WO2023085604A1 - Composition for prevention or treatment of inflammatory diseases containing cartilage tissue-derived extracellular matrix as active ingredient - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of inflammatory diseases, containing a cartilage tissue-derived extracellular matrix as an active ingredient. More specifically, when the cartilage tissue-derived extracellular matrix or extracellular vesicles in the extracellular matrix were pre-treated with inflammatory cytokine Interleukin-1β and non-steroidal anti-inflammatory drug celecoxib in an osteoarthritis animal model, it was confirmed that an enhanced anti-inflammatory effect was exhibited in the osteoarthritis model, and thus the present invention is intended to provide a composition containing as an active ingredient pre-treated cartilage tissue-derived extracellular matrix or extracellular vesicles, as a therapeutic agent for inflammatory diseases exhibiting an enhanced anti-inflammatory effect.

Description

Composition for preventing or treating inflammatory diseases containing cartilage tissue-derived extracellular matrix as an active ingredient

The present invention relates to a composition for preventing or treating inflammatory diseases, which contains, as an active ingredient, an extracellular matrix derived from cartilage tissue pretreated with an inflammatory cytokine, a non-steroidal anti-inflammatory agent, or a combination thereof.

Various intracellular inflammatory regulators activate inflammatory cells such as macrophages in vivo by inducible nitric oxide synthase (iNOS) or cyclo-oxygenase-2 (COX-2). inflammatory mediators such as nitric oxide (NO), prostaglandin (PG), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF- α), cytokines, etc. are produced in large quantities. In addition, inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are induced, which also affects tissues such as muscles, tendons, and ligaments, resulting in severe pain. cause

Non-steroidal anti-inflammatory drugs (NSAIDS), a widely used agent for the treatment of most inflammatory diseases, release prostaglandin from arachidonic acid called cyclooxygenase (COX). By inhibiting the enzyme activity involved in the biosynthesis, it exhibits anti-inflammatory action, but in addition to the main therapeutic action, it causes serious side effects such as gastrointestinal disorders, liver disorders, and renal disorders, so long-term use is limited. Therefore, although studies have been conducted on compositions for treating inflammatory diseases using plant extracts derived from nature, development of new anti-inflammatory analgesics excellent in anti-inflammatory efficacy without side effects and long-term use in place of the non-steroidal anti-inflammatory drugs has been conducted. is desperately needed.

An object of the present invention is to provide a composition containing, as an active ingredient, a cartilage tissue-derived extracellular matrix pretreated with an inflammatory cytokine, a non-steroidal anti-inflammatory agent, or a combination thereof, as a preventive or therapeutic agent for inflammatory diseases.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing, as an active ingredient, cartilage tissue-derived extracellular matrix pre-treated with inflammatory cytokines, non-steroidal anti-inflammatory agents, or a combination thereof.

In addition, the present invention comprises the steps of pre-treating an inflammatory cytokine, a non-steroidal anti-inflammatory agent or a combination thereof to the cartilage tissue isolated from the subject;

Culturing the pre-treated cartilage tissue for 1 to 5 days; and

Provided is a method for enhancing the anti-inflammatory function of the extracellular matrix comprising the step of separating the extracellular matrix from the cultured cartilage tissue.

According to the present invention, the extracellular matrix derived from cartilage tissue pretreated with the inflammatory cytokine Interleukin-1β and the non-steroidal anti-inflammatory drug celecoxib or the extracellular vesicles in the extracellular matrix were prepared in an osteoarthritis animal model. When treated, as it was confirmed that an improved anti-inflammatory effect was exhibited in an osteoarthritis model, the composition containing the pre-treated cartilage tissue-derived extracellular matrix or extracellular vesicles as an active ingredient exhibits an enhanced anti-inflammatory effect. can be provided with

Figure 1 is a schematic diagram of the production and principle of cartilage tissue-derived extracellular vesicles after pretreatment.

Figure 2 is a result confirming the effectiveness of the pretreatment in chondrocytes, Figure 2A is the result of confirming the anti-inflammatory factor gene expression level, Figure 2B is the result of confirming the anti-inflammatory factor protein expression level.

Figure 3 is the result of confirming the effectiveness of pretreatment in cartilage tissue, Figure 3A is the result of confirming the anti-inflammatory factor gene expression level, Figure 3B is the result of confirming the anti-inflammatory factor protein expression level, Figure 3C is the signal of anti-inflammatory enhancement induction modeled the route.

