WO2018056706A1

WO2018056706A1 - Composition comprising thioredoxin-interacting protein-derived peptide or polynucleotide encoding same as effective ingredient for reverse-ageing of aged stem cell and use thereof

Info

Publication number: WO2018056706A1
Application number: PCT/KR2017/010358
Authority: WO
Inventors: 최인표; 정해용; 김동오; 김미정; 변재은
Original assignee: 한국생명공학연구원
Priority date: 2016-09-22
Filing date: 2017-09-20
Publication date: 2018-03-29
Also published as: KR101987354B1; KR20180032506A

Abstract

The present invention relates to a pharmaceutical composition comprising a thioredoxin-interacting protein (TXNIP)-derived peptide or a polynucleotide coding therefor as an effective ingredient for reverse ageing of stem cells. In mice competitively transplanted with aged hematopoietic stem cells (HSC), a TXNIP-derived peptide was observed to increase the engraftment of aged HSC, decrease lineage skewing, reduce a long-term HSC (LT-HSC) ratio, and inhibit p38 and ROS, thus suppressing features of aged HSC, compared to a control untreated with the peptide. In acute leukopenia models, administration of the TXNIP-derived peptide was found to increase the leukocyte proliferation of aged mice to a level of young mice. In addition, it was found that the TXNIP-derived peptide causes recovery from Cdc42 polarization in aged HSC, downregulates the expression of the senescence-associated genes p16, p19, p21, and Wnt5a, and increases the homing of HSC. Therefore, the TXNIP-derived peptide of the present invention can be useful as a composition for reverse ageing of hematopoietic stem cells.

Description

Composition for reverse aging of senescent stem cells containing thioredoxin binding protein-derived peptide or polynucleotide encoding the same as an active ingredient and use thereof

The present invention relates to a pharmaceutical composition for reverse aging of senescent stem cells containing a thioredoxin-interacting protein (TXNIP) -derived peptide or a polynucleotide encoding the same as an active ingredient.

Aging is a natural phenomenon of life in living organisms, and most older people suffer from aging-associated diseases. In order to treat these aging-related diseases, there is a method of rejuvenating stem cells. Recently, studies on aging of stem cells have been conducted, and methods for rejuvenating stem cells have been reported.

Stem cells are divided into embryonic stem cells (ES cells) and adult stem cells according to their differentiation potential. Embryonic stem cells have the potential to differentiate into cells of all tissues. Adult stem cells, on the other hand, are stem cells that are specific to each organ obtained from the placenta at the stage of the development of each organ of an adult or embryo, and its differentiation capacity is generally limited to cells that make up the tissue (multipotent). . These adult stem cells remain in most organs after adulthood and compensate for the loss of normal or pathological cells. As the stem cells age, the tissues age and the ability to maintain homeostasis decreases, leading to reduced tissue regeneration.

Hematopoietic stem cells (hmatopoietic stem cells, hematopoietic stem cells, HSCs) are representative adult stem cells, capable of differentiating into all kinds of hematopoietic cells to provide blood cells for life. Hematopoietic stem cells produce blood throughout life and show high turn over. As hematopoietic stem cells age, their immune function decreases and aging-related diseases occur.

HSCs are differentiated into different types of hematopoietic progenitor cells according to differentiation stages, and LT-HSC (long-term HSC) located at the top of HSC differentiation hierarchy is all blood cells present in the body with self-replicating ability. It is possible to differentiate and to maintain hematopoiesis to permanently replenish the hematopoietic system. Short-term HSC (ST-HSC) has self-replicating ability to differentiate into all blood cells present in the body, but unlike LT-HSC, the duration of hematopoietic action is short, and multipotent progenitor (MPP) is present in the body. They have the ability to differentiate into all blood cells, but unlike LT-HSC and LT-HSC, they do not have self-replicating capacity. That is, LT-HSC, ST-HSC, MPP in order to reduce the self-replicating capacity as a stem cell can be said to be more differentiated cells. These three types of hematopoietic progenitor cells are characterized by expressing Sca1 and c-Kit without Lineage (Lineage ^- Sca1 ⁺ c-kit ⁺ ), and can be distinguished from other stages of HSC and are collectively called LSK cells. In addition, when MPP is further differentiated, it differentiates into myeloid progenitor cells CMP (common myeloid progenitor) and lymphocytic progenitor cells CLP (common lymphoid progenitor), and CMP is again megamegaloyocyte-erythroid progenitor (MEP) and granulocyte-monocyte progenitor (GMP) After differentiation into), finally differentiate into myeloid cells such as erythrocytes, giant cells, eosinophils, CLP is differentiated into lymphoid cells such as B cells, T cells, NK cells.

However, in the case of adult stem cells, stem cells also age with the aging of the body, and their functional activity or homeostasis cannot be maintained. The characteristics of aging HSCs are known to increase lineage skew, increase LT-HSC, decrease B-cells, increase myeloids, and increase ROS (Florian et al., 2012, Cell stem cell, 10). , 520-530; Montecino-Rodriguez et al., 2013, The Journal of clinical investigation, 123, 958-965). As HSC ages, the production of blood cells decreases, leading to a decrease in immune function, which can cause various diseases. Therefore, if the aging HSC can be reverse aging to maintain self-replicating ability and hematopoiesis, it will be the basis of a healthy immune system, and it is also expected to be of great help in the treatment or prevention of diseases.

In several studies, old mice have reported significant changes in the hematopoietic system. ^{^{CD34 - / Flk2 - / LSK (}} Lineage - / C-kit / + Sca-1 +) cells (LT-HSC) increases, and occur a lineage skewing (line deflection), increased reactive oxygen species (ROS), and peripheral Leukocytes decrease in the blood vessels. Older HSCs result in mitochondrial DNA damage, increased ROS and p38, DNA damage, telomere shortening, epigenetic alteration, loss of Cdc 42 polarity, increased Wnt-5a, and replication stress. Recently, a method of inhibiting Cdc42 activity and rejuvenating HSC has been reported.

Thioredoxin-interacting protein (TXNIP) is an inhibitor of thioredoxin and is known as a tumor suppressor because it prevents cell cycle progression (Han et al., 2003, Oncogene, 22, 4035). -4046), its use as a therapeutic agent for active oxygen-related diseases by increasing its resistance to free radicals (WO013159879). In addition, the relationship between TXNIP and hematopoietic activity has been reported to increase TXNIP by decreasing p38 and JNK to induce final differentiation of red blood cells (Gasiorek J. et al. 2015, Experimental Hematology, 43, 393-403). A decrease in the number of hematopoietic stem cells has been reported in knockout mice, but there is no disclosure of stem cell de-aging of TXNIP.

Therefore, the present inventors are trying to find a way to restore the function of the aged HSC peptides derived from thioredoxin-interacting protein (TXNIP) competitively transplant the old HSC (competitively transplantation) ), Increased engraftment of aged HSCs, decreased myeloid and decreased B cell trends, decreased LT-HSC ratio, decreased p38 activity and ROS compared to untreated TXNIP. In addition, in the acute leukopenia model, the administration of TXNIP-derived peptides increased leukocyte proliferation in old mice to the level of young mice, and also restored the polarization of Cdc42 in aged HSCs, and aging-related genes. It was confirmed that the expression of p16, p19, p21 and Wnt5a is reduced, and the homing of HSC is increased.

The present inventors found that thioredoxin-binding protein (hereinafter referred to as TXNIP) interacts with p38 mitogen-activated protein kinase (MAPK) to regulate aging of HSCs. In particular, the TXNIP-p38 interaction motif peptide was found to inhibit the activity of p38 to reduce reactive oxygen species (hereinafter referred to as ROS) and to convert aged HSCs to young HSCs in vivo and in vitro. The present invention was completed by discovering that the peptide can be used as an agent for preventing and treating aging-related diseases.

An object of the present invention is to provide a pharmaceutical composition for the reverse aging of stem cells containing a thioredoxin-interacting protein (TXNIP) -derived peptide or polynucleotide encoding the same as an active ingredient.

In order to achieve the above object, the present invention provides a peptide comprising any one selected from the amino acid sequence set forth in SEQ ID NO: 2, 3, 4 and 5.

The present invention also provides a fusion peptide of a TAT peptide consisting of a peptide comprising any one selected from the amino acid sequence of SEQ ID NO: 2, 3, 4 and 5 and the sequence of SEQ ID NO: 9.

In addition, the present invention is a thioredoxin binding protein-derived peptide that interacts with Thioredoxin-interacting protein (TXNIP) or p38 mitogen-activated protein kinase (MAPK), or a polynucleotide encoding them as an active ingredient. It provides a composition for reverse aging containing.

In another aspect, the present invention provides a stem cell anti-aging composition containing a thioredoxin binding protein derived from a thioredoxin binding protein or a p38 MAPK, or a polynucleotide encoding them as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of senile diseases containing thioredoxin binding protein or a thioredoxin binding protein-derived peptide interacting with p38 MAPK, or a polynucleotide encoding them as an active ingredient. .

The present invention also provides a method for screening a stem cell aging inhibitor comprising the following steps.

1) treating a test substance to stem cells deficient in thioredoxin-interacting protein (TXNIP); And

2) selecting a test substance exhibiting any one or more characteristics selected from the group consisting of a to d compared to a control not treated with the test substance;

a. reduced expression or activity of the p16, p19, p21 or Wnt5a genes;

b. reduced phosphorylation of p38;

c. Reduced reactive oxygen species (ROS) levels; And

d. Increased polarity of Cdc42 (Cell division control protein 42 homolog).

The present invention also provides an anti-aging health functional food containing a thioredoxin binding protein derived from a thioredoxin binding protein or a p38 MAPK-derived peptide, or a polynucleotide encoding them as an active ingredient.

In addition, the present invention provides an anti-aging cosmetic composition containing a thioredoxin binding protein derived from a thioredoxin binding protein or p38 MAPK, or a polynucleotide encoding them as an active ingredient.

The present invention also provides a method for treating senile disease by administering to a patient a pharmaceutically effective amount of a thioredoxin binding protein or a thioredoxin binding protein-derived peptide interacting with p38 MAPK, or a composition containing a polynucleotide encoding them. Provide a method.

