Use of GRP78-like peptides

Abstract

The present invention relates to a use of GRP78, and a similar peptide thereof or a nucleic acid encoding same. The peptide or nucleic acid according to the present invention can increase the expression of a biomarker having reduced expression in senescent cells, and thus is useful for restoring functions, and the like, of muscle tissue of which functions are reduced by aging.

Description

Uses of GRP78-like peptides

[0001] The present invention relates to the use of GRP78 and its analogue peptides or nucleic acids encoding them, and more particularly, to a composition comprising GRP78 and its analogue peptides or nucleic acids encoding them for inhibiting or reversing aging of cells, tissues or organs, or restoring or regenerating the function of aged or damaged cells, tissues or organs, a pharmaceutical composition for preventing or treating muscle diseases, and a cosmetic composition for preventing or reversing aging.

[0002]

[0003] Aging refers to the gradual deterioration of the body's structure and functions as we age. First, differentiation and proliferation decrease at the cellular level, leading to changes in the structure of specific molecules and altered response pathways. This, in turn, leads to inevitable anatomical and physiological changes that extend beyond the cellular and molecular levels. Homeostasis in organs and systems deteriorates, dramatically increasing susceptibility to external stress, disease, and death (Cynthia J Kenyon, Nature. 2010 Mar 25;464(7288):504-12).

[0004] Currently, developed countries around the world, including Korea, are facing rapid social structural changes due to an aging population. Beyond a passive response to societal aging, we need to understand the biological principles of aging and, furthermore, develop new technologies to control and reverse it. In 2018, the World Health Organization (WHO) included aging in its 2018 International Classification of Diseases-11 (ICD-11) classification, including its disease code, among preventable and treatable conditions.

[0005] Telomeres are DNA-protein regions located at the ends of chromosomes, protecting their ends. When a parent cell divides to produce daughter cells, there is a limit to how fully it can replicate the ends of its DNA molecules. This leads to gradual loss of telomeres with each cell division. This phenomenon is specific to aging, and unless other mechanisms exist to prevent telomere attrition, telomeres will continue to shorten with each cell division.

[0006] When telomeres shorten below a certain length, cells reach senescence and stop dividing, resulting in cell death, which accelerates human aging (Saliha Rizvi, et al., Curr Aging Sci. 2014;7(3):161-7). In humans, a deficiency of telomerase, which is involved in telomere end formation, can cause various diseases, such as pulmonary fibrosis, dyskeratosis congenita, and aplastic anemia, which are caused by a lack of the ability of specific tissues to regenerate (Joao Pedro de Magalhaes, Joao F Passos, Mech Ageing Dev. 2018 Mar:170:2-9). The ends of long, connected genomes are broken, and when cells recognize this as DNA damage, the DNA repair system can cause incorrect genome ligation. To prevent this, telomeres are protected by a protein complex called shelterin, which binds to the ends of DNA. Even if telomeres do not shorten, problems with shelterin can disrupt the protective structure, potentially affecting cell aging and survival. Problems with shelterin can also damage the telomere protective structure and lead to chromosomes mispairing. These problems can also lead to decreased tissue resilience, accelerated aging, and impacted cell survival. The causal relationship between telomere loss, cellular aging, and individual-level aging has been studied through genetic modification experiments using animal models. In particular, mice engineered to have shorter or longer telomeres showed shorter or longer lifespans, respectively (Bruno Bernardes de Jesus et al., EMBO Mol Med. 2012 Aug;4(8):691-704).

[0007] Telomere length related factor-1 (TRF-1) encodes a telomere-specific protein that regulates telomere length and acts as a telomerase inhibitor, and its expression is reduced in senescent cells (Keiji Okamoto, et al., J Biol Chem. 2008 Aug 29;283(35):23981-8). Telomere length related factor-2 (TRF-2) exists at telomeres, forms the shelterin nucleoprotein complex, and plays a role in regulating telomere length, and its expression is reduced in senescent cells (Giacomo Buscemi, et al., Curr Biol. 2009 May 26;19(10):874-9). Protection of Telomeres 1 (POT1) encodes a nuclear protein involved in telomere maintenance, binds to TTAGGG repeats to regulate telomere length, and its expression is reduced in senescent cells (Tomas Aramburu, et al., Comput Struct Biotechnol J. 2020 Jul 3:18:1939-1946). Human telomerase reverse transcriptase (hTERT) is a component of the telomerase complex, a human telomerase reverse transcriptase, and its expression is reduced in senescent cells (Anastasia I Palamarchuk, et al., Biomedicines. 2023 Apr 4;11(4):1091). Glucose-regulated protein 78 (GRP78) plays a role in maintaining protein folding and state when newly synthesized proteins are translocated to the endoplasmic reticulum. In young cells, it is abundant on the cell surface, in aging cells, it is abundantly distributed in the nucleus, and in aging cells, its expression decreases (Da Hyeon Choi, et al., Biomaterials. 2021 Nov:278:121156).

[0008]

[0009] Accordingly, the present inventors have conducted extensive research to discover novel substances capable of reversing the transformation of senescent cells into young cells. As a result, they have discovered peptides that have the effect of increasing biomarkers whose expression is reduced in senescent cells. Surprisingly, when GRP78-like peptides are expressed in senescent cells, the expression pattern of senescent biomarkers changes to that of young cells. Furthermore, when GRP78-like peptides are administered to aged mice, the expression pattern of senescent biomarkers changes from that of young tissues to that of aged tissues, and tissue function is restored, thereby completing the present invention.

[0010]

[0011] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0012]

[0013] [Prior Art Literature]

[0014] (Non-patent literature 1) Cynthia J Kenyon, Nature. 2010 Mar 25;464(7288):504-12

[0015] (Non-patent document 2) Da Hyeon Choi, et al., Biomaterials. 2021 Nov:278:121156

[0016]

[0017] Summary of the invention

[0018] The purpose of the present invention is to provide a peptide and its use that can inhibit or reverse aging and restore or regenerate the function of cells, tissues or organs related to aging by increasing biomarkers whose expression is reduced in aging cells.

[0019]

[0020] To achieve the above purpose, the present invention provides a composition for inhibiting or reversing aging of a cell, tissue or organ, or restoring or regenerating the function of an aged or damaged cell, tissue or organ, comprising GRP78, a similar peptide thereof or a nucleic acid encoding the same as an active ingredient.

[0021]

[0022] Figure 1 shows the plasmid map of the GRP78 expression vector and the control vector.

[0023] Figure 2 shows the level of GRP78 mRNA expression after injecting the GRP78 expression vector into different cells. NT indicates the untreated experimental group, Mock indicates the experimental group introduced only with the control vector, and GRP78 indicates the experimental group introduced with the GRP78 expression vector.

[0024] Figure 3 shows the results of confocal microscopy after transfection of cells (young and aged cells) with a vector carrying GRP78-EGFP. NT is the untreated experimental group, Mock is the experimental group introduced with only the control vector, and GRP78 is the experimental group introduced with the GRP78-EGFP expression vector (blue: nucleus, red: F-actin, green: GRP78-EGFP).

[0025] Figure 4 shows the results of measuring the cluster ratio of cells expressing GRP78-EGFP using FACS after injecting the GRP78-EGFP expression vector into young and aged cells.

[0026] Figure 5 is a graph showing the change in mRNA expression of TERT, a factor involved in telomere length, when GRP78 was expressed in dental stem cells, DPSC and PDLSC. NT represents the untreated experimental group, pcDNA represents the experimental group introduced with only the control vector, and GRP78 represents the experimental group introduced with the GRP expression vector (*p<0.05, **p<0.01).

[0027] Figure 6 shows the overall process of measuring the histological changes in muscles when a GRP78 expression vector was introduced into the muscles of mice of each age group.

[0028] Figure 7 is a confocal micrograph showing the expression of GRP78 confirmed by fluorescence when the GRP78-EGFP expression vector was injected into the muscle of a mouse.

[0029] Figure 8 is a graph showing the results of confirming the mRNA expression level of GRP78 when a GRP78 expression vector was introduced into mice of each age group (****p<0.0001).

[0030] Figure 9 shows the results of confirming the level of GRP78 protein expression in tissues using Western blotting in mice of each age group.

[0031] Figure 10 shows the results comparing the changes in muscle bundle thickness when GRP78 was expressed in mice of each age group.

[0032] Figure 11 shows the results of confirming the changes in cell characteristics according to aging in periodontal ligament tissue-derived cells of young rats (11 weeks old, control group) and aged rats (96 weeks old, aged group). Figure 11A shows the results of measuring the cell length and width of the control group and aged group (**p<0.01). Figure 11B shows the results of measuring the absorbance using the SA-β gal staining method for the expression of beta galactosidase, which is highly expressed in aged cells (***p<0.001). Figure 11C shows the results of measuring the gene expression levels of aging-related biomarkers, telomere length related factor-1 (TRF-1), telomere length related factor-2 (TRF-2), protection of telomeres 1 (POT1), and glucose-regulated protein 78 (GRP78), by RT-PCR (**p<0.01, ***p<0.001). Figure 11D shows the results of measuring cell proliferation (***p<0.001).

[0033] Figure 12 shows the results of observing the cell permeability of peptides of sequence numbers 1 to 4 using a confocal microscope. Figure 12A shows the cell permeability results of young rPDL, and Figure 12B shows the cell permeability results of old rPDL. Sequence 1 is the group treated with the peptide of sequence number 1, Sequence 2 is the group treated with the peptide of sequence number 2, Sequence 3 is the group treated with the peptide of sequence number 3, and Sequence 4 is the group treated with the peptide of sequence number 4. The above descriptions apply equally to the subsequent drawings.

[0034] Figure 13 shows the results of measuring changes in the expression of aging-related biomarkers by peptides of sequence numbers 1 to 4. Figure 13A shows the results of measuring changes in the expression of aging-related biomarkers TRF-1, TRF-2, and human telomerase reverse transcriptase (hTERT) by peptides of sequence numbers 1 to 4 in TMSC human tonsil tissue-derived stem cells (passage 20) aged by continuous culture, measured by RT-PCR (*p<0.05, **p<0.01, ***p<0.001). Figure 13B shows the results of RT-PCR measurements of changes in the expression of aging-related biomarker genes TRF-1, TRF-2, POT1, and GRP78 by peptides of SEQ ID NOS: 1 to 4 in periodontal ligament tissue-derived cells extracted from young rats (12 weeks old) and aged rats (96 weeks old) ($p<0.001, compared to Young rPDL_control and Old rPDL_control, p<0.001, *** p<0.001, ** p<0.01, *p<0.05, compared to the control when peptide treated). Seq 1 is the group treated with the peptide of SEQ ID NO: 1, Seq 2 is the group treated with the peptide of SEQ ID NO: 2, Seq 3 is the group treated with the peptide of SEQ ID NO: 3, and Seq 4 is the group treated with the peptide of SEQ ID NO: 4. The above descriptions also apply equally to the subsequent drawings.