4 is a result of confirming various pretreatment methods using NSAIDs and inflammatory cytokines, FIG. 4A is the result of confirming the IL-10 gene expression level, and FIG. 4B is the result of confirming the IL-10 protein expression level.

Figure 5 is a result of confirming the anti-inflammatory effectiveness of cartilage tissue components changed after pretreatment. Figure 5A is a schematic diagram of a method for separating components in cartilage tissue, and Figure 5B shows the distribution of proteins included by analyzing the separated components by SDS-PAGE. This is the result of checking

Figure 6 is a result of confirming the anti-inflammatory effectiveness after pretreatment of the synovial cell inflammation model before and after treatment with cartilage tissue extracellular matrix, Figures 6A and 6B are the results of confirming the gene expression level of inflammatory mediators, and Figure 6C is protein expression This is the result of confirming the level, and FIG. 6D is the result of quantitatively analyzing the expression of COX-2, a major inflammatory mediator.

Figure 7 is a result of confirming the separation of cartilage tissue-derived extracellular vesicles (CEV) and the characteristics of the extracellular vesicles, Figure 7A is a schematic diagram of a method for isolating cartilage tissue-derived extracellular vesicles, Figure 7B is a SEM image , Figure 7C is the result of confirming the size distribution, Figure 7D is a fluorescence image after cell penetration of CEV with PKH detection attached, Figure 7E is the result of confirming the protein expression of CD81 and CD9 used as markers of CEV.

Figure 8 is a result of confirming the anti-inflammatory efficacy according to the treatment of extracellular vesicles after pretreatment with cartilage tissue-derived extracellular vesicles in a human synovial cell in vitro inflammatory model, Figures 8A and 8B are the results of confirming the gene expression level of inflammatory cytokines , Figure 8C is the result of confirming the protein expression level of the main inflammatory mediator, Figure 8D is the result of quantitatively analyzing the expression of the main inflammatory mediator COX-2, Figure 8E is the inflammatory mediator PGEd released outside the cell This is the result of analyzing the protein quantification of

Figure 9 confirms the immune cell regulatory ability according to the extracellular matrix (P_CECM) and extracellular vesicles (P_CEV) treatment after pretreatment of cartilage tissue-derived extracellular matrix (CECM) and extracellular vesicles (CEV) in human monocyte cell in vitro experiments. As a result, FIG. 9A is the result of confirming the M1 Macrophage and M2 Macrophage markers with fluorescence images through an ICC experiment, and FIG. 9B is the result of quantitatively analyzing the expression of the identified markers.

Figure 10 shows the anti-inflammatory efficacy of extracellular matrix (P_CECM) and extracellular vesicles (P_CEV) treatment after pretreatment of cartilage tissue-derived extracellular matrix (CECM) and extracellular vesicles (CEV) in an in vivo experiment in rats induced with osteoarthritis disease As a result of confirmation, FIG. 10A is the result of confirming the cartilage protection function and the increase or decrease of the inflammatory response by performing Safranin-O, H&E, and CD68-IHC staining, and FIG. 10B is the result of CD68 IHC analysis capable of quantitative analysis of the inflammatory response.

Hereinafter, the present invention will be described in more detail.

The present invention may provide a pharmaceutical composition for preventing or treating inflammatory diseases, which contains, as an active ingredient, cartilage tissue-derived extracellular matrix pre-treated with inflammatory cytokines, non-steroidal anti-inflammatory agents, or a combination thereof.

The pre-treated cartilage tissue-derived extracellular matrix is a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that it comprises extracellular vesicles with enhanced anti-inflammatory properties.

The inflammatory cytokine may be one or more selected from the group consisting of interleukin-1β, TNF-a, and interferon-γ.

The non-steroidal anti-inflammatory agent is one or more selected from the group consisting of salicylic acid, propionic acid, celecoxib, naproxen, acetic acid and fenamic acid can

The inflammatory diseases include dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus , It may be any one selected from the group consisting of fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, and multiple sclerosis.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating inflammatory diseases is prepared in the form of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or solutions according to conventional methods. Any one formulation selected from the group consisting of may be used.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating inflammatory diseases is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, flavoring agent, antioxidant commonly used in the manufacture of pharmaceutical compositions , It may further include one or more additives selected from the group consisting of a buffer, a bacteriostatic agent, a diluent, a dispersing agent, a surfactant, a binder, and a lubricant.

Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

According to one embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject as

The preferred dosage of the stone maple extract may vary depending on the condition and weight of the subject, the type and severity of the disease, the type of drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

In addition, the present invention comprises the steps of pre-treating an inflammatory cytokine, a non-steroidal anti-inflammatory agent or a combination thereof to the cartilage tissue isolated from the subject;

Culturing the pre-treated cartilage tissue for 1 to 5 days; and

It is possible to provide a method for enhancing the anti-inflammatory function of the extracellular matrix comprising the step of separating the extracellular matrix from the cultured cartilage tissue.

More specifically, after harvesting the cartilage tissue using a surgical blade, the cartilage tissue was washed again with physiological saline and then made into cartilage pieces having a size of about 5 mm × 5 mm × 2 mm using a blade. In the same way as in the pre-treatment of chondrocytes, the tissue was washed with 1× PBS and treated with chondrogenic DMEM culture, and then the pre-treatment group was treated with 0.1 ng/ml of Interleukin-1β and 10 μM celecoxib. ) was added and treated for 3 days.

The anti-inflammatory function enhancing method is a pre-treatment method for enhancing anti-inflammatory function of cartilage tissue-derived extracellular matrix or extracellular vesicles, and various inflammatory cytokines and non-steroidal anti-inflammatory drugs may be used.

The inflammatory cytokine may be one or more selected from the group consisting of interleukin-1β, TNF-a, and interferon-γ, but is not limited thereto.

The non-steroidal anti-inflammatory agent is one or more selected from the group consisting of salicylic acid, propionic acid, celecoxib, naproxen, acetic acid and fenamic acid It may be, but is not limited thereto.

The extracellular matrix obtained by the anti-inflammatory function enhancing method may include extracellular vesicles having enhanced anti-inflammatory properties.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Confirmation of biochemical characteristics after isolation and pretreatment of porcine chondrocytes

1. Isolation and culture of porcine chondrocytes

Porcine cartilage tissue was harvested using a surgical blade from a porcine knee joint. Cartilage tissue was washed with physiological saline and treated with 0.1% Type 2 Collagenase for 16 hours to obtain a suspension. The suspension was filtered through a 100 μm size nylon mesh, centrifuged at 600 × g for 10 minutes, the supernatant was discarded, and the remaining cells were collected in DMEM medium containing 10% FBS, 1% penicillin and streptomycin (penicillin/streptomycin). cultured in. The medium was replaced every 2-3 days, and passages were performed when the confluency was 7-80%.

2. Pretreatment of chondrocytes with inflammatory cytokines and non-steroidal anti-inflammatory drugs

After attaching the chondrocytes to the culture dish, they were cultured until they had 6-70% confluency. After removing the culture medium and washing the cells with 1× PBS (phosphate buffered saline), the cells were treated with FBS-free chondrogenic DMEM culture medium. Chondrogenic DMEM cultures were supplemented with Insulin-Transferrin-Selenium (ITS), 50 μg/ml L-ascorbic acid, 100 nM Dexamethasone, 40 μg/ml L-proline, 1.25 mg/ml Bovine Serum Albumin (BSA), 100 μg/ml L-proline in DMEM. μg/ml Sodium pyruvate and 1% penicillin/streptomycin were included. The pretreatment group was treated for 72 hours by adding 0.1 ng/ml interleukin-1β and 10 μM celecoxib to the chondrogenic DMEM culture medium.

3. Analysis of biological characteristics of chondrocytes after pretreatment

Inflammation-related factors in the pre-treated chondrocytes were quantitatively analyzed using real-time RT-PCR. In addition, protein expression of anti-inflammatory factors was evaluated by Western blot. As a result, it was confirmed that the expression of interleukin-10 (IL-10) and TGF-β, known as anti-inflammatory factors, were higher in the pre-treated chondrocytes than in non-pre-treated chondrocytes, as shown in FIGS. 2A and 2B.

