WO2018088821A1 - Composition for promoting stem cell activity, comprising histone deacetylase inhibitor and priming factor as active ingredients - Google Patents

Abstract

The present invention relates to a composition for promoting stem cell activity, comprising a histone deacetylase inhibitor and a priming factor as active ingredients. When stem cells were treated with a low-concentration histone deacetylase inhibitor and priming factor, the activity of the stem cells significantly improved. In particular, primed MSCs promote a cell movement, colony-forming activity, and anti-inflammatory capability in terms of cell culturing conditions and thereby improve hypertension, inflammatory disease, or immune disease treatment effects and thus are expected to become a useful treatment strategy.

Description

Stem cell activity promoting composition comprising a histone deacetylation inhibitor and a priming factor as an active ingredient

The present invention relates to a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient.

Mesenchymal stem-cells (MSCs) are isolated from bone-marrow (BM), fat, dental pulp, umbilical-cord (UC) and umbilical cord blood (UCB). , Multipotent progenitor cells. Administration of MSCs repairs organ tissues of damaged organs to provide a therapeutic effect. The efficacy of MSCs is due to cytoprotective effects regulated by cytokines and paracrine factors such as pro-angiogenic and pro-arteriogenic effects. However, current MSC treatments are limited due to their low therapeutic effectiveness, particularly in vivo engraftment and survival of transplanted cells. Most (≥99%) intravenously inject MSCs near the lungs, of which only 2-3% are released into the circulation. Thus, there is a need to understand the exact mechanisms for the migration and engraftment of MSCs during tissue repair.

Some chemokines such as stromal cell derived factor-1 (SDF-1) and growth factors such as vascular endothelial growth-factor (VEGF), platelet- Platelet-derived growth-factor (PDGF) or hepatocyte growth-factor (HGF) plays an important role in the migration and engraftment of adult MSCs. Of these, the SDF-1 and its receptor, the CXCR4 axis, are important factors for homing / engraftment of stem cells. In fact, the engraftment of hematopoietic stem / progenitor cells (HSPCs) according to the SDF-1 gradient in the bone marrow is a prerequisite for transplantation (e.g. systemic irradiation and bone marrow ablation). After chemotherapy) in the bone marrow. The sensitivity / responsiveness of HSPCs to the SDF-1 gradient is positively influenced (“primed”) by a rich set of molecules in the injured tissue. These include bioactive lipids (eg, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P)), neutrophil-derived cationic peptide caseycides. Cathelicidin (LL-37), β2-defensin and soluble membrane attack complex (sMAC) (C5b-9).

We recently confirmed that the priming phenomena observed in HSPCs process similarly occurs in MSCs primed with LL-37, S1P and CP1 bioactive lipids and cationic peptides. In particular, primed MSCs promote cell migration, colony-forming activity and anti-inflammatory capacity in cell culture conditions, which enhances the effect of treating pulmonary arterial hypertension (PAH). However, primed MSCs still exhibit limited in vivo engraftment in injured tissue. Moreover, despite the intensive washing process prior to MSC administration, it failed to completely eliminate residual priming factors that trigger a negative inflammatory response. Therefore, it is necessary to develop a priming strategy at low concentration.

Disclosure of Invention An object of the present invention is to provide a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient, and a method for promoting stem cell activity through the same.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hypertension, including a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof as an active ingredient.

In addition, another object of the present invention to provide a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases, including stem cells treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient. Is in.

The present invention provides a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient.

In addition, the present invention provides a method for promoting stem cell activity comprising the step of treating a histone deacetylation inhibitor and priming factor to the isolated stem cells.

The present invention also provides a pharmaceutical composition for preventing or treating hypertension, comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.

The present invention also provides the use of histone deacetylation inhibitors and priming factors for promoting stem cell activity.

In addition, in the manufacture of a medicament for preventing or treating hypertension, there is provided the use of a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture thereof.

The present invention also provides the use of a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture thereof in the manufacture of a medicament for preventing or treating an inflammatory disease or an immune disease.

The present invention relates to a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient. When the stem cells are treated with a low concentration of histone deacetylation inhibitor and a priming factor, the activity of the stem cells is increased. Excellent improvement. In particular, primed MSCs promote cell migration, colony-forming activity, and anti-inflammatory capacity in cell culture conditions, which may be useful therapeutic strategies because they enhance the effect of treating hypertension, inflammatory or immunological diseases. do.

1 shows the results for the negative effect of 5-azacytidine (5-Aza) on UC-MSCs primed with S1P. (A) RQ-PCR analysis of CXCR4 in human UC-MSCs treated with 1-μM 5-Aza or 0.5-mM VPA for 24 hours. Relative expression levels of the indicated genes were expressed as change in magnification compared to the values of untreated MSCs (NT) and were indicated by means ± SEM (n = 4), *** p <0.001, one-way ANOVA test. (B) Expression of indicated MSC surfaces (CD29, CD73 and CD90) and hematopoietic lineage (CD14, CD34, and CD45) proteins in UC-MSCs primed for 24 hours with 1-μΜ 5-Aza and 50-nM S1P Flow cytometry results. (C) Representative images of chondrocyte, osteocytic or adipocyte differentiation analysis using UC-MSCs primed with 5-Aza + S1P are shown. Chondrocyte production, osteoblast production and adipocyte production were measured through Alcian Blue, Alizarin Red S and Oil Red O staining, respectively. (D) Cell proliferation (n = 3), (E) Chemotaxis (n = 6) and (F) CFU-F analysis of UC-MSCs primed with 5-Aza + S1P for 24 hours (n) n≥6) result is shown. All results are expressed as means ± SEM. ** p <0.01, *** p <0.001, compared to non-primed cells (non-parametric Mann Whitney test). Representative images of migrated cells (E) or stained colonies (F) are shown in the right panel.

