotube cells using succinylated antibodies, followed by detection with TBK1 antibody, showing an increase in TBK1 levels in SIRT5- / - human myotube cells.

[0135] In one embodiment of the present invention, knocking out or knocking down SIRT5 protein in skeletal muscle cells differentiated from human embryonic stem cells or pluripotent stem cells alters the SIRT5-TBK1-RelA signaling pathway. Specifically, after SIRT5 protein is knocked out and / or knocked down in cells, cellular TBK1 and / or RelA phosphorylation is significantly upregulated. The expression of inflammatory factors IL-6, IL-8, TNFα, and MCP1 is increased.

[0136] In one embodiment of the present invention, the SIRT5-TBK1-RelA signaling pathway is altered in aging or aged tissues, specifically manifested as: significant downregulation of SIRT5 protein in skeletal muscle tissue; and significant increase in phosphorylation levels of TBK1 and / or RelA proteins in skeletal muscle tissue.

[0137] In one embodiment of the invention, knocking down TBK1 protein can delay the senescent phenotype of SIRT5 knockout skeletal muscle cells.

[0138] Specifically, in SIRT5 knockout skeletal muscle cells, knocking down TBK1 protein expression can increase muscle fiber diameter and / or reduce the proportion of SA-β-gal positive cells, and reduce the transcriptional or protein expression levels of inflammatory factors IL-6, IL-8, TNFα, and MCP1.

[0139] In one embodiment of the present invention, knocking down RelA protein can delay the senescent phenotype of SIRT5 knockout skeletal muscle cells. Specifically, in SIRT5 knockout skeletal muscle cells, knocking down the expression of TBK1 protein can increase muscle fiber diameter and / or reduce the proportion of SA-β-gal positive cells, as well as reduce the transcriptional or protein expression levels of inflammatory factors IL-6, IL-8, TNFα, and MCP1.

[0140] In one embodiment of the present invention, the reintroduction of SIRT5 protein into skeletal muscle cells can delay the senescence phenotype of SIRT5 knockout cells. Specifically, in SIRT5 knockout cardiomyocytes, the reintroduction of SIRT5 protein expression through retrovirus methods can increase myofiber diameter and / or reduce the proportion of SA-β-gal positive cells, as well as reduce the transcriptional or protein expression levels of inflammatory factors IL-6, IL-8, TNFα, and MCP1.

[0141] In any of the above-described applications, products, or methods, the organism may be a mammal.

[0142] Furthermore, the mammal may be a primate or a mouse.

[0143] Furthermore, the primates mentioned can be human or non-human primates.

[0144] Furthermore, the non-human primate mentioned could be a cynomolgus monkey.

[0145] The organ, tissue, or cell may be an isolated organ, tissue, or cell.

[0146] Furthermore, the organ may be skeletal muscle.

[0147] The tissue may be the skeletal muscle of the quadriceps femoris.

[0148] The cells may be skeletal muscle cells.

[0149] Furthermore, the skeletal muscle cells may be skeletal muscle cells derived from embryonic stem cells or induced pluripotent stem cells.

[0150] In any of the above-described applications, products, or methods, the SIRT5 protein, the TBK1 protein, or the RelA protein may be a natural protein, such as a protein derived from mammals, or a non-natural protein, such as a recombinant protein. The recombinant protein only needs to have the function of the natural protein.

[0151] The SIRT5 protein may be human SIRT5 protein, cynomolgus monkey SIRT5 protein, or mouse SIRT5 protein. The GenBank accession number for the amino acid sequence of the human SIRT5 protein is NP_036373.1, the GenBank accession number for the amino acid sequence of the cynomolgus monkey SIRT5 protein is XP_065400465.1, and the GenBank accession number for the amino acid sequence of the mouse SIRT5 protein is XM_006516951.

[0152] The SIRT5 gene can be the human SIRT5 gene, the cynomolgus monkey SIRT5 gene, or the mouse Sirt5 gene. The human SIRT5 gene has the GenBank accession number NM_012241.5, and its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 339-1271 of NM_012241.5; the cynomolgus monkey SIRT5 gene has the GenBank accession number XM_065544393.1, and its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 1601-2647 of XM_065544393.1; the mouse Sirt5 gene has the GenBank accession number XM_006516951.3, and its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 208-1260 of XM_006516951.3.

[0153] The TBK1 protein may be human TBK1 protein, cynomolgus monkey TBK1 protein, or mouse TBK1 protein. The GenBank accession number for the amino acid sequence of the human TBK1 protein is NP_037386.1, the GenBank accession number for the amino acid sequence of the cynomolgus monkey TBK1 protein is XP_045230844.2, and the GenBank accession number for the amino acid sequence of the mouse TBK1 protein is NP_062760.3. The TBK1 gene may be human TBK1 gene, cynomolgus monkey TBK1 gene, or mouse TBK1 gene. The human TBK1 gene has a GenBank number of NM_013254.4, and its coding sequence is a DNA molecule whose nucleotide sequence is shown in positions 100-2289 of NM_013254.4; the cynomolgus monkey TBK1 gene has a GenBank number of XM_045374909.2, and its coding sequence is a DNA molecule whose nucleotide sequence is shown in positions 167-2014 of XM_045374909.2; the mouse Tbk1 gene has a GenBank number of NM_019786.4, and its coding sequence is a DNA molecule whose nucleotide sequence is shown in positions 160-2349 of NM_019786.4.

[0154] The RelA protein may be human RelA protein, cynomolgus monkey RelA protein, or mouse RelA protein.

[0155] The amino acid sequence of the human RelA protein is represented by the GenBank accession number NP_068810.3, the amino acid sequence of the cynomolgus monkey RelA protein is represented by the GenBank accession number XP_005577305.3, and the amino acid sequence of the mouse RelA protein is represented by the GenBank accession number NP_033071.1.

[0156] The RelA gene can be the human RelA gene, the cynomolgus monkey RelA gene, or the mouse RelA gene. The human RelA gene has GenBank accession number NM_021975.4, and its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 85-1740 of NM_021975.4; the cynomolgus monkey RelA gene has GenBank accession number XM_005577248.4, and its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 87-1721 of XM_005577248.4; the mouse RelA gene has GenBank accession number NM_009045.5, and its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 295-1944 of NM_009045.5.

[0157] None of the applications or methods described above are intended for the diagnosis and treatment of disease.

[0158] The present invention also provides senescent cells prepared according to the method described in F4 above.

[0159] The beneficial technologies and / or therapeutic effects achieved by this invention are as follows:

[0160] 1) This invention establishes, for the first time, proteomic and transcriptomic atlases of skeletal muscle tissue from young and aged healthy cynomolgus monkeys, and for the first time, a transcriptomic atlas of skeletal muscle cells differentiated from SIRT5 knockout human embryonic stem cells. Based on these datasets, the regulatory networks of important biological events, key proteins, and / or genes in the skeletal muscle of non-human primates, as well as the gene regulatory network of senescence in SIRT5 knockout human cardiomyocytes, providing a rich protein / gene database related to skeletal muscle aging.

[0161] 2) This invention, through proteomic data of cynomolgus monkey skeletal muscle tissue, for the first time discovered a significant decrease in SIRT5, an important protein resisting skeletal muscle aging, in non-human primate skeletal muscle tissue and human skeletal muscle tissue. SIRT5 knockout human myotube cells exhibited the same phenotype as cynomolgus monkey skeletal muscle aging. Further bioinformatics and proteomic analysis revealed TBK1, a key transcription factor potentially regulating SIRT5. Through in-depth exploration of the mechanism, this invention discovered that SIRT5 can regulate skeletal muscle cell aging by modulating succinylation modification of TBK1, thereby regulating its phosphorylation activation and affecting the downstream target gene RelA. This invention also found that inhibiting the levels of TBK1 and / or RelA proteins or their phosphorylation-activated proteins, or by reintroducing SIRT5, can delay skeletal muscle cell aging.

[0162] 3) This invention is the first to inject SIRT5 virus into the skeletal muscle tissue of aged mice and found that it can effectively improve the motor ability of aged mice and significantly increase the area of ​​muscle fibers. The proportion of fast-twitch muscle fibers suggests that it significantly improves the skeletal muscle function of aged mice.

[0163] 4) This invention can promote the understanding of changes in skeletal muscle aging and the development of novel aging intervention strategies, including the development of therapeutic strategies targeting the SIRT5 protein and / or the SIRT5-TBK1-RelA signaling pathway by employing agonists and / or gene therapy, in order to delay and / or improve and / or treat skeletal muscle aging or age-related skeletal muscle diseases. Attached Figure Description

[0164] Figure 1 shows the phenotypic changes of skeletal muscle tissue in aged cynomolgus monkeys.

[0165] Figure 2 shows that inflammation is a prominent feature of skeletal muscle aging in primates.

[0166] Figure 3 shows how SIRT5 protein deficiency induces myotube aging in humans.

[0167] Figure 4 shows the interaction between SIRT5 and TBK1, which causes TBK1 to desuccinylate.

[0168] Figure 5 shows how SIRT5-mediated desuccinylation of TBK1 at lysine 137 promotes its dephosphorylation and thus accelerates myotube aging in humans.

[0169] Figure 6 shows how knocking down the TBK1 cascade slows down aging and inflammation in human myotubules.

[0170] Figure 7 shows how SIRT5 gene therapy improves skeletal muscle dysfunction in aged mice.

[0171] Figure 8 shows a summary of information on cynomolgus monkeys and an analysis of skeletal muscle phenotypes in juvenile and older monkeys.

[0172] Figure 9 shows the transcriptomic and quantitative proteomic analysis of skeletal muscle in young and old monkeys based on sex.

[0173] Figure 10 shows the generation and characterization of wild-type and SIRT5-deficient hESCs.

[0174] Figure 11 shows the functional analysis of myotubular mitochondria in SIRT5-deficient individuals.

[0175] Figure 12 shows the interaction between SIRT5 and TBK1, and the localization analysis of both in myotube cells.

[0176] Figure 13 shows the expression of AAV9-SIRT5 and control AAV9-Luc in HEK293 cells (a), their expression in mouse muscle after transfection (b), functional analysis (c), and the analysis results of their effect on mouse grip strength (d). Detailed Implementation

[0177] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0178] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0179] All animal experiments described in the examples below were approved by the Animal Protection and Utilization Committee of the Chinese Academy of Sciences.

[0180] The experimental data in the following examples were processed using GraghPad Prism 8 statistical software. The experimental results are presented in the figures below.

[0181] Mean ± standard deviation is expressed. A two-tailed t-test is used. P < 0.05 indicates statistical significance, P < 0.01 indicates significant difference, and P < 0.001 indicates highly significant difference.

[0182] The biological materials used in the following examples are from the following sources: Cynomologus Macaque is a product of Beijing Xieerxin Biological Resources Research Institute Co., Ltd.

[0183] The C57BL6 / J mice were products of Beijing Spefolk Biotechnology Co., Ltd. The NOD / SCID mice were products of Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals used in this study were approved by the Ethics Review Committee of the Institute of Zoology, Chinese Academy of Sciences. The human muscle tissue samples involved were approved by the Ethics Committee of Peking University Third Hospital.

[0184] Methods and culture media for skeletal muscle cell differentiation:

[0185] Human embryonic stem cells (hESCs) were cultured in differentiation medium for approximately 10 days to generate human myotube progenitor cells. This differentiation medium contained MEMα (Thermo Fisher Scientific), 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 1% penicillin / streptomycin (Thermo Fisher Scientific), 10 ng / mL bFGF (Joint Protein Central), and 5 ng / mL TGFβ (StemImmune). Subsequently, early-passaged hESCs were infected with the lentiviral vector MyoD-ER(T) and transferred to pre-coated culture dishes. When cell confluence reached 70%-80%, 1 μM 4-oh-tamoxifen (Sigma-Aldrich) was added to the myotube progenitor cell culture medium, and the cells were maintained at 37°C, 5% CO2, and 3-5% O2. 24 hours later, the culture medium was replaced with fresh hMyotube differentiation medium (high glucose DMEM medium containing 2% horse serum), and 1 μM 4-oh-tamoxifen was continued to be supplemented, with the medium changed every 2 days. Day 1 was defined as the first day of 4-oh-tamoxifen administration. Mature myotubes usually formed on day 5 or 6.

[0186] HEK293T cell culture medium: 89 parts DMEM high glucose medium (Hyclone), 10 parts fetal bovine serum (Gibco), 1 part penicillin / streptomycin (Gibco).

[0187] The biological materials used in the following examples are from the following sources:

[0188] Human embryonic kidney 293T cells (HEK293T cells): ATCC, CRL-3216.

[0189] The human embryonic stem cell line hESCsH9 is a product of the WiCell Research Institute in Madison, Wisconsin, USA.

[0190] Human pluripotent stem cells (iPSCs) are described in the literature “Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells. Ling C, et al., Protein Cell. 2019 Apr; 10(4):249-271.” The public can obtain this biological material from the applicant only for the purpose of repeating the relevant experiments of this invention and it may not be used for other purposes.

[0191] Lentiviral packaging vector psPAX2 (plasmid psPAX2 for short): Addgene product, #12260.

