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WO2018056706A1 - Composition comprenant un peptide dérivé d'une protéine interagissant avec la thiorédoxine ou un polynucléotide codant pour celle-ci en tant que principe actif pour le vieillissement inverse d'une cellule souche âgée et son utilisation - Google Patents

Composition comprenant un peptide dérivé d'une protéine interagissant avec la thiorédoxine ou un polynucléotide codant pour celle-ci en tant que principe actif pour le vieillissement inverse d'une cellule souche âgée et son utilisation Download PDF

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WO2018056706A1
WO2018056706A1 PCT/KR2017/010358 KR2017010358W WO2018056706A1 WO 2018056706 A1 WO2018056706 A1 WO 2018056706A1 KR 2017010358 W KR2017010358 W KR 2017010358W WO 2018056706 A1 WO2018056706 A1 WO 2018056706A1
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Prior art keywords
peptide
txnip
seq
hsc
set forth
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Korean (ko)
Inventor
최인표
정해용
김동오
김미정
변재은
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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.
  • TXNIP thioredoxin-interacting protein
  • 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.
  • Embryonic stem cells have the potential to differentiate into cells of all tissues.
  • Adult stem cells 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 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
  • ST-HSC Short-term HSC
  • MPP multipotent progenitor
  • LT-HSC 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.
  • LSK cells LSK cells
  • MPP myeloid progenitor cells
  • CMP common myeloid progenitor
  • CLP lymphocytic progenitor cells
  • MMP megamegaloyocyte-erythroid progenitor
  • GMP granulocyte-monocyte progenitor
  • myeloid cells such as erythrocytes, giant cells, eosinophils
  • CLP is differentiated into lymphoid cells such as B cells, T cells, NK 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).
  • HSC 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.
  • LT-HSC Lineage - / C-kit / + Sca-1 + cells
  • ROS reactive oxygen species
  • 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.
  • TXNIP Thioredoxin-interacting protein
  • -4046 its use as a therapeutic agent for active oxygen-related diseases by increasing its resistance to free radicals (WO013159879).
  • 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.
  • 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.
  • 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.
  • TXNIP thioredoxin-binding protein
  • MAPK mitogen-activated protein kinase
  • 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.
  • ROS reactive oxygen species
  • 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.
  • TXNIP thioredoxin-interacting protein
  • 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.
  • 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.
  • 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.
  • 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.
  • TXNIP thioredoxin-interacting protein
  • 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;
  • ROS reactive oxygen species
  • 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.
  • 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.
  • 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.
  • 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.
  • TXNIP thioredoxin-interacting protein
  • 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.
  • FIG. 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
  • 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.
  • FIGS. 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
  • HSC hematopoietic stem cell
  • FIG. 3c is a diagram confirming the LT-HSC, ST-HSC, MPP ratio in various ages by flow cytometry.
  • FIGS 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) .
  • FIG. 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.
  • FIGS. 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.
  • FIGS. 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
  • FIG. 7b is a diagram showing that TXNIP and p38 bind to HSC through in situ proximity ligation (PLA) results.
  • PLA in situ proximity ligation
  • 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).
  • FIGS. 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
  • 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
  • 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.
  • FIGS. 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.
  • TXNIP thiredoxin interacting protein
  • 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-1 ⁇ 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.
  • FIG. 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.
  • TRAP titanium dioxide
  • 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.
  • OVX osteoporosis induced osteoporosis after Vehicle
  • 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.
  • 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.
  • 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).
  • MAPK mitogen-activated protein kinase
  • TXNIP thioredoxin-interacting protein
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the senile disease is osteoporosis or degenerative arthritis.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • TXNIP -/- mice were used to investigate the function of senescence of hematopoietic stem cells (HSCs) of TXNIP.
  • HSCs hematopoietic stem cells
  • 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.
  • ROS reactive oxygen species
  • 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.
  • domain fragments of four thioredoxin-interacting protein were prepared to confirm that the peptide binds to p38 (see FIG. 1), and to invade the cells.
  • TXNIP thioredoxin-interacting protein
  • 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).
  • 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.
  • TXNIP thioredoxin-interacting protein
  • ROS reactive oxygen species
  • 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.
  • 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 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.
  • 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 "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.
  • 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.
  • 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).
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • TXNIP Thioredoxin-Interacting Protein
  • TXNIP construct GST-TXNIP-T, was constructed to inhibit the phosphatase activity of p38.
  • 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.
  • 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).
  • HRP-attached antibody to confirm that His-p38a selectively binds to GST-TXNIP (150-317).