< Example 2> Pre-treatment of cartilage tissue using inflammatory cytokines and non-steroidal anti-inflammatory drugs

1. Isolation of porcine cartilage tissue and pretreatment with inflammatory cytokines and non-steroidal anti-inflammatory drugs

As in the cartilage tissue separation method, cartilage tissue was harvested using a surgical blade. The cartilage tissue was washed again with physiological saline and then made into pieces of cartilage with a size of about 5 mm × 5 mm × 2 mm using a blade. In the same way as in the pre-treatment of chondrocytes, the tissue was washed with 1× PBS and treated with chondrogenic DMEM culture, and then the pre-treatment group was treated with 0.1 ng/ml of Interleukin-1β and 10 μM celecoxib. ) and cultured for 3 days.

As an anti-inflammatory enhancing pretreatment method of cartilage tissue, various inflammatory cytokines and non-steroidal anti-inflammatory drugs can be used. Interleukin-1β or TNF-a may be used as an inflammatory cytokine, and salicylic acid, propionic acid, or Coixb series may be used as a non-steroidal anti-inflammatory agent, but is not limited thereto.

2. Analysis of biological characteristics of pre-treated cartilage tissue

Protein expression of anti-inflammatory factors was confirmed by Western blotting in the pre-treated cartilage tissue. As a result, it was confirmed that the pre-treated cartilage tissue, as shown in FIGS. 3A and 3B, showed higher expression of genes and proteins of anti-inflammatory factors such as IL-10 and TGF-β compared to the control group. The mechanism of action of the expression of anti-inflammatory factors in cells in cartilage tissue by the pretreatment process is as shown in FIG. 3C, since inflammatory cytokines regulate the SMAD pathway and non-steroidal anti-inflammatory agents such as celocoxib regulate other pathways such as NFkB. Synergistic effects such as enhanced anti-inflammatory effects may be suggested.

In addition, the tissue pretreatment efficacy of various inflammatory cytokines and non-steroidal anti-inflammatory agents other than interleukin-1 and celecoxib was evaluated through RT-PCR and Western blotting. As a result, as shown in FIGS. 4A and 4B, it was confirmed that the expression of genes and proteins of anti-inflammatory factors was significantly increased compared to the control group in various combinations of inflammatory cytokines and non-steroidal anti-inflammatory agents.

< Example 3> Analysis of anti-inflammatory properties after separating soluble and insoluble factors from pre-treated cartilage tissue

1. Separation of soluble/insoluble factors from pre-treated cartilage tissue

In order to specify the components changed in the cartilage tissue according to the pretreatment, as shown in FIG. 5A, the biological properties were evaluated by separating the cartilage tissue into soluble signal molecules (small molecules) and insoluble structural proteins (structural molecules) through pepsin solubilization treatment. .

As a result of SDS-PAGE analysis, as shown in FIG. 5B, it was confirmed that a large amount of large structural molecules such as collagen were present in the structural components, and a large amount of relatively small-sized proteins were present in the soluble components.

2. Confirmation of anti-inflammatory efficacy among Soluble ECM and Insoluble ECM components

In the model in which inflammation was induced by treating human synovial cells (SW982) with 10 ng/ml of interleukin-1β (IL-1β) and 25 ng/ml of tumor necrosis factor-alpha (TNF-α) for 24 hours, The separated soluble ECM components and insoluble ECM components were treated for 24 hours at a concentration of 100 μg/ml, respectively.

As a result, as shown in FIGS. 6A and 6B, the expression of IL-1B and IL-6, which are major mediators of inflammation, decreased in all pretreatment groups, as well as COX-2 and p-IκB-α as shown in FIGS. 6C and 6D. The expression of was significantly decreased compared to the inflammation induction group, and in particular, a more significant decrease was confirmed in the soluble ECM components treatment group after pretreatment.

<Example 4> Isolation and biochemical characterization of cartilage tissue-derived extracellular vesicles

1. Isolation of cartilage tissue-derived extracellular vesicles

After the pretreatment, the cartilage tissue was recovered after washing with 1 × PBS. This was cooled in a cryogenic freezer at -80 ° C and freeze-dried for 3 days. After subdividing the same weight of dried cartilage tissue, it was treated with 0.1 mg/ml Type 2 Collagenase solution (in 200 mM NaCl, 5 mM CaCl2, 50 mM Tris buffer) for 2 days to obtain a suspension. The suspension was centrifuged at 500 × g for 10 minutes, 2500 × g for 20 minutes, and 10000 × g for 30 minutes using a high-speed centrifuge to remove cells and cell debris, followed by 0.45 μm filter tube, 0.2 μm filter tube, and 3kD filter tube in sequence. was concentrated using After recovering the concentrate, extracellular vesicles were separated using ExoQuick-TC (System Biosciences, USA), subdivided into equal amounts of protein through Bradford assay, and stored in a cryogenic freezer at -80°C.