The present invention also provides the use of a thioredoxin binding protein derived from a thioredoxin binding protein or p38 MAPK, or a polynucleotide encoding the same, in the manufacture of a medicament for the treatment of senile diseases.

The present invention also provides thioredoxin binding protein-derived peptides that interact with thioredoxin binding protein or p38 MAPK, or polynucleotides encoding them, for use in the treatment of senile diseases.

In the present invention, when a peptide derived from thioredoxin-interacting protein (TXNIP) competitively transplants old HSCs, the engraftment of old HSCs is increased compared to the control group without the peptides. It has been shown to reduce the tendency of series bias, decrease the LT-HSC ratio, decrease p38 activity and ROS, and administration of TXNIP-derived peptides promotes leukocyte proliferation in older mice to the level of young mice in acute leukopenia model. Thiore by restoring the polarization of Cdc42 in aged HSCs, decreasing the expression of p16, p19, p21 and Wnt5a, and increasing the homing of HSCs. Peptide binding protein-derived peptides can be usefully used as a composition for the reverse aging of hematopoietic stem cells.

Figures 1a to 1d is a diagram showing the inhibitory effect of p38 through the interaction of p38 with a peptide derived from thioroxox interacting protein (TXNIP). 1A is a diagram showing GST-full down analysis of GST-TXNIP-T and p38, and FIG. 1B is a diagram illustrating the inhibition of phosphatase activity of p38 by GST-TXNIP-T treatment through in vitro kinase assay. Figure 1c shows the reduction of phosphorylation of ATF-2, a substrate protein of the substrate, Figure 1c is a diagram showing the GST-full down analysis of TXNIP-derived peptides (TN12, TN13, TN14, TN15) and p38, Figure 1d Figure showing the binding site of TN13 and p38.

Figure 2a to 2c is a diagram showing the regulation of activity by the mutual binding of p38 of TAT-TN13. Figure 2a shows the interaction of p38 with TAT-TN13 through isothermal titration calorimetry (ITC) analysis, Figure 2b shows that TAT does not bind to p38 as a control, Figure 2c in aged bone marrow cells P38 activity of TAT-TN13 and inhibitory effect of ATF-2 phosphorylation, a substrate protein of p38.

3A to 3C are diagrams illustrating bone marrow cell senescence of TXNIP knockout (TXNIP ^{− / −} ) mice. Figure 3a is a diagram confirming the mRNA expression of TXNIP in the cells constituting the bone marrow, Figure 3b is a representative road TXNIP ^{+ / +} showing the effect of TXNIP on hematopoietic stem cell (HSC) ratio in 12 months old mice And TXNIP ^-/- bone marrow in the LT-HSC, ST-HSC, MPP ratio is confirmed by flow cytometry, Figure 3c is a diagram confirming the LT-HSC, ST-HSC, MPP ratio in various ages by flow cytometry.

4a to 4e are diagrams showing the expression of ROS (a) or p16 (b), p19 (c), p21 (d) or Wnt5a (e) mRNA which are HSC aging markers in hematopoietic stem cells (HSC) .

5a to 5g is a diagram showing the anti-aging effect of hematopoietic stem cells of TXNIP. Figure 5a is a diagram showing the change in the number of leukocytes in the blood after induction of acute leukopenia, Figure 5b is a diagram showing the survival rate after induction of acute leukopenia, Figures 5c to 5g are competitive in TXNIP ^{+ / +} and TXNIP ^-/- peripheral blood (c) and bone marrow (d) the engraftment of cell-based deflection (e), LSK transplantation (BMT) in the ^{graft-configuration} of ^{(Lin / Sca-1 + /} c-Kit +) cells (f), free radicals ( g) is confirmed.

6a to 6e are diagrams showing the interaction between TXNIP and p38 kinase. Figure 6a is a diagram confirming the mRNA expression of p38 isoproteins in bone marrow cells, Figures 6b to 6d is a flow cytometry (b), immunofluorescence staining (c) for p-p38 expression in TXNIP ^{+ / +} and TXNIP ^-/- , Western blot (d) is confirmed, Figure 6e is a diagram confirming the expression of TXNIP according to the age of LT-HSC by immunofluorescence staining.

7A to 7G are diagrams showing the mutual binding and binding sites of TXNIP and p38 kinase. FIG. 7a is a diagram showing that TXNIP and p38 are mutually coupled by immunoprecipitation analysis using TXNIP antibody, and FIG. 7b is a diagram showing that TXNIP and p38 bind to HSC through in situ proximity ligation (PLA) results. Figure 7c is a diagram showing the expression changes of TXNIP and p-p38 (p38 phosphorylation) by active oxygen, Figure 7d is the result of in situ PLA showing the interaction of p38 and TXNIP by age and reactive oxygen in HSC 7E is a GST-full down result confirming that the increase in the interaction of TXNIP and p38 by reactive oxygen is not related to p38 kinase dead. FIGS. 7F to 7G are TXNIP essential for the mutual binding of TXNIP and p38. Fig. 3 shows the amino acid residues of (f) and p38 (g).

8A to 8D are diagrams showing the activity inhibitory effect of p38 kinase of TAT-TN13. FIG. 8A shows that p38 phosphorylation is reduced when TAT-TN13 peptide is treated in aged HSC. FIG. 8B and FIG. 8C show the binding action of MKK3 (C) and MKK6 (D) and p38 to phosphorylate p38. The TAT-TN13 peptide is shown to be reduced, and FIG. 8D is a diagram showing the decrease in the cross-linking of p38 and TXNIP when immunotreated with TAT-TN13 peptide in aged bone marrow.

Figures 9a to 9g is a diagram showing the anti-aging effect of hematopoietic stem cells by inhibiting the activity of p38 kinase. Figure 9a is a diagram showing the engraftment of CD45.2 ⁺ in the peripheral blood after transplantation competitive, Figure 9b is a diagram of analyzing the series after deflection competitive transplantation, bone marrow cells, in Figure 9c ^{LSK (Lin - / Sca-1} + / c-Kit ⁺ ) is a diagram showing the configuration of cells, Figure 9d is a diagram showing the expression of p-p38, Figure 9e is a diagram showing the change in free radicals, Figure 9f is a series bias of 2 months and 12 months old mice Figure 9g is a diagram showing the survival rate after acute leukopenia induction.

Figures 10a to 10h is a diagram showing the hematopoietic stem cell de-aging effect by the inhibition of p38 kinase in vitro of TXNIP-derived peptide TAT-TN13. 10a to 10h are diagrams showing p-p38 expression (a), free radical levels (b), and Cdc42 polarity (c) after treatment of aged hematopoietic stem cells with TAT-TN13, p16 (d), p19 Figures (e), p21 (f) and Wnt5a (g) show mRNA expression and show short-term homing analysis (h) of hematopoietic stem cells in vivo.

11A to 11E are diagrams showing the in vivo HSC reverse aging effect of TXNIP-derived peptide TN13 expressed in plasmid gene. 11a to 11e show competitive grafting of CD45.2 ⁺ hematopoietic stem cells transduced with TN13 peptide followed by engraftment of transplanted cells in peripheral blood (a), lineage deflection (b), and LSK (Lin ⁻ / Sca) in bone marrow cells Fig. 1 shows the composition (c), p-p38 expression (d), and free radical levels (e) of -1 ⁺ / c-Kit ⁺ ) cells.

12a to 12f are diagrams showing the HSC reverse aging effect of TXNIP-derived peptide TAT-TN13 in vivo. 12a to 12e show the engraftment of transplanted cells in peripheral blood (a), lineage deflection (b) and bone marrow cells after competitive transplantation of old CD45.2 ⁺ hematopoietic stem cells treated with TAT-TN13 peptide in vitro Fig. 12F shows the composition (c), p-p38 expression (d) and free radical levels (e) of LSK (Lin ⁻ / Sca-1 ⁺ / c-Kit ⁺ ) cells. FIG. 12F shows TAT after induction of acute leukopenia. Figure showing the leukocyte number change by -TN13 treatment.

FIG. 13 shows Western blot inhibition of p38 MAPK activity in bone marrow cells of TN13, a peptide fragment derived from a thiredoxin interacting protein (TXNIP) -derived peptide that binds to p38 MAPK.

Figure 14a to 14c is a result of measuring the amount of inflammatory cytokine secreted by LPS-induced Raw264.7 macrophages according to the TN13 administration concentration (a) IL-1β cytokine production amount (b) IL- 6 Cytokine production amount (c) It is a figure which shows the result of having confirmed the change of the TNF-alpha cytokine production amount.

15 is a diagram showing the results of confirming that the TN13 peptide treatment also effectively inhibits p38 MAPK activity, which also causes the inhibition of NF-kB (p65) and c-Jun, which are inflammation regulatory transcription factors.

Figure 16 shows the results of effectively inhibiting iNOS and COX-2 protein expression in Raw264.7 macrophages induced inflammation by LPS by TN13 peptide treatment.

Fig. 17 shows the results of inhibition of NO production in macrophages by TN13 peptide treatment.

Figure 18a and Figure 18b shows the results of differentiation into mature osteoclasts by M-CSF and RANKL stained with TRAP (tartrate resistant acid phosphatase) solution to show the inhibition of osteoclast differentiation by TN13 peptide treatment. Differentiation into Mature Osteoclasts from Macrophage Host Raw 264.7 Cells FIG. 18B shows the results of differentiation from mouse bone marrow-derived osteoclast precursors to mature osteoclasts.

Figure 19a shows the experimental schedule of the experiment using the estrogen-deficient ovarian extraction osteoporosis model in the mouse, Figure 19b is a three-dimensional image of the inside of the femur of the above experimental model Sham group, osteoporosis induced osteoporosis after Vehicle ( OVX) and the group which received TN13 after osteoporosis induction.

20A to 20E are numerical representations of histological morphological examination of tissue samples of the left femur in an estrogen deficient ovarian osteoporosis model using mice. (b) the number of shochu bones. (c) bone surface. (d) Bone volume / total volume. (e) A diagram showing the results of confirming bone surface / total volume.