2 shows the results for the effect of VPA on UC-MSCs primed with S1P. (A and B) Flow cytometry and pluripotency (B) for surface antigen (A) of UC-MSCs primed for 24 hours with 0.5-mM VPA alone (VPA) or in combination with 50-nM S1P (VPA + S1P) The analysis results are shown.

3 shows the results for improved reactivity to SDF-1 in UC-MSCs primed with VPA + S1P. (A and B) 150-ng / ml SDF-1 of MSCs exposed for 24 hours to 0.5-mM VPA alone (VPA), 50-nM S1P alone (S1P) or combination with 50-nM S1P (VPA + S1P) The results of the chemotaxis analysis for. (A) Representative images of transwell insets are shown in the migration assay. (B) Relative amounts for migration were quantified as a change in magnification for the number of migrated cells and expressed as means ± SEM (n = 6). ** p <0.01, *** P <0.001 compared to nonprime cells (NT) (one-way ANOVA with Bonferroni post-test). (C) Western blot analysis of phosphorylated- (p-) or total AKT and MAPK ^{p42 / 44} is shown. MSCs primed with VPA + S1P were treated for 12 hours in serum-deficient state and then for the time indicated as 150-ng / ml SDF-1.

4 shows the results for the effect of VPA on UC-MSCs primed with S1P. (A and B) Cell proliferation assay of UC-MSCs primed for 24 hours with VPA, S1P alone or VPA + S1P (A, n = 3) and CFU-F analysis (B, n = 6) Results are shown. For CFU-F analysis, 60 cells were seeded in 6-well culture plates, incubated for 14 days, and colony numbers were quantified. Representative stained colonies of adherent cells are shown in the right panel. (C) shows quantitative results of TNF-α protein (n = 4) secreted from rat alveolar macrophage lines stimulated with LPS for 5 hours, depending on the presence of a conditioned medium (CM) harvested from the indicated cells. . All results are means ± SEM, * p <0.05, ** p <0.01, *** p <0.001 compared to nonprime cells (NT), #p <0.05, ### p <0.001 compared to VPA + S1P primed cells One-way ANOVA test.

5 shows VPA + S1P upregulated genes involved in the therapeutic effect of MSCs. (MP) MMP12 in UC-MSCs primed for 24 h with 0.5-mM VPA alone (VPA), 50-nM S1P alone (S1P) or in combination with 50-nM S1P (VPA + S1P) (A), growth factors (B), angiogenesis (C) and anti-inflammatory (D) related gene expression levels. Expression levels were calculated as the numerical ratio of primed UC-MSCs to unprimed UC-MSCs (NT). The result is means ± SEM (n ≧ 4); * p <0.05, ** p <0.01, *** p <0.001 compared to NT group, #p <0.05, ### p <0.001 compared to VPA + S1P (one-way ANOVA with Bonferroni post-test) Indicated.

6 shows the results of Chemotaxis analysis for SDF-1a according to various VPA and S1P concentrations.

FIG. 7 shows the results of improving the treatability of VPA + S1P primed UC-MSCs with respect to bladder function recovery in the HCl-IC rat model. (a) Results of awake cystometry under non-anesthetic. (b) Bladder urination parameters one week after injection of control or VPA + S1P primed UC-MSCs (5 independent animals per group) with 2.5 × 10 ⁵ (250 K) cell count (K = 1,000) The quantitative results of IVP; Bladder internal pressure, IAP; Intra-abdominal pressure. Sham: Sham-operated. All results are means ± SEM; * p <0.05, ** p <0.01, *** p <0.001 compared to the HCl-IC group; #p <0.05, ## p <0.001, ### p <0.001 compared to the naive UC-MSC group with Bonferroni post-test.

8 shows the histological analysis of the effect of UC-MSC administration on HCl-induced bladder injury. (a) 1 week after administration of PBS or 250 K cell number, (i) cytokeratin immunostaining in bladder tissue of UC-MSCs HCl-IC mice (magnification × 40, scale bar = 100 μm), (ii) Toluidine blue (magnification × 200, scale bar = 100 μm), (iii) Masson's trichrome (magnification × 200, scale bar = 100 μm) and (iv) TUNEL analysis (magnification) X400, scale bar = 100 μm) The results are shown. Arrows indicate infiltrated mast cells. Sham: Sham-operated. Nuclei were stained with Mayer's hematoxylin (i, ii and iii) or DAPI (blue, iv). (b) Results of quantification of histological experiments. Data was normalized from sham group (n = 15). The results were mean ± SEM, * p <0.05, ** p <0.01, *** p <0.001 compared to the HCl-IC group; #p <0.05, ## p <0.001, ### p <0.001 compared to the naive UC-MSC group with Bonferroni post-test.

9 shows the effect of UCB-MSCs and VPA + S1P primed MSCs (VPA + S1P-MSCs) in MCT induced PAH rat model. (a) MCT induced an increase in RVSP levels and significantly reduced this elevation as a result of administration of VPA + S1P primed UC-MSC two weeks after MCT infusion (left panel). MCT increased the weight ratio of RV / (LV + S) and VPA + S1P-UC-MSC significantly reduced this effect (right panel). (b) MCT increased lung tissue inflammation and medial wall thickness index (vascular wall thickness per vessel diameter), and VPA + S1P-MSC significantly reduced this increase (top panel). Unprimed UCB-MSC showed little improvement in RV / (LV + S) and media thickness index. It shows an immunofluorescence staining of α-SMA ⁺ smooth muscle cells in the pulmonary artery (lower panel) (magnification × 400). RVSP, right ventricular systolic pressure; CTL, control; MSC, human cord blood mesenchymal stem cell; S1P-MSC, sphingosine-1-phosphate primed human umbilical cord mesenchymal stem cells; RV, right ventricle; LV + S, left ventricle and interventricular septum. * p <0.05, ** p <0.01 compared to MCT alone (one-way ANOVA with Bonferroni post-test).