[0035] Figure 14 shows the results of measuring the time that 8-week-old, 48-week-old, and 72-week-old mice remained on a rotating rod without falling. NT is the untreated experimental group, sequence 3 is the group treated with the peptide of sequence number 3, and sequence 4 is the group treated with the peptide of sequence number 4.

[0036] Figure 15 shows the peptide administration schedule for mice at each age. Seq 19 represents the muscle regeneration peptide of sequence number 19. The above description also applies to subsequent drawings.

[0037] Figure 16 shows the results of H&E staining and measurement of muscle fiber diameter of mouse muscle tissue administered with peptides of sequence number 3 or sequence number 4 for 4 weeks.

[0038] Figure 17 shows the results of H&E staining and measurement of muscle fiber diameter of mouse muscle tissues that were injected with the peptide of sequence number 3 or sequence number 4 for 4 weeks, and then additionally administered with the peptide of sequence number 3 or sequence number 4 alone or in combination with the muscle regeneration peptide (SEQ ID NO: 19) for 4 weeks (*p<0.05, **p<0.01).

[0039] Figure 18 shows the results of measuring fluorescence intensity after confocal microscopic observation of the expression of MyoD1 in muscle tissue of mice administered peptides of sequence numbers 3 and 4 for 4 weeks (**p<0.01, ****p<0.0001).

[0040] Figure 19 shows the results of measuring fluorescence intensity after confocal microscopic observation of the expression of MYH in muscle tissue of mice administered peptides of sequence numbers 3 and 4 for 4 weeks (***p<0.001, ****p<0.0001).

[0041] Figure 20 shows the results of confocal microscopic observation of the expression of MyoD1 in the muscles of mice that were injected with the peptide of sequence number 3 or sequence number 4 for 4 weeks, and then administered the peptide of sequence number 3 or sequence number 4 alone for an additional 4 weeks, or administered the peptide of sequence number 3 or sequence number 4 in combination with a muscle regeneration peptide (SEQ ID NO: 19) (*p<0.05, ***p<0.001, ****p<0.0001).

[0042] Figure 21 shows the results of measuring fluorescence intensity after confocal microscopic observation of MYH expression in the muscles of mice that were injected with the peptide of sequence number 3 or sequence number 4 for 4 weeks, and then administered the peptide of sequence number 3 or sequence number 4 alone for an additional 4 weeks, or administered the peptide of sequence number 3 or sequence number 4 in combination with a muscle regeneration peptide (SEQ ID NO: 19) (****p<0.0001).

[0043] Figure 22a is a graph showing changes in human telomerase gene expression when young TMSC cells and aged TMSC cells were treated with a GRP78-like peptide.

[0044] Figure 22b shows the activity of telomerase compared in young and aged cells using the TRAP assay, which can visually compare the length of telomerase, an enzyme that elongates telomeres (***p<0.001).

[0045] Figure 22c shows the length of telomeres, a representative indicator of aging, measured by the TRF assay, which measures the length of TRF (telomere restriction fragment) in young and aged cells (*p<0.05).

[0046] Figure 22d shows the results of Southern blotting to determine the length of visible telomeres in young and aged cells.

[0047] Figure 23a shows the results of changes in mitochondrial protein expression by treating young and aged TMSC cells with a GRP78-like peptide labeled with a fluorescent material (Rhodaime B).

[0048] Figure 23b is a graph showing the results of quantitative analysis of the protein expression results shown in Figure 12A using the Image J program.

[0049] Figure 23c is a graph showing the results of measuring changes in the copy number of mtDNA using qRT-PCR to confirm quantitative changes in mitochondrial DNA (mtDNA).

[0050] Figure 23d shows the results of changes in protein expression of factors related to mitochondrial activity and biogenesis in young and aged cells (*p<0.05, **p<0.01, ***p<0.001).

[0051] Figure 23e shows the results of changes in gene and protein expression caused by GRP78-like peptides of PPARγ, PGC1α, and p53, which are factors associated with mitochondria and telomeres (**p<0.01, ****p<0.0001).

[0052]

[0053] Detailed description of the invention and preferred embodiments

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0055]

[0056] As used herein, the term "aging" refers to changes in the body's functions and conditions that occur over time, such as a decline in cell differentiation or proliferation capacity due to the increase in an individual's age. While the onset of aging varies from individual to individual, aging typically refers to a decline in the normal functions of cells, tissues, or organs over time. Aging progresses independently of disease. Aging increases the incidence of disease, and in individuals with disease, it can accelerate the functional decline of damaged cells, tissues, or organs.

[0057] In an in vitro experimental setting, “aging” can refer to a decrease in a cell’s differentiation or proliferation capacity due to an increase in passage number. As used herein, the term “passage” refers to a method of continuously culturing cells in a healthy state for a long period of time by periodically transferring a portion of the cells to a new culture vessel and then changing the culture medium, thereby continuing the cell generation. This refers to replacing the culture vessel or dividing the cell population and culturing it. One culture vessel replacement or one cell population division and culturing is referred to as one passage.

[0058]

[0059] In one embodiment of the present invention, it was confirmed that the expression of aging-related biomarkers TRF-1, TRF-2, POT1, hTERT, and GRP78 genes was decreased in aged periodontal ligament (PDL) cells and tonsil-derived mesenchymal stem cells (TMSC), and it was confirmed that when the GRP78-like peptide according to the present invention was treated to the aged cells, the expression of the biomarker genes whose expression had been decreased was increased again. In addition, when the GRP78-like peptide according to the present invention was administered to a natural aging mouse model, it was confirmed that the muscle function of the aged mice was restored, and when administered to aged TMSC, the effect of restoring the expression of factors related to telomere length and restoring mitochondrial activity was confirmed. All of these are surprising effects that could not have been predicted at all in the past, separate from the usefulness of GRP78 as an aging-related biomarker.

[0060]

[0061] Accordingly, the present invention relates, in one aspect, to a composition for inhibiting or reversing aging of a cell, tissue or organ, or for restoring or regenerating the function of an aged or damaged cell, tissue or organ, comprising GRP78, a similar peptide thereof or a nucleic acid encoding the same as an active ingredient.

[0062]

[0063] GRP78, a 78-kD glucose-regulated protein, is an endoplasmic reticulum (ER) chaperone protein, also known as BiP or HSPA5. GRP78 is known to play a crucial role not only in protein folding but also in regulating the unfolded protein response (UPR). Under ER homeostasis, GRP78 normally binds to three UPR transmembrane sensors—ATF6, PERK, and IRE1—and maintains them in an inactive state. Under ER stress conditions, where unfolded proteins accumulate in the ER lumen, GRP78 dissociates from the UPR sensors and contributes to their activation. The activated UPR alleviates ER stress by decreasing protein translation and increasing ER folding capacity, a process in which GRP78 is upregulated. If ER homeostasis is not restored, the UPR can induce apoptosis.

[0064] GRP78 functions in a wide range of protein folding processes through two domains: the nucleotide binding domain (NBD), which binds and hydrolyzes ATP, and the substrate binding domain (SBD), which binds substrate proteins. The NBD consists of two subdomains (I and II), each of which is further divided into two smaller subdomains (A and B). These subdomains are separated by a cleft that binds a nucleotide, one Mg2+ ion, and two K+ ions. The SBD is also divided into two subdomains (alpha and beta). The SBD alpha subdomain provides a pocket for substrate protein binding, and the SBD beta subdomain acts as a lid covering the binding pocket.

[0065] GRP78 activity is regulated by an allosteric ATPase cycle. When ATP binds to the NBD, the lid (SBD beta subdomain) opens, causing a conformational change in the SBD that reduces GRP78's binding affinity to the substrate protein. When ATP is hydrolyzed, ADP binds to the NBD, closing the lid on the bound substrate. This increases the substrate protein's binding affinity and prevents the bound substrate protein from folding improperly. When ADP is replaced by ATP, the lid reopens, releasing the substrate protein. This ATPase cycle is tightly regulated by nucleotide binding and substrate protein binding. Substrate protein binding promotes ATP hydrolysis, whereas nucleotide binding influences either the binding or release of the substrate protein.

[0066] In the present invention, the amino acid sequence of GRP78 and the base sequence encoding it can be obtained from a known database such as NCBI's GenBank (e.g., GenBank Accession AAA52614.1).

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] The GRP78-like peptide according to the present invention may be a peptide that includes a part of the GRP78 amino acid sequence represented by SEQ ID NO: 21, and includes amino acid residues at which a phosphorylation or oxidation reaction of GRP78 may occur. The amino acid residues may preferably be selected from the group consisting of serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), methionine (M), tryptophan (W), glutamic acid (D), and proline (P), and the GRP78-like peptide according to the present invention may be characterized by including one or more, two or more, preferably three or more of the above amino acids. The peptide preferably has a length of 40-mer or less, 35-mer or less, 30-mer or less, or 25-mer or less.

[0074]

[0075] The GRP78-like peptide according to the present invention may be a peptide comprising an amino acid sequence identical or similar to the NBD represented by SEQ ID NO: 22 or a portion thereof. The portion of the NBD may be, for example, the IA, IB, IIA, or IIB subdomain or a portion thereof, but is not necessarily limited thereto. The similar amino acid sequence refers to an amino acid sequence having a sequence identity of at least 70%, at least 80%, at least 90%, or at least 95% with a reference amino acid sequence.

[0076]

[0077] The GRP78-like peptide according to the present invention may be a peptide comprising an amino acid sequence identical or similar to the SBD represented by SEQ ID NO: 23 or a portion thereof. The portion of the SBD may be, for example, an alpha or beta subdomain or a portion thereof, but is not necessarily limited thereto. The similar amino acid sequence refers to an amino acid sequence having a sequence identity of at least 70%, at least 80%, at least 90%, or at least 95% with a reference amino acid sequence.

[0078]

[0079] The GRP78-like peptide according to the present invention may be characterized by including an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 18 or a portion thereof, but is not limited thereto.

