KR20210052366A - Pharmaceutical composition for preventing or treating inflammatory disease comprising mitochondria isolated form muscle - Google Patents

Abstract

Muscle-derived mitochondria according to the present invention have the ability to regulate inflammatory cytokines and Treg/Th17 cells in osteoarthritis animal models, encephalomyelitis models, Sjogren's syndrome animal models and transplant rejection models, thereby being able to be used for various inflammatory or immune diseases.

Description

Composition for preventing or treating immune diseases including muscle-derived mitochondria {PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING INFLAMMATORY DISEASE COMPRISING MITOCHONDRIA ISOLATED FORM MUSCLE}

The present invention relates to a composition for the prevention or treatment of inflammatory diseases or immune diseases such as osteoarthritis, transplant rejection disease, Sjogren's syndrome, and autoimmune diseases of the nervous system comprising muscle-derived mitochondria as an active ingredient.

Mitochondria are organelles of eukaryotic cells that are involved in the synthesis and regulation of adenosine triphosphate (ATP), a source of energy in cells. Mitochondria are involved in various metabolic pathways in vivo, such as cell signaling, cell differentiation, apoptosis, as well as the control of cell cycle and cell growth. Mitochondria have their own genome and are organelles that play a pivotal role in the energy metabolism of cells. Mitochondria produce energy through electron transfer and oxidative phosphorylation, and play an important role, such as being involved in apoptosis signaling pathways.

It has been reported that the decrease in energy production due to the decrease in mitochondrial function causes various diseases. When the function of the electron transport chain reaction is deteriorated according to the mutation of the mitochondrial genome and protein, the ATP production decreases, the active oxygen is excessively generated, and the calcium regulation function decreases. In this case, a change in the membrane permeability of mitochondria occurs, causing apoptosis function to be abnormal, resulting in cancer and intractable diseases.

Such human diseases caused by mitochondrial dysfunction include mitochondrial-related genetic diseases (Wallace DC 1999), diabetes (Maechler P 2001), heart disease (Sorescu D 2002), and senile dementia such as Parkinson's disease or Alzheimer's disease (Lin MT 2006). ), and the occurrence of various cancers (Petros JA, 2005) and cancer metastasis (Ishikawa K, 2008) have been reported. In addition, the characteristics commonly found in more than 200 different types of cancer consisted of impaired apoptosis, increased inflammatory response, and increased abnormal metabolic action. In addition, studies on the relationship between mitochondria and immune diseases are currently being conducted.

If there are signaling mechanisms in the mitochondria that have a close relationship with cell death and can interpret and control these signals, it can be applied to disease treatment (Biochimica et Biophysica Acta, 2006; Cell, 2005; Trends Biochem Sci, 2005; Nature Med , 2005; Nature, 2005; J Clin Invest, 2005; Heart Lung Circ, 2005; J Nat Cancer Inst, 2005, Science, 2005). Mitochondrial decline is not a single symptom or disease, but can appear as a variety of diseases in various organs in the human body.Therefore, the possibility of specific treatments for lesions and diseases related to diseased mitochondria is being raised. State. In particular, the increase in oxidative stress caused by increased intracellular reactive oxygen species has recently been suggested as a fundamental cytological and molecular biological mechanism in cardiovascular and metabolic diseases as well as degenerative neurological diseases. The effectiveness of antioxidant treatment is still controversial. Gene therapy as micro-targeting therapy capable of controlling the expression of membrane proteins and matrix proteins that can directly control mitochondrial function is a promising strategy as a permanent or semi-permanent therapy.

Therefore, it is necessary to develop immune modulators and treatments that control not only immune regulation, but also infection and strengthening of immune regulatory cell functions through improvement of mitochondrial function. In addition, there is a need to develop a new therapeutic agent for inflammatory immune disease and a treatment method that is inexpensive and has excellent therapeutic effects without side effects.

On the other hand, immune disease refers to a disease in which components of the immune system cause, mediate, or otherwise contribute to a pathology. In particular, inflammatory disorders are one of the most important health problems in the world. Inflammation generally refers to the localized protective response of body tissues to host invasion by foreign substances or harmful stimuli. Causes of inflammation include infectious causes such as bacteria, viruses, and parasites; Physical causes such as burns or irradiation; Chemicals such as toxins, drugs or industrial agents; Immune responses such as allergic and autoimmune reactions, or conditions associated with oxidative stress.