Expression of CXCR4, a major target of stem cell priming, is upregulated by DNA-demethylating agent 5-azacytidine (5-Aza) and histone deacetylation inhibitor valproic acid (VPA). do. The present inventors have identified the role of the epigenetic modulators in a strategy for promoting MSC priming to promote a therapeutic effect and completed the present invention.

The present invention provides a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient.

Preferably, the histone deacetylation inhibitor may be valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) or sirtinol, but is not limited thereto. It doesn't happen.

Preferably, the priming factor is sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37) or It may be a pioglitazone, but is not limited thereto.

Preferably, the VPA may be included in a concentration of 0.1 to 1.0 mM, the S1P may be included in a concentration of 10 to 50 nM, but is not limited thereto.

As used herein, the term "priming" refers to a phenomenon in which reactivity (activity) is improved to enhance the therapeutic efficacy of stem cells, and in the present invention, a histone deacetylation inhibitor and a priming factor that induce the priming. (priming factor) to promote the activity of the stem cells.

In the present invention, "stem cell activity" refers to cell mobility, cell proliferation ability, pluripotency (in vitro differentiation into chondrocytes, osteocytes or adipocyte lineage), colony forming ability or anti-inflammatory activity of stem cells, etc. .

In the present invention, the activity promotion or priming induction is not only induced priming of stem cells when the stem cells are directly treated with histone deacetylation inhibitors and priming factors, but also by the priming treatment (pre-treatment). Other differentiation may be induced by using stem cells with improved stem cell activity.

In addition, the composition for promoting activity can be enhanced by injecting in vivo by mixing with a cell therapy agent for treatment, to enhance the in vivo effect of the cell therapy, and after treatment of the present composition to the stem cells themselves cell therapy agent increased function It can also be used as a method for implanting in vivo.

For example, the priming of the present invention is to enhance stem cell function, including cell mobility, colony forming ability, and anti-inflammatory activity of stem cells.

As used herein, the term "stem cell" refers to a cell having the ability to differentiate into two or more cells while having a self-replicating ability, totipotent stem cells, pluripotent stem cells It can be classified into multipotent stem cells.

Stem cells of the present invention may be selected without appropriate limitation depending on the purpose, and may be derived from adult cells, such as all known tissues, cells, etc. derived from mammals, including humans, preferably humans, for example, Bone marrow, umbilical cord blood, placenta (or placental tissue cells), fat (or adipose tissue cells) and the like.

For example, the stem cells are restricted from bone marrow, adipose tissue, muscle tissue, ex vivo cultured autologous mesenchymal stem cells, allogeneic mesenchymal stem cells, umbilical cord blood, embryonic yolk sac, placenta, umbilical cord, periosteum, fetal and adolescent skin, and blood It may be a stem cell obtained without, and may be a stem cell derived from the fetus or shortly after birth or adult.

In a preferred embodiment of the present invention, the stem cells are neural stem cells, liver stem cells, hematopoietic stem cells, cord blood stem cells, epidermal stem cells, gastrointestinal stem cells, endothelial stem cells, muscle stem cells, mesenchymal stem cells and It is selected from the group consisting of pancreatic stem cells, and more preferably may be selected from the group consisting of liver stem cells, hematopoietic stem cells, cord blood stem cells and mesenchymal stem cells, but is not limited thereto.

In addition, the present invention provides a method for promoting stem cell activity comprising the step of treating a histone deacetylation inhibitor and priming factor to the isolated stem cells.

Preferably, the histone deacetylation inhibitor may be valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) or sirtinol, but is not limited thereto. It doesn't happen.

Preferably, the priming factor is sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37) or It may be a pioglitazone, but is not limited thereto.

Preferably, the VPA may be included in a concentration of 0.1 to 1.0 mM, the S1P may be included in a concentration of 10 to 50 nM, but is not limited thereto.

The present invention also provides a pharmaceutical composition for preventing or treating hypertension, comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.

On the other hand, the activation of stem cells through priming has an effect on cardiovascular diseases including hypertension, and there are several reports on the mechanisms of cardiovascular disease related priming stem cells (Stem Cell Research & Therapy (2015) 6: 218; Stroke. 2011; 42: 2932-2939; J. Cell. Mol. Med. Vol 17, No 5, 2013 pp. 617-625; Stem Cells International 2015 Article ID 685383).

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.

On the other hand, when stem cells are activated through priming, they can be applied to various inflammatory diseases and immune diseases by participating in immune regulation and inflammatory response, and there are various reports on the mechanisms of immune control and inflammatory response of primed stem cells. (J Orthop Res. 2016 Apr 6, 1-12; Stem Cell Rev and Rep (2014) 10: 351-375; Stem Cells International 2016 Article ID 9364213).

Preferably, the histone deacetylation inhibitor may be valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) or sirtinol, but is not limited thereto. It doesn't happen.

Preferably, the priming factor is sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37) or It may be a pioglitazone, but is not limited thereto.

In the present invention, the "culture medium" includes a culture medium capable of supporting stem cell growth and survival in vitro, secretion of cultured stem cells contained in the medium, and the like. The medium used for culturing includes all conventional mediums used in the art suitable for culturing stem cells. Depending on the type of cells, medium and culture conditions can be selected. The medium used for the culturing is preferably a cell culture minimum medium (CCMM), and generally includes a carbon source, a nitrogen source and a trace element component. Such cell culture minimal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI1640, F-10, F-12, α Minimal Essential Medium (GMEM) (Glasgow's Minimal essential Medium) and Iscove's Modified Dulbecco's Medium, but are not limited to these.

In addition, the medium may include antibiotics such as penicillin, streptomycin, gentamicin, and the like.