Hereinafter, the present invention will be described in detail.

The present invention provides a peptide comprising any one selected from the amino acid sequences set forth in SEQ ID NOs: 2, 3, 4 and 5. Preferably, a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 is provided.

The peptide according to the present invention is a peptide sequence that binds and interacts with p38 mitogen-activated protein kinase (MAPK) as part of the thioredoxin-interacting protein (TXNIP). According to the present invention, a sequence of a thioredoxin binding protein capable of interacting with p38 was first identified and has an effect such as anti-aging, inhibition of senescence of stem cells, proliferation of leukocytes, and reduction of aging-related genes through the sequence. . Through these effects, it is possible to prevent and treat senile diseases, inhibit aging of stem cells, and have an anti-aging effect.

The present invention also provides a fusion peptide of a TAT peptide consisting of a peptide comprising any one selected from the amino acid sequence of SEQ ID NO: 2, 3, 4 and 5 and the sequence of SEQ ID NO: 9. Preferably, a fusion peptide comprising a peptide comprising an amino acid sequence as set out in SEQ ID NO: 3 and a TAT peptide consisting of a sequence as set out in SEQ ID NO: 9 is provided.

The peptide may be a peptide derived from thioredoxin-interacting protein (TXNIP).

The thioredoxin binding protein-derived peptide used in the present invention may be derived from an animal, a plant or a microorganism, and is preferably a human-derived thioredoxin binding protein, but is a heterologous species having an activity equivalent to that of a human-derived thioredoxin binding protein. May be a derived protein.

The protein may additionally have modifications such as phosphorylation, acetylation, methylation, glycosylation, etc., but may be combined with other proteins, but may be regarded as the same as the protein before modification unless it is changed enough to lose the function of the protein. .

Preferably, the TAT peptide binds to an end of the peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, 3, 4 or 5, and the end is an amino terminus (5 'terminus, N-terminus) or carboxy terminus (3). Any of the 'terminal, C-terminus) is possible, but most preferably the amino terminus (5' terminus, N-terminus).

The thioredoxin binding protein is a protein as set forth in SEQ ID NO: 1, but at least one or several amino acids of the protein have the same activity as the protein or at the same gene position encoding the thioredoxin binding protein on a chromosome. It can consist of sequences that are added, deleted, or substituted.

The thioredoxin binding protein is at least 80% homology to the amino acid sequence of SEQ ID NO: 1, more specifically at least 90% homology, most specifically at least 95%, 96%, 97%, 98%, 99% or 99.5% It is composed of, but not limited to, sequences having homology.

Preferably, the thioredoxin binding protein-derived peptides that interact with thioredoxin-interacting protein (TXNIP) or p38 mitogen-activated protein kinase (MAPK) in the reverse aging composition are SEQ ID NOs: 2, 3, 4 and 5 It is a peptide comprising any one selected from the amino acid sequence described, and more preferably a fusion peptide further comprising a TAT peptide consisting of the sequence described in SEQ ID NO: 9.

Preferably, the thioredoxin binding protein-derived peptide interacting with thioredoxin-interacting protein (TXNIP) or p38 mitogen-activated protein kinase (MAPK) in the stem cell anti-aging composition is SEQ ID NO: 2, 3, It is a peptide comprising any one selected from amino acid sequences described in 4 and 5, more preferably a fusion peptide further comprising a TAT peptide composed of the sequence set forth in SEQ ID NO: 9.

Preferably, the peptide derived from thioredoxin binding protein interacting with thioredoxin-interacting protein (TXNIP) or p38 mitogen-activated protein kinase (MAPK) in the pharmaceutical composition for the prevention and treatment of senile diseases is SEQ ID NO: It is a peptide comprising any one selected from amino acid sequences described in 2, 3, 4 and 5, more preferably a fusion peptide further comprising a TAT peptide consisting of the sequence set forth in SEQ ID NO: 9.

The senile disease is preferably one or more selected from the group consisting of dementia, hypertension, Parkinson's disease, diabetes, cataracts, osteoporosis, stroke, periodontal disease and degenerative arthritis, but is not limited thereto. Preferably the senile disease is osteoporosis or degenerative arthritis.

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating inflammatory diseases comprising a thioredoxin binding protein derived from a thioredoxin binding protein or a p38 MAPK-derived peptide, or a polynucleotide encoding them as an active ingredient. The inflammation is one of biological tissue's defense response to a certain stimulus, and is a biological defense mechanism that attempts to restore the original state by removing various harmful stimuli. Inflammatory diseases include, for example, inflammation and gastritis, colitis, arthritis, nephritis, hepatitis, atherosclerosis, or degenerative diseases, and the like.

The thioredoxin binding protein-derived peptide preferably includes an amino acid sequence described by any one of SEQ ID NOs selected from the group consisting of SEQ ID NOs: 2 to 5, but is not limited thereto.

The thioredoxin binding protein-derived peptide is preferably a TAT peptide described in SEQ ID NO: 9 at the N-terminus, but is not limited thereto.

The thioredoxin binding protein-derived peptide is preferably encoded by a polynucleotide sequence described by any one of SEQ ID NOs selected from the group consisting of SEQ ID NOs: 42 to 45, but is not limited thereto.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. Pharmaceutically acceptable carriers are not particularly limited so long as they are suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components can be mixed and used as needed. Conventional additives can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into main dosage forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions. The composition of the present invention comprises 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.

The compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and more preferably administered once to several times a day.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, such as the method of administration, the site of interest, the condition of the patient, and the like. Therefore, when used in humans, the dosage should be determined in an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from an effective amount determined through animal testing. Such considerations when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

In the present invention, TXNIP ^-/- mice were used to investigate the function of senescence of hematopoietic stem cells (HSCs) of TXNIP. The senescence of TXNIP ^-/- HSC is caused by reactive oxygen species (ROS) or p38 activity, and the present invention is that TXNIP binds to p38 and inhibits the activity of p38, thereby inhibiting aging of HSC and deaging senescent HSC. Said. In addition, by making TXNIP-derived TN13 peptide and TAT-TN13 peptide, it was confirmed that inhibition of p38 and inhibition of aging of HSC can be usefully used as a composition for preventing senile disease and inhibiting aging by desensitizing stem cells younger. It was.

In a specific embodiment of the present invention, domain fragments of four thioredoxin-interacting protein (TXNIP) were prepared to confirm that the peptide binds to p38 (see FIG. 1), and to invade the cells. In order to increase the TXNIP domain fragment TN13 to produce a fusion peptide combining the HIV TAT transduction domain sequence was confirmed to inhibit the activity of p38 in combination with p38 (see Figure 2), hematopoietic and peripheral bone marrow in TXNIP knockout mice Flow cytometry of the hematopoietic stem cell (HSC) confirmed the senescence of HSCs (see FIG. 3), the expression of aging genes p16, p19, p21 and Wnt5a, p38 phosphorylation and free radical levels (see FIG. 4). , Confirming that hematopoiesis is inhibited (see FIG. 5), suggesting that TXNIP has an effect of preventing aging. In addition, TXNIP interacts with p38 and oxidative stress confirms that TXNIP enhances p38 interaction (see FIGS. 6 and 7). TXNIP ^{− / −} / p38 ^AF ^{/ +} mice (TXNIP knockout / p38) Inactivation) inhibits the senescence of HSC by inhibiting p38 (see FIG. 8), and confirms that TXNIP-derived peptides inhibit the activity of p38 by docking with p38 (see FIG. 9). TAT-TN13 fusion peptide Treatment in vitro with aged HSC to restore the polarization of Cdc42 (see FIG. 10C), reduce the expression of aging related genes p16, p19, p21 and Wnt5a (see FIGS. 10D-10G), The homing was increased (see FIG. 10H), and in vivo mice were treated with TN13 peptide or TAT-13 peptide to confirm the effect of reverse aging of HSCs (see FIGS. 11 and 12).

Therefore, TXNIP has the effect of preventing the aging of HSC and reverse aging of HSC by inhibiting activity by interacting with p38, TXNIP-derived peptides or polynucleotides encoding the same, the pharmaceutical composition for aging stem cell reverse aging, stem It can be usefully used as a composition for inhibiting cell aging and a pharmaceutical composition for preventing and treating senile diseases.

In addition, the present invention

2) provides a screening method for a drug for inhibiting stem cell aging, comprising the step of selecting a test substance having any one or more characteristics selected from the group consisting of a to d compared to a control not treated with a test substance. .

a. reduced expression or activity of the p16, p19, p21 or Wnt5a genes;

b. reduced phosphorylation of p38;

c. Reduced reactive oxygen species (ROS) levels; And

d. Increased polarity of Cdc42 (Cell division control protein 42 homolog).

In a specific embodiment of the present invention, the TXNIP-derived peptide recovers the polarization of Cdc42 in the HSC of old age (see FIG. 10C), and the expression of p16, p19, p21 and Wnt5a genes related to aging. By reducing (see Figures 10D-10G) and confirming increasing homing of HSCs (see Figure 10H), the present invention is derived from thioredoxin binding proteins that interact with thioredoxin binding proteins or p38 MAPKs. Peptides can be usefully used for the screening method of drugs for inhibiting stem cell aging.

The "health functional food" of the present specification is manufactured by using nutrients or ingredients (functional raw materials) having useful functions to the human body, which are easily deficient in a daily meal, and maintaining health through physiological functions or maintaining normal functions of the human body. The food is to maintain and improve the food as defined by the Commissioner of Food and Drug Safety, but is not limited to this and is not used to exclude the health food in the normal sense.

The health functional food of the present invention may be added as it is or used with other food or food ingredients, and may be appropriately used according to conventional methods.

In addition, the health functional food of the present invention further includes a food supplement acceptable food supplement. Food acceptable acceptable food supplement additives which can be used in the present invention include sugars such as glucose, fructose, maltose, sucrose, dextrin, cyclodextrin and natural carbohydrates such as sugar alcohols such as xylitol, sorbitol, erythritol, tau Natural flavors such as martin and stevia extract, synthetic flavors such as saccharin, aspartame, colorants, pectic acid or salts thereof, alginic acid or salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin , Alcohols, carbonating agents, and the like, but are not limited thereto.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring agents and neutralizing agents (such as cheese and chocolate).