Inflammation is characterized by symptoms such as pain, redness, swelling, fever, and eventual loss of function in the affected area. These symptoms are the result of a complex series of interactions between cells in the immune system. Cellular responses result in several groups of inflammatory mediators [proteins (e.g. cytokines, enzymes (e.g. proteases, peroxidase)), major basic proteins, adhesion molecules (ICAM, VCAM), lipid mediators (e.g. For example, an interactive network of eicosanoids, prostaglandins, leukotrienes, platelet activating factor (PAF)), and reactive oxygen species (eg, hydroperoxide, superoxide anion, and nitric oxide (NO)) is created. However, most of these mediators are also modulators of normal cellular activity, so a lack of inflammatory response leads to uncontrolled damage to the host (ie, infection), and thus chronic inflammation in part due to several of the aforementioned mediators. This overproduction leads to mediated inflammatory diseases.

In addition, autoimmune diseases, which are one of the immune diseases, are characterized in that the immune system attacks its own organs and causes a spontaneous reaction. These responses are due to the recognition of auto-antigens by T lymphocytes, which induces humoral (autoantigen production) and cellular (lymphocyte and macrophage cytotoxic activity increase) immune responses. Autoimmune diseases include rheumatoid disease, psoriasis, systemic dermatomyositis, multiple sclerosis, lupus erythematosus, or worsening immune responses by antigens, ie, asthma, allergies to drugs or food. All of these diseases are limited and chronic diseases, and in some cases, they are fatal, and there is no effective treatment method for treating the diseases to date. Therefore, any drug, medicine, or medium that can alleviate or alleviate the disease during the progression of the disease will be said to be an important solution for the health of the patient.

We have been making intensive efforts to find suitable drugs and methods by searching for treatment methods for autoimmune diseases. Treatment of autoimmune diseases today is primarily based on the use of immunosuppressive drugs such as glucocorticoids, calcineurin inhibitors and antiproliferatives-antimetabolites. However, since such drugs act on various targets, they can reduce immune function as a whole. In addition, long-term use of these pharmacological therapies may result in several cytotoxic effects, and by suppressing the immune system in a non-specific manner, the patient may suffer from infection or cancer. Since calcineurin and glucocorticoids present another problem due to their nephrotoxicity and diabetic properties, their use is limited in some cases of clinical symptoms (eg renal insufficiency, diabetes, etc.). Therefore, as a substance capable of treating immune diseases such as autoimmune diseases and inflammatory diseases, there is a need to develop a new therapeutic agent for immune diseases that has no side effects and has excellent therapeutic effects.

Korean Patent Registration No. 10-1723265

An object of the present invention is to provide a composition for preventing or treating inflammatory diseases or immune diseases such as osteoarthritis, transplant rejection disease, Sjogren's syndrome, and autoimmune diseases of the nervous system, comprising muscle-derived mitochondria as an active ingredient.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases mediated by IL-17, comprising mitochondria isolated from muscles as an active ingredient.

According to an embodiment of the present invention, the immune disease or inflammatory disease may be one or more diseases selected from osteoarthritis, transplant rejection, Sjogren syndrome, and autoimmune diseases of the autonomic nervous system.

In addition, according to an embodiment of the present invention, the osteoarthritis is

1) an increase in the number of cells expressing IL-1β, MMP13 and MCP1;

2) COX4 is reduced; And

3) It may be osteoarthritis with an increased condition of Th17 cells in the spleen tissue.

In addition, according to an embodiment of the present invention, the transplant rejection reaction may be at least one transplant rejection reaction selected from the group consisting of cells, blood, tissues, and organs.

The transplant rejection may be one or more rejection reactions selected from the group consisting of rejection reactions of bone marrow transplantation, heart transplantation, corneal transplantation, bowel transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney transplantation, and skin transplantation.

The graft rejection reaction may be a graft-versus-host disease (GVHD).

According to an embodiment of the present invention, autoimmune diseases of the nervous system include multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, ascending myelitis, and central myelitis. myelitis, descending myelitis, transverse myelitis, myasthenia gravis, guillain-barre syndrome, autoimmuneuveitis, autoimmune encephalopathy, It may be one or more selected from the group consisting of encephalomyelitis and chronic inflammatory demyelinating polyneuropathy.