[0192] Lentiviral packaging vector pMD2.G (pMD2.G for short): Addgene product, #12259.

[0193] The pLE4 vector is described in the literature “A genome-wide CRISPR-based screen identifies KAT7 as a driver of cellular senescence. Wang W, et al. Sci Transl Med. 2021 Jan 6;13(575):eabd2655.” The public can obtain this biological material from the applicant for the purpose of repeating the relevant experiments of this invention only, and it may not be used for other purposes.

[0194] FLAG-SIRT5-WT vector: Constructed on pLE4 vector, the specific construction method is detailed in the examples below.

[0195] FLAG-SIRT5-H158Y vector: The vector obtained by mutating the 472nd position of the SIRT5 protein encoding gene sequence on the FLAG-SIRT5-WT vector from base C to base U.

[0196] HA-SIRT5-WT vector: Constructed on pLE4 vector, the specific construction method is detailed in the examples below.

[0197] HA-SIRT5-H158Y vector: The vector obtained by mutating the 472nd position of the SIRT5 protein encoding gene sequence on the HA-SIRT5-WT vector from base C to base U.

[0198] FLAG-TBK1-WT vector: Constructed on pLE4 vector, the specific construction method is detailed in the examples below.

[0199] FLAG-TBK1-K137R vector: A vector obtained by mutating base A to base G at position 410 of the TBK1 protein-coding gene sequence in the FLAG-TBK1-WT vector. FLAG-TBK1-K451R vector: A vector obtained by mutating base A to base G at position 1352 of the TBK1 protein-coding gene sequence in the FLAG-TBK1-WT vector.

[0200] FLAG-TBK1-K460R vector: A vector obtained by mutating base A to base G at position 1379 of the TBK1 protein-coding gene sequence in the FLAG-TBK1-WT vector. FLAG-TBK1-K567R vector: A vector obtained by mutating base A to base G at position 1700 of the TBK1 protein-coding gene sequence in the FLAG-TBK1-WT vector.

[0201] FLAG-TBK1-K615R vector: The vector obtained by mutating base A to base G at position 1844 of the TBK1 protein encoding gene sequence on the FLAG-TBK1-WT vector.

[0202] FLAG-Luciferase (Luc) vector: Constructed on pLE4 vector, which is described in the literature "Stabilization of heterochromatin by CLOCK promotes stem cell rejuvenation and cartilage regeneration. Liang C, et al., Cell Res. 2021 Feb; 31(2):187-205" (the name in the literature is FLAG-Luc). The public can obtain this biomaterial from the applicant for the purpose of repeating the relevant experiments of this invention only, and it cannot be used for other purposes.

[0203] The reagents used in the following examples were sourced from the following sources: anti-Myosin heavy chain Type I antibody (DSHB, catalog number: BA-D5), anti-Myosin heavy chain Type IIa antibody (DSHB, catalog number: SC-71), anti-Myosin heavy chain Type IIx antibody (DSHB, catalog number: 6H-1), anti-MyHC antibody (DSHB, catalog number: MF20), anti-LAP2 antibody (Cell Signaling Technology, catalog number: 2497S), anti-GAPDH antibody (Santa Cruz, catalog number: sc-365062), anti-Neurofilament antibody (Millipore, catalog number: MAB5256A5), anti-PAX7 antibody (Abcam, catalog number: ab34360), anti-CD31 antibody (R&D Systems, catalog number: AF3628), anti-HP1g antibody (Cell Signaling Technology, catalog number: 2619S), and anti-Lamin antibody. Anti-B1 antibody (Abcam, catalog number: ab16048), anti-Histone H3 (trimethyl K9) antibody (Abcam, catalog number: ab8898), anti-CD68 antibody (Abcam, catalog number: ab125212), anti-CD68 antibody (Abcam, catalog number: ab955), anti-CD45 antibody (Abcam, catalog number: ab8216).

[0204] Anti-S100A8 antibody (Abcam, catalog number: ab180735), Anti-S100A9 antibody (Abcam, catalog number: ab22506), Anti-CD163 antibody (Abcam, catalog number: ab182422), Anti-CD206 antibody (Abcam, catalog number: ab64693), Anti-CD86 antibody (Abcam, catalog number: ab239075), Anti-SOX2 antibody (Abcam, catalog number: ab239218), Anti-OCT4 antibody (Santa Cruz, catalog number: sc-5297), Anti-NANOG antibody (Abcam, catalog number: ab21264), Anti-TUJ1 antibody (Abcam, catalog number: ab78078), Anti-SMA antibody (Zhongshan Jinqiao Company, catalog number: ZM-0003), Anti-FOXA2 antibody (Cell Signaling) Anti-Ki67 antibody (Zhongshan Jinqiao Company, catalog number: ZM-0166), anti-IL6 antibody (Abcam Company, catalog number: ab9324), anti-SIRT5 antibody (Cell Signaling Technology Company, catalog number: 8782S), anti-β-Tubulin antibody (Immunoway Company, catalog number: YM3030), anti-TBK1 antibody (Cell Signaling Technology Company, catalog number: 3504S), anti-TBK1 (phospho S172) antibody (Cell Signaling Technology Company, catalog number: 5483S), anti-RelA antibody (Cell Signaling Technology Company, catalog number: 8242S), anti-RelA (phospho S536) antibody (Cell Signaling Technology Company, catalog number: 3033S), anti-FLAG antibody (Sigma Company, catalog number: F1804), anti-HA antibody (Cell Signaling... Technology Company (Catalog No.: 3724S), Anti-β-Actin Antibody (Santa Cruz Company, Catalog No.: sc-69879), Anti-Succinyllysine Antibody (Jingjie Company, Catalog No.: PTM-401), Anti-TOM20 Antibody (Abcam Company, Catalog No.: ab289670), Anti-4-HNE Antibody (Abcam Company, Catalog No.: ab46545), Anti-OXPHOS Antibody (Abcam Company, Catalog No.: ab110413), Anti-SOD1 Antibody (ABclonal Company, Catalog No.: A0274), Anti-Fbx32 Antibody (Abcam Company, Catalog No.: ab168372), Anti-MuRF1 ... ab168372), Anti-MuRF1 Antibody (Santa Cruz Company, Catalog No.: sc-69879), Anti-4-HNE Antibody (Abcam Company, Catalog No.: ab46545), Anti-OXPHOS Antibody (Abcam Company, Catalog No.: ab110413), Anti-SOD1 Antibody (Abclonal CompanyProduct number: sc-398608). Alexa, 568 Donkey Anti-Rabbit IgG (H+L) Secondary Antibody (Catalog No.: A10042), Alexa 488 Donkey Anti-Mouse IgG (H+L) Secondary Antibody (Catalog No.: A21202) Alexa 647Goat anti-Mouse IgM (Heavy Chain) secondary antibody (Catalog No.: A21238) Alexa 488Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody (Catalog No.: A21121) Alexa 594Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody (Catalog No.: A21145), Alexa 568Donkey Anti-Rat IgG (H+L) Secondary Antibody (Catalog No.: A78946), Alexa 647 Goat anti-Rabbit IgM (Heavy Chain) secondary antibody (Catalog No.: A31573). All secondary antibody reagents are from Invitrogen. Horseradish peroxidase conjugated goat anti-rabbit IgG (H+L) (Zhongshan Jinqiao Company, Catalog No.: ZB-2301), Horseradish peroxidase conjugated goat anti-mouse IgG (H+L) (Zhongshan Jinqiao Company, Catalog No.: ZB-2305).

[0205] Example 1: Phenotypic Analysis of Skeletal Muscle Aging in Cynomolgus Monkeys

[0206] 1. Experimental grouping and material collection

[0207] The experimental grouping and sex information of the two groups of cynomolgus monkeys are as follows:

[0208] Young group: 8 crab-eating macaques aged 4-6 years (4 females and 4 males). Older group: 8 crab-eating macaques aged 18-21 years (4 females and 4 males).

[0209] Sampling: Cynomolgus monkeys were anesthetized and perfused. After perfusion, skeletal muscle tissue was rapidly isolated, and peripheral adipose tissue was carefully removed. Based on the muscle's anatomical structure, the quadriceps femoris muscle was carefully isolated. The isolated tissue was fixed in 4% paraformaldehyde and could be embedded to prepare paraffin tissue blocks or prepared as OCT cryopreserved samples. The remaining tissue was directly and rapidly cryopreserved in liquid nitrogen for subsequent RNA extraction, protein extraction, or other analyses.

[0210] 2. Experimental Methods

[0211] (1) Masson staining

[0212] Masson staining was performed using the Masson trichrome staining kit (Solarbio, G1346) according to its instructions. After paraffin embedding, tissue sections were cut into 5 μm thick sections, dewaxed with xylene, and hydrated with 100% ethanol, 95% ethanol, 70% ethanol, and tap water. The sections were then stained overnight with potassium dichromate solution. After rinsing with tap water for 10 minutes, the sections were stained in azurite blue solution for 3 minutes, rinsed with tap water, stained in hematoxylin working solution for 5 minutes, rinsed with tap water, differentiated with 1% hydrochloric acid ethanol for 1 second, rinsed with tap water for 5 minutes, and then stained in virgin red and fuchsin solution for 10 minutes. Differentiation was then performed in phosphomolybdic acid solution for 10 minutes, stained with aniline blue solution for 5 minutes, and differentiated in 1% acetic acid solution for 2 minutes. The sections were then dehydrated twice with 95% ethanol and twice with anhydrous ethanol, cleared with xylene, covered with coverslips, and mounted with neutral resin.

[0213] (2) Immunofluorescence staining of tissues

[0214] After OCT embedding, tissues were sectioned to a thickness of 10 μm, fixed with 4% paraformaldehyde for 10 minutes, gently washed three times with PBS, permeabilized in 0.4% Triton X-100 / PBS solution for 15 minutes, and washed twice with PBS. Sections were blocked with PBS containing 10% donkey serum at room temperature for 1 hour, then incubated overnight at 4°C with primary antibody. The next day, they were incubated with fluorescent secondary antibody at room temperature for 1 hour, and the cell nuclei were stained with the DNA-specific dye Hoechst 33342 (Thermo Fisher Scientific). Finally, the sections were mounted with a fluorescent antiquencher (Vector Laboratories). Imaging was performed using laser confocal microscopy (Leica TCS SP5 II or Zeiss LSM 900).

[0215] (3) Western blotting experiment

[0216] Skeletal muscle tissue was removed and rapidly frozen in liquid nitrogen in cryovials. The tissue was then removed, homogenized in liquid nitrogen, and thoroughly lysed with 1×SDS lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2% (wt / vol) SDS). The lysate was then boiled at 105°C for 10 minutes. After centrifugation at 13000 rpm and 4°C for 15 minutes, the protein supernatant was carefully collected, and protein concentration was determined using a BCA kit. The protein lysis buffer was then subjected to SDS-PAGE. After electrophoresis and transfer, the PVDF (Millipore) membrane was blocked with TBST containing 5% skim milk powder (BBI Life Sciences), washed three times with TBST for 10 minutes each time, incubated overnight at 4°C with primary antibody, and then incubated at room temperature for 1 hour with enzyme-labeled secondary antibody (Jackson ImmunoResearch). After three TBST washes, each lasting 10 minutes, the images were developed and photographed using the ChemiDocXRS system (Bio-Rad) and Image Lab software. The acquired images were then quantified using ImageJ software.

[0217] 3. Experimental Results

[0218] The skeletal muscle aging phenotypes of cynomolgus monkeys (including females and males) were examined by comparing those of young (4-6 years old, equivalent to approximately 16 years old in humans) and older (18-21 years old, equivalent to approximately 65 years old in humans) individuals (Figure 1a, Figure 8a). It was found that older cynomolgus monkeys exhibited decreased cross-sectional area (CSA) of muscle fibers, increased levels of the muscle atrophy marker molecule Fbx32, and an increased number of muscle fibers with centrums (Figure 1b and c, Figure 8b and c). Simultaneously, an age-related shift from fast to slow muscle fiber types was also identified, characterized by a decrease in MYH2-positive type IIA fibers and an increase in MYH7-positive type I fibers (Figure 1b, Figure 8c). The inventors also detected phenotypic changes in aging skeletal muscle tissue, including increased fibrotic areas as shown by Masson staining, increased lipid deposition as shown by BODIPY staining, and decreased axonal regions and terminal clasps labeled with neurofilament proteins, PAX7-positive muscle stem cells, and the number of CD31-labeled capillaries (Figure 1d to Figure 1h). Furthermore, the inventors detected multiple biomarkers of cellular senescence, including decreased expression of the nuclear membrane and its associated proteins (such as Lamin B1 and LAP2), and heterochromatin-associated proteins H3K9me3 and HP1γ (Figure 1i to Figure 1j, Figure 8d to Figure 8e). In summary, these findings provide a detailed description of the diverse age-related changes at the tissue, cellular, and subcellular levels in non-human primate skeletal muscle during physiological aging.

[0219] Example 2: Increased immune cells and significantly upregulated inflammation-related factors are prominent features of skeletal muscle aging in non-human primates.