Cosmetics prepared with the cosmetic composition of the present invention can be prepared in the form of general emulsion formulations and solubilized formulations. Cosmetics of the emulsified formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations are flexible lotion. In addition to cosmetics containing the extract of the light wood and conical extract of the present invention, by containing a dermatologically acceptable medium or base can be prepared in the form of adjuvants that can be applied topically or systemically applied commonly used in the field of dermatology.

In addition, the cosmetic composition of the present invention, in addition to thioredoxin binding protein-derived peptides, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances , Surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic or dermatological arts, such as any other ingredients commonly used in the art. And the above ingredients may be introduced in amounts generally used in the field of dermatology.

The cosmetic composition of the present invention is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, soap, shampoo It can be applied in the form of cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, press powder, loose powder, eye shadow and the like.

The present invention provides a method for treating senile disease by administering to a patient a pharmaceutically effective amount of a thioredoxin binding protein or a thioredoxin binding protein-derived peptide interacting with p38 MAPK, or a composition containing a polynucleotide encoding them. To provide. Preferably, thioredoxin binding protein-derived peptides that interact with thioredoxin-interacting protein (TXNIP) or p38 mitogen-activated protein kinase (MAPK) are amino acids of SEQ ID NOs: 2, 3, 4, and 5. A peptide comprising any one selected from the sequences, and more preferably a fusion peptide further comprising a TAT peptide composed of the sequence set forth in SEQ ID NO: 9.

The present invention provides the use of a thioredoxin binding protein derived from a thioredoxin binding protein or p38 MAPK, or a polynucleotide encoding the same, in the manufacture of a medicament for the treatment of senile disease. Preferably, thioredoxin binding protein-derived peptides that interact with thioredoxin-interacting protein (TXNIP) or p38 mitogen-activated protein kinase (MAPK) are amino acids of SEQ ID NOs: 2, 3, 4, and 5. A peptide comprising any one selected from the sequences, and more preferably a fusion peptide further comprising a TAT peptide composed of the sequence set forth in SEQ ID NO: 9.

The present invention provides a thioredoxin binding protein-derived peptide that interacts with a thioredoxin binding protein or p38 MAPK, or a polynucleotide encoding them, for use in the treatment of senile disease. Preferably, thioredoxin binding protein-derived peptides that interact with thioredoxin-interacting protein (TXNIP) or p38 mitogen-activated protein kinase (MAPK) are amino acids of SEQ ID NOs: 2, 3, 4, and 5. A peptide comprising any one selected from the sequences, and more preferably a fusion peptide further comprising a TAT peptide composed of the sequence set forth in SEQ ID NO: 9.

The present invention provides a method for treating an inflammatory disease by administering to a patient a pharmaceutically effective amount of a thioredoxin binding protein or a thioredoxin binding protein derived peptide that interacts with p38 MAPK, or a composition containing a polynucleotide encoding them. To provide. Preferably, thioredoxin binding protein-derived peptides that interact with thioredoxin-interacting protein (TXNIP) or p38 mitogen-activated protein kinase (MAPK) are amino acids of SEQ ID NOs: 2, 3, 4, and 5. A peptide comprising any one selected from the sequences, and more preferably a fusion peptide further comprising a TAT peptide composed of the sequence set forth in SEQ ID NO: 9.

The present invention provides the use of a thioredoxin binding protein derived from a thioredoxin binding protein or p38 MAPK, or a polynucleotide encoding the same, in the manufacture of a medicament for the treatment of an inflammatory disease. Preferably, thioredoxin binding protein-derived peptides that interact with thioredoxin-interacting protein (TXNIP) or p38 mitogen-activated protein kinase (MAPK) are amino acids of SEQ ID NOs: 2, 3, 4, and 5. A peptide comprising any one selected from the sequences, and more preferably a fusion peptide further comprising a TAT peptide composed of the sequence set forth in SEQ ID NO: 9.

The present invention provides thioredoxin binding protein-derived peptides that interact with thioredoxin binding protein or p38 MAPK, or polynucleotides encoding them, for use in the treatment of inflammatory diseases. Preferably, thioredoxin binding protein-derived peptides that interact with thioredoxin-interacting protein (TXNIP) or p38 mitogen-activated protein kinase (MAPK) are amino acids of SEQ ID NOs: 2, 3, 4, and 5. A peptide comprising any one selected from the sequences, and more preferably a fusion peptide further comprising a TAT peptide composed of the sequence set forth in SEQ ID NO: 9.

The matters mentioned in the uses, compositions and methods of treatment of the invention apply equally unless they contradict each other.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only for illustrating the present invention, the present invention is not limited by the following Examples and Experimental Examples.

[[ 실시예Example 1] One]

Preparation of Thioredoxin-Interacting Protein (TXNIP) -derived Peptide

<1-1> GST-TXNIP-T 제작<1-1> GST-TXNIP-T production

To confirm the interaction between TXNIP and p38 in-vitro , a TXNIP construct, GST-TXNIP-T, was constructed to inhibit the phosphatase activity of p38.

Specifically, His-labeled 150-317th amino acid fragment (GST-TXNIP-T; SEQ ID NO: 1) of TXNIP fused with GST to prepare a TXNIP construct that inhibits the phosphatase activity of p38 p38 protein was prepared, and the interaction with p38 was confirmed by GST-full down analysis and western blot. Recombinant protein, GST, GST-TXNIP (150-317) and His-p38 expressed in E. coli were purified by affinity chromatography. Dilute each 5 ug of protein with GST + His-p38 or GST-TXNIP (150-317) + His-p38 in a 1.5 ml tube containing cell lysis solution (500 ul) and add 20 ul of GST-4B bead. After reacting for 2 hours at ° C, the bead was washed three times with 1 ml of lysis solution. All supernatants were removed from the washed bead, lysis solution and 5X SDS sample solution were added, heated at 100 ° C. for 3 minutes, and electrophoresed on SDS-PAGE gel, followed by western blot. The primary antibody was -His and the secondary antibody was exposed through ECL solution using HRP-attached antibody to confirm that His-p38a selectively binds to GST-TXNIP (150-317). In order to confirm the relative amount by using the primary antibody α-GST as described above as above.

As a result, GST-TXNIP-T binds to p38 (FIG. 1A), and confirms that GST-TXN inhibits phosphorylation (p-ATF-2) of ATF-2, a lower signaling molecule of p38 (FIG. 1B). It was confirmed that TXNIP-T inhibited the phosphatase activity of p38.

<1-2> 4가지의 TXNIP의 도메인 단편을 제작<1-2> Four TXNIP Domain Fragments

In addition, we have prepared four domain fragments of TXNIP that interact with p38 as shown in Table 1 (TN12; SEQ ID NO: 2, TN13; SEQ ID NO: 3, TN14; SEQ ID NO: 4, and TN15; SEQ ID NO: 5). Among them, TN13 binds most strongly to P38 by GST-full down analysis (FIG. 1C). TN13 binds to the docking region of P38 as well as MKK6 (SEQ ID NO: 6), MKK3b (SEQ ID NO: 7), and MEF2A (SEQ ID NO: 8), which are known to bind p38 (Fig. 1D).

<1-3> 펩타이드의 침투효율을 증가시키기 위한 TAT-TN13의 제작<1-3> Fabrication of TAT-TN13 to Increase Penetration Efficiency of Peptides

In addition, the present inventors coupled the HIV TAT transduction domain sequence (SEQ ID NO: 9) to the N-terminus of TN13 prepared in Example <1-2> to efficiently deliver the synthesized protein into cells. In order to confirm the penetration efficiency of the peptide, a TAT-TN13 peptide was prepared in which FITC was bound to the end of the TAT sequence. Isothermal titration calorimetry (ITC) confirmed that the TAT-TN13 peptide binds to P38 (FIGS. 2A and 2B), and TAT-TN13 binds p38 and ATF-2 phosphorylation (p-p38 and p-ATF-2). By confirming the inhibition by Western blot (FIG. 2C), it was confirmed that TAT-TN13 effectively inhibited phosphatase activity of p38.

[[ 실시예Example 2] 2]

TXNIP와 p38의 변이체 제작Variant Construction of TXNIP and p38

To confirm the interaction between TXNIP and p38, variants of TXNIP and p38 were prepared. Was subjected to man TXNIP (NM_006472.5, SEQ ID NO: 10) or p38α (NM_139012, SEQ ID NO: 11) was used as a template using the primers shown in Table 2, the site-specific mutation (site-directed mutagenesis), the FLAG-CMV vector Introduced into the cells.

Experimental Example 1 Aging of TXNIP ^-/- HSC

<1-1> Confirmation of HSC cell composition of TXNIP ^-/-

In order to confirm the functionality of TXNIP from Stem Cell (Hematopoietic stem cells, HSC), bone marrow (bone marrow, BM) of the subset to configure and bone marrow ^{^{[Lin +, Lin -, MPP}} (multipotent progenitor), ST-HSC ( quantitative polymerase chain reaction in short-term HSC (LT-HSC), long-term HSC (LT-HSC), common lymphoid progenitor (CLP), common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), or megakeryocyte-erythroid progenitor (MEP) mRNA expression of TXNIP was confirmed by (qPCR). Total RNA of cells was isolated using RNeasy Micro Kit (Qiagen), and quantitative real-time PCR was performed using SYBR Premix ExTaq (Takara Bio) and Thermal Cycler Dice Real-Time System TP800 instrument (Takara Bio). Primers for amplification of gene fragments are shown in Table 3.

As a result, it was confirmed that the expression of TXNIP was significantly increased in LT-HSC (Fig. 3A).