The muscle-derived mitochondria according to the present invention have the ability to modulate the ability to regulate inflammatory cytokines and Treg/Th17 cells in osteoarthritis animal models, encephalomyelitis models, Sjogren's syndrome animal models, and transplant rejection models. Available for

1 is a diagram showing the pain measurement results of a patient with osteoarthritis caused by muscle-derived mitochondria according to an embodiment of the present invention.
Figure 2 shows the safranin o staining image and clinical arthritis evaluation index in the group administered with muscle-derived mitochondria in an embodiment of the present invention.
3 is a result of confirming the IHC staining image and the expression of IL-1β, MMP13, MCP1 and cox4 in an animal to which muscle-derived mitochondria were administered in an embodiment of the present invention.
4 is an analysis result confirming the ability to simultaneously regulate TH17/Treg cells by muscle-derived mitochondria in an embodiment of the present invention.
5 is an analysis result confirming the ability to regulate TH17 cells by muscle-derived mitochondria in an embodiment of the present invention.
6 is an analysis result confirming the ability to simultaneously regulate IL-17/IL-10 by administration of muscle-derived mitochondria in an embodiment of the present invention.
7 shows the effect of regulating metabolic factors of osteoarthritis chondrocytes by administration of muscle-derived mitochondria in an embodiment of the present invention.
8 is a diagram illustrating the therapeutic effect on autoimmune diseases of the nervous system such as encephalomyelitis by administration of muscle-derived mitochondria according to an embodiment of the present invention.
9 is a diagram illustrating the therapeutic effect of Sjogren syndrome by administration of muscle-derived mitochondria in an embodiment of the present invention.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention. However, the following embodiments are presented as examples of the present invention, whereby the present invention is not limited, and the present invention is capable of various modifications and applications within the scope of equality interpreted from the description of the claims to be described later. .

It provides a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases mediated by IL-17, comprising mitochondria isolated from muscles as an active ingredient.

The mitochondria may be obtained from a mammal or may be obtained from a human. In addition, the mitochondria may be isolated from cells or cell culture. However, it is preferable that the mitochondria are isolated from muscle tissue or muscle cells. In addition, the mitochondria may be normal mitochondria obtained from cells having normal mitochondrial biological activity, or mitochondria derived from patients. In addition, the mitochondria may be cultured in vitro.

Meanwhile, when the mitochondria are separated from a specific cell, the mitochondria may be separated through various known methods, such as using a specific buffer solution or using a potential difference and a magnetic field.

In terms of maintaining mitochondrial activity, the mitochondrial separation may be performed by disrupting cells and performing centrifugation. In one embodiment, the steps of culturing the cells, and the first centrifugation of a composition containing such cells to produce a pellet, resuspending the pellet in a buffer solution and homogenizing, the homogenized solution in a second Secondary centrifugation may be performed to prepare a supernatant, and the supernatant may be subjected to a third centrifugation to purify mitochondria. At this time, it is preferable in terms of maintaining cell activity that the time for the second centrifugation is performed to be shorter than the time for the first and third centrifugation to be performed, and the third centrifugation in the first centrifugation As you go to, you can increase the speed.

In addition, the mitochondria can be obtained in a culture medium obtained after culturing the cells. Such a culture medium may contain mitochondria such as cytokines, chemokines, exosomes and microvesicles secreted by cells. In one embodiment, the mitochondrial separation is a step of culturing cells, removing cell debris, etc. by first centrifuging a composition containing the obtained culture medium to obtain a supernatant, and second centrifuging the supernatant to obtain mitochondria. It can be carried out in a step of purification. It can be appropriately adjusted according to the number of centrifugation and the content of the sample.

The muscle-derived mitochondria may be isolated from the muscles of a patient with osteoarthritis, but are not limited thereto.

By the composition, the number of cells expressing IL-1β, MMP13, MCP1, etc., which are known factors that aggravate the condition of osteoarthritis, can be reduced, thereby alleviating and ameliorating the symptoms of osteoarthritis. In addition, cox4, which measures the amount of Mitochondria, is increased by the composition of the present invention, and osteoarthritis can be improved by activation of mitochondria.

By the composition, the expression of TH17/Treg cells is simultaneously regulated, so that the Treg cells increase, while the expression of TH17 may decrease.

By the composition, the expression of IL-17/IL-10 is simultaneously regulated, so that IL-17 decreases and IL-10 increases.