On the other hand, the present invention is the form containing all of the stem cells, their secretions, media components, forms containing only secretions and media components, only secretion to separate or used in combination with stem cells, or by administering only stem cells It is also possible to use it in a form that produces secretions in the body.

The stem cells can be obtained using any method known in the art.

Stem cells treated with histone deacetylation inhibitors and priming factors of the present invention can be used as a cell therapy for the treatment of certain diseases, and the treatment can be direct or pre-treatment of the molecules. .

The term "cell therapy" means proliferating, screening, or otherwise altering the biological properties of cells in vitro to autologous, allogenic, and xenogenic cells to restore the function of cells and tissues. It refers to medicines used for the purpose of treatment, diagnosis and prevention through a series of actions.

The cell therapeutic agent may be administered to the human body via any general route as long as it can reach the desired tissue.

In a preferred embodiment of the present invention, the hypertension is idiopathic pulmonary arterial hypertension; Familial pulmonary arterial hypertension; Pulmonary arterial hypertension associated with collagen vascular disease, congenital pulmonary shortness, portal hypertension, HIV infection, drugs or toxins; Pulmonary hypertension associated with thyroid disorders, glycogen storage disease, Gaucher disease, hereditary hemorrhagic capillary dilatation, hemochromatosis, myeloproliferative disorder or splenectomy; Pulmonary arterial hypertension associated with pulmonary capillary angiomatosis; Persistent pulmonary hypertension in newborns; Pulmonary hypertension associated with chronic obstructive pulmonary disease, interstitial lung disease, hypoxia induced alveolar hypoventilatory disorder, hypoxia induced sleep disorder breathing or chronic exposure to high altitudes; Pulmonary hypertension associated with developmental abnormalities; And pulmonary hypertension by thromboembolic obstruction of distal pulmonary artery, more preferably idiopathic pulmonary arterial hypertension, familial pulmonary arterial hypertension or chronic obstructive pulmonary disease, most preferably idiopathic pulmonary arterial hypertension.

In a preferred embodiment of the present invention, the inflammatory disease or immune disease is osteoarthritis, rheumatoid arthritis, cystitis, interstitial cystitis, asthma, dermititis, atopy, psoriasis, cystic fibrosis (Cystic Fibrosis), Post transplantation late and chronic solid organ rejection, Graft-versus-host disease, Graft rejection disease, Multiple Sclerosis, Systemic lupus erythematosus Systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia , Glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Diesese, Crohns disease, Autoimmune Diabetes, Diabetes Diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary disease (COPD), Graves disease (Graves disease), gastrointestinal allergy, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis, arterial disease or mitochondrial disease May be, but is not limited thereto.

The pharmaceutical compositions of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable auxiliaries in addition to the active ingredients, which may include excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants. Or solubilizers such as flavoring agents can be used. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition by containing one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Pharmaceutical formulation forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds, and the like. Can be. The pharmaceutical compositions of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered. An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required to prevent or treat a disease. Thus, the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet, sex and diet, time of administration, route of administration and composition of the patient. It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently.

The present invention also provides the use of histone deacetylation inhibitors and priming factors for promoting stem cell activity.

In addition, in the manufacture of a medicament for preventing or treating hypertension, there is provided the use of a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture thereof.

The present invention also provides the use of a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture thereof in the manufacture of a medicament for preventing or treating an inflammatory disease or an immune disease.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Experimental Example

The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

1. Culture of Human Umbilical Cord-derived MSCs

Human UCs were obtained from healthy normal term newborns with written informed parental consent in accordance with guidelines approved by the Bioethics Review Board of Asan Hospital. All mothers received informed consent prior to UC collection. UC-derived MSCs (UC-MSCs) are 2-mM L-glutamine, 20-mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) (pH 7.3), minimal-required medium ( minimum-essential medium (MEM) non-essential amino acid solution, penicillin / streptomycin (Corning Cellgro, Pittsburgh, PA), 1-mg / ml ascorbic acid (Sigma-Aldrich), 10% heat-inactivated fetal bovine serum ( fetal bovine serum; FBS) (HyClone), 5-ng / mL human epidermal growth factor (Sigma-Aldrich, St. Louis, MO), 10-ng / ml basic fibroblast growth factor and 50-mg / ml long- R3 insulin-like growth factor -1 (ProSpec, Rehovot, Israel) are added to the low-glucose Dulbecco's modified Eagle's medium (DMEM) (HyClone, Pittsburgh, PA) 5% CO ₂ at atmospheric conditions, were incubated in 37 ℃. In order to maintain pluripotency, UC-MSCs extended to 5 times or less were used. Expression of surface proteins was analyzed as previously reported (Stem Cells and Dev. 24 (2015) 1658-1671).

2. Cell migration assay

An 8-μm polycarbonate membrane was coated with 50 μl of 1.0% gelatin (Sigma) for 1 hour. UC-MSCs were primed for 1 day with VPA (0.5-mM; Sigma-Aldrich) or 5-Aza (1-μM; Sigma-Aldrich) alone or mixed with 50-nM S1P. UC-MSCs are TrypLE solution (Thermo Scientific, Pittsburgh PA) as was separated, washed and resuspended in DMEM containing 0.5% bovine serum albumin (BSA) , 3 X 10 4 trans-well inserts with cells / well density ( Transwell inserts) (Corning Costar, Pittsburgh, Pa.) Were inoculated into the upper chamber. The lower chamber was filled with 150-ng / ml SDF-1 (R & D Systems) in DMEM containing 0.5% BSA. After 1 day, the inserts were removed from the transwell plates. Cells remaining in the upper chamber were scraped with cotton wool, and the migrated cells were fixed in 4% paraformaldehyde (PFA) solution dissolved in phosphate-buffered saline (PBS), followed by 0.5% crystal violet ( Sigma-Aldrich). Digital images were analyzed with Image Pro 5.0 software (Media Cybernetics, Rockville, MD, USA) to quantify the stained cells on the underside of the membrane.