In addition, the distribution of leukocytes was analyzed by flow cytometry in peripheral blood of TXNIP ^{+ / +} (wild type) and TXNIP ^-/- (TXNIP konckout) mice. For flow cytometry, femurs, tibias, hipbones or shoulder bones of mice were crushed to extract bone marrow cells and suspended in RPMI1640 medium containing 2% fetal bovine serum (FBS). It was. Cultured bone marrow cells were flow cytometrically analyzed using FACSCanto II (BD Biosciences), and cells were isolated using FACSAria cell sorter (BD Biosciences). The following antibodies were used as cell surface markers or lineage markers for flow cytometry or cell separation: anti-CD11b-biotin (clone M1 / 70, BD biosciences), anti-Gr-1-biotin (clone RB6). -8C5, BD biosciences), anti-B220-biotin (clone RA3-6B2, BD biosciences), anti-NK1.1-biotin (clone PK136, BD biosciences), anti-CD2-biotin (RM2-5) anti-TER119 -biotin (BD biosciences), anti-Streptavidin-APC-eFluor780 (eBiosciences), anti-c-kit-PE (clone 2B8, BD biosciences), anti-c-kit-PE-Cy7 (clone 2B8, BD biosciences), Anti-Sca-1-PE-Cy7 / PE / BV421 (clone D7, BD biosciences), anti-Sca-1-APC (clone D7, eBiosciences), anti-CD34-FITC / Alexa Fluor647 / PE (clone RAM34, BD biosciences), anti-CD135-APC / BV421 (clone A2F10.1, BD biosciences), anti-CD135-PE-Cy7 (clone A2F10.1, eBiosciences), anti-CD45.1 eFluor450 (clone A20, eBiosciences) or anti CD45.2-V500 (clone 104, BD biosciences). For peripheral blood analysis, anti-B220-PE (clone RA3-6B2, BD biosciences), anti-CD3e-APC-efluor780 (clone 17A2, eBiosciences), anti-CD3e-BV421 / PE-Cy7 (clone 17A2, 145-2C11 , BD biosciences), anti-Gr-1-Alexa Fluor 488 / eFluor 660 (clone RB6-8C5, eBiosciences), anti-CD11b-PE-Cyanine7 (clone M1 / 70, eBiosciences) or anti-CD45.2-APC ( clone 104, BD biosciences). ^{^{Also, Lineage - / Sca-1 +}} / c-kit + (LSK) to the cells was prepared using the MACS purification, TXNIP or p-p38, wherein -TXNIP (clone D5F3E, Cell Signaling) to the cells in the dyeing, Anti-rabbit IgG Alexa Fluor 647 (Life technology) or anti-phospho-p38-APC (clone 4NIT4KK, eBiosciences) was used.

LT-HSC is a top-level blood-stem cell, capable of self-replicating and differentiating into all blood cells in the body and continuing hematopoiesis to permanently replenish the hematopoietic system. ST-HSC has a self-replicating ability to differentiate into all blood cells in the body, but unlike LT-HSC, ST-HSC has a short duration of hematopoietic action and MPP has the ability to differentiate into all blood cells in the body. Unlike LT-HSC and ST-HSC, it does not have self-replicating capability. That is, LT-HSC, ST-HSC, MPP in order to reduce the self-replicating capacity as a stem cell can be said to be more differentiated cells.

In normal rats, as the age increases, the number of LT-HSC increases, the ratio of LT-HSC in Lin (lineage) ^- / Sca-1 ⁺ / c-kit ⁺ ) increases, and the percentage of MPP decreases. In the flow cytometry of TXNIP ^-/- mice, the ratio of LT-HSC was increased compared to TXNIP ^{+ / +} (12 months of age, Figure 3b), LSK (Lin (lineage) ^- / Sca-1 ⁺ / c-kit) ⁺⁾ cells, i.e., LT-HSC, ST-HSC , MPP, the 12 months of age TXNIP ^{- / -} the ratio of the LT-HSC in mice is increased to roughly TXNIP ^{+ / +} 24 months of age similarly the ratio of MPP It was confirmed that the HSC of TXNIP ^{− / −} was prematurely aged (FIG. 3C).

<1-2> Expression of HSC aging factor of TXNIP ^-/-

To determine whether TXNIP inhibits the expression of aging-related factors in HSCs, we measured the levels of reactive oxygene species (ROS) known to increase with aging in HSCs of TXNIP ^-/- mice and the p16, mRNA expression of p19, p21 and Wnt5a was confirmed using qPCR. Bone marrow cells were stained with cell surface markers immediately after extraction for measurement of free radicals and then reacted with free radical-specific probes CM-DCF-DA (Molecular Probes / Thermofisher Scientific) or Dihydroethidium (DHE, Molecular Probes / ThermoFisher Scientific). The flow was analyzed by FACSCanto II (BD Biosciences).

As a result, the HSCs of 12-month ^-old TXNIP ^-/- mice compared to the control (TXNIP ^{+ / +} ) It was confirmed that free radicals were increased (FIG. 4A), and the expression of p16, p19, p21 and Wnt5a increased similarly to the 24 month old TXNIP ^{+ / +} HSC which had already advanced (FIG. 4B to FIG. 4E). Thus, the results suggest that the genetic deficiency of TXNIP is the cause of aging of HSCs.

<1-3> Confirmation of hematopoietic inhibition of TXNIP ^-/-

To identify the role of TXNIP in hematopoiesis, 5-FU (5-fluoracil, 150 mg / kg), which targets cells circulating in the blood and induces acute leukopenia, is expressed in TXNIP ^{+ / +} and TXNIP ^{− /} -Mice were administered and the number of white blood cells in the blood was checked for 17 days.

As a result, the number of leukocytes gradually decreased in both TXNIP ^{+ / +} and TXNIP ^-/- mice, resulting in the lowest number on day 6. TXNIP ^{+ / +} mice recovered normal state in 14 days, while TXNIP ^-/- Mice failed to recover and on day 17 all TXNIP ^{− / −} mice died (FIG. 5A, FIG. 5B).

In addition, a competitive transplantation assay was performed to confirm the autonomous function of HSC cells. For competitive bone marrow transplantation analysis, bone marrow cells were extracted from young mice (2 months old) ( CD45.2 ⁺ ) and LT-HSCs were isolated. Number of pseudogenes 6-8 weeks old lethal irradiated (9Gy) by mixing 400-500 LT-HSCs with 1 x 10 ⁶ to 1.5 x 10 ⁶ competitor bone marrow cells (CD45.1 ⁺ ) The tail vein of the congenic recipient (CD45.1 ⁺ ) mice was injected. After 16 weeks, the engrafted LT-HSC proliferation was subjected to flow cytometry of peripheral blood or bone marrow cells obtained from the tail vein. In addition, in order to check the active oxygen level of LT-HSC, bone marrow cells were stained with cell surface markers immediately after extraction, followed by active oxygen-specific probe CM-DCF-DA (Molecular Probes / Thermofisher Scientific) or Dihydroethidium (DHE, Molecular Probes). / ThermoFisher Scientific) and flow cytometry using FACSCanto II (BD Biosciences).

As a result, the engraftment of CD45.2 ⁺ HSCs in the peripheral blood and bone marrow of the recipients transplanted with HSCs derived from TXNIP ^{− / −} mice was significantly reduced (FIG. 5C, FIG. 5D). As in the above experiment, when the old HSC is transplanted into a young donor, the engraftment of B lymphocytes is suppressed and the differentiation into myeloid series is called lineage skewing. Known. In the competitive transplantation, the HSC series bias was confirmed by flow cytometry. As a result, TXNIP ^-/- CD45.2 ⁺ cells derived from HSC of mice were identified as TXNIP ^{+ / +} . Compared to CD45.2 ⁺ cells, the differentiation into B lymphocytes (B220 ⁺ ) and T lymphocytes (CD3 ⁺ ) decreased and differentiation to myleoid was observed, confirming that HSC of TXNIP ^{− / −} was aged (FIG. 5E). ). In addition, it was confirmed that the cells derived from HSC of TXNIP ^{− / −} mice showed a decrease in MPP and an increase in LT-HSC among CD45.2 ⁺ LSK cells compared to TXNIP ^{+ / +} (FIG. 5F), and TXNIP ^{− / −} mice. In the HSC-derived LT-HSC increased free radicals (Fig. 5g), it can be seen from the results that TXNIP is a regulator of hematopoietic action and the lack of TXNIP aging HSC.

Experimental Example 2 Confirmation of p38 Activation in TNXIP ^-/- HSC

p38 is known as an enzyme that plays an important role in cell signaling pathways induced by oxidative stress and aging-related gene expression of HSCs. Therefore, in order to examine the relationship between p38 and aging of HSC, the present inventors have found that p38 isoforms (aform, α, mRNA expression of β, γ, and δ) was confirmed.

As a result, it was confirmed that p38α is mainly expressed among the four p38 isoforms, and is most predominantly expressed in LT-HSC (FIG. 6A).

In addition, to determine the relationship between p38 and TXNIP in HSC aging, p38 activity was identified by age (2, 12 and 24 months) in TXNIP ^{+ / +} and TXNIP ^{− / −} mice. For immunofluorescence staining, LT-HSC isolated from bone marrow cells was dropped on a cover glass coated with fibronectin, reacted at 4 ° C for 10 minutes, adhered to the membrane, and then punctured in a membrane with 0.2% Triton X-100 solution. Block in PBS containing BSA for 30 minutes, react with anti-p38 or anti-TXNIP primary antibody for 1 hour at room temperature, wash with PBS three times for 5 minutes, then secondary antibody, Alexa Fluor 647 or Alexa Fluor 488 After treatment for 1 hour at room temperature and then washed three times for 5 minutes and then put the mounting solution containing the DAPI on the slide glass was fixed and confirmed by the confocal microscope image.

As a result, the phosphorylation of p38 protein (p-p38) was increased by increasing age and decreasing TXNIP (TXNIP ^-/- ) by flow cytometry (Fig. 6b), immunofluorescence staining (Fig. 6c), western blot (Fig. 6d). In addition, by confirming that the expression of TXNIP in LT-HSC is increased at 24 months compared to 2 months (FIG. 6E), the results suggested that TXNIP and p38 are associated with aging in HSC.