[0080]

[0081] Peptides of sequence numbers 1, 8, and 9 are peptides having amino acid sequences predicted to be susceptible to phosphorylation or oxidation among the entire sequence of the GRP78 protein based on protein structure and sequence analysis.

[0082] The peptides of SEQ ID NOS: 2 to 4 and 10 to 14 are peptides having amino acid sequences corresponding to a portion of the NBD of GRP78. The NBD is divided into sequences IA, IB, IIA, and IIB. The peptides of SEQ ID NOS: 2 and 3 include the amino acid sequence of IA, the peptides of SEQ ID NOS: 10 and 11 include the amino acid sequence of IB, the peptides of SEQ ID NOS: 12 and 13 include the amino acid sequence of IIA, and the peptides of SEQ ID NOS: 4 and 14 include the amino acid sequence of IIB. The above peptides recognize and bind to ATP, a substrate of GRP78.

[0083] The peptides of SEQ ID NOS: 15 to 18 are peptides having an amino acid sequence corresponding to a portion of the SBD of GRP78. The SBD is divided into the SBD alpha domain and the SBD beta domain, and the peptides of SEQ ID NOS: 15 and 16 include the amino acid sequence of the SBD alpha domain, and the peptides of SEQ ID NOS: 17 and 18 include the amino acid sequence of the SBD beta domain. The peptides bind to peptides that bind to other substrates, which are breakdown products generated when ATP, which is regulated by GRP78, is broken down.

[0084] Preferably, the GRP78-like peptide according to the present invention may be characterized by comprising an amino acid sequence having a sequence identity of 70% or more, 80% or more, 90% or more, 95% or more, or 100% or more with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 18, and more preferably, the GRP78-like peptide according to the present invention may be characterized by comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 18.

[0085] Preferably, the GRP78-like peptide according to the present invention may be characterized by comprising an amino acid sequence having a sequence identity of 70% or more, 80% or more, 90% or more, 95% or more, or 100% or more with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, and more preferably, the GRP78-like peptide according to the present invention may be characterized by comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

[0086] The GRP78-like peptide according to the present invention is interpreted to mean a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18, but also including variants or fragments thereof in which amino acid residues are conservatively substituted at specific amino acid residue positions. As used herein, “conservative substitution” means a modification of a peptide that includes substituting one or more amino acids with amino acids having similar biochemical properties that do not cause loss of the biological or biochemical function of the peptide.

[0087] A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues with similar side chains are well-defined and known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0088] It is expected that the GRP78 or a similar peptide thereof of the present invention may still retain activity even if it has conservative amino acid substitutions.

[0089]

[0090] Without being bound by theory, the GRP78-like peptide of the present invention may act as a substance that mimics the effect of the GRP78 protein by regulating the unique biochemical interactions of the GRP78 protein (e.g., interactions for regulating homeostasis of ATP and calcium, binding to substrate proteins for regulating protein unfolding, etc.) by including amino acid residues that are likely to undergo phosphorylation or oxidation reaction of GRP78, including amino acid residues included in the NBD of GRP78, or including amino acid residues included in the SBD of GRP78.

[0091] The active ingredient according to the present invention may preferably be characterized by increasing the expression of GRP78 when delivered to a cell. Here, increasing the expression of GRP78 includes directly or indirectly increasing the expression of GRP78. Such a substance may be a peptide comprising a portion of the GRP78 amino acid sequence represented by SEQ ID NO: 21, or an NBD represented by SEQ ID NO: 22, as described above, or a portion thereof, or an SBD represented by SEQ ID NO: 23, or a portion thereof, and a peptide comprising three or more amino acids selected from the group consisting of serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), methionine (M), tryptophan (W), glutamic acid (D), and proline (P).

[0092] The above peptide preferably has a length of 40-mer or less, 35-mer or less, 30-mer or less, or 25-mer or less.

[0093]

[0094] When the active ingredient according to the present invention is a nucleic acid encoding GRP78, the nucleic acid may be DNA, mRNA, plasmid DNA, etc., and may be delivered into cells by a cell-penetrating functional nanocarrier. The nucleic acid may be used with various delivery vehicles such as lipid nanoparticles (LNPs) and liposomes, which are known to effectively deliver oligonucleotides into cells, but are not limited thereto.

[0095]

[0096] GRP78 according to the present invention, a peptide analog thereof or a nucleic acid encoding the same may be useful for inhibiting or reversing aging of cells, tissues or organs, or for restoring or regenerating the function of aged or damaged cells, tissues or organs.

[0097] In the present invention, the cell, tissue or organ may be a cell, tissue or organ derived from a patient suffering from a disease or condition selected from the group consisting of muscle disease, bone disease, joint disease, skin disease, metabolic disease, urinary system disease, neurological disorder, cardiovascular disease, pulmonary dysfunction, cancer, immune disease, dental disease, periodontal disease and oral tissue dysfunction. The patient may be an adult patient, and may be characterized as being, for example, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older or 70 years of age or older.

[0098] Muscle mass decreases after the age of 30, and is affected by a decrease in growth hormone or androgen. Muscle diseases for which the GRP78 or a peptide analog thereof according to the present invention may be useful include, but are not limited to, sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, muscular dystrophy, atony, amyotrophic lateral sclerosis, cachexia, masticatory muscle disorder, temporomandibular disorder, or inflammatory myopathy.

[0099] Bone loss (osteoporosis) typically begins in men in their 50s and in women after menopause. The ligaments connecting joints lose elasticity, and arthritis develops due to long-term cartilage wear and inflammation, also a common joint disease associated with aging. Bone or joint diseases for which the GRP78 or a peptide analog thereof according to the present invention may be useful include osteoporosis, Paget's disease, rickets, osteomalacia, osteolysis, renal osteodystrophy in patients with renal failure, fractures, degenerative bone diseases, osteogenesis imperfecta, osteopenia, bone defects and hip dysplasia, osteoarthritis, degenerative arthritis, osteochondritis dissecans, postmeniscal injury arthrosis or arthritis, joint malalignment, avascular necrosis, arthroses, isolated chondral defects, chondromalacia patellae, synovitis, bursitis, traumatic effusion, ligamentous deficiency arthroses, osteochondritis dissecans (OCD), patellar instability, This may include, but is not limited to, rheumatoid arthritis, juvenile idiopathic arthritis, juvenile arthritis, post-traumatic arthritis, inflammatory arthritis, septic arthritis, lupus, scleroderma, tendonitis, fibromyositis, fibromyositis, or polymyositis.

[0100] As we age, our skin turnover cycle lengthens and subcutaneous fat decreases. Vitamin D deficiency is common because the skin's ability to synthesize vitamin D3 decreases. Skin becomes thinner, less elastic, drier, and more wrinkled. Other conditions associated with aging include cataracts, retinal degeneration, hearing loss, and decreased taste bud sensitivity. Skin diseases for which the GRP78 or a peptide analog thereof according to the present invention may be useful include, but are not limited to, pruritus erythematosus, seborrheic dermatitis, contact dermatitis, xeroderma, urticaria, acne, psoriasis, eczema, exfoliative dermatitis, hidradenitis, atopic dermatitis, drug allergies, diabetic dermatitis, bacterial dermatitis, fungal dermatitis, hidradenitis, allergic dermatitis, or neurodermatitis.

[0101] Metabolic diseases for which GRP78 or a peptide analog thereof according to the present invention may be useful include, but are not limited to, obesity, diabetes, hyperlipidemia, hypertriglyceridemia, liver disease, arteriosclerosis, stroke, myocardial infarction, cardiovascular disease, hyperglycemia, insulin resistance disease, or hyperinsulinemia.

[0102] As aging progresses, kidney weight and the number of glomeruli decrease and harden. Furthermore, renal vascular resistance increases, leading to decreased blood flow to the kidneys and a decreased glomerular filtration rate, which can lead to renal failure. The incidence of electrolyte abnormalities, glomerulonephritis, and chronic renal failure also increases. Decreased bladder storage capacity or weakened bladder muscles can lead to urinary incontinence, and in men, benign prostatic hyperplasia is a common complication. Urinary system diseases for which GRP78 or a peptide analog thereof according to the present invention may be useful include, but are not limited to, urinary incontinence, cystitis, nephritis, benign prostatic hyperplasia, prostatitis, overactive cystitis, and urinary stones.

[0103] As people age, various neurological and brain diseases, such as depression, delirium, mood and behavioral disorders, dementia, cerebrovascular disease, and Parkinson's disease, are observed. With aging, the number and weight of brain neurons decrease by approximately 10%, and the ventricular area increases. Fatty brown pigment accumulation in neurons, amyloid deposition in cerebral blood vessels, senile plaques, and neurofibrillary tangles increase, and reactive oxygen species production increases, all of which are associated with nervous system damage. Furthermore, there is a decrease in enzymes and receptors involved in neurotransmitter production, as well as a decrease in cerebral blood flow and glucose metabolism. Neurological disorders for which the GRP78 or analogue peptides thereof according to the present invention may be useful include, but are not limited to, spinal muscular atrophy, Friedreich's ataxia, CLN3 Batten, CLN6 Batten, CLN7 Batten, epileptic encephalopathy, Leigh syndrome, Charico-Marie-Tooth disease, giant axonal neuropathy, Lafora disease, SLC13A5 epileptic encephalopathy, congenital glycosylation disorder, Type Iq, Carridge syndrome, Angelman syndrome, Rett syndrome, spastic paraplegia, childhood alternating hemiplegia or Zellweger spectrum disorder.

[0104] With aging, the prevalence of cardiovascular diseases such as hypertension, aortic disease, heart failure, ischemic heart disease, arrhythmia, peripheral vascular disease, and valvular disease increases. The heart exhibits left ventricular hypertrophy and left atrial dilation with increasing age. This can be attributed to cardiomyocyte necrosis and enlargement, other factors such as decreased growth hormone levels, and localized collagen deposition. In addition, vascular elasticity decreases, which is due to decreased production of vascular endothelial cells and a decrease in nitric oxide (NO), a vasodilator, during the aging process. Cardiovascular diseases for which the GRP78 or a peptide analog thereof according to the present invention may be useful include, but are not limited to, myocardial infarction, angina pectoris, hypertension, heart failure, cardiomyopathy, primary cardiac arrest, congestive heart disease, ischemic heart failure, arteriosclerosis, arrhythmia, coronary artery disease, stroke, or peripheral vascular disease.