3. In vitro Characterization of MSCs

Cell proliferation of MSCs, colony-forming unit-fibroblast (CFU-F), pluripotency (in vitro differentiation into chondrocytes, osteocytes or adipocyte lineages) and in vitro anti-inflammatory assays This was done as reported in Stem Cells and Dev. 24 (2015) 1658-1671.

4. Western blot and real-time quantitative PCR (RQ-PCR)

UC-MSCs primed with 50-nM S1P and 0.5-mM VPA were starvated for 12 hours at 37 ° C. in DMEM containing 0.5% BSA and 5, 10, 20 with 150-ng / ml SDF-1. Or after 30 min of stimulation, phosphorylation of mitogen-activated protein kinase (MAPK) ^{p42 / 44} and AKT (Ser473) using a 30 μg-cell extract was analyzed via Western blot. For gene expression analysis, total RNA from indicated cells was reverse transcribed and the indicated transcripts were quantified via RQ-PCR.

5. Statistical Analysis

To identify statistical significance differences, the data were analyzed using a non-parametric Mann Whitney test or one-way ANOVA with the Bonferroni post-hoc test. We performed all analyzes using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, Calif.), And statistical significance was defined as p <0.05, 0.01, or 0.001.

6. Experimental design

An object of the present invention is to develop an umbilical cord-derived MSCs derived from human umbilical cord-derived MSCs primed with V1P + S1P to treat interstitial cystitis / bladder pain syndrome (IC / BPS) in a rat model. In addition to measuring the effects of -MSCs), the in vivo cellular properties of the transplanted cells were also widely observed. Control or V1P + S1P primed UC-MSCs were administered to rats with bladder injuries and affected the effects on bladder urination function, urothelium denudation, mast cell infiltration, tissue fibrosis, apoptosis and tumor formation. Measured. For all experiments, two independent experiments were performed on 5 independent animals per group. They were randomly assigned to the injured group, cell transplant or vehicle administration group, and cystometry group for treatment. Researchers involved in the surgical procedure were not informed of the type and dosage of cells administered. All bladder pressure measurements, histology and gene expression measurements were performed by researchers who did not know information about treatment groups. All animals unexpectedly killed by bladder injury or catheter insertion were excluded from all analysis. All animal experiments were conducted in accordance with the guidelines and regulations of the Animal Experimental Ethics Committee of the University of Ulsan College of Medicine (IACUC-2014-14-167).

7. Transplantation of Animal Models and UC-MSCs

HCl infused IC / BPS rat models have been previously reported (Stem Cells and Development 24, 2015, 1648-1657). HCl week after injury, the abdomen was cut, using a 500 ㎛ syringe and 26-gauge needle and a ^{2.5 × 10 5 (250 K)} UC-MSCs or PBS was administered directly to the front wall outer layer and the ceiling of the bladder. From one day prior to administration of stem cells, indomethacin (PMG Pharm Co., Ltd. Ansan, Korea; every 12 h at 2.5 mg / kg) or gefitinib (Gefitinib; to block Wnt or IGF-mediated signaling); Santa Cruz Biotechnology, Santa Cruz, CA, USA; every day at 5 mg / kg) was injected subcutaneously.

8. Non-anesthetic ( Unanesthetized ) And Non-inhibition (unrestrained) Bladder breakdown voltage chart  Acquisition (non-anesthetic bladder pressure measurement)

Cystometrograms were performed in non-anesthesia and non-suppressed mice in metabolic cages. Catheter was inserted simultaneously 3 days prior to bladder pressure measurement for measurement of intravesical pressure (IVP) and intra-abdominal pressure (IAP) recordings. Briefly, after anesthesia induction, a cuffed polyethylene catheter (PE-50; Becton-Dickinson, Parsippany, NJ, USA) was inserted into the bladder ceiling via an intraperitoneal incision. To record the IAP, an abdominal balloon (Latex; Daewoo Medical, Incheon, Korea) near the cuff of the catheter tip was placed proximal to the bladder and tied to another catheter with a silk thread. The polyethylene catheter (PE-50) was heated in warm water and the tip of the insert was extended to ~ 1.5 times its original length and filled with heparinized saline (100 IU / mL). When the bladder catheter was implanted, the extended catheter was inserted into the femoral vein. The catheter then penetrated through the subcutaneous space, exited through the back of the animal, and attached to the back skin. After surgery, each rat was raised individually and maintained in the same way.

For awake cystometric analysis, a T-tube connected to a pressure transducer (Research Grade Blood Pressure Transducer; Harvard Apparatus, Holliston, Mass., USA) and a microinjection pump (PHD22 / 2000 pump; Harvard Apparatus) An indwelling catheter was connected to the bladder via a double valve. To record the IAP, another indwelling catheter with a fluid-filled abdominal balloon was connected to another pressure transducer. Sterile saline was injected into the bladder at a rate of 0.4 mL / min and urine volume was continuously recorded through a fluid collector connected to a force displacement transducer (Research Grade Isometric Transducer; Harvard Apparatus). IVP, IAP and urination volume were continuously recorded using an MP150 data acquisition system (Biopac Systems, Goleta, CA, USA) equipped with Acq Knowledge 3.8.1 software at a sampling rate of 50 Hz. All urination cycle values measured for 8 minutes from each animal were used for the evaluation.

Without urine drainage, an increase in IVP above 15 cmH ₂ O from baseline was measured by non-voiding contraction (NVC). BP means the lowest bladder pressure while the bladder is filled, MP means the highest bladder pressure during the urination cycle, MV means the urine volume during urine drainage, and RV means the amount of urine remaining after urine drainage. BC is defined as MV + RV, MI means the interval between urination contractions.