<< 실험예Experimental Example 3> 3> HSC에서From HSC TXNIP와With TXNIP p38의 직접 상호작용 확인 Confirm direct interaction of p38

In addition, we performed immunoprecipitation and in situ proximity ligation (PLA) analysis to determine whether TXNIP regulates p38 activity by p38 interaction. For in situ PLA analysis, HSC was treated with H ₂ O ₂ , sprinkled on fibronectin-coated coverslips, and reacted for 4 to 10 minutes to fix. In situ binding of TXNIP and p38 was carried out according to the manufacturer's instructions using a Duolink assay kit (Sigma), stained using TXNIP antibody (MBL) and p38 antibody (Cell signaling), and LSM510 confocal microscope (Carl Zeiss). Got an image. Immunoprecipitation isolates bone marrow cells, breaks them down with cell lysis solution, performs high-speed centrifugation, separates supernatants, dilutes proteins to 500 ug / 500 ul, and then dilutes anti-TXNIP antibodies. 10 ug / 500 ul concentration, 20 ul of protein A agarose bead that can bind to the primary antibody and reacted at 4 ℃ for 4 hours, washed three times with 1 ml of lysis solution and remove all lysis solution 25 ul of lysis solution and 5X SDS sample solution were added and boiled at 100 ° C. for 3 minutes, followed by electrophoresis on SDS-PAGE gel, followed by Western blotting.

As a result, it was confirmed that TXNIP and p38 were mutually bound from the results of immunoprecipitation (FIG. 7a), and it was confirmed that TXNIP and p38 were mutually bound from the PLA result (Fig. 7b).

In addition, to investigate the effect of free radicals (ROS) on the interaction between TXNIP and p38, the expression of TXNIP and p-p38 was confirmed by Western blot after treatment with H ₂ O ₂ , and in situ PLA analysis was performed. The binding of TXNIP and p38 was confirmed.

As a result, TXNIP in bone marrow cells increased very rapidly by H ₂ O ₂ (0.5 mM), peaked at 15 minutes, then gradually decreased, and p-p38 increased by H ₂ O ₂ (0.5 mM). Up to 60 minutes (FIG. 7C). In addition, in situ PLA analysis, H ₂ O ₂ treatment in young HSC increased the PLA signal to the HSC level of old, it was confirmed that the interaction of TXNIP and p38 increased (Fig. 7d). In addition, GST-full down assay was performed in HEK293T cells overexpressing TXNIP and p38, and the binding of TXNIP and p38 was increased by treatment with H ₂ O ₂ (0.5 or 1 mM). It was confirmed that not related to, and through these results it was confirmed that the oxidative stress has an effect of enhancing the interaction of TXNIP and p38 (Fig. 7e).

Next, four site-directed mutagenesis were applied to the hydrophobic residues of TXNIP, which are considered potential docking domains, in order to examine the essential amino acid residues of the TXNIP-p38 interaction. ) And confirmed binding to p38 by GST-full down analysis.

As a result, the positional mutants of the L290 and L292 residues completely reduced the binding to p38 (FIG. 7F), indicating that L290 and L292 of TXNIP are important for the interaction with p38. In addition, all binding domain variants of p38 were shown to have reduced cross-linking with TXNIP (FIG. 7G). The results suggest that TXNIP modulates the activity of p38 of HSC by directly binding to p38.

<< 실험예Experimental Example 4> in 4> in vivo에서in vivo p38의 활성을 억제하고 inhibit the activity of p38 ROS를ROS 감소시켜 Reduce HSC의HSC 역노화Aging 확인 Confirm

To investigate the role of p38 in the rejuvenation of aged HSCs, we used competitive transplantation using either TXNIP ^-/- / p38 ^AF ^{/ +} mice (TXNIP knockout / p38 inactivation) or mice treated with p38 inhibitor SB203580. And engraftment of CD45.2 ⁺ LT-HSC in peripheral blood vessels was analyzed.

As a result, the characteristics of aging (ie LT-HSC engraftment) in young TXNIP ^-/- (TXNIP ^-/- 2M) Decreased, series-biased analysis showed that B lymphocytes and T lymphocytes decreased and increased myeloid, but the inhibition of p38 activity (TXNIP ^-/- / p38 ^AF ^{/ +} or TXNIP ^-/- / SB) suppressed the characteristics of aging. It was confirmed (Fig. 9a. B). In addition, analysis of the composition of CD45.2 ⁺ LSK cells after competitive transplantation showed that in TXNIP ^-/- / p38 ^AF ^{/ +} or TXNIP ^-/- / SB, LT-HSC decreased and MPP increased (FIG. 9C). By confirming that p38 activity and free radical levels are low (FIG. 9D, FIG. 9E), it was demonstrated that inhibition of p38 prevents senescence of hematopoietic stem cells.

In addition, TXNIP ^{+ / +} , TXNIP ^{+ / +} / p38 ^AF ^{/ +} , Analysis of leukocyte distribution in peripheral blood of TXNIP ^-/- and TXNIP ^-/- / p38 ^AF ^{/ +} mice showed aging series bias in 12-month-old TXNIP ^-/- mice, but 12-month-old TXNIP ^-/- / p38 ^It was confirmed that the ^{AF / +} mice recovered to TXNIP ^{+ / +} mouse levels (FIG. 9F).

In addition, when 5-FU was administered to induce acute leukopenia, the survival rate of TXNIP ^-/- mice gradually decreased and died after 18 days, and the inhibition of p38 activity significantly increased the survival rate of TXNIP ^-/- mice. Confirmation (FIG. 9G) shows that p38 ages HSCs and that inhibition of p38 activity induces rejuvenation of HSCs.

<< 실험예Experimental Example 5> 5> TNXIPTNXIP 유래 origin 펩타이드의Peptide p38 활성 억제 확인 Confirmation of p38 activity inhibition

As a result of confirming the inhibitory effect of P38 phosphorylation of TAT-TN13 peptide on aged bone marrow cells or HSC, the expression of p-P38 and p-ATF2 was inhibited in a concentration-dependent manner of TAT-TN13 peptide, so that TAT-TN13 peptide effectively phosphorylated P38. It was confirmed to inhibit the activity of the enzyme (Fig. 8a).

To investigate the p38 inhibitory mechanism of the TAT-TN13 peptide, TAT-TN13 peptide was overexpressed with another p38 upper kinase MKK3 or MKK6 that binds to p38 and treated with p38 protein and GST-full down assay. It was confirmed that the binding of p38 and MKK3 or p38 and MKK6 was inhibited by -TN13 peptide (FIGS. 8B and 8C).

The results indicate that the TAT-TN13 peptide shares the p38 docking site with MKK3 and MKK6, and inhibits the activity of the p38 kinase by interfering with the upper phosphatase through mutual interaction with p38. In addition, as shown in FIG. 8D, p38 protein could be identified by immunoprecipitation of TXNIP in the group not treated with TAT-TN13 peptide, whereas when TAT-TN13 10 uM was treated, p38 protein sedimentation was significantly inhibited. Peptides and TXNIP were confirmed to bind p38 competitively. From the above results, it can be seen that TXNIP directly binds to p38 to inhibit activity.

<< 실험예Experimental Example 6> 6> in vitroin vitro 에서 TAT-TN13로 인한 Caused by TAT-TN13 HSC의HSC 역노화Aging 확인 Confirm

To determine if inhibition of p38 with TAT-TN13 could return the aged HSC back to the young phase, 10 h of TAT-TN13 was treated for 16 hours in the aged HSC.

As a result, it was confirmed that TAT-TN13 inhibited p-p38 (FIG. 10A) and free radicals (FIG. 10B) to a level similar to that of p38 inhibitor (SB203580) treatment in aged HSC.

According to Florian et al., The polarity of Cdc42 is an indicator of the age of HSCs (the polarity is lost when aging), and pharmaceutical inhibition of Cdc42 leads to reverse aging of aged HSCs (2013, Nature, 503, 392-). 396).

In this experimental example, the polarity of Cdc42 was confirmed by immunofluorescence staining as an indicator of aging. Aged HSC was sprayed onto fibronectin-coated coverslips and incubated for 4 to 10 minutes to fix, followed by treatment with 10 uM of TAT, TAT-TN13 or SB203580 for 16 hours, followed by Cdc42 antibody (Cell signaling). , Alexa Fluor 546 antibody (Life technology), DAPI containing mounting reagent (Molecular Probes). Confocal microscope confirmed the polarization trend of Cdc42 of 50 to 60 HSC for polarity analysis.

As a result, most of the aged HSCs are depolarized (Old HSC-No, Old HSC-TAT), but when P38 is inhibited with TAT-TN13 or SB203580, HSC is reverse aged and young HSCs (2M HSC) Similarly, the polarity is increased (FIG. 10C).

In addition, to determine whether p38 activity inhibition induced HSC reverse aging, TAT-TN13 or SB203580 was treated in older HSCs and the expressions of p16, p19, p21 and Wnt5a were confirmed by qPCR, and short-term homing analysis (Short -term homing assay). For short-term homing analysis, CD45.2 ⁺ HSCs were treated with 10 uM of TAT, TAT-TN13 (synthetic from Peptron) or SB203580 (Selleckchem) immediately after extraction and incubated at 37, 5% CO ₂ conditions for 16 hours. 6-8 week old congenic recipients (CD45.1) lethal radioactively irradiated (9 Gy) to 10,000 TSCs, TAT-TN13 or SB203580 treated HSCs or no drug treated HSCs ⁺ ) Were injected into the tail vein of the mice. Eighteen hours later, bone marrow was collected from the recipient mice, stained with CD45.1 or CD45.2 antibodies, and flow cytometric analysis was performed to analyze the relative frequency of CD45.2 ⁺ cells in total bone marrow cells.

As a result, TAT-TN13 or SB203580 significantly reduced the expression of the aging related genes p16, p19, p21 and Wnt5a (FIGS. 10D-10G) and confirmed that increasing homing of HSCs (FIG. 10H). Inhibition of p38 activity by TAT-TN13 was found to induce reverse aging of HSCs.