[0220] Experimental methods:

[0221] (1) Transcriptome sequencing and analysis

[0222] Total RNA was extracted from cynomolgus monkey heart tissue and human cardiomyocytes using TRIzol reagent. After assessing RNA quality, libraries were constructed and high-throughput sequencing was performed on each sample. In short, using... UltraTM RNA Library Prep Kit for Libraries were prepared at NEB (USA), and each sample was indexed. The libraries were then sequenced at 150-base-pair reads on an Illumina paired-end sequencing platform (Novogene Bioinformatics Technology Co., Ltd.). Raw transcriptome data from cynomolgus monkey skeletal muscle tissue samples were processed using Trim Galore software (version 0.4.5) to remove low-quality reads and adapter sequences. Subsequently, the cynomolgus monkey skeletal muscle sample data were aligned to the cynomolgus monkey MacFas 5.0 reference genome using HISAT2 software (version 2.0.4). HTSeq (version 0.11.0) was used to determine the expression level of each gene. The fragment-per-thousand (FPKM) of each gene was calculated using StringTie (version 2.1.4). The R package DESeq2 (version 1.22.2) was used to identify differentially expressed genes (DEGs). Referring to previous studies involving transcriptomes of different tissues in cynomolgus monkeys, the thresholds for DEG between the hearts of aged and young cynomolgus monkeys were P<0.01 and |log2(fold change)|>0.5. Pathway enrichment analysis was performed using Metascape.

[0223] (2) Protein extraction and trypsin digestion

[0224] Cynomolgus monkey skeletal muscle tissue samples were placed in a mortar pre-cooled with liquid nitrogen and ground thoroughly into powder. Four times the volume of lysis buffer (8M urea, 1% protease inhibitor, and 2mM EDTA) was added to each sample, and the mixture was sonicated. The samples were centrifuged at 12000g for 10 min at 4°C to remove cell debris. The supernatant was transferred to a new centrifuge tube, and protein concentration was determined using a BCA assay kit. Dithiothreitol was added to the protein solution to a final concentration of 5mM, and the mixture was reduced at 56°C for 30 min. Iodoacetamide was then added to a final concentration of 11mM, and the mixture was incubated at room temperature in the dark for 15 min. Finally, the urea concentration of the samples was diluted to below 2M. Trypsin was added at a 1:50 mass ratio (trypsin:protein), and the mixture was incubated overnight at 37°C. Trypsin was then added again at a 1:100 mass ratio (trypsin:protein), and the mixture was incubated for another 4 h.

[0225] (3) TMT quantitative proteomics sequencing

[0226] Peptides digested by trypsin were desalted using a Strata X C18 (Phenomenex) column and then freeze-dried under vacuum. The peptides were dissolved in 0.5M TEAB and labeled according to the TMT kit instructions. The procedure was as follows: the labeling reagent was thawed, dissolved in acetonitrile, mixed with the peptides, and incubated at room temperature for 2 hours. The labeled peptides were then desalted and freeze-dried under vacuum. The peptides were fractionated using high-pH reversed-phase HPLC on an Agilent 300 Extend C18 column (5 μm particle size, 4.6 mm inner diameter, 250 mm length). Q Exactive was used for further analysis. TM LC-MS / MS analysis was performed using the Plus (Thermo) ultra-high performance liquid chromatograph.

[0227] (4) Quantitative proteomics data analysis

[0228] Tandem mass spectrometry data were analyzed using the Maxquant search engine (v.1.5.2.8). Tandem mass spectra were matched against the UniProt cynomolgus monkey database. Enzyme specificity was set to Trypsin / P, allowing a maximum of two undigested sites. The mass tolerances for precursor ions in the first and main searches were set to 20 ppm and 5 ppm, respectively. The mass tolerance for fragment ions was 0.02 Da. The false discovery rate threshold was less than 0.01, and the minimum peptide score was set to 40. Protein abundance was normalized by row mean and column median, followed by a two-tailed unpaired Student's t-test. The threshold for differentially expressed proteins (DEPs) was: an absolute value of log2 (fold change) greater than 0.26, and a Benjamini-Hochberg corrected P < 0.05.

[0229] (5) Immunohistochemical staining

[0230] After paraffin embedding, tissue sections were cut into 5 μm thick sections, dewaxed with xylene, and hydrated with 100% ethanol, 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 50% ethanol, and tap water. Sections were microwaved five times for 3 minutes each in 10 mM sodium citrate buffer (pH 6.0). After cooling to room temperature (RT), sections were gently washed three times with PBS, permeabilized in 0.4% Triton X-100 / PBS solution for 1 hour, and then washed twice with PBS buffer for 5 minutes each time. They were then incubated in 3% H2O2 for 10 minutes to block endogenous peroxidase activity. Sections were blocked with PBS containing 10% donkey serum at room temperature for 1 hour, then incubated overnight at 4°C with primary antibody. The next day, they were incubated with enzyme-labeled secondary antibody at room temperature for 1 hour, and then detected using a DAB staining kit (ZSJQ-BIO). Sections were reverse stained with hematoxylin, differentiated with 1% hydrochloric acid-ethanol, dehydrated with fractionated ethanol (50%, 70%, 80%, 90%, 100%, and 100%) and xylene, and then mounted with neutral resin. Images were taken using a Vectro Polaris instrument (PerkinElmer) or under a microscope (NikonEclipse).

[0231] (6) RT-qPCR

[0232] Total RNA was extracted from skeletal muscle tissue using TRIzol reagent (Thermo Fisher Scientific, #15596018), and then cDNA was synthesized by reverse transcription using the GoScript reverse transcription kit (Promega, #A5001). Subsequently, RT-qPCR was performed using Thunderbird qPCR Mix (TOYOBO, #QPS-201) reagent and a Real-Time System (Bio-Rad Laboratories, Inc. or Thermo Fisher Scientific).

[0233] Table 1. Primer sequences for monkey RT-qPCR

[0234] Experimental results:

[0235] To elucidate the molecular mechanisms of skeletal muscle aging in primates, the inventors performed transcriptomic and quantitative proteomic analyses on young and aged NHP skeletal muscle (Figure 2a-f, Figure 9a-e). They found that most differentially expressed genes (DEGs) / proteins (DEPs) showed age-related downregulation, with 1,091 DEGs and 156 DEPs showing decreased expression in aged skeletal muscle compared to young skeletal muscle (Figure 2e). These downregulated molecules are associated with the muscle cytoskeleton, muscle development, and metabolic pathways such as the citric acid (TCA) cycle and electron transport chain, reflecting a disruption of structural and metabolic homeostasis in aged muscle (Figure 2b, 2d, and 2f, Figure 9a-e). Conversely, 585 DEGs and 89 DEPs were upregulated in aged muscle, primarily involving immune responses and inflammatory pathways, including adaptive immunity, interleukins, and the NF-κB signaling pathway (Fig. 2b, Fig. 2d, and Fig. 2f; Fig. 9a-Fig. 9e). At the tissue level, an increase in CD45-positive immune cells, CD68-positive macrophages, and CD86-positive pro-inflammatory M1 macrophages was found in aged skeletal muscle tissue (Fig. 2g-Fig. 2i). Furthermore, signs of increased inflammation were evident in aged skeletal muscle, manifested by an increase in S100A8 and S100A9-positive cells and elevated levels of pro-inflammatory cytokines such as IL-6, IL-1, and IFNB (Fig. 2j-Fig. 2m). These experimental results are consistent with histological analysis, indicating that increased immune cells and significantly upregulated inflammation-related factors are prominent features of skeletal muscle aging in cynomolgus monkeys.

[0236] Example 3: The loss of SIRT5 protein leads to myotube aging in humans.

[0237] 1. Experimental Methods:

[0238] (1) Obtaining SIRT5 knockout human embryonic stem cells

[0239] SIRT5 protein knockout was performed in human embryonic stem cells using CRISPR / Cas9 technology. The specific steps are as follows: annealing sgSIRT5-F and sg-SIRT5-R to form nucleic acid molecules (containing sequences homologous to the vector and sgRNA target sequences targeting exon 5 of the SIRT5 gene; the sgRNA target sequence was ligated into the pCAG-mCherry-gRNA plasmid (Addgene, plasmid number: 87110) via homologous recombination to obtain the SIRT5 knockout plasmid.

[0240] sgSIRT5-F: 5'-TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCAGGGCCGGCGATGTC-3'; (SEQ ID NO: 23)

[0241] sgSIRT5-R: 5'-GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACGACATCGCCGGCCCT-3' (SEQ ID NO: 24).

[0242] Table 2. sgRNA target sequences

[0243] SIRT5+ / + hESCs (H9 hESCs (female)) (WiCell Research) were digested and cultured in mTeSR medium in Matrigel-coated plates for 24 hours with the ROCK inhibitor Y-27632 (Selleck) added to the medium. Then, using a Lonza 4D nucleofector transducer, the constructed SIRT5-targeting plasmid (SIRT5 knockout plasmid) along with the Cas9 nuclease expression vector (pCAG-1BPNLS-Cas9-1BPNLS-2AGFP, plasmid number: 87109; Addgene) was electroporated into SIRT5+ / + hESCs cells. The electroporated cells were then cultured in fresh mTeSR medium in Matrigel-coated plates for 48 hours with the ROCK inhibitor Y-27632 added to the medium. After culture, GFP+ / mCherry+ cells were sorted using a flow cytometer (BD Influx) and cultured in MEF feeder cells for ESC. Following culture, genomic DNA was extracted and analyzed from clones derived from individual cells.

[0244] (2) Differentiating human embryonic stem cells and human induced pluripotent stem cells into myotube cells

[0245] hESCs were cultured for approximately 10 days in differentiation medium (MEMα (Thermo Fisher Scientific), 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 1% penicillin / streptomycin (Thermo Fisher Scientific), 10 ng / mL bFGF (Joint Protein Central), and 5 ng / mL TGFβ (StemImmune)) to generate human myotube progenitor cells. Early-passaged hESCs were infected with the lentiviral vector MyoD-ER(T) (Addagen, catalog number: 26809) and transferred to matrix-coated culture dishes. When the cells reached 70-80% confluence, 1 μM 4-oh-tamoxifen (Sigma-Aldrich) was added to the myotube progenitor cell culture medium, and the cells were maintained at 37°C, 5% CO2, and 3-5% O2. After 24 hours, the culture medium was replaced with fresh hMyotube differentiation medium (high glucose DMEM medium containing 2% horse serum), supplemented with 1 μM 4-oh-tamoxifen, and refreshed every 2 days. Day 1 was the first day of 4-oh-tamoxifen administration. Mature human myotube cells formed on day 5 or 6.

[0246] (3) Cell immunofluorescence staining

[0247] Cultured cells were fixed in 4% PFA for 20 minutes, washed twice with PBS, and permeabilized in 0.4% Triton X-100 / PBS for 1 hour, followed by two washes with PBS. Cells were blocked in PBS containing 10% donkey serum at room temperature for 1 hour, then incubated overnight at 4°C with primary antibody, washed three times with TBST, incubated for 1 hour with fluorescent secondary antibody at room temperature, and the nuclei were stained with the DNA-specific dye Hoechst 33342 (Thermo Fisher Scientific). Finally, the cells were mounted with a fluorescent antiquencher (Vector Laboratories). Imaging was performed using laser confocal microscopy (Leica TCS SP5 II or Zeiss LSM 900).

[0248] (4) Human myotube cell transcriptome sequencing

[0249] Total RNA was extracted from human myotube cells using TRIzol reagent. After assessing RNA quality, libraries were constructed and each sample was subjected to high-throughput sequencing. In short, using... RNA Library Prep Kit for I Libraries were prepared at NEB (USA), and each sample was indexed. The libraries were then sequenced at 150-base-pair reads on the Illumina paired-end sequencing platform (Novogene Bioinformatics Technology Co., Ltd.). Raw transcriptome data from human myotube cell samples were processed using Trim Galore software (version 0.4.5) to remove low-quality reads and adapter sequences. Human myotube cell sample data were aligned to the human hg19 genome using HISAT2 software (version 2.0.4). HTSeq (version 0.11.0) was used to determine the expression level of each gene. FPKM (fractions per thousand bases) for each gene was calculated using StringTie (version 2.1.4). The R package DESeq2 (version 1.22.2) was used to identify differentially expressed genes (DEGs). Referring to previous studies involving transcriptomes of different cynomolgus monkey tissues, the thresholds for DEG between the hearts of aged and young cynomolgus monkeys were P < 0.01 and |log2(fold change)| > 0.5. For DEG between SIRT5-deficient human cardiomyocytes and wild-type controls, the criteria were set as Benjamini-Hochberg corrected P < 0.01 and |log2(fold change)| > 0.5. Pathway enrichment analysis was performed using Metascape.

[0250] (5) RT-qPCR

[0251] Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific), and then cDNA was synthesized by reverse transcription using the GoScript reverse transcription kit (Promega). Subsequently, RT-qPCR was performed using Thunderbird qPCR Mix reagent and a Real-Time System (Bio-Rad Laboratories, Inc. or Thermo Fisher Scientific).