9. Pulmonary artery hypertension (PAH) animal model

Monocrotaline (MCT) induced PAH rat models were established according to previous reports (Stem Cells and Development 24, 2015, 1658-1671). Male specific-pathogen-free Lewis rats (8 weeks old, 250-280 g) were reared without restriction of food and water, under the control of breeding temperature and light (12 hour contrast cycle). PAH was induced through subcutaneous administration of MCT (60 mg / kg, Sigma). Mice in the control group received the same volume of PBS. Two weeks after MCT or PBS administration, priming-induced UC-MSCs or UC-MSCs pretreated with VPA + S1P were administered via the tail vein at a density of 2.5 × 10 ⁵ cells per 200 μl PBS. A solvent-only vehicle control was administered with 200 μl PBS without cells. Prior to injection, cells were washed twice with warm PBS and viability of the administered cells was observed using FACS analysis via 7-AAD (BD Biosciences) exclusion staining.

10. Systolic Right ventricular pressure (right ventricular systolic pressure; RVSP ) And right ventricular hypertrophy (RVH) measurements

 Four weeks after MCT or PBS administration, patients under breathing maintenance through a ventilator (Harvard Apparatus, Holliston, MA) and under anesthesia with zoletil (40 mg / kg) and rompun (10 mg / kg) The systolic right ventricular pressure was measured by direct puncture of the diaphragm using a 26G needle connected to an MDE Escort II patient monitor (Arleta). Right ventricular pressure (RVP) maintained breathing through the ventilator. To measure RVH, the right ventricle of the heart was isolated from the interventricular septum and the weight of the left ventricle (LV + S), including the right ventricle (RV) and the ventricular septum, was measured.

11. Histology and Gene Expression Analysis

For histological analysis of bladder tissues, epithelial cell detachment via cytokeratin immunostaining, mast cell infiltration and torsion tricolor through toluidine blue staining (8544-4125; Daejung Chemicals & Metals, Seoul, Korea) Apoptosis was measured through tissue fibrosis and TUNEL staining (1 684 795; Roche, Mannheim, Germany) via staining (Masson's trichrome staining; Junsei Chemical, Tokyo, Japan). For lung tissue, at least for an arbitrarily selected region of randomly selected vessels having a thickness of 4 μm and a diameter of 25-100 μm at H & E- or α-smooth muscle actin (α-SMA) -stained sites More than 5 times, capture at 400 × magnification. Anti-α-SMA (1: 100, Abcam) was applied according to the manufacturer's recommended protocol and reacted. Nuclei were counterstained with 4 ', 6'-diamino-2-phenylindole (4', 6'-diamino-2-phenylindole; D9542; DAPI, Sigma-Aldrich). Medial wall thickness was measured using the NIH ImageJ program (http://rsbweb.nih.gov/ij/). The media thickness index is defined as the ratio of the outer diameter to the inner diameter to the outer diameter.

For gene expression analysis, total RNA was prepared using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA), reverse transcription was performed using TaqMan Reverse Transcription Reagents (Applied Biosystems), and PikoReal Real-Time PCR System ( Thermo Scientific) and iQ SYBR Green PCR Master Mix (Bio-Rad, Hercules, Calif.) Were used to perform real-time quantitative PCR (RQ-PCR). For five independent animals per treatment group, a randomly selected three-part area (n = 15) from each slide was used to quantify the digital image. Similarly, RQ-PCR was repeated twice (n = 10) from five randomly selected animals per group to obtain gene expression data.

Example 1 Influence of 5-Aza on Priming of Human UC-MSCs by S1P

Low concentration treatment (1-μM and 0.5-mM) of 5-Aza and VPA greatly increased the expression of CXCR4 in UC-MSCs (FIG. 1A). Next, the inventors confirmed the effect of the epigenetic modulator on the basic characteristics of UC-MSCs. Independent of the priming of 50-nM S1P, both 5-Aza and VPA did not significantly affect the expression of surface marker proteins (CD29, CD73 and CD90 positive, CD34 and CD45 negative) (FIG. 1B and FIG. 2A). In addition, S1P priming treated with 5-Aza (5-Aza + S1P) or VPA (VPA + S1P) had little effect on the multi-system differentiation capacity, but the multi-system differentiation capacity was determined by glycosaminoglycans; Based on in vitro differentiation analysis of chondrocytes, osteocytes and adipocyte lineages by measuring levels of alcian blue), mineral deposition (Alizarin Red S) and lipid accumulation (oil red O staining), respectively. (FIG. 1C and FIG. 2B).

Contrary to upregulation of CXCR4 (FIG. 1A), UC-MSCs primed with 5-Aza + S1P in transwell migration assays reduced migration activity for SDF-1 responses by about 30% compared to unprimed cells. (FIG. 1E). In addition, 5-Aza + S1P priming severely impaired the activity of clonal CFU-F, which indicates the frequency of self-renewal into clonal progenitor cells (FIG. 1F). Cell proliferation of MSCs was little changed by the priming of 5-Aza + S1P (FIG. 1D).

Example 2 VPA Enhancing the Priming of UC-MSCs at Low S1P Treatment

Next, the present inventors investigated the effect of VPA on the priming of UC-MSCs at low concentrations of S1P treatment. To this end, we applied 50-nM S1P four times less than the optimal dose (200-nM) used for priming of fat- and UCB-derived MSCs. Unlike VPA alone and S1P alone, UC-MSCs priming increased VT + S1P treatment two to three times higher chemotactic activity for SDF-1 than unprimed cells (FIGS. 3A and 3A). 3B). The response to SDF-1 enhanced by VPA + S1P priming appeared completely different from that of 5-Aza + S1P priming. Next, we identified the state of the signaling pathway associated with the movement of HSPCs. Consistent with the results of chemotaxis assay, UC-MSCs exposed to VPA + S1P increased the phosphorylation of MAPK ^{p42 / 44} and AKT proteins, indicating activation of signaling pathways (FIG. 3C). The results indicate that 5-Aza unlike VPA is even lower concentrations of S1P MAPK ^{p42 / 44} and activate the AKT signaling pathway can promote the priming of the UC-MSCs.