The muscle-derived mitochondria reduce the invasion of inflammatory factor-expressing immune cells and reduce the activity of IL-17-expressing inflammatory Th17 cells, thereby controlling not only joint inflammation but also systemic immune system pathogenesis, but is not limited thereto.

The muscle-derived mitochondria inhibit the activity of metabolic factors of cartilage cells, thereby controlling cartilage cell damage/cartilage tissue damage, and thus protect joints, but are not limited thereto.

Therefore, the muscle-derived mitochondria of the present invention

1) an increase in the number of cells expressing IL-1β, MMP13 and MCP1;

2) COX4 is reduced; And

3) Th17 cells in the spleen tissue can have a therapeutic effect on osteoarthritis with increased conditions.

The term "transplantation rejection" of the present invention refers to a reaction that the recipient's immune system recognizes as non-self after transplantation and attacks the transplanted organ and rejects the reaction to remove it. It is called. The most important factor involved in transplant rejection is the major histocompatibility complex (MHC), and it is known that the minor histocompatibility complex is also related. Rejection reaction involves both cellmediated immune response and humoral immune response. In the case of a cell-mediated reaction, the recipient's lymphocytes encounter the donor MHC in the transplant organ (CD4 T cell-type II MHC molecule, or CD8 T cell-type I MHC molecule). Activated T cells secrete cytokine, increase blood vessel permeability, and invade monocytes such as macrophages. As a result, damage to microvessels, tissue ischemia, and destruction of transplanted tissues and cells occur.

The transplant rejection reaction is one or more transplant rejection reactions selected from the group consisting of cells, blood, tissues and organs, preferably bone marrow transplant, heart transplant, corneal transplant, intestine transplant, liver transplant, lung transplant, pancreas transplant, kidney At least one selected from the group consisting of transplantation and skin graft rejection, but is not limited thereto.

Graft rejection includes, but is not limited to, graft-versus-host disease (GVHD).

Sjogren's syndrome is an autoimmune disease in which inflammatory cells infiltrate into the lacrimal glands and salivary glands, causing chronic inflammation of unknown origin. This disease is known to be caused by loss of control of the immune system, which is the body's defense system. As a result, inflammatory cells infiltrate into the lacrimal glands and salivary glands, which are the secretory glands that maintain the humidity of the body, destroying normal cells, making it impossible to maintain their functions anymore.

In the present invention, the autoimmune diseases of the nervous system are multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, ascending myelitis, central myelitis, and descending myelitis. (descending myelitis) transverse myelitis, myasthenia gravis, guillain-barre syndrome, autoimmuneuveitis, autoimmune encephalopathy, and chronic inflammatory demyelinating polyneuropathy The group consisting of chronic inflammatory demyelinating polyneuropathy, but is not limited thereto.

The composition according to the present invention can be used as a pharmaceutical composition capable of preventing and treating inflammatory diseases or immune diseases such as osteoarthritis, transplant rejection disease, Sjogren's syndrome and neurological autoimmune diseases, and the pharmaceutical composition includes a pharmaceutically acceptable carrier. Can be included as. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause an allergic reaction such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, a carrier for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like, and for parenteral administration such as water, suitable oil, saline, aqueous glucose and glycol. Carrier, etc., and may further include a stabilizer and a preservative. Suitable stabilizers are antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed, Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and as a representative formulation for parenteral administration, an isotonic aqueous solution or suspension is preferable as a dosage form for injection. Formulations for injection can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be formulated for injection by dissolving it in saline or buffer. In addition, formulations for oral administration include powders, granules, tablets, pills, and capsules, but are not limited thereto.

In addition, the present invention includes the step of administering a composition for preventing or treating osteoarthritis, transplant rejection disease, Sjogren's syndrome and nervous system autoimmune diseases to an individual in need of treatment, including osteoarthritis, transplant rejection disease, Sjogren's syndrome and nervous system autoimmune diseases. It provides a method of preventing or treating an inflammatory disease selected from the group consisting of.

The treatment method of the present invention includes administering a composition for preventing or treating osteoarthritis, transplant rejection disease, Sjogren's syndrome, and autoimmune diseases of the nervous system in a therapeutically effective amount to an individual. A specific therapeutically effective amount for a specific individual is the type and extent of the reaction to be achieved, the specific composition, including whether or not other agents are used in some cases, the individual's age, weight, general health status, sex and diet, administration time, It is preferable to apply differently according to various factors including the route of administration and the secretion rate of the composition, the treatment period, drugs used with or concurrently with the specific composition, and similar factors well known in the field of medicine. Therefore, it is preferable to determine an effective amount of a composition suitable for the purposes of the present invention in consideration of the foregoing.