[0252] Table 3. Primer sequences for human RT-qPCR

[0253] (6) Cell SA-β-gal staining

[0254] Cultured cells were washed twice with PBS and fixed with 2% formaldehyde and 0.2% glutaraldehyde for 3–5 min at room temperature, then stained overnight at 37°C in 1 mg / mL X-gal staining solution. Images were acquired under a microscope (Nikon Eclipse or Olympus), and the percentage or intensity of SA-β-gal positive areas or cells was calculated using ImageJ.

[0255] (7) Western blotting experiment

[0256] For the extraction of cellular proteins, cultured cells were directly lysed using 1×SDS lysis buffer. The lysis was performed at 13000 rpm.

[0257] After centrifugation at 4°C for 15 minutes, the protein supernatant was carefully collected, and the protein concentration was determined using a BCA kit. The protein lysis buffer was then subjected to SDS-PAGE. After electrophoresis and transfer, the PVDF membrane (Millipore) was blocked with TBST containing 5% skim milk powder (BBI Life Sciences), washed three times with TBST for 10 minutes each time, incubated overnight at 4°C with primary antibody, and then incubated at room temperature for 1 hour with enzyme-labeled secondary antibody. After washing three times with TBST for 10 minutes each time, the images were developed and photographed using the ChemiDoc XRS system (Bio-Rad) and Image Lab software. The acquired images were quantified using ImageJ software.

[0258] (8) ELISA detection

[0259] The protein levels of IL-8 in the culture medium of SIRT5+ / + and SIRT5- / - deficient human cardiomyocytes were measured using an ELISA kit, following the instructions in the manufacturer's manual (R&D, #D8000C). Data were read at 450 nm using a BioTek Synergy H1 multi-functional microplate reader.

[0260] (9) Detection of mitochondrial membrane potential and superoxide anion detection

[0261] Mitochondrial membrane point potential was evaluated using 2.5 μM nonylacridine orange (TMRM).

[0262] (10) Detection of teratoma

[0263] In the teratoma experiment, SIRT5-deficient human embryonic pluripotent stem cells cultured on Matrigel were digested into single cells using TrypLE and counted. Three × 10⁶ SIRT5-deficient human embryonic pluripotent stem cells were resuspended in a Matrigel / mTeSR (1:4) mixture and injected subcutaneously into the groin of NOD / SCID mice (6-8 weeks old, male). After 8-12 weeks, the teratomas were removed for frozen sectioning and immunostaining analysis.

[0264] (11) Karyotype analysis

[0265] Karyotype analysis was performed by San Valley Diagnostics. Human embryonic pluripotent stem cells were cultured in cell culture flasks to a density of 70%, and then treated with KaryoMAX Colcemid Solution in HBSS (Thermo Fisher Scientific, #15210040) to arrest the cells in the M phase. After trypsin digestion, the cells were ready for further karyotype analysis.

[0266] 2. Experimental Results

[0267] To further investigate the molecular drivers of skeletal muscle aging in NHP, SIRT5, a member of the Sirtuin family, is well-known for its role in regulating aging and lifespan in various organisms and is one of the most significantly downregulated proteins in aged NHP skeletal muscle (Figure 3a). Transcriptional analysis also revealed that SIRT5 is the most significantly downregulated Sirtuin during NHP skeletal muscle aging (Figure 3b, Figure 10a). Decreased SIRT5 protein expression levels in aged skeletal muscle tissue were observed, and a similar age-dependent decline was found in human muscle biopsies (Figure 3c, Figure 3d). These findings suggest that decreased SIRT5 protein expression may be a key gene contributing to skeletal muscle aging.

[0268] To explore the role of SIRT5 in aging, SIRT5-deficient human embryonic stem cells (SIRT5- / -hESCs) were constructed using CRISPR / Cas9 technology and differentiated into myotubes (SIRT5- / -hMyotubes) (Fig. 3e, Extended Data Fig. 3b-h). The absence of SIRT5 protein was identified as not affecting the expression of other Sirtuins (Fig. 3f, Fig. 10d, Fig. 11a) or the differentiation efficiency of myotube cells (Fig. 11b). However, increased aging-related β-galactosidase (SA-β-gal) activity was observed in SIRT5- / - human myotubes (Fig. 3g), along with decreased myotube diameter and elevated expression levels of muscle atrophy markers Fbx32 and MuRF1 (Fig. 3h-i). Furthermore, SIRT5 deficiency leads to a series of impairments in myotube mitochondrial function, including decreased mitochondrial membrane potential, impaired oxidative respiratory chain, increased production of mitochondrial superoxide anion and hydrogen peroxide, elevated expression of the oxidative stress marker 4-HNE, and downregulation of the antioxidant protein SOD1 in myotubes (Figure 3j to Figure 3k, Figure 11d to Figure 11e). Simultaneously, the upregulation of pro-inflammatory genes in SIRT5- / - human myotube cells reflects the intensified inflammatory response in aged NHP skeletal muscle, including genes related to the aging-associated secretory phenotype (SASP) (Figure 3l to Figure 3m). Transcriptome sequencing revealed that SIRT5- / - human myotube cells exhibit gene expression changes similar to those in aged skeletal muscle, such as upregulation of pro-inflammatory genes associated with interferon and tumor necrosis factor (TNF) signaling pathways, and downregulation of genes crucial for myocyte differentiation (Figure 3n to Figure 3o). These findings indicate that SIRT5 deficiency induces senescence in human myotube cells, characterized by increased chronic inflammation and impaired mitochondrial function.

[0269] Example 4: SIRT5 interacts with TBK1-Lys137 to cause desuccinylation modification.

[0270] 1. Experimental Method:

[0271] (1) Construction of plasmids

[0272] Human SIRT5 cDNA was obtained from human skeletal muscle cells via PCR amplification (total RNA was extracted from cells, reverse transcribed to obtain total cDNA, and human FLAG-SIRT5 cDNA was obtained by PCR amplification using FLAG-SIRT5-OE-F and FLAG-SIRT5-OE-R as templates). The plus One-step PCR cloning kit (Novoprotein, #NR005) specifies the location of the human FLAG-SIRT5 cDNA pLE4 vector. Specifically, the FLAG-SIRT5-WT vector is a recombinant vector obtained by replacing the small fragment between the BamHI and MluI restriction sites of pLE4 with a recombinant gene, while maintaining other nucleotide sequences unchanged. The recombinant gene is obtained by adding a FLAG tag encoding gene to the 5' end of the SIRT5 cDNA nucleotide sequence.

[0273] The human FLAG-SIRT5-H158Y vector was obtained using the Fast Mutagenesis System (Transgen biotech, #FM111-02) with the human FLAG-SIRT5-WT vector. The human FLAG-SIRT5-H158Y vector was obtained by mutating the 472nd position of the SIRT5 protein-coding gene sequence on the human FLAG-SIRT5-WT vector from a C base to a U base.

[0274] Human TBK1 cDNA was obtained from human cells via PCR amplification (total RNA was extracted from cells, and total cDNA was obtained by reverse transcription; PCR was performed using FLAG-TBK1-OE-F and FLAG-TBK1-OE-R as templates).

[0275] Human FLAG-TBK1 cDNA was amplified and then used... The human FLAG-TBK1 cDNA was ligated into the pLE4 vector using the plus One-step PCR cloning kit (Novoprotein, #NR005) to obtain the human FLAG-TBK1-WT vector. The human FLAG-TBK1-K137R, FLAG-TBK1-K451R, FLAG-TBK1-K460R, FLAG-TBK1-K567R, and FLAG-TBK1-K615R vectors were then obtained using the Fast Mutagenesis System (Transgen biotech, #FM111-02) with the human FLAG-TBK1-WT vector. The human FLAG-TBK1-K137R, FLAG-TBK1-K451R, FLAG-TBK1-K460R, FLAG-TBK1-K567R, and FLAG-TBK1-K615R vectors are obtained by mutating base A to base G at positions 410, 1352, 1379, 1700, and 1844 of the TBK1 protein encoding gene sequence on the human FLAG-TBK1-WT vector.

[0276] FLAG-SIRT5-OE-F:5’-ACTCTAGAGGATCCCTCGAGATGCGACCTCTCCAGATTGTC-3’;(SEQ ID NO:34)

[0277] FLAG-SIRT5-OE-R:5’-TCCAGAGGTTGATTGTCGACTTAGCCCTTGTCGTCGTCGTCCTTGTAGTCAGAAACAGT-3’;(SEQ ID NO:35)

[0278] HA-SIRT5-OE-F:5’-ACTCTAGAGGATCCCTCGAGATGCGACCTCTCCAGATTGTC-3’;(SEQ IDNO:34)

[0279] HA-SIRT5-OE-R:5’-TCCAGAGGTTGATTGTCGACTTAAGCGTAGTCTGGGACGTCGTATGGGTAAGAAACAGT-3’;(SEQ ID NO:36)

[0280] SIRT5-H158Y-F:5’-CAAGAACCTTCTGGAGATCTATGGTAGCTTAT-3’;(SEQ ID NO:37)

[0281] SIRT5-H158Y-R:5’-AGATCTCCAGAAGGTTCTTGGTGCCAGCCTTG-3’;(SEQ ID NO:38)

[0282] FLAG-TBK1-F:5’-GCACTCTAGAGGATCCATGCAGAGCACTTCTAAT-3’;(SEQ ID NO:39)

[0283] FLAG-TBK1-R:5’-GATTGTCGACACGCGTCTACTTGTCGTCGTCGTCCTTGTAGTCAAGACAGTCAACGTTGCG-3’;(SEQ ID NO:40)

[0284] FLAG-TBK1-K137R-F:5’-ATGGTATAGTGCACCGTGATATCAGGCCAGGAAATATCATGCGTGTTAT-3’;(SEQ ID NO:41)

[0285] FLAG-TBK1-K137R--R:5’-TAACACGCATGATATTTCCTGGCCTGATATCACGGTGCACTATACCAT-3’;(SEQ ID NO:42)

[0286] FLAG-TBK1-K451R--F:5’-GGGGATACGATGGCTGATTGAATTAATTAGAGATGATTACAATGAAACTGTTCA-3’;(SEQ ID NO:43)

[0287] FLAG-TBK1-K451R--R:5’-GTGAACAGTTTCATTGTAATCATCTCTAATTAATTCAATCAGCCATCGTAT-3’;(SEQ ID NO:44)

[0288] FLAG-TBK1-K460R--F:5’-TGATTACAATGAAACTGTTCACAGAAAGACAGAAGTTGTGATCACATT-3’;(SEQ ID NO:45)

[0289] FLAG-TBK1-K460R--R:5’-CAATGTGATCACAACTTCTGTCTTTCTGTGAACAGTTTCATTGTAATCA-3’;(SEQ ID NO:46)

[0290] FLAG-TBK1-K567R--F:5’-GCATGACAGAGATTTACTATCAGTTCAGAAAAGACAAAGCAGAACGTAGATTA-3’;(SEQ ID NO:47)

[0291] FLAG-TBK1-K567R--R:5’-TCTACGTTCTGCTTTGTCTTTTCTGAACTGATAGTAAATCTCTGTCATGC-3’;(SEQ ID NO:48)

[0292] FLAG-TBK1-K615R--F:5’-AAAGTATGAGGCATTTTTGAATAGGTCAGAAGAATGGATAAGAAA-3’;(SEQ ID NO:49)

[0293] FLAG-TBK1-K615R--R: 5'-CTTTCTTATCCATTCTCTGACCTATTCAAAAATGCCTCATACT-3'; (SEQ ID NO: 50)

[0294] (2) LC-MS / MS analysis and peptide identification

[0295] To identify the interacting proteins of SIRT5, this invention performed LC-MS / MS analysis. The specific steps are as follows: First, HEK293T cells were transfected with plasmids expressing FLAG-luciferase (Luc) or FLAG-SIRT5. Then, FLAG-Luc or FLAG-SIRT5 was purified and incubated with cynomolgus monkey skeletal muscle lysate. The proteins eluted by immunoprecipitation were run on 10% SDS-PAGE and stained with Coomassie Brilliant Blue (Beijing Dingguo Changsheng Biotechnology Co., Ltd., #WB-0101). The gel bands containing the protein bands were carefully removed, cut into small pieces, dehydrated (100% acetonitrile), reduced (10 mM DTT in 25 mM NH4HCO3, 56°C, 45 min), and alkylated (40 mM iodoacetamide in 25 mM NH4HCO3, room temperature, protected from light, 45 min). Next, the small gel strips were dried and digested overnight in 25 mM NH4HCO3 at 37°C with sequencing-grade modified trypsin (40 ng per strip). Digestion was terminated by adding formic acid to a final concentration of 1%. The digested solution was then carefully transferred to sample vials for LC-MS / MS analysis. High-resolution mass spectrometry data of 70,000 (m / z 200) were obtained using a Q Exactive mass spectrometer (Thermo Scientific). Protein identification was performed using Proteome Discovery (version 2.2.0.388), and peptide data were searched using the UniProtMacacafascicularis database (updated October 2017).