Example 3 Improvement of Treatment Capability of UC-MSCs Primed with VPA + S1P

We tested the effect of VPA + S1P priming on other cellular activities related to the therapeutic effect of MSCs. Unlike 5-Aza + S1P (FIG. 1), UC-MSCs primed with VPA + S1P improved both cell proliferation (FIG. 4A) and clonal CFU-F activity (FIG. 4B). However, no increase in cell proliferation and CFU-F activity was observed in UC-MSCs treated with VPA alone and S1P alone (FIGS. 4A and 4B). Since MSCs affect anti-inflammatory and immunomodulatory capacity, we tested whether VPA + S1P priming affected the anti-inflammatory effects of UC-MSCs. Accordingly, the present inventors prepared a conditioned medium collected from unprimed UC-MSCs or VPA alone or from UC-MSCs primed with VPA + S1P, and the conditioned medium stimulated with lipopolysaccharide (LPS). Tumor necrosis factor-α (TNF-α) secretion was inhibited from MH-S cells, alveolar macrophages cells. Conditional medium collected from UC-MSCs was effective in reducing the secretion of TNF-α from LPS stimulated MH-S cells (FIG. 4C). In particular, the conditioned media collected from VPA + S1P primed UC-MSCs inhibited TNF-α secretion more than unprimed UC-MSCs or UC-MSCs primed with VPA alone. We used CM collected with IMR90, a human lung fibroblast, as a control, which was shown to further increase TNF-α secretion in vitro anti-inflammatory analysis.

To confirm the molecular mechanism for the effect of VPA + S1P priming, we tested the expression of growth factors, pro-inflammatory cytokines and anti-inflammatory factors secreted by MSCs. Consistent with the results of the above-mentioned cytological experiments, UC-MSCs primed with VPA + S1P showed growth factors and their receptors [eg, PDGFB, PDGFRB, cMET] (FIG. 5B), pre-angiogenesis- [eg , VEGFB, VEGFC, ANGPT1, ANGPT2] (FIG. 5C), anti-inflammatory- [eg, LIF, TSG6, IDO1, IDO2] (FIG. 5D) and stem cell migration-[eg, MMP12] (FIG. 5A) Expression was significantly upregulated. However, no significant changes were observed in the aforementioned gene expression in UC-MSCs treated with VPA alone and S1P alone (FIGS. 4A and 4B). In addition, 5-Aza + S1P priming downregulated the aforementioned genes affected by VPA + S1P priming. Taken together, the results indicate that priming of MSCs with minimal concentrations of VPA and S1P treatment effectively promotes the migration, proliferation, self-renewal and anti-inflammatory capacity of MSCs, which plays an important role in the therapeutic potential of MSCs.

< Example  3> various VPA  And S1P  According to concentration SDF Chemotaxis Analysis of -1a

The present inventors attempted to determine the lowest concentration of VPA and S1P by performing a chemotaxis analysis on SDF-1a according to various VPA and S1P concentrations.

As shown in FIG. 6, VPA was fixed at 0.5 mM, and the S1P concentration was tested at 5-50 nM. As a result, the lowest concentration of S1P was determined to be 10 nM (FIG. 6A). In addition, S1P was fixed at 50 nM and the VPA concentration was 0.05-0.5 mM. As a result, the lowest concentration of VPA was determined to be 0.1 nM (FIG. 6B).

< Example  4> In IC / BPS therapy, VPA + S1P Primed UC - MSc  Confirmation of in vivo treatment potential

Next, we confirmed the in vivo effects of UC-MSCs using an IC / BPS animal model established by HCl injection. As a result of bladder function analysis using awake cystometry, the HCl-induced IC / BPS mice (HCl-IC group) showed irregular urination and urination compared to sham-operated (sham) mice. Not only did the MI (turturation interval; MI) decrease, but also the micturition volume (MV), maximum pressure, micturition pressure (MP), and bladder capacity (BC) were also shown to be lower (FIGS. 7A and FIG. 7b). Single transplantation of control UC-MSCs (UC-MSC 250K) or VPA + S1P primed cells (UC-MSC 250K + VPA + S1P) with 2.5 × 10 ⁵ cell numbers improved impaired urination parameters. Importantly, VPA + S1P primed UC-MSCs showed a better effect than control cells (FIGS. 7A and 7B). In particular, in animals in the HCl-IC group, contraction frequency was increased during non-voiding periods (NVC), which was significantly improved in VPA + S1P primed UC-MSCs, but in control UC. Not so much improved in MSCs (FIGS. 7A and 7B). Were Bima take cystometry alcohol (awake cystometry) results are consistent with the functional effect of improving the control or VPA + S1P primed 2.5 × 10 ⁵ UC-MSCs severe urinary tract epithelial detachment in a rat model because of the administration of (urothelium denudation), Abnormal histological phenomena such as mast cell infiltration, fibrosis, and cell death have been shown to recover (FIGS. 8A and 8B). These abnormal histological phenomena are also characteristic of human IC / BPS bladder. In particular, recovery of histological alterations showed a better improvement in bladder tissues implanted with VPA + S1P primed UC-MSCs compared to control cells. Taken together, the results showed that for IC bladder treatment, VPA + S1P priming can significantly improve the therapeutic potential of UC-MSCs.