The individual is applicable to any mammal, and the mammal includes humans and primates, as well as livestock such as cattle, pigs, sheep, horses, dogs and cats.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of muscle-derived mitochondria

The joint muscle tissue from the osteoarthritis patient was finely cut on IBM buffer, and the tissue by-products were filtered with a 100um filter, and then centrifuged at 200g for 3 minutes at 4°C. The pellet was suspended in IBM buffer and the cell membrane was burst using a homogenizer. Then, it was centrifuged to obtain a mitochondrial pellet (reference: Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements).

Example 2. Confirmation of the therapeutic effect of muscle-derived mitochondria on osteoarthritis

1.Improve pain level and check arthritis index

In order to confirm the therapeutic effect of muscle-derived mitochondria on osteoarthritis, the present inventors performed an experiment to analyze the therapeutic effect after injecting the muscle-derived mitochondria into an animal model in which osteoarthritis was induced.

One day after MIA induction to the animal model, the muscle-derived mitochondria isolated as in Example 1 were injected into the joint cavity of the MIA model (i.a) once a week at a concentration of 10 ug/100 ul for a total of 3 weeks. The weight bearing evaluation was evaluated after the MIA induction, and was measured once a week.

Then, 3 weeks after MIA induction, the mice were sacrificed, and the cartilage of the joint was stained with safranin O to analyze the degree of damage to the articular cartilage tissue.

As a result, it was confirmed that when the muscle-derived mitochondria were injected into the mice in which osteoarthritis was induced, the pain value and weight bearing were improved compared to the MIA group, which is an osteoarthritis-inducing model (FIG. 1). In addition, it was confirmed that when muscle-derived mitochondria were injected into mice in which osteoarthritis was induced, the tissue was improved and the clinical arthritis index was lowered compared to the MIA group, which is an osteoarthritis-inducing model (FIG. 2).

2. Inhibitory effect of osteoarthritis-related factors by muscle-derived mitochondria

The present inventors conducted an experiment to confirm whether the antibody-modulating effect by muscle-derived mitochondria in an animal model in which osteoarthritis was induced.

In animal models, mice were sacrificed 3 weeks after MIA induction, and IHC immunostaining was performed to analyze the invasion of immune cells expressing inflammatory factors in the synovium joint tissue. Cells expressing IL-1β, mmp13, mcp-1, and cox4 in the synovium tissue were analyzed using each specific antibody.

As a result, in mice injected with muscle-derived mitochondria, the number of cells expressing IL-1β, MMP13, and MCP1, which are known to aggravate the condition of osteoarthritis, decreased compared to the MIA group, which is an osteoarthritis-inducing model, thereby reducing invasion (Fig. 3). ). In addition, cox4, a marker for measuring the amount of mitochondria, rather showed an increase in the muscle-derived mitochondrial injection group (FIG. 3), which is thought to be an effect due to the increased mitochondria.

3. Th17/Treg cell regulation effect by muscle-derived mitochondria

The present inventors analyzed whether TH17/Treg cells are controlled by muscle-derived mitochondria in an osteoarthritis-induced animal model.

The mice were sacrificed at 3 weeks after induction of MIA to the animal model, and cells were isolated from the spleen tissue of each animal model, and Th17 and Treg cell activities were analyzed by flow cytometry. For flow cytometric analysis, after staining with CD4-PerCP, IL-17 APC, Foxp3-ECD, and CD25-FITC, the stained cells were analyzed by flow cytometry.

As a result, the activity of IL-17-expressing Th17 cells in the spleen tissue of the MIA-induced osteoarthritis animal model was increased, and the activity of IL-17-expressing inflammatory Th17 cells in the spleen tissue was decreased by intraarticular injection of muscle-derived mitochondria. Was confirmed (Fig. 4). These results suggest that muscle-derived mitochondria can regulate not only joint inflammation but also systemic immune system pathogenesis.

4. Simultaneous regulation of IL-17/IL-10 by muscle-derived mitochondria

After inoculating osteoarthritis chondrocytes at 3×10 ⁵ cells in a 24 well plate, 5 ug of muscle-derived mitochondria isolated as in Example 1 were air-cultured for 1 day, and RNA samples were obtained. The obtained RNA was synthesized with cDNA, and the amounts of MMP1, MMP3, and MMP13, which are catabolic markers of chondrocytes, were measured and analyzed at the mRNA level using QPCR.