<< 실험예Experimental Example 7> 7> in in vivovivo 에서in GFPGfp -TN13 및 TAT-TN13에 의한 노화된 -Aged by TN13 and TAT-TN13 HSC의HSC 역노화Aging 확인 Confirm

In order to confirm that the TN13 peptide is also applied to animals, CD45.2 ⁺ HSC was extracted from the mouse, infected with GFP-bound GFP-TN13-expressing lenti-viral vector three times for 36 hours, and then GFP ⁺ HSC. Bay was extracted and mixed with CD45.1 ⁺ bone marrow cells for competitive transplantation. As a control, a lenti-viral vector expressing only GFP without TN13 binding was used.

As a result, TN13 inhibits aging in young TXNIP ^-/- mice (TXNIP ^-/- 2M-GFP) and old TXNIP ^{+ / +} mice (TXNIP ^{+ / +} Old-GFP). Specifically, peripheral blood was analyzed to compare the ratio of CD45.2 ⁺ cells. As a result, young TXNIP ^-/- mice (TXNIP ^-/- 2M-GFP) and aged TXNIP ^{+ / +} mice (TXNIP ^{+ / +} Old-GFP) were compared. Engraftment of CD45.2 ⁺ was decreased, and the decrease of CD45.2 ⁺ was increased by TN13 expression (TXNIP ^{− / −} 2M-GFP-TN13 or TXNIP ^{+ / +} Old-GFP-TN13) (FIG. 11A). ). Series bias analysis also inhibited myeloid increase and decreased B220 cells in TXNIP ^-/- 2M-GFP-TN13 and aged TXNIP ^{+ / +} Old-GFP-TN13 in CD45.2 ⁺ cells of peripheral blood. (FIG. 11B), it was confirmed that TN13 inhibited the aging tendency by inhibiting the increase of LT-HSC and increasing the decreased MPP in CD45.2 ⁺ LSK cells (FIG. 11C). It was also confirmed that TN13 inhibits p38 and free radicals in TXNIP ^-/- 2M-GFP-TN13 and TXNIP ^{+ / +} Old-GFP-TN13 (FIGS. 11D, 11E). The results suggest that TN13 can inhibit aging of HSCs.

in vivo To confirm the utility of TAT-TN13 as a reverse aging drug against aging HSC, aged HSC (TXNIP ^{+ / +} Old-TAT-TN13) treated with TAT-TN13 or TXNIP ^-/- HSC (12 months old) TXNIP ^-/- 12M-TAT-TN13) was competitively transplanted.

As a ^{result, TXNIP + / + Old-TAT} -TN13 or TXNIP ^{- / -} control group ^{12M-TAT-TN13 (TXNIP +} / + Old-TAT or ^{TXNIP - / - 12M-TAT)} CD45.2 + engraftment of the cells as compared to 12 (b), the aging series bias tended to recover similar to young TXNIP ^{+ / +} (Fig. 12b), and the LT-HSC ratio of CD45.2 ⁺ LSK cells decreased and MPP ratio increased (Fig. 12a). 12c), p-p38 and free radicals are reduced (Fig. 12d, e), it was confirmed that all the aging characteristics appearing in the control group is changed to reverse aging characteristics. In addition, 5-FU (100 mg / kg) was administered to aged TXNIP ^{+ / +} mice to induce acute leukopenia, and when TAT-TN13 (25 mg / kg) was administered daily for 4 days after 1 day, the control group (24M-TXNIP ^{+ / +} -TAT) showed a rapid decrease in the white blood cell count, and there was almost no white blood cell on the 10th, and all died.In contrast, the TAT-TN13 group (24M-TXNIP ^{+ / +} TAT-TN13) rapidly increased the white blood cell count. It was observed that the number of young TXNIP ^{+ / +} than, and was stabilized after 10 days to maintain the same level as the young TXNIP ^{+ / +} (TXNIP ^{+ / +} 2M) on day 13 (Fig. 12f). The results suggest that TAT-TN13 can be used as a drug to reverse aging HSC.

<< 실험예Experimental Example 8>TN13에8> with TN13 의한 p38 By p38 MAPKMAPK 활성 억제 확인 Confirmation of Active Suppression

LGTSFKGKYGCVD (SEQ ID NO: 46) corresponding to amino acid sequence 110-122 of TXNIP at a position not involved in TAT-TXNIP-derived or P38 interaction to identify selective P38 kinase activity inhibition of TAT-TN13 peptide in bone marrow cells Peptide TAT-TN13C containing the sequence was prepared and tested as a control. The result confirmed by Western blot is shown in FIG. As confirmed in FIG. 13, it was confirmed that TAT-TN13 reduced the expression of p-P38 in a concentration-dependent manner. As a result, it was confirmed that the TAT-TN13 peptide selectively and effectively inhibited the activity of P38 kinase.

<< 실험예Experimental Example 9>TN13에9> with TN13 의한 항염증 효과 확인 Anti-inflammatory effects

<9-1> 염증성 사이토카인 분비 억제 확인<9-1> Confirmation of Inflammatory Cytokine Secretion

Enzyme-linked immunosorbent assay (ELISA) confirmed whether TN13 peptide inhibited the secretion of inflammatory cytokines (IL-1β / IL-6 / TNF-α) out of cells. Raw264.7 macrophages were aliquoted into 1 × 10 ⁶ cells / well in a 12-well plate and incubated for 2 hours. Thereafter, TN13 peptides were treated by concentration, and then cultured for 1 hour, LPS was treated at 100 ng / mL in each well, and the supernatant cultured for 18 hours was taken to measure the amount of IL-1β, IL-6, and TNF-α. . Cytokine was measured by dispensing 100 μL of standard and samples prepared in 96 well plates coated with antibodies reacting to each cytokine according to the manufacturer's analysis method, incubating at room temperature for 2 hours, washing three times, and then using the biotinylated antibody reagent well. 50 μL of the solution was incubated at room temperature for 2 hours, washed three times, and 100 μL of the Streptavidin-HRP solution was incubated for 20 minutes. After washing three times, 100 μL of each TMB substrate solution was finally used. Aliquots were made and reacted at room temperature in the dark. The reaction was stopped by adding 50 μL of stop solution (0.16 M sulfuric acid), and the absorbance was measured and measured at 450 nm using a UV / VIS spectrophotometer (SpectraMax i3x, Molecular Device, USA).

The results are shown in Figs. 14A to 14B.

IL-1β, IL-6 and TNF-α, which are representative of inflammatory cytokine, are mediators of inflammatory responses and are known to be particularly involved in early inflammatory responses. As shown in FIG. 14, TN13 peptide treatment effectively reduced the production of increased IL-1β, IL-6 and TNF-α in LPS-induced Raw264.7 macrophages, as measured in the amount of inflammatory cytokine production. I was. In particular, when treated with 20 μM concentration of TN13 peptide, the production of IL-1β, IL-6 and TNF-α was suppressed by about 50% compared to the LPS-treated group. As a result, it was confirmed that the TN13 peptide effectively inhibits the secretion of inflammatory cytokines.

<9-2> p38 MAP <9-2> p38 MAP KinaseKinase , , NFNF -- kBkB (p65) 및 c- (p65) and c- Jun의Jun's 활성 억제 확인 Confirmation of Active Suppression

To determine whether p38 MAP Kinase activation in RAW264.7 cells via LPS stimulation is inhibited by TN13 peptide, RAW264.7 cells were suspended in DMEM containing 10% FBS and then 5 × in 6 well plates (Corning, USA). 2 ml of each well was dispensed into 10 ⁵ cells / ml and stabilized for 2 hours in a 37 ° C 5% CO ₂ incubator. After exchange with fresh DMEM medium, TN13 peptides were treated to cells for 1 hour at concentration (1/5/10/20 μM). After the reaction of the TN13 peptide was terminated by adding LPS to the existing medium at a concentration of 100 ng / ml for 30 minutes. After LPS stimulation, the media was removed and washed with Cold PBS. Cell lysates were extracted with Lysis buffer (+ 1% Triton X-100). Protein content was quantified by BCA protein assay (Thermo, USA) to electrophorese 40 ug of protein and transferred to Immobilon-PVDF membrane (Merck, USA). The transferred membrane was blocked with 5% skim milk dissolved in Phosphate-buffered saline Tween-20 (PBST) (20 mM Tris, pH 7.6, 136 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature, followed by anti-phospho overnight reaction at 4 ° C with -p65, anti-p65, anti-phospho-p38, anti-p38 MAP kinase, anti-phospho-c-Jun, anti-c-Jun and β-actin primary antibody (1: 1000 dilution) After washing three times with PBST, and reacted with HRP-conjugated secondary antibody (1: 1000 dilution) for 1 hour at room temperature. After washing three times with PBST, immunoreactive protein bands were detected using WSE-6200 LuminoGraph II (ATTO, Japan).

The results are shown in FIG.

The activity of p38 MAP Kinase is known to induce the activity of NF-kB (p65) and c-Jun, which are important transcription factors in the inflammatory response. As confirmed in FIG. 15, it was confirmed that the TN13 peptide inhibited the activities of NF-kB (p65) and c-Jun by effectively inhibiting p38 MAP Kinase activity.

<9-3> TN13 <9-3> TN13 펩타이드에On peptides 의한 대식세포에서 Caused by macrophages iNOSiNOS 및 COX-2 단백질 발현을 억제 확인 And inhibits COX-2 protein expression

When inflammatory reactions occur, inflammatory factors such as excess nitric oxide (NO) and prostaglandin E2 (PGE 2) are formed by inducible NO synthase (iNOS) and cyclooxygenase (COX-2). To determine the effect of TN13 peptides on these two proteins, RAW264.7 cells were suspended in DMEM containing 10% FBS, followed by 2.5 × 10 ⁵ cells / ml in 6 well plates (Corning, USA). 2 ml of each well was stabilized for 2 hours at 37 ℃ 5% CO2 incubator. After exchange with fresh DMEM medium, TN13 peptides were treated with the cells by concentration (1/5/10/20 μM) for 1 hour.

The results are shown in FIG.