[0105] Age-related lung disease is associated with decreased lung function, including weakening of the respiratory muscles and diaphragm, decreased elastic recoil of the lungs, and decreased diffusing capacity (the rate of gas exchange between the lungs and the blood). The increased risk of airway obstruction in the elderly increases mortality from chronic obstructive pulmonary disease and cardiovascular disease, as well as the incidence of lung cancer. Diseases that may be useful for lung dysfunction, such as pulmonary hypertension, chronic obstructive pulmonary disease, pulmonary fibrosis, acute respiratory distress syndrome, bronchial asthma, inflammatory lung disease, pneumonia, or emphysema, include, but are not limited to, these diseases.

[0106] Cancer is sometimes called a disease of aging, and for example, it is known that the probability of developing cancer increases by more than 10 times after the age of 65. As we age, cell death decreases, which may increase the incidence of cancer. Cancers or immune-related diseases for which the GRP78 or a peptide analog thereof according to the present invention may be useful include, but are not limited to, cancer, rheumatoid arthritis, insulin-dependent diabetes, juvenile diabetes, systemic lupus erythematosus, atopic dermatitis, Crohn's disease, psoriasis, multiple sclerosis, hyperthyroidism, anemia, autoimmune diseases including Behcet's disease and autoimmune encephalomyelitis, and transplant rejection diseases including graft-versus-host disease.

[0107] Aging occurs throughout the body, but oral tissues, which play a crucial role in maintaining independent living, are particularly affected by aging. This can lead to functional decline, tooth extraction, recession, and degenerative loss of function in teeth, surrounding tissues, and secretory glands (such as salivary glands), threatening the quality of life and lives of older adults. Periodontal disease, which damages periodontal tissues due to factors such as periodontitis, weakens oral muscles (masticatory muscles), and causes loss of teeth, gum bone, and masticatory function, are common ailments associated with the elderly. Furthermore, aging leads to an overall decline in skeletal muscle mass, which can lead to various complications, including a decline in oral muscles (masticatory muscles), falls, fractures, and metabolic syndrome. Aging of oral tissues not only directly impacts tooth loss, gingival tissue damage and inflammatory destruction, and degenerative changes in secretory tissues such as the salivary glands, but also directly and indirectly impacts metabolic diseases such as heart disease, obesity, and diabetes, and neurological aging disorders such as Alzheimer's and Parkinson's disease. Dental or periodontal diseases for which the GRP78 or a peptide analog thereof according to the present invention may be useful include, but are not limited to, gingivitis, periodontitis, gingival pain, gingival bleeding, severe bad breath, gingival discoloration, tooth loosening, tooth movement, gingival recession, gingival edema, periodontal pocket formation, pericoronitis, or periodontal abscess.

[0108] The dental or periodontal disease for which the GRP78 or analogue peptide according to the present invention may be useful may be characterized by impaired function of oral tissues, and may have one or more characteristics selected from the group consisting of, but not necessarily limited to, tooth wear, tooth loss, gingival recession, loss of periodontal ligament attachment, alveolar bone loss, and decreased saliva secretion. Such dental or periodontal disease may be, but is not necessarily limited to, periodontitis, gingivitis, pulpitis, mucositis, exfoliative oral disorder, oral lichen planus, pemphigus vulgaris, stomatitis, root caries, alveolar bone defect, inflammatory fibrous hyperplasia, temporomandibular joint disorder, peri-implantitis, xerostomia, or oral cancer.

[0109] Diseases or conditions for which the GRP78 or analogue peptides thereof according to the present invention may be useful may be characterized by aged muscle cells or muscle tissue, and may be accompanied by one or more features selected from the group consisting of, but not limited to, muscle dysfunction, sarcopenia, muscle wasting, muscle wasting, and muscle degeneration. Such diseases or conditions may be selected from the group consisting of, but not necessarily limited to, dystonia, muscular atrophy, muscular degeneration, muscular rigidity, amyotrophic lateral sclerosis, myasthenia gravis, cachexia, masticatory muscle disorders, temporomandibular joint disorders, and sarcopenia.

[0110]

[0111] The GRP78-like peptide according to the present invention, particularly a peptide comprising an NBD represented by SEQ ID NO: 22 or a part of an amino acid thereof, or a peptide comprising an SBD represented by SEQ ID NO: 23 or a part of an amino acid thereof, or a peptide comprising a part of a GRP78 amino acid sequence represented by SEQ ID NO: 21, wherein the peptide comprises three or more amino acids selected from the group consisting of serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), methionine (M), tryptophan (W), glutamic acid (D) and proline (P), or a peptide capable of increasing the expression of GRP78, is thought to potentially reduce endoplasmic reticulum stress in a manner similar to the GRP78 protein that regulates endoplasmic reticulum stress, and thereby exhibits utility in inhibiting or reversing aging of cells, tissues or organs, or in restoring or regenerating the function of aging or damaged cells, tissues or organs. Therefore, the GRP78-like peptide according to the present invention, particularly the peptide comprising the NBD represented by SEQ ID NO: 22 or a part of its amino acid, or the peptide comprising the SBD represented by SEQ ID NO: 23 or a part of its amino acid, or the peptide comprising a part of the GRP78 amino acid sequence represented by SEQ ID NO: 21, wherein the peptide comprises three or more amino acids selected from the group consisting of serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), methionine (M), tryptophan (W), glutamic acid (D), and proline (P), or the peptide capable of increasing the expression of GRP78, may be useful for the prevention or treatment of various diseases associated with endoplasmic reticulum stress through such a mechanism.

[0112] In the present invention, the term “endoplasmic reticulum” refers to a very important cellular organelle where protein folding occurs during protein biosynthesis, and plays a very important role in maintaining cellular homeostasis, and the endoplasmic reticulum stress response occurring in the endoplasmic reticulum is closely related to the survival or death of cells, similar to the autophagy phenomenon.

[0113] The endoplasmic reticulum (ER) is a branched membrane structure extending from the nuclear envelope. It comes in two types: the rough ER, which has attached ribosomes, and the smooth ER, which does not have ribosomes. Approximately one-third of the proteins in a cell are transformed into active protein structures through post-translational modifications, such as folding, assembly, glycosylation, and disulfide bond formation, in the rough ER. The smooth ER is also a site for the synthesis of lipids and steroid hormones, and serves as a calcium storage facility, playing a crucial role in regulating intracellular calcium concentrations.

[0114] In this specification, the term "endoplasmic reticulum stress (ER stress)" refers to a disorder in the function of the ER due to the influx of immature proteins into the cell body beyond the capacity of the ER to process due to a physiological or pathological environment, or due to depletion of calcium within the ER. In other words, in a state of ER stress, abnormal unfolded proteins accumulate within the ER, which can cause various pathological conditions. Causative factors of ER stress include environmental changes within the cell due to diseases or the addition of chemical reagents, decreased glucose concentration, or the addition of inhibitors of sugar chain repair enzymes, and hyperphosphorylation of viral proteins due to viral infection. When ER stress occurs, cells develop a defense mechanism to survive, which is called the ER stress response.

[0115] The ER stress response is triggered by signals transmitted through ER transmembrane receptors located in the ER membrane, including pancreatic ER kinase (PKR)-like ER kinase (PERK), activation transcription factor 6, and inositol-requiring enzyme 1 (IRE1). In normal cells, these three ER stress receptors are inactivated by binding to the ER chaperone GRP78 / BiP. When unfolded proteins accumulate in the ER, GRP78 / BiP dissociates from the receptors, activating them and ultimately inducing the ER stress response. This induced ER stress response restores ER function and reduces the accumulation of unfolded proteins. However, if the accumulation of unfolded proteins continues and ER stress is not alleviated, the apoptosis pathway is activated, ultimately resulting in cell death.

[0116] The ER stress response is linked to mitochondrial function. For example, increased calcium release from the ER due to ER stress leads to mitochondrial dysfunction, including increased mitochondrial reactive oxygen species (ROS) production. Accordingly, the degree of ER stress can be identified by measuring changes in mitochondrial copy number and the rate of mitochondrial synthesis.

[0117] The “endoplasmic reticulum stress-related disease” referred to in this specification refers to a disease caused by the accumulation of unfolded proteins in the endoplasmic reticulum due to endoplasmic reticulum stress, and includes neurodegenerative diseases, chronic metabolic diseases, cancer, viral infections, etc.

[0118] Specifically, in the case of neurodegenerative diseases, it is known that neurodegenerative diseases are caused by protein tangles that generally occur with the progression of aging, and endoplasmic reticulum stress is frequently found in these diseases. Examples of the above neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, hypoxia of the brain and heart, cardiac hypertrophy, and arteriosclerosis. Regarding the relationship between the above diseases and endoplasmic reticulum stress, in the case of Alzheimer's disease, the PERK (PKR Like ER Kianse) pathway, which plays an important role in the expression and regulation of the GRP78 protein, and its downstream substrate eIF2a (eukaryotic translation initiation factor 2 alpha) pathway are activated in the patient's tissue samples, and this corresponds to the mechanism that explains the phenomenon of reducing endoplasmic reticulum stress by blocking protein synthesis within the cell as a whole by inactivating eIF2a. In the case of Parkinson's disease, Parkin, an E3 ligase protein, is known to suppress cell death caused by endoplasmic reticulum stress, and endoplasmic reticulum stress itself is known to increase the expression of Parkin. In the case of Huntington's disease, a part of the gene is amplified due to a mutation, and glutamine polymers that appear form protein tangles that are too large for the cell's decomposition capacity to handle. If this situation persists for a long time, endoplasmic reticulum stress occurs and nerve cell death is induced. Cerebral and cardiac hypoxia: Hypoxia (ischemia) symptoms that occur in the brain and heart cause endoplasmic reticulum stress by disrupting the cell's redox regulation function and increasing unfolded proteins.In addition, in the case of cerebral and cardiac hypoxia, hypoxia (ischemia) that occurs in the brain and heart causes an increase in unfolded proteins while disrupting the redox regulation function of cells, thereby inducing endoplasmic reticulum stress, and the reperfusion situation after hypoxia increases the amount of nitric oxide, which disrupts the balance of reactive oxygen species and s-nitrosylation in cells, thereby increasing oxidative stress within the cells again.

[0119] In the case of diabetes, which is a representative chronic metabolic disease, both type 1 diabetes and type 2 diabetes are known to be related to endoplasmic reticulum stress. In particular, in the case of type 2 diabetes, it is known that stress enzymes (stress kinases) activated by endoplasmic reticulum stress disrupt cell signaling initiated by insulin, thereby making cells insensitive to insulin and preventing normal glucose uptake.