[0296] (3) Co-IP experiment

[0297] For exogenous Co-IP experiments, HEK293T cells were transfected with plasmids expressing FLAG-luciferase (Luc), FLAG-SIRT5-WT, FLAG-SIRT5-H158Y, or FLAG-TBK1-WT. Transfected HEK293T cells were lysed using CHAPS lysis buffer (120 mM NaCl, 0.3% CHAPS, 40 mM HEPES (pH 7.5), 1 mM EDTA, and a complete protease inhibitor mixture (Roche)).

[0298] For exogenous succinylation Co-IP experiments, HEK293T cells were transfected with the FLAG-TBK1-WT plasmid, along with HA-Luc, HA-SIRT5-WT, or HA-SIRT5-H158Y plasmids. Transfected HEK293T cells were lysed using whole-cell lysis buffer (50 mM Tris–HCl (pH 7.8), 137 mM NaCl, 1 mM NaF, 1 mM NaVO3, 1% Triton X-100, 0.2% sarkosyl, 1 mM DTT, 10% glycerol, a complete protease inhibitor mixture (Roche), 5 mM nicotinamide (NAM, Sigma, #N0636), and 10 μM trichostatin A (TSA, Sigma, #V900931)). Lysed cells were lysed by rotation at 4°C for 2 hours, followed by centrifugation at 12,000 × g for 30 minutes at 4°C. The resulting supernatant was quantified and gently mixed with an anti-FLAG M2 affinity gel (Sigma, #A2220), and incubated overnight at 4°C by rotation. The interacting protein complexes captured by the anti-FLAG affinity gel were competitively eluted with FLAG-peptide and then analyzed by Western blotting.

[0299] For endogenous Co-IP, human myotube cells (SIRT5+ / + or SIRT5- / - human myotube cells) or monkey skeletal muscle tissue (young or old) were lysed using NP40 lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% NP40, 1 mM PMSF, and a complete protease inhibitor mixture (Roche)). For endogenous succinylated Co-IP, 20 mM NAM and 4 μM TSA were added to the above NP40 lysis buffer, and the mixture was lysed by rotation at 4°C for 2 hours, followed by centrifugation at 12,000 × g for 30 minutes at 4°C. The resulting quantified supernatant was incubated with anti-succinylated lysine antibody and rotated overnight at 4°C. After overnight lysis, the protein-antibody mixture was incubated with Protein A / G-PLUS agarose beads (Santa Cruz, #sc-2003) by rotation for 2 hours at 4°C. Finally, protein samples were collected for Western blotting analysis, with samples from the same group of cynomolgus monkeys being merged together for analysis.

[0300] (4) Western blotting experiment

[0301] For cellular protein extraction, cultured cells were directly lysed using 1×SDS lysis buffer. After centrifugation at 13000 rpm and 4°C for 15 minutes, the protein supernatant was carefully collected, and protein concentration was determined using a BCA kit. The protein lysis buffer was then subjected to SDS-PAGE. After electrophoresis and transfer, the PVDF membrane (Millipore) was blocked with TBST containing 5% skim milk powder (BBI Life Sciences), washed three times with TBST for 10 minutes each time, incubated overnight at 4°C with primary antibody, and then incubated with enzyme-labeled secondary antibody at room temperature for 1 hour. After washing three times with TBST for 10 minutes each time, images were developed and photographed using a ChemiDoc XRS system (Bio-Rad) and ImageLab software. The acquired images were quantified using ImageJ software.

[0302] 2. Experimental Results

[0303] To elucidate the mechanism of SIRT5's role in skeletal muscle aging, protein-protein interactions were investigated using FLAG-tagged SIRT5 protein with NHP skeletal muscle lysate, followed by co-immunoprecipitation (Co-IP) and mass spectrometry analysis (Figure 4a and Extended Data Table 3). Mass spectrometry results showed that proteins interacting with SIRT5 were functionally enriched in inflammatory responses, including cytokine signaling and the TNF-α / NF-κB signaling complex (Figure 4b). Among the identified interacting proteins, in addition to known proteins (e.g., SDHA, succinate dehydrogenase complex flavoprotein subunit A), TBK1 is well-known for its regulatory function in inflammatory responses (Figure 4b and Figure 12a). The interaction between SIRT5 and TBK1 was validated by Co-IP experiments, pulling FLAG-tagged SIRT5 or TBK1 from HEK293T cells or hMyotubes (Figure 4c-4f). Immunostaining revealed the co-localization of SIRT5 and TBK1 within the mitochondrial region (Figure 12b).

[0304] Given that SIRT5, as an NAD+-dependent lysine deacylase, exhibits significant desuccinylation specificity, overexpression of wild-type SIRT5 leads to a decrease in TBK1 succinylation levels, a change that cannot be mimicked by the desuccinylation-deficient mutant SIRT5H158Y (Figure 4 g-h). This observation is further confirmed by the increased succinylated TBK1 levels in SIRT5- / - human myotube cells (Figure 4 i-k).

[0305] Previous research revealed that SIRT5 inhibits TBK1 phosphorylation activation by desuccinylating TBK1. To further elucidate the site of SIRT5 succinylation of TBK1, a TBK1 lysine succinylation map was constructed using three bioinformatics databases. Analysis of common site information identified five candidate sites: K137, K451, K460, K567, and K615 (Figure 5a). Site-directed mutagenesis replacing lysine residues with arginine (a mutation leading to persistent succinylation) revealed that the mutation at Lys137 (TBK1K137R) resulted in a reduced SIRT5 desuccinylation capacity (Figure 5b-5c). These experimental results suggest that Lys137 may serve as a SIRT5-targeted TBK1 desuccinylation site.

[0306] Example 5: SIRT5-mediated TBK1 desuccinylation promotes its dephosphorylation and inhibits human myotube cell senescence.

[0307] 1. Experimental Methods

[0308] (1) Lentiviral packaging and infection of myotube cells

[0309] HEK293T cells in a 10cm dish were co-transfected with 10 μg of lentiviral plasmid, 10 μg of psPAX2 plasmid, and 3 μg of pMD2.G plasmid using a Lipo3000 transfection kit (Thermo Fisher Scientific). After 8 hours of culture, the culture medium was replaced. Supernatants were collected at 24h, 48h, and 72h, filtered through a 0.22μm filter, and stored at 4°C. The supernatants were centrifuged at 19400 rpm for 2.5h at 4°C, the supernatant was discarded, and the cells were resuspended in skeletal muscle medium to obtain recombinant lentiviruses, which were then aliquoted and stored at -80°C. Viral titration was performed using immunostaining to detect overexpressed virus. After titration, the cells were infected with the predetermined viral titer, and subsequent phenotypic analysis was conducted.

[0310] (2) Cell immunofluorescence staining

[0311] Cultured cells were fixed in 4% PFA for 20 minutes, washed twice with PBS, and permeabilized in 0.4% Triton X-100 / PBS for 30 minutes, followed by two washes with PBS. After blocking with PBS containing 10% donkey serum at room temperature for 1 hour, primary antibody was added and incubated overnight at 4°C. The next day, cells were incubated with fluorescent secondary antibody at room temperature for 1 hour, and the nuclei were stained with the DNA-specific dye Hoechst 33342 (Thermo Fisher Scientific). Finally, the cells were mounted with a fluorescent antiquencher (Vector Laboratories). Imaging was performed using laser confocal microscopy (Leica TCS SP5 II or Zeiss LSM 900).

[0312] (3) Cell SA-β-gal staining

[0313] Cultured cells were washed twice with PBS and fixed with 2% formaldehyde and 0.2% glutaraldehyde for 3–5 min at room temperature, then stained overnight at 37°C in 1 mg / mL X-gal staining solution. Images were acquired under a microscope (Nikon Eclipse or Olympus), and the percentage or intensity of SA-β-gal positive areas or cells was calculated using ImageJ.

[0314] (4) RT-PCR

[0315] Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific), and then cDNA was synthesized by reverse transcription using the GoScript reverse transcription kit (Promega). RT-qPCR was then performed using Thunderbird qPCR Mix reagent and a Real-Time System (Bio-Rad Laboratories, Inc. or Thermo Fisher Scientific). The primer sequences for RT-qPCR are shown in Table 4.

[0316] Table 4. Primer sequences for human RT-qPCR

[0317] 2. Experimental Results

[0318] Overexpression of SIRT5 in SIRT5- / - human myotube cells revealed decreased phosphorylation at the TBK1Ser172 site (a key site for TBK1 activation), significantly different from the phosphorylation level at the TBK1Ser172 site in the desuccinylation SIRT5H158Y mutant (Fig. 5d). This result is consistent with increased p-TBK1S172 levels in aged NHP skeletal muscle (Fig. 5e). The TBK1K137R mutant exhibited complete deletion of p-TBK1S172, revealing a correlation between SIRT5 succinylation at Lys137 and phosphorylation at Ser172 (Fig. 5b and 5c). TBK1 structural analysis showed that Lys137, located within the TBK1 kinase domain, is closer to the phosphorylation site Ser172 than other predicted desuccinylation sites (Fig. 5f-5h), suggesting that changes at this site may affect TBK1 phosphorylation.

[0319] To further investigate the effects of TBK1 desuccinylation on its downstream pathways, FLAG-Luc, FLAG-TBK1WT, or FLAG-TBK1K137R were overexpressed in human myotubes (Figure 5i). The TBK1K137R mutant exhibited reduced p-TBK1S172 levels (Figure 5i), decreased expression of pro-inflammatory cytokines (IL6, IL8, MCP1) (Figure 5j), and senescence-related phenotypes in human myotube cells (Figure 5k-Figure 5l). These results suggest that SIRT5 regulation of TBK1 desuccinylation plays a crucial role in TBK1 activity and its associated inflammatory pathways.

[0320] Example 6: Inactivation of the TBK1 signaling pathway reduces aging and inflammation in human myotubules.

[0321] 1. Experimental Methods

[0322] (1) Lentiviral packaging and infection of myotube cells

[0323] HEK293T cells in a 10cm dish were co-transfected with 10 μg of lentiviral plasmid, 10 μg of psPAX2 plasmid, and 3 μg of pMD2.G plasmid using a Lipo3000 transfection kit (Thermo Fisher Scientific). After 8 hours of culture, the culture medium was replaced. Supernatants were collected at 24h, 48h, and 72h, filtered through a 0.22μm filter, and stored at 4°C. The supernatants were centrifuged at 19400 rpm for 2.5h at 4°C, the supernatant was discarded, and the cells were resuspended in skeletal muscle medium to obtain recombinant lentiviruses, which were then aliquoted and stored at -80°C. Viral titration was performed using immunostaining to detect overexpressed virus. After titration, the cells were infected with the predetermined viral titer, and subsequent phenotypic analysis was conducted.

[0324] (2) siRNA method to knock down TBK1 or RelA

[0325] To reduce TBK1 expression, the corresponding siRNA or non-target siRNA in human myotubes was transfected using RNAiMAX (Invitrogen). 25 μM of TBK1 negative control or siRNA was mixed with 100 μL Opti-MEM (Gibco) and 2 μL Lipofectamine. TM The RNAiMAX transfection reagent (Thermo Fisher Scientific) was mixed and then added to one well of a 12-well plate. After culturing for 8 hours, the medium was replaced with fresh medium, and cells were collected for analysis 72 hours after transfection. TBK1 siRNA and RelA siRNA were synthesized by Ribot Biotech, China.

[0326] Table 5 siRNA sequences

[0327] (3) Cell immunofluorescence staining

[0328] Cultured cells were fixed in 4% PFA for 20 minutes, washed twice with PBS, and permeabilized in 0.4% Triton X-100 / PBS for 30 minutes, followed by two washes with PBS. After blocking with PBS containing 10% donkey serum at room temperature for 1 hour, primary antibody was added and incubated overnight at 4°C. The next day, cells were incubated with fluorescent secondary antibody at room temperature for 1 hour, and the nuclei were stained with the DNA-specific dye Hoechst 33342 (Thermo Fisher Scientific). Finally, the cells were mounted with a fluorescent antiquencher (Vector Laboratories). Imaging was performed using laser confocal microscopy (Leica TCS SP5 II or Zeiss LSM900).

[0329] (4) Cell SA-β-gal staining

[0330] Cultured cells were washed twice with PBS and fixed with 2% formaldehyde and 0.2% glutaraldehyde for 3–5 min at room temperature, then stained overnight at 37°C in 1 mg / mL X-gal staining solution. Images were acquired under a microscope (Nikon Eclipse or Olympus), and the percentage or intensity of SA-β-gal positive areas or cells was calculated using ImageJ.

[0331] (5) RT-PCR

[0332] Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific), and then cDNA was synthesized by reverse transcription using the GoScript reverse transcription kit (Promega). Subsequently, RT-qPCR was performed using Thunderbird qPCR Mix reagent and a Real-Time System (Bio-Rad Laboratories, Inc. or Thermo Fisher Scientific). The primer sequences for RT-qPCR are shown below.

[0333] Table 6. Primer sequences for human RT-qPCR

[0334] (6) ELISA detection

[0335] The protein levels of IL-8 in the culture medium of SIRT5+ / + and SIRT5- / - deficient human cardiomyocytes were measured using an ELISA kit, following the instructions in the manufacturer's manual (R&D, #D8000C). Data were read at 450 nm using a BioTek Synergy H1 multi-functional microplate reader.