2. Analysis of biochemical characteristics of extracellular vesicles

The shape and size of the extracellular vesicles isolated from the pretreated cartilage tissue were analyzed using a scanning electron microscope. Then, the particle size distribution was measured using a dynamic light scattering method (ELSZ-2000, Otsuka Electronics, Osaka, Japan).

As a result, it was confirmed that the size of the extracellular vesicles had a size of 100 to 200 nm, as shown in FIGS. 7B and 7C.

In addition, to confirm the cell penetration ability of extracellular vesicles, the extracellular vesicles were labeled with PKH-26, treated with SW982, and confirmed through confocal laser scanning. As a result, as shown in FIG. 7D, it was confirmed that extracellular vesicles penetrated near the nucleus within the cell. In addition, as shown in FIG. 7E, expression of CD9 and CD81, which are surface antigens specific to extracellular vesicles, was confirmed by Western blotting.

< Example 5> extracellular Validation of biological properties and anti-inflammatory function before and after pretreatment

One. cartilage tissue origin extracellular vesicle and after pretreatment cartilage tissue origin extracellular Anti-inflammatory validation

In order to confirm the anti-inflammatory efficacy level of cartilage tissue-derived extracellular vesicles and cartilage tissue-derived extracellular vesicles after pretreatment, human synovial cells (SW982) were treated with 10 ng/ml of interleukin-1β (IL-1β) and 25 ng/ml Inflammation was induced for 24 hours using tumor necrosis factor-alpha (TNF-α), and Western blotting was performed to compare the extracellular vesicles treated group and the pretreated extracellular vesicles treated group.

As a result, when gene expression was confirmed using RT-qPCR as shown in FIGS. 8A and 8B, the inflammatory mediators IL-1β and IL-6 were increased in the inflammatory group but decreased in the pre-treated extracellular vesicles-treated group. could

In addition, as shown in FIGS. 8C and 8D, the expression of COX-2, p-IκB-α, and phospho p65, which are major inflammatory mediators, increased in the inflammatory group, whereas in the group treated with extracellular vesicles, it was confirmed that the expressions all decreased. .

In addition, the culture medium of each treatment group was collected and the expression of inflammatory factors in the culture medium was confirmed by ELISA.

As a result, as shown in FIG. 8E, the expression of PGE2, an inflammatory mediator, increased in the inflammatory group but decreased in the extracellular vesicles-treated group. From the above results, it was confirmed that cartilage tissue-derived extracellular vesicles have an anti-inflammatory function that reduces the degree of inflammation of human synovial cells.

< Example 6> Derived from cartilage tissue extracellular matrix and extracellular Confirmation of M2 Macrophage differentiation inducing ability

To confirm the level of induction of M2 Macrophage differentiation of cartilage tissue-derived extracellular matrix (CECM) and extracellular vesicles (CEV) and cartilage tissue-derived extracellular matrix (P_CECM) and extracellular vesicles (P_CEV) after pretreatment, M0 Macrophage The group was treated with a concentration of 10 μg/ml, and ICC analysis for CD86 (M1 marker) and CD163 (M2 marker) was performed. As a positive control group, 100 ng/ml of LPS and 20 ng/ml of IFN-γ were used to induce differentiation of M1 macrophage, and 20 ng/ml of IL-4 and 20 ng/ml of IL-13 were used for M2 macrophage. were treated with M0 Macrophage for 24 hours to induce differentiation.

As a result, when confirming the expression of the M1 and M2 markers in the ICC analysis as shown in FIG. 9A, it was confirmed that the expression of the M2 marker was dominant in the pretreated extracellular matrix and extracellular vesicles treated groups.

In addition, when the ratio of M1 / M2 markers was quantitatively analyzed as shown in FIG. 9B, it was confirmed that the induction of differentiation of M2 marcrophages was significantly increased in both the extracellular matrix and the extracellular vesicles by the pretreatment process.