[0120] In addition to the above diseases, immunoglobulin light chain amyloidosis, amyotrophic lateral sclerosis (ALS), hemodialysis-related amyloidosis, reactive amyloidosis, cystic fibrosis, sickle cell anemia, Creutzfeldt-Jakob disease, familial hypercholesterolaemia, alpha1 antitrypsin deficiency, cirrhosis, emphysema systemic, cerebral hereditary amyloidoses, Wolcott-Rallison syndrome, Wolfram syndrome, inflammatory Endoplasmic reticulum stress is known to be involved in the development of various diseases, including intestinal diseases, coronary artery disease, ulcerative colitis, breast cancer, and prostate cancer.

[0121] Therefore, it is suggested that if endoplasmic reticulum stress is controlled, the occurrence of endoplasmic reticulum stress-related diseases such as the above can be prevented and treated.

[0122]

[0123] The GRP78 or a similar peptide thereof or a nucleic acid encoding the same according to the present invention may be used for medical or cosmetic purposes. In this regard, the term "composition" herein may refer to a pharmaceutical composition or a cosmetic composition.

[0124]

[0125] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating muscle diseases, comprising GRP78, a peptide analog thereof, or a nucleic acid encoding the same.

[0126] In another aspect, the present invention relates to a method for preventing or treating muscle disease, comprising a step of administering the peptide or nucleic acid.

[0127] In another aspect, the present invention relates to the use of the peptide or nucleic acid for preventing or treating muscle diseases.

[0128] In another aspect, the present invention relates to the use of the peptide or nucleic acid for the manufacture of a medicament for preventing or treating muscle diseases.

[0129] In the present invention, the muscle disease may be characterized by being selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, muscular dystrophy, atony, amyotrophic lateral sclerosis, cachexia, masticatory muscle disorder, temporomandibular disorder, and inflammatory myopathy, but is not limited thereto.

[0130] The pharmaceutical composition of the present invention may include pharmaceutically acceptable additives. The additives include, but are not limited to, stabilizers, surfactants, lubricants, solubilizers, buffers, sweeteners, bases, adsorbents, maturing agents, binders, suspending agents, curing agents, antioxidants, brighteners, flavoring agents, flavoring agents, pigments, coating agents, humectants, moisture regulators, fillers, antifoaming agents, refreshing agents, chewing agents, antistatic agents, coloring agents, sugar coating agents, isotonic agents, softeners, emulsifiers, adhesives, thickeners, foaming agents, pH regulators, excipients, dispersants, disintegrants, waterproofing agents, preservatives, preservatives, solubilizing agents, solvents, fluidizing agents, and the like.

[0131] The pharmaceutical composition of the present invention may include pharmaceutically acceptable carriers, excipients, and diluents. The carriers, excipients, and diluents are commonly used and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0132] The composition of the present invention can be formulated into an injectable formulation such as an aqueous solution, suspension, emulsion, etc., a pill, a capsule, a granule, or a tablet, and thus, the composition of the present invention can be an injection, a patch, a liquid, a capsule, a granule, a tablet, a powder, a spray, an ointment, a gel, a mucosal administration formulation, a suppository, etc. These formulations can be prepared by a conventional method used for formulation in the art or by a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA, and can be formulated into various formulations depending on each disease or ingredient.

[0133] The composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

[0134]

[0135] In another aspect, the present invention relates to a cosmetic composition for preventing or reversing aging comprising GRP78, a peptide analog thereof or a nucleic acid encoding the same.

[0136] In the present invention, the composition may be characterized by having at least one function selected from the group consisting of improving skin wrinkles, changing skin or hair color, and alleviating or preventing hair loss and reversing such changes, but is not limited thereto.

[0137] The cosmetic composition of the present invention may be any cosmetic composition commonly used in the art, but preferably, it can be provided for use as a skin ointment, basic cosmetic, makeup cosmetic, body cosmetic, or shaving cosmetic.

[0138] In addition, the cosmetic composition according to the present invention may be a cosmetic composition mixed with other cosmetic compositions, specifically, a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, an essence, a nourishing essence, a pack, a soap, a shampoo, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, an emulsion, a pressed powder, a loose powder, an eye shadow, etc., and may be used after being diluted, if necessary.

[0139] In addition, the cosmetic composition of the present invention may further include, in addition to including the peptide according to the present invention as an active ingredient, a functional material having an effect of improving wrinkles or whitening, or in one embodiment, a functional material having a UV blocking or moisturizing effect, or a material such as an excipient or diluent for improving physical properties. In one embodiment, the cosmetic composition may further include a fragrance, a pigment, a sterilizer, an antioxidant, a preservative, a moisturizer, a thickener, an inorganic salt, or a synthetic polymer material for improving physical properties. In addition, the compounding ingredients that may be added may include a fat component, an emollient, a surfactant, an organic pigment, an inorganic pigment, an organic powder, an ultraviolet absorber, a pH adjuster, alcohol, a blood circulation promoter, a cooling agent, an antiperspirant, or purified water.

[0140]

[0141] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0142]

[0143] Example 1: Evaluation of the ability of aged cells to revert to young cells by inducing increased GRP78 expression in aged cells.

[0144] We constructed a GRP78 overexpression vector containing the nucleic acid sequence of SEQ ID NO: 20 and performed experiments. The GRP78 gene was overexpressed in aged model stem cells. 72 hours after transfection, intracellular GRP78 overexpression and changes in aging- and stem cell potential-related gene expression following GRP78 overexpression were examined using qRT-PCR. The constructed vector is shown in Figure 1.

[0145] The experimental results confirmed that when the GRP78 expression vector was introduced into young and aged cells, the expression level of GRP78 mRNA significantly increased. Specifically, compared to the control group that did not introduce the GRP78 vector, the expression level of mRNA in the experimental group that introduced the GRP78 vector increased by a minimum of 60-fold and a maximum of 4,000-fold (Fig. 2). These results are consistent with the results observed by FACS in Example 2 below, in which more vector was introduced into aged cells when comparing the gene introduction efficiency between young and aged cells, confirming that the GRP78 overexpression vector was effectively introduced into aged cells.

[0146]

[0147] Example 2: Evaluation of molecular genetic changes through the introduction of intracellular anti-aging factors.

[0148] In order to evaluate molecular genetic changes by introducing the pcDNA3.1 vector containing the GRP78 gene, which was confirmed to function as a senescence regression factor in Example 1, into stem cells, an experiment was conducted using a GRP78-EGFP expression vector with a fluorescent material attached, provided by Chungbuk National University. After establishing young stem cells (under 10 passages) and aged stem cells (over 15 passages) through subculture of DPSCs, the vector was introduced into each cell using a transfection reagent. 72 hours after transfection, the cells were fixed and fluorescently stained, and confocal microscopy was used to confirm whether the introduced GRP78-EGFP was expressed in the cells. Confocal microscopy confirmed fluorescence in cells introduced only with the GRP78-EGFP vector (Fig. 3), confirming that the GRP78-EGFP vector was normally introduced into the cells. In addition, it was confirmed that the gene was introduced equally into young and aged cells.

[0149]

[0150] Additionally, to confirm the efficiency of gene expression vector introduction into young and aged cells, FACS was used for additional analysis. The GRP78-EGFP expression vector was transfected into young or aged DPSCs, and 72 hours after transfection, cells were completely detached from the cell culture plate with trypsin-EDTA and clumped cells were filtered using a FASC strainer. When analyzing with FACS, the gate was set based on untreated cells, and the cells were analyzed using a 488 nm wavelength fluorescence.

[0151] As a result, it was confirmed that about 21% of the cells expressed GRP78-EGFP when the vector was introduced into young cells. This means that the vector introduction efficiency into young stem cells was about 21%. On the other hand, when analyzing aged stem cells, the cell population expressing fluorescence was confirmed to be about 83%. It was confirmed that the transfection rate of the GRP78 expression vector was higher in aged stem cells than in young stem cells, and it was confirmed that the protein was normally expressed by the GRP78 expression vector in aged DPSCs as well (Fig. 4).

[0152]

[0153] Additionally, to confirm the expression of GRP78 mRNA by the GRP78 vector transfected into cells, the GRP78 expression vector was transfected into DPSC and PDLSC, which are dental stem cells, and 72 hours later, qPCR was performed using Sybr green to confirm the amount of TERT mRNA, a factor involved in telomere length, expressed.

[0154] As a result, it was confirmed that the expression of TERT in aged DPSCs and PDLSCs by the introduced GRP78 vector significantly increased compared to that in young DPSCs and PDLSCs (Fig. 5, **p<0.01 compared to young GRP78 and old NT in DPSCs, respectively, *p<0.05 compared to young GRP78 and old NT in PDLSCs, respectively). This suggests that the expression of TERT, a senescence factor marker, significantly increased by the expression of the GRP78 protein introduced into aged cells, and that the senescence regression effect by GRP78 could also be confirmed (Fig. 5).

[0155]

[0156] Example 3: Evaluation of tissue changes through introduction of anti-aging factors into an aging animal model.

[0157] To evaluate tissue changes through the introduction of aging-related factors in an aging animal model, we performed an experiment to confirm increased GRP78 mRNA and protein levels after the introduction of a GRP78 expression vector at the cellular level. To confirm whether tissue delivery of the GRP78 expression vector also increases GRP78 mRNA and protein expression levels in tissues at the animal level, the GRP78 expression vector was transfected into the gastrocnemius muscle of mice using an in vivo transfection reagent. The experimental groups were divided into three groups: young (8-week-old), middle-aged (20-week-old), and naturally aged (48-week-old) mice (Fig. 6). After mixing the transfection reagent and vector, 50 μl was injected per gastrocnemius muscle.

[0158] First, to confirm whether the vector was transfected into the tissue, the transfection reagent-vector was injected into the gastrocnemius muscle using the GRP78-EGFP expression vector. 72 hours after injection, the mice were sacrificed, and the gastrocnemius muscles were obtained and cryosectioned using an OCT compound. Afterwards, the tissues were stained for nuclei with DAPI and examined using a confocal microscope. As a result, fluorescence was confirmed in the gastrocnemius muscles of animals injected with the GRP78-EGFP vector, confirming that the GRP78 expression vector could be normally injected using the transfection reagent (Fig. 7).