  • 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.
  • 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).
  • 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.
  • a TAT-TN13 peptide was prepared in which FITC was bound to the end of the TAT sequence.
  • ITC Isothermal titration calorimetry
  • TAT-TN13 binds p38 and ATF-2 phosphorylation (p-p38 and p-ATF-2).
  • FIG. 2C Western blot
  • TXNIP TXNIP
  • p38 NM_139012, SEQ ID NO: 11
  • Table 2 the site-specific mutation (site-directed mutagenesis), the FLAG-CMV vector Introduced into the cells.
  • 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)
  • LT-HSC quantitative polymerase chain reaction in short-term HSC
  • LT-HSC long-term HSC
  • CLP common lymphoid progenitor
  • CMP common myeloid progenitor
  • GMP granulocyte-monocyte progenitor
  • MMP megakeryocyte-erythroid progenitor
  • 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.
  • 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).
  • anti-CD11b-biotin (clone M1 / 70, BD biosciences), anti-Gr-1-biotin (clone RB6).
  • 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
  • anti-CD45.2-APC clone 104, BD biosciences
  • 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.
  • 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.
  • TXNIP + / + and TXNIP ⁇ / -Mice were administered and the number of white blood cells in the blood was checked for 17 days.
  • 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).
  • a competitive transplantation assay was performed to confirm the autonomous function of HSC cells.
  • 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.
  • CM-DCF-DA Molecular Probes / Thermofisher Scientific
  • DHE Dihydroethidium
  • FACSCanto II FACSCanto II
  • 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, ⁇ , mRNA expression of ⁇ , ⁇ , and ⁇ ) was confirmed.
  • p38 ⁇ is mainly expressed among the four p38 isoforms, and is most predominantly expressed in LT-HSC (FIG. 6A).
  • p38 activity was identified by age (2, 12 and 24 months) in TXNIP + / + and TXNIP ⁇ / ⁇ mice.
  • 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.
  • 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.
  • TXNIP and p38 were expressed 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.
  • 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).
  • 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).
  • 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).
  • mice TXNIP knockout / p38 inactivation mice
  • mice treated with p38 inhibitor SB203580 mice treated with p38 inhibitor SB203580.
  • engraftment of CD45.2 + LT-HSC in peripheral blood vessels was analyzed.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • Cdc42 antibody Cell signaling
  • Alexa Fluor 546 antibody Life technology
  • DAPI containing mounting reagent Molecular Probes
  • 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).
  • 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.
  • 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.
  • 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.
  • TN13 inhibits aging in young TXNIP -/- mice (TXNIP -/- 2M-GFP) and old TXNIP + / + mice (TXNIP + / + Old-GFP).
  • peripheral blood was analyzed to compare the ratio of CD45.2 + cells.
  • young TXNIP -/- mice TXNIP -/- 2M-GFP
  • aged TXNIP + / + mice TXNIP + / + Old-GFP
  • 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).
  • TAT-TN13 as a reverse aging drug against aging HSC
  • aged HSC TXNIP + / + Old-TAT-TN13
  • TAT-TN13 TAT-TN13
  • TXNIP -/- HSC 12 months old
  • 5-FU 100 mg / kg was administered to aged TXNIP + / + mice to induce acute leukopenia
  • TAT-TN13 25 mg / kg was administered daily for 4 days after 1 day
  • the control group 24M-TXNIP + / + -TAT
  • the TAT-TN13 group 24M-TXNIP + / + TAT-TN13 rapidly increased the white blood cell count.
  • TAT-TN13 can be used as a drug to reverse aging HSC.
  • 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.
  • Enzyme-linked immunosorbent assay confirmed whether TN13 peptide inhibited the secretion of inflammatory cytokines (IL-1 ⁇ / IL-6 / TNF- ⁇ ) 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-1 ⁇ , IL-6, and TNF- ⁇ . .
  • Cytokine was measured by dispensing 100 ⁇ L 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 ⁇ L of the solution was incubated at room temperature for 2 hours, washed three times, and 100 ⁇ L of the Streptavidin-HRP solution was incubated for 20 minutes. After washing three times, 100 ⁇ L 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 ⁇ L 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).
  • IL-1 ⁇ , IL-6 and TNF- ⁇ which are representative of inflammatory cytokine, are mediators of inflammatory responses and are known to be particularly involved in early inflammatory responses.
  • TN13 peptide treatment effectively reduced the production of increased IL-1 ⁇ , IL-6 and TNF- ⁇ in LPS-induced Raw264.7 macrophages, as measured in the amount of inflammatory cytokine production. I was.
  • the production of IL-1 ⁇ , IL-6 and TNF- ⁇ was suppressed by about 50% compared to the LPS-treated group.
  • the TN13 peptide effectively inhibits the secretion of inflammatory cytokines.
  • 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 ⁇ M). 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.
  • 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 ⁇ -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).
  • PBST Phosphate-buffered saline Tween-20
  • p38 MAP Kinase 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.
  • inflammatory factors such as excess nitric oxide (NO) and prostaglandin E2 (PGE 2) are formed by inducible NO synthase (iNOS) and cyclooxygenase (COX-2).
  • NO nitric oxide
  • PGE 2 prostaglandin E2
  • COX-2 cyclooxygenase
  • 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 ⁇ M 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.
  • the reddish violet TRAP thyroid-resistant acid phosphatase positive cells were identified as osteoclasts, and it was confirmed that osteoclast differentiation was inhibited by TN13 treatment.
  • BMMs bone marrow macrophages
  • Macrophages were aliquoted into a 24well plate at a density of 5 ⁇ 10 4 / well and TN13 and ⁇ -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 ⁇ M 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 ⁇ M and stained with TRAP solution on day 5 to identify osteoclasts.
  • 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.
  • 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.
  • FIG. 3a bone density
  • Fig. 3b number of bone bones
  • Fig. 3c bone surface
  • Fig. 3d bone surface / total volume
  • FIG. 3e bone surface / total volume
  • TN13 can be used as a therapeutic material for the treatment of osteoporosis.