[0336] 2. Experimental Results

[0337] The NF-κB signaling pathway is a major downstream cascade of TBK1, responsible for coordinating inflammatory responses. Knockdown of TBK1 in SIRT5- / - human myotube cells detected decreased phosphorylated RelA (p-RelA) (Figure 6a). Transcriptional analysis integrating differentially upregulated genes in skeletal muscle aging and SIRT5- / - and SIRT5+ / + human myotube cells suggests that the RelA-NF-κB complex plays a crucial role in skeletal muscle and SIRT5- / - myotube cell aging. Consistent with this, p-RelA levels were significantly elevated in SIRT5- / - human myotube cells and aged skeletal muscle (Figure 6d-6e). Knockdown of RelA in SIRT5- / - human myotube cells using siRNA significantly improved aging-related phenotypes, namely a decreased proportion of SA-β-gal positive cells, an increased cell diameter, and decreased expression of pro-inflammatory cytokines (IL6, IL8, MCP1, TNFA) (Figure 6i-Figure 6m). These results indicate that the TBK1-RelA axis is a key mediator of the protective effect of SIRT5 against aging in human myotube cells.

[0338] Example 7: Gene therapy based on lentivirus-mediated SIRT5-TBK1-RelA signaling axis to alleviate skeletal muscle degeneration and atrophy

[0339] 1. Experimental Methods

[0340] (1) Plasmid construction

[0341] The method for constructing FLGA-SIRT5 and FLAG-Luc plasmids is described in Example 4.

[0342] (2) Packaging of lentiviruses

[0343] 10 μg of lentiviral plasmid, 10 μg of psPAX2 plasmid, and 3 μg of pMD2.G plasmid were co-transfected into one 10 cm dish of 293T cells using a Lipo3000 transfection kit (Thermo Fisher Scientific). After 8 hours of culture, the medium was replaced. Supernatants were collected at 24 h, 48 h, and 72 h, filtered through a 0.22 μm filter, and stored at 4 °C. The supernatants were centrifuged at 19400 rpm at 4 °C for 2.5 h, the supernatants were discarded, and the cells were resuspended in myocardial inoculum to obtain recombinant lentiviruses, which were then aliquoted and stored at -80 °C. For viral titration, immunostaining was used to detect overexpressed virus.

[0344] (3) Intramuscular injection of lentivirus into mouse skeletal muscle tissue

[0345] Experimental groups and test mice:

[0346] Young-lenti-Luc group: 14 male C57BL6 / J mice aged 4 months;

[0347] The aged-lenti-Luc group consisted of 14 male C57BL6 / J mice aged 23 months.

[0348] The aged-lenti-SIRT5 group consisted of 14 male C57BL6 / J mice aged 23 months.

[0349] Mice in each group were anesthetized by inhalation of 2% isoflurane. Hair on the chest was removed with depilatory cream, and then lentiviruses expressing luciferase (Luc) or SIRT5 were injected at two sites in the quadriceps femoris and tibialis anterior muscles. Behavioral and molecular level analyses were performed 5 weeks after injection.

[0350] (4) Skeletal muscle tissue transcriptome sequencing

[0351] Total RNA was extracted from mouse skeletal muscle tissue using TRIzol reagent. After assessing RNA quality, libraries were constructed and each sample was subjected to high-throughput sequencing. In short, using... RNA Library Prep Kit for Libraries were prepared at NEB (USA), and each sample was indexed. The libraries were then sequenced at 150-base-pair reads on an Illumina paired-end sequencing platform (Novogene Bioinformatics Technology Co., Ltd.). The obtained raw transcriptome data were processed using Trim Galore software (version 0.4.5) to remove low-quality reads and adapter sequences. Subsequently, the genomes were aligned with a mouse MacFas 5.0 reference genome using HISAT2 software (version 2.0.4). HTSeq (version 0.11.0) was used to determine the expression level of each gene. The fragment-per-thousand (FPKM) count for each gene was calculated using StringTie (version 2.1.4). The R package DESeq2 (version 1.22.2) was used to identify differentially expressed genes (DEGs). Referring to previous studies involving transcriptomes of different tissues, the thresholds for DEG between groups were P < 0.01 and |log2(fold change)| > 0.5. Pathway enrichment analysis was performed using Metascape.

[0352] (5) Behavioral testing

[0353] Netting Experiment: A square grid was securely mounted on an elevated frame. A mat was placed beneath the grid to protect the mice during the test. Each mouse was gently placed in the center of the grid. The grid was then carefully inverted. The time from the moment of inversion until the mouse fell was recorded.

[0354] Rotating bar experiment: The rotating bar experiment was conducted using the Rota bar system (Yiyan Tech, YLS-4C). Mice underwent initial training for three days. In each experiment, each mouse was placed in a separate channel on the bar. The speed of pulling the bar was gradually increased from 4 revolutions per minute (rpm) to 44 revolutions per minute (rpm), with the acceleration always maintained at 8 revolutions per minute squared (rpm² / min). This continued until each mouse fell off the bar three times. The average time of each mouse's fall was carefully recorded and subsequently used for statistical analysis.

[0355] Exhaustion test: Before the formal experiment, mice were trained for three days on a treadmill (SANS Bio Instrument, SA101) at a 5° incline. During the training, 2 mA of electrical stimulation was used. Each test lasted 20 minutes, with the speed gradually increased from 5 to 20 m / min. On the test day, the mice ran on the treadmill at an acceleration of 2 rpm / min. When the mice could no longer run on the treadmill and remained on the electrodes for more than 10 seconds, the duration and distance were recorded.

[0356] (6) Immunofluorescence staining

[0357] Refer to Example 1.

[0358] (7) RT-qPCR

[0359] Total RNA was extracted from mouse skeletal muscle tissue using TRIzol reagent (Thermo Fisher Scientific, #15596018), and then cDNA was synthesized by reverse transcription using the GoScript reverse transcription kit (Promega, #A5001). Subsequently, RT-qPCR was performed using Thunderbird qPCR Mix (TOYOBO, #QPS-201) reagent and a Real-Time System (Bio-Rad Laboratories, Inc. or Thermo Fisher Scientific).

[0360] Table 7. Primer sequences for mouse RT-qPCR

[0361] 2. Experimental Results

[0362] Overexpression of FLAG-Luc, FLAG-SIRT5WT, and FLAG-SIRT5H158Y (desuccinylase inactive) in SIRT5- / - human myotube cells revealed that overexpression of wild-type SIRT5, rather than mutant variants, alleviated age-related phenotypes in myotube cells, such as reduced myotube cell diameter and the proportion of SA-β-gal positive cells (Figure 7a-Figure 7b).

[0363] To further investigate the role of SIRT5 in skeletal muscle aging in vivo, the expression level of SIRT5 protein in the skeletal muscle tissue of aged mice was first examined. The results showed a significant decrease in SIRT5 protein levels in the skeletal muscle of aged mice (Figure 7c). Compared with mice injected with luciferase-expressing lentivirus (control), mice treated with the SIRT5 expression vector showed increased SIRT5 levels in skeletal muscle (Figure 7d-E). SIRT5 intervention resulted in increased net-grabbing time, increased hanging time, and enhanced endurance in aged mice (Figure 7f-h). Furthermore, overexpression of SIRT5 in aged skeletal muscle delayed age-related phenotypes, including the alleviation of muscle atrophy, manifested as an increase in muscle fiber cross-sectional area, reversal of fast-twitch muscle fiber loss, reduced immune cell infiltration, and decreased expression levels of pro-inflammatory factors (Figure 7i-l, Figure 12e). RNA transcriptome sequencing results showed that SIRT5 gene therapy reset the skeletal muscle transcriptome to a more youthful state, characterized by reduced inflammation and improved muscle function (m-o in Figure 7, f-g in Figure 12). These results indicate that SIRT5-based gene therapy alleviates age-related muscle dysfunction in aged male mice, providing a novel therapeutic approach for skeletal muscle aging.

[0364] Example 8: Gene therapy based on adeno-associated virus (AAV)-mediated SIRT5-TBK1-RelA signaling axis to alleviate skeletal muscle degeneration and atrophy.

[0365] Experimental methods:

[0366] (1) Plasmid construction and optimization

[0367] The whole-genome human SIRT5 gene and the sequence-optimized SIRT5 gene, or TBK1 shRNA or siRNA, or RelA shRNA or siRNA, were synthesized and constructed using enzyme digestion and ligation to create corresponding AAV plasmids. The AAV plasmids were transfected into HEK293 cells using transfection reagents. After 72 hours, proteins were extracted, and the expression levels of the target proteins were compared using Western blotting. The optimal expression vector was selected.

[0368] (2) Packaging and purification of AAV

[0369] rAAV virus was packaged using a mature three-plasmid co-transfection system. The viral genome plasmid containing the Cβ promoter-driven human SIRT5 (hSIRT5) cDNA expression cassette, the pAAV2 / 9Rep-Cap packaging plasmid, and the pHelper helper plasmid were introduced into HEK293 cells via PEI transfection. 60-72 hours post-transfection, cells were collected and lysed using repeated freeze-thaw cycles with detergent to release viral particles. The cell lysates were clarified and purified using iodixanol density gradient ultracentrifugation. The purified virus was then transferred to PBS buffer containing 5% (w / v) D-sorbitol to obtain a high-titer rAAV9-Cβ-hSIRT5 viral stock solution, which was aliquoted and stored at -80°C for later use. The viral genome titer was determined to be no less than 1 × 10^13 vg / mL.

[0370] (3) Delivery of AAV virus in mouse bone

[0371] In the muscle tissue of mice with physiological aging and muscle atrophy models, control AAV and experimental AAV were delivered. The dosage and treatment time of the virus were optimized during the process, and behavioral and molecular level detection and analysis were performed after injection.

[0372] The experimental design for the intervention of physiological skeletal muscle aging is as follows:

[0373] Young-AAV9-Luc group: 20 male C57BL6 / J mice aged 3 months;

[0374] The aged-AAV9-Luc group consisted of 19 male C57BL6 / J mice aged 19 months.

[0375] The aged-AAV9-SIRT5 group consisted of 19 male C57BL6 / J mice aged 19 months.

[0376] Mice in each group were anesthetized by inhalation of 2% isoflurane. Hair on the chest was removed with depilatory cream, and then AAV expressing luciferase (Luc) or SIRT5 was injected at two points in each quadriceps femoris and tibialis anterior muscle. Each mouse received 5 x 10 injections per leg. 10 VG viral load. Behavioral and molecular-level analyses were performed 5 weeks after injection.

[0377] (4) Behavioral testing

[0378] Netting Experiment: A square grid was securely mounted on an elevated frame. A mat was placed beneath the grid to protect the mice during the test. Each mouse was gently placed in the center of the grid. The grid was then carefully inverted. The time from the moment of inversion until the mouse fell was recorded.

[0379] Rotating bar experiment: The rotating bar experiment was conducted using the Rota bar system (Yiyan Tech, YLS-4C). Mice underwent initial training for three days. In each experiment, each mouse was placed in a separate channel on the bar. The speed of pulling the bar was gradually increased from 4 revolutions per minute (rpm) to 44 revolutions per minute (rpm), with the acceleration always maintained at 8 revolutions per minute squared (rpm² / min). This continued until each mouse fell off the bar three times. The average time of each mouse's fall was carefully recorded and subsequently used for statistical analysis.

[0380] Exhaustion test: Before the formal experiment, mice were trained for three days on a treadmill (SANS Bio Instrument, SA101) at a 5° incline. During the training, 2 mA of electrical stimulation was used. Each test lasted 20 minutes, with the speed gradually increased from 5 to 20 m / min. On the test day, the mice ran on the treadmill at an acceleration of 2 rpm / min. When the mice could no longer run on the treadmill and remained on the electrodes for more than 10 seconds, the duration and distance were recorded.

[0381] Grasp force test: Grasp force was tested using a digital force measurement system. Mice were placed on the testing platform, allowing their paws to naturally grasp the grid bars. The mice were then pulled backward at a constant speed until they released their grip. The peak grip force was recorded in real time using an integrated sensor. Each mouse underwent 10 tests consecutively, and the average of the 10 tests was used for statistical analysis.

[0382] (5) Skeletal muscle tissue transcriptome sequencing

[0383] Total RNA was extracted from mouse skeletal muscle tissue using TRIzol reagent. After assessing RNA quality, libraries were constructed and each sample was subjected to high-throughput sequencing. In short, using... UltraTM RNA Library Prep Kit for Libraries were prepared at NEB (USA), and each sample was indexed. The libraries were then sequenced at 150-base-pair reads on an Illumina paired-end sequencing platform (Novogene Bioinformatics Technology Co., Ltd.). The obtained raw transcriptome data were processed using Trim Galore software (version 0.4.5) to remove low-quality reads and adapter sequences. Subsequently, the genomes were aligned with a mouse MacFas 5.0 reference genome using HISAT2 software (version 2.0.4). HTSeq (version 0.11.0) was used to determine the expression level of each gene. The fragment-per-thousand (FPKM) count for each gene was calculated using StringTie (version 2.1.4). The R package DESeq2 (version 1.22.2) was used to identify differentially expressed genes (DEGs). Referring to previous studies involving transcriptomes of different tissues, the thresholds for DEG between groups were P < 0.01 and |log2(fold change)| > 0.5. Pathway enrichment analysis was performed using Metascape.