[0159] To measure the expression levels of GRP78 mRNA and protein in the tissues 7 days after injecting the GRP78 expression vector into the gastrocnemius muscle of mice using a transfection reagent, total RNA was obtained from the tissues using TRIzol, and cDNA was synthesized through reverse transcription for the mRNA, and the expression level of GRP78 mRNA was quantified using the synthesized cDNA. As a result, it was confirmed that the expression level of GRP78 RNA was significantly increased compared to the untreated experimental group (Fig. 8; ****p<0.0001, compared to 8-week-old NT).

[0160] Additionally, to determine the protein expression level of GRP78 within the tissue, gastrocnemius muscle lysates were prepared using RIPA buffer, and protein expression levels were determined using Western blot techniques. As a result, it was confirmed that protein expression levels increased by up to 50% or more in the GRP78 expression vector-injected experimental group compared to the other experimental groups (Fig. 9).

[0161]

[0162] Additionally, tissue sections were prepared using the gastrocnemius muscle to histologically examine changes in fascicle thickness. To prepare tissue sections, the tissue was dehydrated and paraffin-embedded, and then embedded in paraffin. After embedding, 5 μm sections were prepared and stained with hematoxylin and eosin (H&E).

[0163] As a result of the experiment, it was confirmed that the muscle bundle thickness increased in the individuals injected with the GRP78 expression vector compared to the control group in each experimental group at each age, and in particular, it increased more than the muscle bundle thickness of the 8-week-old control group (Fig. 10).

[0164]

[0165] Example 4: Synthesis of GRP78-like peptides

[0166] Peptides of sequence numbers 1 to 18 in Table 1 above were synthesized sequentially from the N-terminus using F-moc solid phase peptide synthesis. The synthesized peptide sequences were cleaved from the resin, washed, lyophilized, and then separated and purified by liquid chromatography. The molecular weights of the purified peptides were confirmed using MALDI-TOF analysis.

[0167]

[0168] Example 5: Comparison of expression of aging-related biomarkers

[0169] Rat periodontal ligament (rPDL) tissue cells were cultured in a 60 mm culture dish at a total density of 4.5 × 10 using culture medium MEM alpha modified 1X. 4Cells were seeded and cultured. After obtaining the amount of cells required for the experiment, the culture medium was removed, washed twice with DPBS, and compared under an optical microscope. SA-β gal staining was performed using the Senescence β-Galactosidase Staining Kit #9860 from Cell Signaling Technology. Quantitative RT-PCR was performed using a Magnetic Induction Cycler (Mic qPCR) from Bio Molecular Systems. Real-time cell proliferation curves were obtained using a Live-Cell Imaging and Analysis Instrument: Incucyte® S3 from Satorius.

[0170] We observed changes in cell characteristics of periodontal ligament tissue-derived cells from young rats (11 weeks old, control group) and aged rats (96 weeks old, aged group) as they aged (Fig. 11). Figure 11A shows the results of measuring cell length and width in the control and aged groups. Cell length significantly increased with aging (**p<0.01).

[0171] Figure 11B shows the results of observing the expression of beta galactosidase, which is highly expressed in senescent cells, using the SA-β gal staining (increased blue staining) method. Beta galactosidase expressed in the cells was stained using the Senescence β-Galactosidase Staining Kit, dissolved in 70% glycerol for 30 minutes, and the absorbance was measured at a wavelength of 405 nm. As the senescent cells increased, the degree of beta galactosidase staining increased, and the absorbance value increased significantly (***p<0.001).

[0172] Figure 11C shows the results of RT-PCR measurement of gene expression levels of aging-related biomarkers, telomere length related factor-1 (TRF-1), telomere length related factor-2 (TRF-2), Protection of Telomeres 1 (POT1), and glucose-regulated protein 78 (GRP78). The expression of all four genes was significantly reduced in the aging group. The expression of TRF-1, TRF-2, and POT1 was significantly reduced to 0.63 ± 0.18, 0.27 ± 0.03, and 0.31 ± 0.21 in the aging group, respectively, compared to the control group (***p<0.001). The expression of GRP78 also significantly decreased to 0.56 ± 0.38 in the aging group (**p<0.01).

[0173] Figure 11D shows the results of measuring the cell proliferation number. The control group had 5.3 × 10 4 After cell division by number, the cell density (cell confluency) was 100% on the 5th day, but the aged group showed a proliferation rate of about 72% on the same period of 5 days due to aging, indicating that the proliferation rate of periodontal ligament cells extracted from aged rats (old rPDL) was significantly lower than that of periodontal ligament cells extracted from young rats (young rPDL) (***p<0.001). In the case of old rPDL, the cell density was investigated to be at the level of 82% even on the 8th day, the end point of the experiment.

[0174] Through the above results, we confirmed that rPDL cells can be used as model cells for aging reversal by showing aging-specific phenomena according to age.

[0175]

[0176] Example 6: Confirmation of cell penetration ability of GRP78-like peptides

[0177] Peptides of sequence numbers 1 to 4 were labeled with Rhodamine B fluorescence. A total of 5.0 × 104 After seeding the cells, the culture medium was removed and washed twice with DPBS. The prepared fluorescent peptide was mixed at a concentration of 200 μg / ml in serum-free culture medium and incubated for 1 hour. After 1 hour of treatment, the cells were washed five times with DPBS and mounted with a mounting solution containing DAPI. The intracellular penetration and distribution of the peptide were examined using an inverted confocal microscope.

[0178] Figure 12 shows the results of observing the cell permeability of peptides of sequence numbers 1 to 4 using a confocal microscope. Figure 12A shows the cell permeability results of periodontal ligament cells (young rPDL) extracted from young rats, and Figure 12B shows the cell permeability results of periodontal ligament cells (old rPDL) extracted from aged rats. The cell permeability of peptides of sequence numbers 1, 3, and 4 was high in both young rPDL and old rPDL, and the degree of permeability of the peptides was observed to be lower in old rPDL than in young rPDL. Therefore, it was confirmed that the cell permeability of aged cells was reduced compared to young cells.

[0179]

[0180] Example 7: Evaluation of anti-aging potential by GRP78-like peptides.

[0181] To confirm the anti-aging ability of GRP78-like peptides, we compared the expression of aging factors induced by the peptides. A total of 4.5 × 10 4 After seeding with canine tonsil-derived stem cells and rat periodontal ligament cells, the culture medium was removed and washed twice with DPBS. The four manufactured peptide candidates were mixed at a concentration of 200 μg / ml in serum-free culture medium and incubated for 1 hour. After 1 hour of treatment, the cells were washed twice with DPBS, and RNA was extracted. The expression of each gene was examined and compared using qRT-PCR.

[0182] Figure 13 shows the results of measuring changes in the expression of aging-related biomarkers by peptides of sequence numbers 1 to 4.

[0183] Figure 13A shows the results of RT-PCR analysis of the expression of senescence-associated biomarkers TRF-1, TRF-2, and hTERT (human telomerase reverse transcriptase) in 20 passages of serially cultured aged human tonsil-derived mesenchymal stem cells (TMSCs). The expression of all genes for senescence-associated biomarkers was significantly reduced in the aged cells. The expression of the GRP78 gene was also significantly reduced in the aged cells.

[0184] When peptides of sequence number 1 and sequence number 2 were treated in aged cells, the expression of TRF-1 was significantly increased by a factor of 1.66±0.30 and 4.32±0.51, respectively, compared to when no peptides were treated (***p<0.001).

[0185] The expression increase rate of TRF-2 was 2.86±0.10 when treated with the peptide of SEQ ID NO: 1, which was significantly increased compared to senescent cells not treated with the peptide (***p<0.001). When treated with the peptide of SEQ ID NO: 3, the expression increase rate was 1.23±0.04, which was significantly increased compared to senescent cells not treated with the peptide (*p<0.05).

[0186] In the case of hTERT expression, when treated with the peptide of SEQ ID NO: 2, there was a significant increase of 2.53±0.48 times compared to senescent cells not treated with the peptide (***p<0.001). When treated with the peptide of SEQ ID NO: 3, there was a significant increase of 2.21±0.16 times compared to senescent cells not treated with the peptide (*p<0.05).

[0187] Figure 13B shows the results of RT-PCR measurements of changes in the expression of aging-related biomarkers TRF-1, TRF-2, POT1, and GRP78 when peptides of SEQ ID NOS: 1 to 4 were treated in periodontal ligament tissue-derived cells extracted from young (12-week-old) and aged (96-week-old) rats. Overall, the expression of all genes was significantly reduced in aged rat cells compared to young rat cells ($p<0.001, comparison of Young rPDL_control and Old rPDL_control).

[0188] When treated with peptides of SEQ ID NO: 2 and 4, TRF-1 expression was significantly increased by 1.34±0.12 and 1.21±0.11 times in the young rPDL group, respectively (***p<0.001). In the old rPDL group, when treated with peptides of SEQ ID NO: 2 and 4, expression was significantly increased by 0.83±0.28 and 0.78±0.18 times, respectively, compared to old rPDL (0.58±0.01) that was not treated with peptides (***p<0.001).

[0189] When the young rPDL group was treated with peptides of sequence numbers 2 and 4, the expression of TRF-2 was significantly increased by 1.25±0.10 and 1.31±0.10 times, respectively (**p<0.01). In the old rPDL group, when the peptides of sequence numbers 2, 3, and 4 were treated, the expression was significantly increased by 0.51±0.21, 0.38±0.11, and 0.74±0.22 times, respectively, compared to old rPDL (0.13±0.01) that was not treated with peptides (***p<0.001).

[0190] When treated with peptides of sequence numbers 2 and 4, the expression of POT1 was significantly increased in the old rPDL group by a factor of 0.34±0.20 (p<0.05) and 0.66±0.31 (**p<0.01), respectively, compared to the old rPDL control group (0.25±0.01) that was not treated with peptides.

[0191] When treated with peptides of SEQ ID NOs: 2, 3, and 4, the expression of GRP78 was significantly increased in the old rPDL group by 0.52±0.18 (p<0.05), 0.65±0.04 (p<0.001), and 0.69±0.30 (p<0.01), respectively, compared to old rPDL (0.56±0.01) that was not treated with peptides.

[0192] In summary, it was demonstrated that peptides of sequence numbers 1 to 4 can re-increase the expression of aging-related biomarker genes that have been decreased due to aging.