As shown in FIG. 16, the LPS-untreated group showed little expression of COX-2 protein, whereas the LPS-treated group showed a marked increase in expression. TN13 peptide treatment showed LPS-induced inflammation in Raw264.7 vs. It was confirmed that the phagocytosis effectively inhibits iNOS and COX-2 protein expression. As a result, the TN13 peptide inhibited NO and PGE2 production by controlling the expression of iNOS and COX-2 proteins. Especially, in the case of 20 μM TN13 peptide, the expression of iNOS and COX-2 proteins was reduced compared to other concentrations. It was confirmed to inhibit to a similar level as the untreated group.

<9-4> TN13 <9-4> TN13 펩타이드에On peptides 의한 대식세포에서의 NO 생성 억제 확인 NO production inhibition in macrophages by

For NO measurement, divide the cells into 1 × 10 ⁵ cells / well in a 48-well plate, incubate for 2 hours, and incubate for 1 hour by treating TN13 peptide with concentration (1/5/10/20 μM). The wells were treated with LPS at a concentration of 100ng / mL and incubated for 24 hours. After incubation, 100 μL of the culture supernatant was taken and tested according to the manual of Nitric Oxide detection kit (IntRON biotech.).

The results are shown in FIG.

As can be seen in Figure 17, LPS-induced inflammation of Raw264.7 macrophages significantly increased NO production (10.433 μM), but Raw264.7 macrophage treated with TN13 peptides by concentration (1-20 μM) NO inhibited the production of NO. In particular, the treatment of 20 μM TN13 peptide showed NO production near the normal group (0.35 μM) without LPS treatment.

As a result, it was confirmed that treatment of TN13 peptide effectively reduced NO production when inflammation was induced.

<< 실험예Experimental Example 10> TN13에 의한 골다공증 치료 효과 확인 10> Confirmation of treatment effect of osteoporosis by TN13

<10-1> TN13에 의한 파골 세포 분화 억제 확인<10-1> Confirmation of inhibition of osteoclast differentiation by TN13

Dispense the raw 264.7 cells into a 12 well plate at a density of 1 × 10 ⁵ / well and add α-minimum essential medium (α-) with 10% Fetal bovine serum (FBS), 1 × antibiotics, and RANKL (40 ng / ml). MEM) medium was incubated with TN13 at 10 μM daily for 4 days. Every 2 days, the medium was exchanged and treated with 10 μM of TN13, and stained with TRAP solution on day 4 to identify osteoclasts.

The result is shown in FIG. 18A.

As shown in FIG. 18A, the reddish violet TRAP (tartrate-resistant acid phosphatase) positive cells were identified as osteoclasts, and it was confirmed that osteoclast differentiation was inhibited by TN13 treatment.

In addition, 8-week-old female C57BL6 / J mice were sacrificed by cervical dislocation and bone marrow was collected from the tibia and femur. Red blood cells were removed from the collected bone marrow cells and cultured in α-MEM medium containing FBS, 1 × antibiotics, and M-CSF (10 ng / ml) for one day. Only non-adherent cells without stromal cells were recovered and incubated in 100 mm culture dish for 3 days in α-MEM medium containing FBS, 1 × antibiotics, and M-CSF (20 ng / ml). After 3 days, non-adherent cells were removed and the remaining adherent cells were used as bone marrow macrophages (BMMs) as osteoclasts. Macrophages were aliquoted into a 24well plate at a density of 5 × 10 ⁴ / well and TN13 and α-MEM medium were added to FBS, 1 × antibiotics, M-CSF (30 ng / ml) and RANKL (50 ng / ml). SB203580, an inhibitor of p38 MAPK, was treated daily with 10 μM and incubated for 5 days. Every 2 days, the medium was exchanged and treated with SB203580, an inhibitor of TN13 and p38 MAPK, daily at 10 μM and stained with TRAP solution on day 5 to identify osteoclasts.

The results are shown in Figure 18b.

As shown in FIG. 18B, it was confirmed that the reddish violet TRAP (tartrate-resistant acid phosphatase) positive cells were osteoclasts, and osteoclast differentiation was inhibited by TN13 treatment, which was similar to the level of the positive control group.

<10-2> 골다공증 동물 모델에서의 치료 효과 확인<10-2> Confirmation of therapeutic effect in animal model of osteoporosis

The therapeutic effect of osteoporosis following TN13 treatment was confirmed.

The experimental schedule is shown in FIG. 19A. The experimental animals were purchased from 8 weeks old C57BL6 / J-based female mice purchased from Duel Co., Ltd. and classified into three groups. To produce a model of osteoporosis, the skin, muscles and peritoneum are cut in the lower abdomen after general anesthesia, exposing both ovaries. OVX), the test material was administered after a week of recovery period, TN13 was administered every two days for two days in a vehicle and 25mg / kg as an intraperitoneal control using a 1cc syringe. After 6 weeks, the mice were sacrificed with cervical vertebra and the femur was fixed with 4% formaldehyde. Three-dimensional images (micro-CT) of the inside of the femur were obtained by using micro-CT. Bone surface, bone volume / total volume and bone surface / total volume were measured morphologically.

The result is shown in FIG. 19B.

As shown in FIG. 19B, it was confirmed that when TN13 was administered intraperitoneally at a dose of 25 mg / kg, bone density reduction of three-dimensional images inside the femur was restored than the vehicle-administered group.

In addition, using the gastric animal model, changes in bone mineral density, the number of small bones, bone surface, bone volume / total volume, and bone surface / total volume of the minor bones were confirmed. After removing the differentiated mature osteoclast medium, the cells were fixed with 4% formaldehyde for 1 minute and washed with distilled water, followed by 2% Fast garnet GBC base solution (Sigma, MO, USA) and sodium nitrite solution (Sigma). , MO, USA) in equal proportions, 5% Naphthol AS-BI phosphoric acid (Sigma, MO, USA), 4% Acetate solution (Sigma, MO, USA) and 2% Tartrate solution (Sigma, MO, USA) USA) solution was fixed to the cells containing the solution, and the dye was cautious for 1 hour in a 37 ℃ water bath while being careful not to enter the light, washed with distilled water and confirmed the cells stained red under a microscope.

The results are shown in Figs. 20A to 20E.

As can be seen from Figure 20, bone density (Fig. 3a, TN13), number of bone bones (Fig. 3b, TN13), bone surface (Fig. 3c, TN13), bone volume / total volume (Fig. 3d, TN13), bone surface / total volume (FIG. 3e, TN13) can be observed to recover significantly.

The above results confirm that TN13 can be used as a therapeutic material for the treatment of osteoporosis.

Claims

A fusion peptide of a TAT peptide consisting of a peptide comprising any one selected from among amino acid sequences set forth in SEQ ID NOs: 2, 3, 4 and 5 and a sequence set forth in SEQ ID NO: 9.
According to claim 1, wherein the fusion peptide is a TAT peptide consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 9
A composition for reverse aging comprising a peptide comprising any one selected from among amino acid sequences set forth in SEQ ID NOs: 2, 3, 4 and 5.
The composition of claim 3, wherein the peptide further comprises a TAT peptide consisting of a sequence set forth in SEQ ID NO: 9.
A composition for inhibiting stem cell senescence containing a peptide comprising any one selected from amino acid sequences set forth in SEQ ID NOs: 2, 3, 4 and 5.
The composition of claim 5, wherein the peptide further comprises a TAT peptide consisting of a sequence set forth in SEQ ID NO: 9.
A pharmaceutical composition for the prophylaxis and treatment of senile diseases containing a peptide comprising any one selected from amino acid sequences set forth in SEQ ID NO: 2, 3, 4 and 5.
According to claim 7, wherein the peptide is a pharmaceutical composition for the prevention and treatment of senile diseases further comprising a TAT peptide consisting of the sequence set forth in SEQ ID NO: 9.
The method of claim 7, wherein the senile disease is any one or more selected from the group consisting of dementia, hypertension, Parkinson's disease, diabetes, cataracts, osteoporosis, stroke, periodontal disease and degenerative arthritis .
A composition for preventing and treating inflammatory diseases comprising a peptide comprising any one selected from amino acid sequences set forth in SEQ ID NOs: 2, 3, 4 and 5.
The composition for preventing and treating inflammatory diseases according to claim 10, wherein the peptide further comprises a TAT peptide consisting of a sequence set forth in SEQ ID NO: 9.
1) treating a test substance to stem cells deficient in thioredoxin-interacting protein (TXNIP); And

2) selecting a test substance exhibiting any one or more characteristics selected from the group consisting of a to d compared to a control not treated with the test substance;

a. reduced expression or activity of the p16, p19, p21 or Wnt5a genes;

b. reduced phosphorylation of p38;

c. Reduced reactive oxygen species (ROS) levels; And

d. Increased polarity of Cdc42 (Cell division control protein 42 homolog);

A method for screening a drug for inhibiting stem cell aging comprising a.
Anti-aging cosmetic composition containing a peptide comprising any one selected from the amino acid sequence of SEQ ID NO: 2, 3, 4 and 5.
A method of treating senile disease by administering to a patient a pharmaceutically effective amount of a composition comprising a peptide comprising any one selected from among amino acid sequences set forth in SEQ ID NOs: 2, 3, 4 and 5.
15. The method of claim 14, wherein the peptide further comprises a TAT peptide consisting of the sequence set forth in SEQ ID NO: 9.
15. The method of claim 14, wherein the senile disease is at least one selected from the group consisting of dementia, hypertension, Parkinson's disease, diabetes, cataracts, osteoporosis, stroke, periodontal disease and degenerative arthritis.
Use of a peptide comprising any one selected from among amino acid sequences set forth in SEQ ID NOs: 2, 3, 4 and 5 in the manufacture of a medicament for the treatment of senile diseases.
The use of a peptide according to claim 14, wherein the peptide further comprises a TAT peptide consisting of the sequence set forth in SEQ ID NO: 9.
A peptide comprising any one selected from amino acid sequences set forth in SEQ ID NOs: 2, 3, 4 and 5 for use in the treatment of senile diseases.
The peptide of claim 19, wherein the peptide further comprises a TAT peptide consisting of the sequence set forth in SEQ ID NO: 9. 21.