< Example 7> Osteoarthritis ( OA ) in the induced rat model extracellular Validation of anti-inflammatory function

1. Administration of cartilage tissue-derived extracellular vesicles in a rat osteoarthritis disease model

As the animal used in the experiment, an 8-week-old male SD-Rat was purchased and used for the experiment at 9 weeks of age after a week of acclimatization. To induce osteoarthritis, 30 μl of Type II collagenase at a concentration of 3 U/ul was injected into the synovial fluid of the left leg of rats, except for the untreated group. The treatment group was forced to treadmill running for 40 minutes every day at a speed of 20 m/min for 2 weeks. Then, the PBS treatment group, the cartilage tissue-derived extracellular matrix treatment group (CECM), the pretreatment post-treatment extracellular matrix treatment group (P_CECM), the cartilage tissue-derived extracellular vesicle treatment group (CEV), the pretreatment post-extracellular vesicle treatment group (P_CEV) 30 μl of silver at a concentration of 2 μg/μl was injected into the synovial fluid of the left leg at weekly intervals for 4 weeks. After treatment with extracellular vesicles, rats were sacrificed and histological analysis was performed.

2. body cartilage tissue origin with extracellular vesicles after pretreatment extracellular vesicle treatment group histological analysis

In order to evaluate the degree of inflammation of the cartilage and synovial membrane in the joint, histological evaluation was performed through safranin-O staining, H&E staining, and immunohistochemical staining of the monocyte-specific surface antigen CD68 as shown in FIG. 10A.

As a result, as a result of safranin-O staining as shown in FIG. 10A, the sGAG content of cartilage was reduced in the PBS-treated group, and the histological morphology of early-stage osteoarthritis was shown. However, the extracellular matrix and extracellular vesicles treated groups showed sGAG content similar to that of the untreated group.

In addition, for quantitative analysis, immunochemical staining of CD68, a monocyte-specific surface antigen expressed when inflammation occurs, was performed.

As a result, as shown in FIG. 10B, the expression of CD68 was increased in the PBS-treated group compared to the untreated group, and after pretreatment, the extracellular matrix and extracellular vesicles-treated group showed a decrease compared to the PBS-treated group.

From the above results, it was confirmed that the group treated with extracellular matrix and extracellular vesicles showed an effect of reducing the degree of synovial inflammation as well as an improved effect of reducing osteoarthritis of cartilage compared to each pretreatment group.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

1. A pharmaceutical composition for the prevention or treatment of inflammatory diseases containing, as an active ingredient, an inflammatory cytokine, a nonsteroidal anti-inflammatory agent, or a pre-treated cartilage tissue-derived extracellular matrix with a combination thereof.
2. The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 1, wherein the pre-treated cartilage tissue-derived extracellular matrix comprises extracellular vesicles with enhanced anti-inflammatory properties.
3. The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 1, wherein the inflammatory cytokine is one or more selected from the group consisting of interleukin-1β, TNF-a and interferon-γ.
4. The method according to claim 1, wherein the non-steroidal anti-inflammatory agent is selected from the group consisting of salicylic acid, propionic acid, celecoxib, naproxen, acetic acid and fenamic acid A pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that one or more are selected.
5. The method according to claim 1, wherein the inflammatory disease is dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout For preventing or treating inflammatory diseases, characterized in that any one selected from the group consisting of ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome and multiple sclerosis pharmaceutical composition.
6. Pre-treating the cartilage tissue isolated from the subject with an inflammatory cytokine, a non-steroidal anti-inflammatory agent, or a combination thereof;

   Culturing the pre-treated cartilage tissue for 1 to 5 days; and

   A method for enhancing the extracellular matrix anti-inflammatory function comprising the step of separating the extracellular matrix from the cultured cartilage tissue.
7. The method according to claim 6, wherein the inflammatory cytokine is selected from the group consisting of interleukin-1β, TNF-a and interferon-γ.
8. The method according to claim 6, wherein the nonsteroidal anti-inflammatory agent is selected from the group consisting of salicylic acid, propionic acid, celecoxib, naproxen, acetic acid and fenamic acid A method for enhancing the extracellular matrix anti-inflammatory function, characterized in that one or more are selected.
9. The method according to claim 6, wherein the extracellular matrix comprises extracellular vesicles with enhanced anti-inflammatory properties.

Images