[0193]

[0194] Example 8: Evaluation of muscle tissue functional recovery by peptides

[0195] To confirm the muscle tissue functional recovery ability of the peptide, muscle function was examined using a natural aging mouse model. C57BL / 6J male mice were used at 8 weeks of age (0.16 years, young), 48 weeks of age (1 year, middle-aged), and 72 weeks of age (1.5 years, old). The peptides of SEQ ID NO: 3 and SEQ ID NO: 4 were injected intramuscularly into the gastrocnemius muscle of the hind limbs of mice at a dose of 10 mg / kg three times a week for 4 weeks. To evaluate the muscle functional recovery by the peptides of SEQ ID NO: 3 and 4, the grip strength test and rotarod test were performed before and for 8 weeks after drug injection. The grip strength test was performed using a grip strength meter (JD-A-22, Jeung Do Bio and Plant Co., Ltd.) to measure the change in grip strength of the mice's forelimbs. The rotarod test was performed using a rotarod (JD-A-07TS, Jeung Do Bio and Plant Co., Ltd.) at a speed of 25 rpm. The time it took for mice to remain on the rotating rod without falling was measured. After the start of administration, the time spent on the rod was measured for up to 8 weeks.

[0196] The retention times on the rod of 48-week-old and 72-week-old mice administered the peptides of SEQ ID NO: 3 and SEQ ID NO: 4 for 8 weeks did not show a significant difference compared to the 8-week-old (8 w NT) mice. Compared to the 48-week-old mice not administered the peptides (48 w NT), the retention times on the rod of 48-week-old mice administered the peptides of SEQ ID NO: 3 and SEQ ID NO: 4 increased. Compared to the 72-week-old mice not administered the peptides (72 w NT), the retention times on the rod of 72-week-old mice administered the peptides of SEQ ID NO: 3 and SEQ ID NO: 4 increased, and the increase was particularly significant with the peptide of SEQ ID NO: 3 (*p<0.05, Fig. 14). The statistical significance between each group was analyzed by one-way ANOVA. Consequently, it means that the muscle function of aged mice was restored by the peptides of SEQ ID NO: 3 and 4.

[0197]

[0198] Example 9: Histological comparison of muscle fibers of naturally aged mice by peptides.

[0199] To confirm the muscle tissue functional recovery ability of GRP78-like peptides, muscle function was examined using a natural aging mouse model. C57BL / 6J male mice were used, and the ages were 8 weeks (0.16 years, young), 48 weeks (1 year, middle-aged), and 72 weeks (1.5 years, old). The peptides of SEQ ID NO: 3 or SEQ ID NO: 4 were injected intramuscularly into the gastrocnemius muscle of the hind limbs of mice at a dose of 4 mg / kg, three times a week for 4 weeks. Thereafter, for an additional 4 weeks, 4 mg / kg of the peptide of SEQ ID NO: 3 or SEQ ID NO: 4, 4 mg / kg of the peptide of SEQ ID NO: 3 and 4 mg / kg of the muscle regeneration peptide (GKMSTRGRKMMRRKK: SEQ ID NO: 19), and 4 mg / kg of the peptide of SEQ ID NO: 4 and 4 mg / kg of the muscle regeneration peptide (SEQ ID NO: 19) were injected intramuscularly into the gastrocnemius muscle of the hind limbs of mice three times a week. After 4 weeks and 8 weeks of administration, mice of each age were sacrificed, and tissue sections were prepared to evaluate muscle tissue recovery and stained with hematoxylin and eosin (H&E). The types, doses, administration periods, and sacrifice dates of the peptides administered to each aged mouse are shown in Table 2 and Figure 15.

[0200]

[0201] Figure 16 shows the results of H&E staining and measurement of muscle fiber diameter of mouse muscle tissue administered with peptides of sequence number 3 or sequence number 4 for 4 weeks.

[0202] Compared to 8-week-old mice that were not administered peptides, we observed an increase in the inter-fiber space in naturally aged 48-week-old and 72-week-old mice that were not administered peptides. In other words, we observed a decrease in the diameter of muscle fibers with aging. In contrast, 48-week-old and 72-week-old mice administered peptides of SEQ ID NO: 3 or SEQ ID NO: 4 exhibited a decrease in the inter-fiber space. Furthermore, the diameter of the muscle fibers was similar to that of 8-week-old mice (Fig. 16). Therefore, we confirmed that the GRP78-like peptide of the present invention can reverse aging.

[0203]

[0204] Figure 17 shows the results of H&E staining and measurement of muscle fiber diameter of mouse muscle tissue that was injected with the peptide of sequence number 3 or sequence number 4 for 4 weeks, and then administered the peptide of sequence number 3 or sequence number 4 alone for an additional 4 weeks, or administered the peptide of sequence number 3 or sequence number 4 in combination with a muscle regeneration peptide.

[0205] Compared to 8-week-old mice that were not administered peptides, the interfiber space in naturally aged 48-week-old and 72-week-old mice that were not administered peptides increased. When the diameter of muscle fibers in each tissue was measured, a decrease in muscle fiber diameter was observed. Similar to the results in Figure 16, the diameter of muscle fibers was confirmed to decrease in aged cells.

[0206] In 48-week-old mice, muscle fiber diameter increased when only the peptide of SEQ ID NO: 3 or SEQ ID NO: 4 was administered compared to when no peptide was administered, and when SEQ ID NO: 3 and the muscle regeneration peptide, or when SEQ ID NO: 4 and the muscle regeneration peptide were co-administered, the muscle fiber diameter increased more significantly (*p<0.05). In addition, in 72-week-old mice, muscle fiber diameter increased when only the peptide of SEQ ID NO: 3 or SEQ ID NO: 4 was administered compared to when no peptide was administered, and when SEQ ID NO: 3 and the muscle regeneration peptide, or when SEQ ID NO: 4 and the muscle regeneration peptide were co-administered, the muscle fiber diameter increased more significantly (*p<0.05) (Fig. 17).

[0207] When only the peptide of sequence number 3 or sequence number 4 was administered, the muscle density and muscle fiber diameter of naturally aging mice increased, and when muscle regeneration peptide was administered together, the effect of increasing the muscle density and muscle fiber diameter of naturally aging mice was found to be greater.

[0208]

[0209] Example 10: Comparison of muscle biomarker expression by peptides

[0210] The tissue sections prepared in Example 9 were subjected to immunofluorescence (IF) staining using MyoD1 antibody (Ab16148, Abcam) and myosin heavy chain (MYH) antibody (sc-376157, Santa Cruz Biotechnology). After paraffin-embedding, the tissue sections were placed in xylene to remove paraffin, and then sequentially immersed in 100%, 95%, and 70% ethanol to hydrate the tissue and then washed with distilled water. The slides were immersed in a citrate buffer-based antigen retrieval buffer (pH 6.0) and heat-treated at 95°C for 15 minutes to perform antigen retrieval. After antigen retrieval, the slides were cooled to room temperature and washed three times with PBS solution for 5 minutes each. The slides were blocked in PBS solution containing 5% BSA (bovine serum albumin) for 1 hour at room temperature, and MYH and MyoD1 primary antibodies were diluted 1:200 each, applied to the slides, and reacted for 16 hours at 4°C. After washing three times with PBS for 5 minutes, the secondary antibody containing a fluorescent label (A-31570, Thermo Fisher Scientific) was diluted 1:500, applied to the slides, and reacted for 1 hour at room temperature, protected from light. The slides were washed three times with PBS for 5 minutes, stained with DAPI (D1306, Thermo Fisher Scientific) for 5 minutes, and then washed with PBS. The slides were then mounted using fluorescent mounting medium (F4680, Sigma-Aldrich) and sealed with coverslips.

[0211] MyoD1 is a marker expressed in the early stages of muscle regeneration, and MYH is a marker expressed in the late stages of muscle regeneration (Montano, M. "Translational models, methods and concepts in studies of muscle tissue repair" Translational Biology in Medicine (2014): 103-128)

[0212]

[0213] Figure 18 shows the results of measuring fluorescence intensity after confocal microscopy of the expression of MyoD1 in muscle tissue of mice administered with peptides of sequence numbers 3 and 4 for 4 weeks. Figure 19 shows the results of measuring fluorescence intensity after confocal microscopy of the expression of MYH in muscle tissue of mice administered with peptides of sequence numbers 3 and 4 for 4 weeks.

[0214] In 8-week-old mice not administered peptides, the expression levels of MyoD1 and MYH in muscle tissue were the highest. In 48-week-old mice, the expression levels of MyoD1 and MYH proteins increased when administered SEQ ID NO: 3 or SEQ ID NO: 4 compared to 48-week-old mice not administered peptides. In 72-week-old mice, the expression levels of MyoD1 and MYH proteins increased when administered SEQ ID NO: 3 or SEQ ID NO: 4 compared to 72-week-old mice not administered peptides (Figs. 18 and 19).

[0215]

[0216] Figure 20 shows the results of measuring fluorescence intensity after confocal microscopic observation of the expression of MyoD1 in the muscles of mice that were injected with the peptide of sequence number 3 or sequence number 4 for 4 weeks, and then further administered with the peptide of sequence number 3 or sequence number 4 for 4 weeks, or co-administered with the peptide of sequence number 3 and a muscle regeneration peptide (SEQ ID NO: 19), or co-administered with the peptide of sequence number 4 and a muscle regeneration peptide (SEQ ID NO: 19). Figure 21 shows the results of measuring fluorescence intensity after confocal microscopic observation of MYH expression in the muscles of mice that were injected with the peptide of sequence number 3 or sequence number 4 for 4 weeks, and then additionally administered the peptide of sequence number 3 or sequence number 4 for 4 weeks, or co-administered the peptide of sequence number 3 and a muscle regeneration peptide (SEQ ID NO: 19), or co-administered the peptide of sequence number 4 and a muscle regeneration peptide (SEQ ID NO: 19).

[0217] The muscle tissue expression levels of MyoD1 and MYH were highest in 8-week-old mice that were not administered peptides. When 48-week-old mice were administered the peptides of SEQ ID NO: 3 or SEQ ID NO: 4, the protein expression levels of MyoD1 and MYH increased compared to 48-week-old mice that were not administered peptides. In addition, when the peptides of SEQ ID NO: 3 and the muscle regeneration peptide, or SEQ ID NO: 4 and the muscle regeneration peptide were co-administered, the expression levels of MyoD1 and MYH significantly increased compared to when the peptides of SEQ ID NO: 3 or SEQ ID NO: 4 were administered alone (**p<0.01). In the case of mice co-administered with the peptides of SEQ ID NO: 3 and the muscle regeneration peptide, or SEQ ID NO: 4 and the muscle regeneration peptide, the expression levels of MyoD1 and MYH were higher than in the youngest 8-week-old mice that were not administered the peptides (Figs. 20 and 21).