Example 5 Enhancement of VPA + S1P-Mediated Treatment Potential of UC-MSC in PAH Animal Model

Next, the inventors confirmed the effect of VPA + S1P priming under in vivo conditions using an MCT-induced PAH animal model. Similar to previous reports, RVSP increased significantly after 4 weeks of MCT administration. Unlike the control UC-MSC (PAH + MSC group), administration of UC-MSC primed with VPA + S1P (PAH + VPA + S1P-MSC group) significantly reduced the elevation of MCT-induced RVSP (FIG. 9a). In addition, after MCT administration, RV / (LV + S) was also increased, with VPA + S1P-MSC significantly reducing RV hypertrophy, and unpriming UC-MSC was ineffective (FIG. 9A). In addition, VPA + S1P-MSC administration reduced lung tissue inflammation, medial thickness index, and α-SMA ⁺ smooth muscle cell induced by MCTs (FIG. 9B). Taken together, the results indicated that priming of UC-MSCs with VPA + S1P can induce angiogenesis and induce a microenvironment that inhibits inflammatory responses.

Having described the specific parts of the present invention in detail, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (20)

1. Stem cell activity promoting composition comprising a histone deacetylation inhibitor and a priming factor (priming factor) as an active ingredient.
2. The method of claim 1, wherein the histone deacetylation inhibitor in the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) and sirtinol Stem cell activity promoting composition, characterized in that any one or more selected.
3. The method according to claim 1, wherein the priming factor is sphingosine-1-phosphate (Sphingosine-1-phosphate (S1P), ceramide-1-phosphate (ceramide-1-phosphate (C1P), cathelicidin (LL-37) ) And pioglitazone (pioglitazone) composition for promoting stem cell activity, characterized in that any one or more selected from the group consisting of.
4. The composition for promoting stem cell activity according to claim 2, wherein the VPA is contained in a concentration of 0.1 to 1.0 mM.
5. The composition for promoting stem cell activity according to claim 3, wherein the S1P is included in a concentration of 10 to 50 nM.
6. The method according to any one of claims 1 to 5, wherein the stem cells are neural stem cells, liver stem cells, hematopoietic stem cells, cord blood stem cells, epidermal stem cells, gastrointestinal stem cells, endothelial stem cells, muscle stem cells, Stem cell activity promoting composition, characterized in that selected from the group consisting of mesenchymal stem cells and pancreatic stem cells.
7. The composition for promoting stem cell activity according to any one of claims 1 to 5, wherein the stem cell activity is cell mobility, colony forming ability and anti-inflammatory activity.
8. Stem cell activity promotion method comprising the step of treating the isolated stem cells histone deacetylation inhibitor and priming factor (priming factor).
9. The method of claim 8, wherein the histone deacetylation inhibitor in the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) and sirtinol Stem cell activity promoting method, characterized in that any one or more selected.
10. The method according to claim 8, wherein the priming factor is sphingosine-1-phosphate (Sphingosine-1-phosphate (S1P), ceramide-1-phosphate (ceramide-1-phosphate (C1P), cathelicidin (LL-37) ) And pioglitazone (pioglitazone) is any one or more selected from the group consisting of stem cell activity promoting method.
11. The method of claim 9, wherein the VPA is treated at a concentration of 0.1 to 1.0 mM.
12. The method of claim 10, wherein the S1P is treated at a concentration of 10 to 50 nM.
13. A pharmaceutical composition for preventing or treating hypertension, comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.
14. The method of claim 13, wherein the histone deacetylation inhibitor in the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) and sirtinol A pharmaceutical composition for preventing or treating hypertension, characterized in that any one or more selected.
15. The method of claim 13, wherein the priming factor is sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and cathelicidin LL-37) and pioglitazone (pioglitazone) is any one or more selected from the group consisting of a pharmaceutical composition for preventing or treating hypertension.
16. The method of claim 13, wherein the hypertension is idiopathic pulmonary arterial hypertension; Familial pulmonary arterial hypertension; Pulmonary arterial hypertension associated with collagen vascular disease, congenital pulmonary shortness, portal hypertension, HIV infection, drugs or toxins; Pulmonary hypertension associated with thyroid disorders, glycogen storage disease, Gaucher disease, hereditary hemorrhagic capillary dilatation, hemochromatosis, myeloproliferative disorder or splenectomy; Pulmonary arterial hypertension associated with pulmonary capillary angiomatosis; Persistent pulmonary hypertension in newborns; Pulmonary hypertension associated with chronic obstructive pulmonary disease, interstitial lung disease, hypoxia induced alveolar hypoventilatory disorder, hypoxia induced sleep disorder breathing or chronic exposure to high altitudes; Pulmonary hypertension associated with developmental abnormalities; And pulmonary hypertension due to thromboembolic closure of the distal pulmonary artery.
17. A pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases, comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.
18. 18. The method of claim 17, wherein the histone deacetylation inhibitor is selected from the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), and sirtinol. Pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases, characterized in that any one or more selected.
19. The method of claim 17, wherein the priming factor is sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and cathelicidin LL-37) and pioglitazone (pioglitazone) pharmaceutical composition for the prevention or treatment of inflammatory or immune diseases, characterized in that any one or more selected from the group consisting of.
20. The method of claim 17, wherein the inflammatory disease or immune disease is osteoarthritis, Rheumatoid Arthritis, cystitis, interstitial cystitis, asthma, dermititis, atopic dermatitis, Psoriasis, cystic fibrosis ), Post transplantation late and chronic solid organ rejection, graft-versus-host disease, graft rejection disease, multiple sclerosis, systemic lupus erythematosus lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis (glomerulonephritis) and pulmonary fibrosis, Inflammatory Bowel Diesese, Crohn's disease, Autoimmune Diabetes, Diabetic retinopathy ), Rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary disease (COPD), Graves disease Selected from the group consisting of gastrointestinal allergy, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis, arterial disease and mitochondrial disease Pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases, characterized in that.

Images