[0384] (6) Immunofluorescence staining

[0385] Refer to Example 1.

[0386] (7) RT-qPCR

[0387] Total RNA was extracted from mouse skeletal muscle tissue using TRIzol reagent (Thermo Fisher Scientific, #15596018), and then cDNA was synthesized by reverse transcription using the GoScript reverse transcription kit (Promega, #A5001). Subsequently, RT-qPCR was performed using Thunderbird qPCR Mix (TOYOBO, #QPS-201) reagent and a Real-Time System (Bio-Rad Laboratories, Inc. or Thermo Fisher Scientific).

[0388] Experimental Results: In HEK293 cells, AAV9-Luc (control) or AAV9-SIRT5 was infected with infection multiples of 1×10⁴, 5×10⁴, and 1×10⁵ vg / cell, respectively. Western blotting results showed that the AAV9-SIRT5 group specifically and efficiently expressed SIRT5 protein, and the expression level increased in a dose-dependent manner, demonstrating successful viral packaging and efficient delivery of the target gene (Figure 13a). Subsequently, in vivo animal experiments were conducted by injecting 1×10¹⁰ vg / leg of AAV9-Luc or AAV9-SIRT5 virus into the bilateral hind limb skeletal muscle of mice. Fourteen days after injection, Western blotting analysis showed that the expression level of SIRT5 protein in the skeletal muscle tissue at the AAV9-SIRT5 injection site was significantly increased, confirming the efficient expression of the vector in vivo (Figure 13b). To further investigate its function, intervention was carried out in aged (19-month-old) mice. Grasp strength test results showed that the grip strength of the young AAV9-Luc control group was significantly higher than that of the aged AAV9-Luc control group, confirming age-related muscle weakness decline. More importantly, the grip strength of the aged AAV9-SIRT5 intervention group was significantly higher than that of the aged AAV9-Luc control group. These results indicate that AAV9-SIRT5-mediated SIRT5 overexpression can effectively improve skeletal muscle function in aged mice and reverse age-related muscle weakness decline (Figure 13c).

[0389] Example 9: Gene therapy based on LNP-mediated SIRT5-TBK1-RelA signaling axis to alleviate skeletal muscle degeneration and atrophy

[0390] Experimental methods:

[0391] (1) LNP construction

[0392] Synthesize a shRNA, siRNA, sgRNA, or ASO targeting TBK1, or a shRNA, siRNA, sgRNA, or ASO targeting RelA. Mix the synthesized sequence with lipids and encapsulate the nucleic acid sequence into the lipids using physical or chemical methods (such as electroporation, sonication, etc.) to form LNPs.

[0393] (2) Purification and characterization of LNP: LNP was purified using techniques such as ultrafiltration, dialysis or chromatographic separation, and characterized using techniques such as dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0394] (3) LNP optimization: The delivery efficiency of LNPs and the expression level of the target protein were verified in a cell model. The preparation process was optimized based on the experimental results to improve the delivery efficiency of LNPs and the expression level of the target protein.

[0395] (4) LNP delivery in mouse bone

[0396] LNPs were delivered to the muscle tissue of mice in physiological aging and muscle atrophy models, targeting either the control or experimental groups. The dosage and processing time of the LNPs were optimized during the process. Behavioral and molecular-level analyses were performed post-injection.

[0397] (5) Behavioral testing

[0398] Netting Experiment: A square grid was securely mounted on an elevated frame. A mat was placed beneath the grid to protect the mice during the test. Each mouse was gently placed in the center of the grid. The grid was then carefully inverted. The time from the moment of inversion until the mouse fell was recorded.

[0399] Rotating bar experiment: The rotating bar experiment was conducted using the Rota bar system (Yiyan Tech, YLS-4C). Mice underwent initial training for three days. In each experiment, each mouse was placed in a separate channel on the bar. The speed of pulling the bar was gradually increased from 4 revolutions per minute (rpm) to 44 revolutions per minute (rpm), with the acceleration always maintained at 8 revolutions per minute squared (rpm² / min). This continued until each mouse fell off the bar three times. The average time of each mouse's fall was carefully recorded and subsequently used for statistical analysis.

[0400] Exhaustion test: Before the formal experiment, mice were trained for three days on a treadmill (SANS Bio Instrument, SA101) at a 5° incline. During the training, 2 mA of electrical stimulation was used. Each test lasted 20 minutes, with the speed gradually increased from 5 to 20 m / min. On the test day, the mice ran on the treadmill at an acceleration of 2 rpm / min. When the mice could no longer run on the treadmill and remained on the electrodes for more than 10 seconds, the duration and distance were recorded.

[0401] (6) Skeletal muscle tissue transcriptome sequencing

[0402] Total RNA was extracted from mouse skeletal muscle tissue using TRIzol reagent. After assessing RNA quality, libraries were constructed and each sample was subjected to high-throughput sequencing. In short, using... RNA Library Prep Kit for Libraries were prepared at NEB (USA), and each sample was indexed. The libraries were then sequenced at 150-base-pair reads on an Illumina paired-end sequencing platform (Novogene Bioinformatics Technology Co., Ltd.). The obtained raw transcriptome data were processed using Trim Galore software (version 0.4.5) to remove low-quality reads and adapter sequences. Subsequently, the genomes were aligned with a mouse MacFas 5.0 reference genome using HISAT2 software (version 2.0.4). HTSeq (version 0.11.0) was used to determine the expression level of each gene. The fragment-per-thousand (FPKM) count for each gene was calculated using StringTie (version 2.1.4). The R package DESeq2 (version 1.22.2) was used to identify differentially expressed genes (DEGs). Referring to previous studies involving transcriptomes of different tissues, the thresholds for DEG between groups were P < 0.01 and |log2(fold change)| > 0.5. Pathway enrichment analysis was performed using Metascape.

[0403] (7) Immunofluorescence staining

[0404] Refer to Example 1.

[0405] (8) RT-qPCR

[0406] Total RNA was extracted from mouse skeletal muscle tissue using TRIzol reagent (Thermo Fisher Scientific, #15596018), and then cDNA was synthesized by reverse transcription using the GoScript reverse transcription kit (Promega, #A5001). Subsequently, RT-qPCR was performed using Thunderbird qPCR Mix (TOYOBO, #QPS-201) reagent and a Real-Time System (Bio-Rad Laboratories, Inc. or Thermo Fisher Scientific).

[0407] Example 10: Alleviating skeletal muscle degeneration and atrophy based on small molecule-mediated activation of the SIRT5-TBK1-RelA signaling axis.

[0408] Experimental methods:

[0409] (1) Selection of candidate small molecules

[0410] One or more small molecules that activate SIRT5 expression, one or more small molecules that inhibit TBK1 succinylation or phosphorylation activation, or one or more small molecules that phosphorylate RelA are used in HEK293 or C2C12 cells at different concentrations and treatment times. The effects of these molecules on SIRT5 activation and / or inhibition of TBK1 and RelA activation are measured at protein expression levels, and the combination of one or more small molecules with the best activation effect is screened.

[0411] (2) Small molecule delivery in mice

[0412] In the muscle tissue of mice with physiological aging and muscle atrophy models, small molecules were delivered to either the control or experimental groups, with the dosage and treatment time of the small molecules optimized during the process. Behavioral and molecular-level analyses were performed after injection.

[0413] (3) Behavioral testing

[0414] Netting Experiment: A square grid was securely mounted on an elevated frame. A mat was placed beneath the grid to protect the mice during the test. Each mouse was gently placed in the center of the grid. The grid was then carefully inverted. The time from the moment of inversion until the mouse fell was recorded.

[0415] Rotating bar experiment: The rotating bar experiment was conducted using the Rota bar system (Yiyan Tech, YLS-4C). Mice underwent initial training for three days. In each experiment, each mouse was placed in a separate channel on the bar. The speed of pulling the bar was gradually increased from 4 revolutions per minute (rpm) to 44 revolutions per minute (rpm), with the acceleration always maintained at 8 revolutions per minute squared (rpm² / min). This continued until each mouse fell off the bar three times. The average time of each mouse's fall was carefully recorded and subsequently used for statistical analysis.

[0416] Exhaustion test: Before the formal experiment, mice were trained for three days on a treadmill (SANS Bio Instrument, SA101) at a 5° incline. During the training, 2 mA of electrical stimulation was used. Each test lasted 20 minutes, with the speed gradually increased from 5 to 20 m / min. On the test day, the mice ran on the treadmill at an acceleration of 2 rpm / min. When the mice could no longer run on the treadmill and remained on the electrodes for more than 10 seconds, the duration and distance were recorded.

[0417] (4) Skeletal muscle tissue transcriptome sequencing

[0418] Total RNA was extracted from mouse skeletal muscle tissue using TRIzol reagent. After assessing RNA quality, libraries were constructed and each sample was subjected to high-throughput sequencing. In short, using... RNA Library Prep Kit for Libraries were prepared at NEB (USA), and each sample was indexed. The libraries were then sequenced at 150-base-pair reads on an Illumina paired-end sequencing platform (Novogene Bioinformatics Technology Co., Ltd.). The obtained raw transcriptome data were processed using Trim Galore software (version 0.4.5) to remove low-quality reads and adapter sequences. Subsequently, the genomes were aligned with a mouse MacFas 5.0 reference genome using HISAT2 software (version 2.0.4). HTSeq (version 0.11.0) was used to determine the expression level of each gene. The fragment-per-thousand (FPKM) count for each gene was calculated using StringTie (version 2.1.4). The R package DESeq2 (version 1.22.2) was used to identify differentially expressed genes (DEGs). Referring to previous studies involving transcriptomes of different tissues, the thresholds for DEG between groups were P < 0.01 and |log2(fold change)| > 0.5. Pathway enrichment analysis was performed using Metascape.

[0419] (5) Immunofluorescence staining

[0420] Refer to Example 1.

[0421] (6) RT-qPCR

[0422] Total RNA was extracted from mouse skeletal muscle tissue using TRIzol reagent (Thermo Fisher Scientific, #15596018), and then cDNA was synthesized by reverse transcription using the GoScript reverse transcription kit (Promega, #A5001). Subsequently, RT-qPCR was performed using Thunderbird qPCR Mix (TOYOBO, #QPS-201) reagent and a Real-Time System (Bio-Rad Laboratories, Inc. or Thermo Fisher Scientific).