[0218] In 72-week-old mice, MyoD1 and MYH protein expression levels increased when administered with SEQ ID NO: 3, SEQ ID NO: 3 plus muscle regeneration peptide, SEQ ID NO: 4, and SEQ ID NO: 4 plus muscle regeneration peptide compared to 72-week-old mice that were not administered peptides. The expression level of MyoD1 was similar when only the peptide of SEQ ID NO: 3 was administered and when SEQ ID NO: 3 plus muscle regeneration peptide was administered, but the expression level of MYH increased more when SEQ ID NO: 3 plus muscle regeneration peptide was administered. The expression level of MyoD1 increased more when SEQ ID NO: 4 plus muscle regeneration peptide was administered than when only the peptide of SEQ ID NO: 4 was administered. On the other hand, the expression level of MYH was similar.

[0219] When only the peptide of sequence number 3 or sequence number 4 was administered, the expression of muscle differentiation biomarkers increased in naturally aging mice, and when muscle regeneration peptides were administered together, the effect of increasing the expression of muscle biomarkers in naturally aging mice was found to be greater.

[0220]

[0221] Example 11: Evaluation of the anti-aging effect through restoration of telomere length regulators.

[0222] In order to evaluate the anti-aging effect of the GRP78-like peptide according to the present invention, an experiment was conducted to determine whether changes in various regulatory factors related to telomeres, which are closely related to aging, are improved by treatment with the GRP78-like peptide and whether the anti-aging effect and telomere length regulatory factors are restored in young TMSC (passage 5) and TMSC (passage 15) that have undergone replicative aging through subculture.

[0223] 60 mm 2 3.5 × 10 in a dish 5TMSCs were seeded, the culture medium was removed, and washed twice with DPBS. The prepared GRP78 peptide (SEQ ID NO: 4) solution was then treated for 24 hours, and samples were obtained. Specific primers were synthesized using the nucleotide sequence of hTERT, and qRT-PCR was performed using these primers. As a result, hTERT expression tended to be lower in the control group of aged TMSCs compared to young TMSCs, and treatment with the peptide of SEQ ID NO: 4 restored hTERT expression (Fig. 22a).

[0224] Also, 60mm 2 3.5 × 10 in a dish 5 TMSCs were divided, and cell culture and peptide treatment were performed in the same manner as in the experiment of Fig. 22a. Afterwards, the experiment was performed according to the method suggested by Sigma-Aldrich using the TRAPeze™ RT Telomerase Detection Kit. As a result, telomerase expression was significantly reduced in the control group of aged TMSCs compared to young TMSCs (***p<0.001), and telomerase expression was found to increase when treated with the peptide of SEQ ID NO: 4 (Fig. 22b).

[0225]

[0226] Next, 60 mm 2 3.5 × 10 in a dish 5TMSCs were seeded and cultured for 2 days until 70-80% confluent. Serum-free culture medium and GRP78 peptide (SEQ ID NO: 4) were treated at various concentrations (0, 100, 200 μg / ml) for 24 hours. Afterwards, TeloTAGGG™ Telomere Length Assay was performed in the same manner as the method suggested by Sigma-Aldrich. As a result, telomere length was reduced 0.87-fold in the control group of aged TMSCs compared to young TMSCs, and when the peptide of SEQ ID NO: 4 was treated in aged TMSCs, telomere length was significantly increased 4.94-fold compared to the aged control group (*p<0.05) (Fig. 22c). Additionally, when the length of visible telomeres was examined using southern blot, it was found that TMSCs that had undergone replicative aging had telomeres of somewhat shorter length compared to young TMSCs (Fig. 22d).

[0227]

[0228] In summary of the above results, it was confirmed that the GRP78-like peptide according to the present invention restores the expression of direct telomere length-related factors decreased due to replicative senescence of TMSCs caused by subculture, which means that it has the ability to improve or reverse aging.

[0229]

[0230] Example 12: Evaluation of the effect of GRP78-like peptide on the recovery of mitochondrial-related factors.

[0231] In order to evaluate the direct quantitative changes in mitochondria and changes and recovery of related factors occurring in TMSC when treated with a GRP78-like peptide according to the present invention, the following experiments were conducted.

[0232] 60 mm 2 3.5 × 10 in a dish 5TMSCs were seeded, the culture medium was removed, and washed twice with DPBS. The cells were treated with the prepared GRP78 peptide (SEQ ID NO: 4) solution and cultured for 24 h. The medium was removed and fixed with 4% paraformaldehyde. After treatment with a mixture of phenol-free DMEM and 1 mM stock mitotracker for 30 min, mitochondria distributed in TMSCs were observed using an inverted confocal microscope.

[0233] As a result, the expression of mitochondria confirmed using mitotracker in replicative senescent TMSCs was observed to be relatively low compared to young TMSCs, and mitochondrial expression was found to be somewhat restored by treatment with the peptide of sequence number 4 (Fig. 23a and Fig. 23b).

[0234] To confirm quantitative changes in mitochondrial DNA (mtDNA), changes in the copy number of mtDNA were measured using qRT-PCR. As a result, a relatively small copy number of mtDNA was confirmed in TMSCs with replication aging compared to young TMSCs, and the copy number was found to increase when treated with the peptide of sequence number 4 (Fig. 23c).

[0235] In addition, as a result of confirming the changes in protein expression of factors related to mitochondrial activity and biosynthesis, when treated with the peptide of sequence number 4, the protein expression of direct GRP78 was significantly increased (*p<0.05), and when peptide was treated to TMSC with replication aging, the expression of AMPKα and CREB was relatively increased compared to the control group with replication aging (Fig. 23d).

[0236] In addition, as a result of measuring the gene and protein expression changes caused by treatment with the peptide of sequence number 4 for PPARγ, PGC1α, and p53, which are factors associated with mitochondria and telomeres, the expression of p53 was found to be higher in TMSCs with replicative senescence compared to young TMSCs (***p<0.001), and when the peptide of sequence number 4 was treated at a concentration of 200 μg / ml, the expression of p53 in TMSCs with replicative senescence was found to be significantly lower (**p<0.01) (Fig. 23e).

[0237]

[0238] Based on the various experimental results related to the above mitochondria, it was found that treatment with the peptide of SEQ ID NO: 4 restored the mitochondrial activity of replicative aging TMSC, and in particular, it suggests that it may be involved in the reversal and recovery of aging by causing changes in the expression of factors related to telomeres. This shows that the peptide of SEQ ID NO: 4 has the function of regulating the endoplasmic reticulum stress response in a manner similar to the GRP78 protein, which potentially regulates endoplasmic reticulum stress, restoring mitochondrial dysfunction, and ultimately reversing and recovering aging.

[0239]

[0240] The GRP78, GRP78-like peptide or nucleic acid encoding the same according to the present invention can increase the expression of a biomarker whose expression is reduced in aging cells, and thus is useful for restoring the function of muscle tissue or the like whose function is reduced due to aging.

[0241]

[0242] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0243]

[0244] Electronic file attached.

Claims

1. A composition comprising GRP78, a peptide analog thereof or a nucleic acid encoding the same as an active ingredient, for inhibiting or reversing aging of a cell, tissue or organ, or for restoring or regenerating the function of an aged or damaged cell, tissue or organ.

2. A composition according to claim 1, characterized in that the effective ingredient is a peptide comprising an amino acid sequence having 80% or more, 90% or more, 95% or more, or 100% sequence identity with the nucleotide binding domain represented by SEQ ID NO: 22 or a part thereof.

3. A composition according to claim 1, characterized in that the effective ingredient is a peptide comprising an amino acid sequence having 80% or more, 90% or more, 95% or more, or 100% sequence identity with the substrate binding domain represented by SEQ ID NO: 23 or a part thereof.

4. In the first paragraph, the effective ingredient is a composition characterized in that it is a peptide having a length of 40-mer or less, which is a part of the GRP78 amino acid sequence represented by the sequence number 21 and contains at least three amino acids selected from the group consisting of serine (S), threonine (T), tyrosine (Y), cysteine ​​(C), methionine (M), tryptophan (W), glutamic acid (D), and proline (P).

5. A composition according to claim 1, wherein the effective ingredient increases the expression of GRP78.

6. A composition according to claim 1, characterized in that the effective ingredient is a peptide comprising an amino acid sequence having 80% or more, 90% or more, 95% or more, or 100% sequence identity with an amino acid sequence selected from the group consisting of sequence numbers 1 to 18 or a part thereof.

7. A composition according to claim 1, characterized in that the effective ingredient is a peptide comprising an amino acid sequence having 80% or more, 90% or more, 95% or more, or 100% sequence identity with an amino acid sequence selected from the group consisting of sequence numbers 1 to 4 or a part thereof.

8. A composition according to claim 1, characterized in that the effective ingredient is a peptide comprising an amino acid sequence of sequence number 1.

9. A composition according to claim 1, characterized in that the effective ingredient is a peptide comprising an amino acid sequence of sequence number 2.

10. A composition according to claim 1, characterized in that the effective ingredient is a peptide comprising an amino acid sequence of sequence number 3.

11. A composition according to claim 1, characterized in that the effective ingredient is a peptide comprising an amino acid sequence of sequence number 4.

12. A composition according to claim 1, characterized in that the cell, tissue or organ is a cell, tissue or organ derived from a patient suffering from a disease or condition selected from the group consisting of muscle disease, bone disease, joint disease, skin disease, metabolic disease, urinary system disease, neurological disorder, cardiovascular disease, pulmonary dysfunction, cancer, immune disease, dental disease, periodontal disease and oral tissue dysfunction.

13. A composition according to claim 1, characterized in that it is used to restore or regenerate the function of aged muscle tissue.

14. A composition according to claim 13, wherein the aged muscle tissue is characterized by decreased muscle function, muscle loss, muscle atrophy, muscle wasting or muscle degeneration.

15. A composition according to claim 13, characterized in that it is used for the prevention or treatment of a muscle disease selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, muscular dystrophy, atony, amyotrophic lateral sclerosis, cachexia, masticatory muscle disorder, temporomandibular disorder, and inflammatory myopathy.

16. A cosmetic composition for preventing or reversing aging, comprising GRP78, a peptide analog thereof, or a nucleic acid encoding the same.

17. A composition according to claim 16, characterized in that it has at least one function selected from the group consisting of improving skin wrinkles, changing skin or hair color, and alleviating or preventing hair loss.

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