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Abstract

La présente invention concerne une composition pharmaceutique comprenant un peptide dérivé d'une protéine interagissant avec la thiorédoxine (TXNIP) ou un polynucléotide codant pour celle-ci en tant que principe actif pour le vieillissement inverse de cellules souches. Chez des souris transplantées de manière compétitive avec des cellules souches hématopoïétiques âgées (HSC), un peptide dérivé de TXNIP a été observé pour augmenter la prise de greffe des HSC âgées, diminuer l'asymétrie de la lignée, réduire un HSC à long terme (LT-HSC) et inhiber p38 et ROS, supprimant ainsi des caractéristiques des HSC âgées, par comparaison avec une commande non traitée avec le peptide. Dans des modèles de leucopénie aiguë, l'administration du peptide dérivé de TXNIP a été trouvée pour augmenter la prolifération des leucocytes de souris âgées à un niveau de jeunes souris. De plus, il a été découvert que le peptide dérivé de TXNIP provoque la récupération de la polarisation de Cdc42 dans des HSC âgées, régule à la baisse l'expression des gènes associés à la sénescence p16, p19, p21, et Wnt5a, et augmente l'écotropisme des HSC. Par conséquent, le peptide dérivé de TXNIP de la présente invention peut être utile en tant que composition pour le vieillissement inverse de cellules souches hématopoïétiques.
PCT/KR2017/010358 2016-09-22 2017-09-20 Composition comprenant un peptide dérivé d'une protéine interagissant avec la thiorédoxine ou un polynucléotide codant pour celle-ci en tant que principe actif pour le vieillissement inverse d'une cellule souche âgée et son utilisation Ceased WO2018056706A1 (fr)

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CN117965712A (zh) * 2023-05-22 2024-05-03 南通大学附属医院 T-5224在制备逆转卵巢衰老药物中的应用
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KR102547965B1 (ko) 2021-12-23 2023-06-29 한국생명공학연구원 혈소판 또는 적혈구 분화도 측정용, 혈소판 분화 유도용 및 혈소판 관련 질환의 예방 또는 치료용 조성물

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WO2020167770A1 (fr) * 2019-02-11 2020-08-20 President And Fellows Of Harvard College Compositions txnip et ldhb et procédés pour le traitement de maladies oculaires dégénératives
AU2022359191B2 (en) * 2021-10-05 2025-07-10 Ingenium Therapeutics Composition for enhancing activity of natural killer cells
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CN117965712A (zh) * 2023-05-22 2024-05-03 南通大学附属医院 T-5224在制备逆转卵巢衰老药物中的应用

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