[0423] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

Application of substances that regulate the SIRT5-TBK1-RelA signaling pathway in any of the following A1)-A5): A1) Delay the aging of the body, organs, tissues, or cells, such as the degeneration or atrophy of muscles, especially skeletal muscles; A2) Prepare products (including drugs) that delay the aging of the body, organs, tissues or cells, such as muscles, especially skeletal muscles, to slow down degeneration or atrophy. A3) Improves dysfunction of aging organisms, organs, tissues, or cells, such as muscle, especially skeletal muscle degeneration or atrophy; A4) Prepare products to improve the functional impairment of aging organisms, organs, tissues or cells, such as muscle, especially skeletal muscle degeneration or atrophy; A5) Prepare products for studying or assisting in the study of the aging of an organism, organ, tissue or cell, such as the mechanisms of muscle, especially skeletal muscle, degeneration or atrophy. The substances that regulate the SIRT5-TBK1-RelA signaling pathway include substance R and / or substance S and / or substance T; The substance R is SIRT5 protein or a substance that upregulates, enhances, or increases the activity and / or content of SIRT5 protein; The substance S is a substance that reduces the succinylation and / or phosphorylation level of TBK1 protein; The substance T is a substance that downregulates, inhibits, or reduces the activity and / or content of RelA protein; The substance R includes, for example, nucleic acids encoding the SIRT5 protein or expression vectors containing nucleic acids encoding the SIRT5 protein, such as retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, plasmids, mRNA-lipid nanoparticles, preferably adeno-associated virus vectors, especially AAV9 vectors, lipid nanoparticles containing mRNA encoding the SIRT5 protein, or lipid nanoparticles containing mRNA encoding enhanced SIRT5 protein expression; or small molecule activators of the SIRT5 protein; The substance S includes, for example, siRNA or ASO targeting TBK1 mRNA or an expression vector of the ASO thereof, or an expression vector of shRNA or sgRNA targeting TBK1, or overexpressed SIRT5 protein, or HDAC6 activators, HDAC3 regulators, p300 / CBP acetyltransferase inhibitors such as C646 and curcumin; IKKε activators, ubiquitination regulators, natural compounds such as resveratrol, MRT67307, BX795, GS-6624, CCX872; The substance T includes, for example, BAY 11-7082, TPCA-1, PRI-724, JSH-23, MLN4924, MZ1, and siRNA or ASO or an expression vector of ASO targeting RelA mRNA, or an expression vector of shRNA or sgRNA targeting RelA, miR-146a mimic, curcumin, resveratrol, propionic acid, acarbose, or a RelA neutralizing antibody such as AMG510. A product whose active ingredient is a substance that regulates the SIRT5-TBK1-RelA signaling pathway; the function of the product is either B1) or B2) below: B1) Delaying the aging of the body, organs, tissues, or cells, such as muscle, especially skeletal muscle degeneration or atrophy; B2) Improves dysfunction of aging organisms, organs, tissues, or cells, such as muscle, especially skeletal muscle degeneration or atrophy; The substances that regulate the SIRT5-TBK1-RelA signaling pathway include substance R and / or substance S and / or substance T; The substance R is SIRT5 protein or a substance that upregulates, enhances, or increases the activity and / or content of SIRT5 protein; The substance S is a substance that reduces the succinylation and / or phosphorylation level of TBK1 protein; The substance T is a substance that downregulates, inhibits, or reduces the activity and / or content of RelA protein; The substance R includes, for example, nucleic acids encoding the SIRT5 protein or expression vectors containing nucleic acids encoding the SIRT5 protein, such as retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, plasmids, mRNA-lipid nanoparticles, preferably adeno-associated virus vectors, especially AAV9 vectors, lipid nanoparticles containing mRNA encoding the SIRT5 protein, or lipid nanoparticles containing mRNA encoding enhanced SIRT5 protein expression; or small molecule activators of the SIRT5 protein; The substance S includes, for example, siRNA or ASO targeting TBK1 mRNA or an expression vector of its ASO, or an expression vector of shRNA or sgRNA targeting TBK1; substance R or overexpressed SIRT5 protein; or HDAC6 activator, HD AC3 regulator, p300 / CBP acetyltransferase inhibitor, such as C646 and curcumin; IKKε activator, ubiquitination regulator, natural compound such as resveratrol; MRT67307, BX795, GS-6624, CCX872; The substance T includes, for example, BAY 11-7082, TPCA-1, PRI-724, JSH-23, MLN4924, MZ1, and siRNA or ASO or an expression vector of ASO targeting RelA mRNA, or an expression vector of shRNA or sgRNA targeting RelA, miR-146a mimic, curcumin, resveratrol, propionic acid, acarbose, or a RelA neutralizing antibody such as AMG510. Use according to claim 1 or product according to claim 2, characterized in that: The organ in question is skeletal muscle; Alternatively, the substance that upregulates, enhances, or increases the activity and / or content of SIRT5 protein is a nucleic acid molecule encoding SIRT5 protein or an expression cassette, recombinant vector, recombinant microorganism, or recombinant cell line containing the nucleic acid molecule; Alternatively, the substance that reduces the activity or content of RelA protein may be a substance that inhibits RelA protein synthesis, promotes RelA protein degradation, or knocks down or eliminates the RelA gene; or, the substance that downregulates, inhibits, or reduces the activity or content of RelA protein may be an RNA molecule that inhibits, reduces, or downregulates the expression of the gene encoding RelA protein, or an RNA molecule that inhibits, reduces, or downregulates the activity or content of the protein, or the gene encoding the RNA molecule, or an expression cassette containing the gene, or a recombinant vector, recombinant microorganism, or recombinant cell line containing the gene or expression cassette. The use of substances for detecting SIRT5 expression levels and / or substances for detecting TBK1 protein succinylation and / or phosphorylation levels and / or substances for detecting RelA expression levels in any of the following C1) or C2): C1) Identify or assist in the identification of aging of an organism, organ, tissue, or cell, such as the level of degeneration or atrophy of muscles, especially skeletal muscles; C2) Prepare products to identify or assist in identifying the level of aging in an organism, organ, tissue, or cell, such as muscle, especially skeletal muscle, degeneration or atrophy. A product for identifying or assisting in the identification of aging of an organism or organ or tissue or cell, or muscle, particularly skeletal muscle, at a level of degeneration or atrophy, comprising substances for detecting SIRT5 expression levels and / or substances for detecting TBK1 protein succinylation or phosphorylation levels and / or substances for detecting RelA expression levels. Application of substances that regulate the SIRT5-TBK1-RelA signaling pathway in any of the following D1)-D6): D1) Constructing senescent cells; D2) Preparation of products for constructing senescent cells; D3) Screening or assisting in the screening of drugs that delay aging, such as muscle, especially skeletal muscle degeneration or atrophy; D4) Products (including cells, senescent cells prepared by the method of F4 of any one of claims 9-11) for screening or assisting in screening for drugs that delay aging, such as muscle, especially skeletal muscle degeneration or atrophy. D5) To study or assist in the study of aging of the body, organs, tissues or cells, such as the mechanisms of muscle, especially skeletal muscle degeneration or atrophy; D6) Prepare products for studying or assisting in the study of the aging of the body, organs, tissues or cells, such as the degeneration or atrophy of muscles, especially skeletal muscles; The substances that regulate the SIRT5-TBK1-RelA signaling pathway include substance X and / or substance Y and / or substance Z; The substance X is a substance that downregulates, inhibits, or reduces the activity and / or content of SIRT5 protein; The substance Y is a substance that increases the succinylation and / or phosphorylation level of TBK1 protein; The substance Z is RelA protein or a substance that upregulates, enhances, or increases the activity and / or content of RelA protein; The substance X includes, for example, a CRISPR / Cas9 gene editing system that targets and knocks out SIRT5 (e.g., which includes a Cas9 endonuclease and an sgRNA that targets the SIRT5 gene, the target sequence of which is, for example, GCGAGTCGTGG TC); small molecule inhibitors that target SIRT5 enzyme activity, such as AK-7, SirReal2, SIRT5-IN-1, C-176, and EX-527 derivatives; substances that reduce SIRT5 protein content, such as SIRT5-PROTAC-1, and molecular glue degraders, such as ML265 analogs; gene silencing tools, such as siRNA and shRNA lentiviral vectors that target the SIRT5 mRNA 3'UTR; siRNA that targets SIRT5 mRNA; and natural compounds and endogenous regulatory molecules, such as quercetin, berberine, α-ketoglutarate, estrogen, and SIRT5 neutralizing antibodies; The substance Y includes, for example, succinic acid, IKKε agonists, MRT67307, berberine, and N-acetylcysteine; The substance Z includes, for example, classical NF-κB pathway activators such as TNF-α, IL-1β, lipopolysaccharide, EBV-LM P1; IKK kinase agonists such as IKKε selective activators such as PS1145; NEMO binding domain peptides; TAK1 agonists such as 5Z-7-oxozeanol; RelA dephosphorylation inhibitors such as okadaic acid and Calyculin A; RelA protein stability enhancers such as MI-63, MG132, emetine, and miR-146a inhibitors; HDAC inhibitors such as trichostatin A and C646; chromatin remodeling agents such as RG108; curcumin, triptolide, and lenalidomide. A product whose active ingredient is a substance that regulates the SIRT5-TBK1-RelA signaling pathway; the function of the product is any one of the following E1)-E3): E1) Constructing senescent cells; E2) Prepare screening or assist screening for drugs that delay aging, such as muscle, especially skeletal muscle degeneration or atrophy; E3) Research or assist in the research of aging of the body, organs, tissues or cells, such as the mechanisms of muscle, especially skeletal muscle degeneration or atrophy; The substances that regulate the SIRT5-TBK1-RelA signaling pathway include substance X and / or substance Y and / or substance Z; The substance X is a substance that downregulates, inhibits, or reduces the activity and / or content of SIRT5 protein; The substance Y is a substance that increases the succinylation and / or phosphorylation level of TBK1 protein; The substance Z is RelA protein or a substance that upregulates, enhances, or increases the activity and / or content of RelA protein; The substance X includes, for example, a CRISPR / Cas9 gene editing system that targets and knocks out SIRT5 (e.g., which includes a Cas9 endonuclease and an sgRNA that targets the SIRT5 gene, the target sequence of which is, for example, GCGAGTCGTGG TC); small molecule inhibitors that target SIRT5 enzyme activity, such as AK-7, SirReal2, SIRT5-IN-1, C-176, and EX-527 derivatives; substances that reduce SIRT5 protein content, such as SIRT5-PROTAC-1, and molecular glue degraders, such as ML265 analogs; gene silencing tools, such as siRNA and shRNA lentiviral vectors that target the SIRT5 mRNA 3'UTR; siRNA that targets SIRT5 mRNA; and natural compounds and endogenous regulatory molecules, such as quercetin, berberine, α-ketoglutarate, estrogen, and SIRT5 neutralizing antibodies; The substance Y includes, for example, succinic acid, IKKε agonists, MRT67307, berberine, and N-acetylcysteine; The substance Z includes, for example, classical NF-κB pathway activators such as TNF-α, IL-1β, lipopolysaccharide, EBV-LM P1; IKK kinase agonists such as IKKε selective activators such as PS1145; NEMO binding domain peptides; TAK1 agonists such as 5Z-7-oxozeanol; RelA dephosphorylation inhibitors such as okadaic acid and Calyculin A; RelA protein stability enhancers such as MI-63, MG132, emetine, and miR-146a inhibitors; HDAC inhibitors such as trichostatin A and C646; chromatin remodeling agents such as RG108; curcumin, triptolide, and lenalidomide. Use according to claim 6 or product according to claim 7, characterized in that: The organ in question is skeletal muscle; Alternatively, the substance that reduces SIRT5 protein activity may be a protein, polypeptide, or small molecule compound that inhibits SIRT5 protein function; or, the substance that reduces SIRT5 protein content may be a substance that inhibits SIRT5 protein synthesis, promotes SIRT5 protein degradation, or knocks down or eliminates the SIRT5 gene; or, the substance that downregulates or inhibits or reduces SIRT5 protein content and / or activity may be an RNA molecule that inhibits or reduces or downregulates the expression of the gene encoding SIRT5 protein, or an RNA molecule that inhibits or reduces or downregulates the activity or content of the protein, or the gene encoding the RNA molecule, or an expression cassette containing the gene, or a recombinant vector, recombinant microorganism, or recombinant cell line containing the gene or expression cassette. Alternatively, the substance that upregulates, enhances, or increases the activity and / or content of RelA protein is a nucleic acid molecule encoding RelA protein or an expression cassette, recombinant vector, recombinant microorganism, or recombinant cell line containing the nucleic acid molecule. Any one of the following methods (F1)-F7): F1) A method for delaying the aging of an organism or organ or tissue or cell, such as muscle, particularly skeletal muscle degeneration or atrophy, comprising the step of contacting the substance that regulates the SIRT5-TBK1-RelA signaling pathway as described in claim 1 with the organism or organ or tissue or cell. F2) A method for improving dysfunction of aging organisms, organs, tissues or cells, such as muscle, especially skeletal muscle degeneration or atrophy, comprising the step of contacting the substance that regulates the SIRT5-TBK1-RelA signaling pathway as described in claim 1 with the aging organism, organ or tissue or cell. F3) A method for identifying or assisting in identifying the level of aging of an organism or organ or tissue or cell, such as muscle, particularly skeletal muscle, degeneration or atrophy, comprising the steps of detecting the expression level of SIRT5 and / or the succinylation and / or phosphorylation level of TBK1 protein and / or the expression level of RelA in said subject samples; F4) A method for constructing senescent cells, comprising: introducing the substance that regulates the SIRT5-TBK1-RelA signaling pathway as described in claim 6 into recipient animal cells to obtain recombinant cells, wherein the recombinant cells are senescent cells; F5) A method for screening or assisting in screening for drugs that delay aging, such as muscle, particularly skeletal muscle degeneration or atrophy, comprising the step of screening for drugs that delay aging using senescent cells constructed by the method described in F4). F6) A method for studying or assisting in the study of the mechanisms of aging of an organism or organ or tissue or cell, such as muscle, particularly skeletal muscle degeneration or atrophy, including the steps of studying the mechanisms of aging of an organism or organ or tissue or cell using senescent cells constructed by the method described in F4); F7) A method for altering the senescent state of isolated cells, comprising the step of introducing a substance that regulates the SIRT5-TBK1-RelA signaling pathway as described in claim 1 or a substance that regulates the SIRT5-TBK1-RelA signaling pathway as described in claim 6 into recipient cells. The method of claim 9, wherein: The organ in question is skeletal muscle. According to the method of claim 10, the substance regulating the SIRT5-TBK1-RelA signaling pathway includes at least one of N1, N2, and N3, wherein N1 is any one of the following: N1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the SIRT5 protein of claim 9, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the protein, or the gene encoding the RNA molecule, or an expression cassette containing the gene, or a recombinant vector, recombinant microorganism, or recombinant cell line containing the gene or expression cassette. N2) Substances that increase the succinylation and / or phosphorylation levels of TBK1 protein; N3) Substances that upregulate, enhance, or increase the activity and / or content of RelA protein are nucleic acid molecules encoding RelA protein or expression cassettes, recombinant vectors, recombinant microorganisms, or recombinant cell lines containing said nucleic acid molecules. A cell model, characterized in that, The cell model is a senescent cell, such as a muscle, in particular skeletal muscle, degenerating or atrophying cell, prepared according to the method of F4) of any one of claims 9-11.

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