As a result, as can be seen in Figure 5, it was confirmed that the activity of metabolic factors of chondrocytes was inhibited by muscle-derived mitochondria, and from these results, it was found that muscle-derived mitochondria could control chondrocyte damage/cartilage tissue damage. Could.

Example 3. Confirmation of the treatment effect of transplant rejection disease by muscle-derived mitochondria

Blood cells (PBMC) and muscle-derived mitochondria from patients with transplant disease and the general population were co-cultured for 3 days, and then only the supernatant of the culture medium was collected and measured for IL-7 and IL-10 by ELISA.

In addition, blood cells (PBMC) and muscle-derived mitochondria were co-cultured for 3 days in male patients with transplantation disease and normal people who had undergone liver transplantation and then observed changes in immune cells. As can be seen in Figure 6, it was confirmed that the expression of Th17 cells was inhibited by the mitochondria derived from muscle.

As a result, it was confirmed that IL-17 decreased and IL-10 increased in the group injected with muscle-derived mitochondria (FIG. 7). Based on the above results, it was confirmed that muscle-derived mitochondria can be used to treat transplant rejection disease.

Example 4. Confirmation of the therapeutic effect of autoimmune diseases of the nervous system by muscle-derived mitochondria

In order to confirm the therapeutic effect of the mitochondria of the present invention in the treatment of autoimmune diseases of the nervous system, an experimental autoimmune encephalomyelitis (EAE) animal model was prepared using MOG peptide. Specifically, as an animal model, 8-week-old female C57BL/6 mice (Narabiotechnology, Republic of Korea; body weight 22-24g) were prepared and stabilized for one week after birth. Mice were mixed with an emulsion containing 200 ug of MOG peptide 35-55 (Sigma-Aldrich, USA) and IFA (Incomplete Freund's adjuvant; Difco, USA) containing 200 ug of mycobacterium tuberculosis (Difco, USA) Thus, a total of 0.1 ml of the emulsion was injected subcutaneously into both waist areas of the EAE group, and at the same time, 500 ng of pertussis toxin (PTX, List Biologic, USA) was injected intraperitoneally. After 48 hours, the same amount of pertussis toxin was injected intraperitoneally again. The scales of clinical symptoms of the chronic EAE animal model induced by the MOG were identified by dividing into eight categories. In other words, no response (step 0), tail tip sagging (step 1), entire tail sagging (step 2), tail sagging and paralysis of one hind limb (step 3), paralysis of both hind legs (step 4), death. (Step 5) Classified.

Thereafter, muscle cell-derived mitochondria were administered to mice twice a week at a concentration of 10 ug/200 ul for a total of 6 times, and the results of confirming the disease score are shown in FIG. 8A. As a result, the effect in the group treated with muscle-derived mitochondria Confirmed

Figure 8B is a flow cytometric analysis of immune cells in the spleen tissue at the time of mouse sacrifice, IL-17 in CD4 T cells (IL-17+ CD4+), IL-17 in CD8 T cells (IL-17+CD8+), IL-17 (IL-17+CD19+) immune cell subtypes in CD19 B cells were analyzed. Results It was confirmed that each T and B cell subtype expressing IL-17 in the muscle-derived mitochondrial transplanted EAE group decreased, and mitochondrial ROS production (mitoSOX) expression was remarkably decreased.

FIG. 8C is an analysis of the degree of destruction of spinal cord tissue at the time of mouse sacrifice through H&E and LFB (Luxol fast blue) staining, and it was confirmed that the white matter and gray matter of the spinal cord tissue in the mitochondrial administration group were clearly distinguished, and tissue damage was suppressed.

Example 5. Confirmation of treatment effect of Sjogren syndrome by muscle-derived mitochondria

1. Confirmation of salivation ability of mitochondria derived from muscle in Sjogren syndrome animal model

In order to confirm the effect of muscle-derived mitochondria for the treatment of Sjogren's syndrome, the present inventors injected muscle-derived mitochondria into NOD (Non-obese diabetic) mice, an animal model of Sjogren's syndrome, and then, a measure of the onset of Sjogren's syndrome in mice. Saliva secretion and secretion maintenance function were investigated. The experiment was conducted by injecting a total of 5 times a week after the onset of Sjogren's syndrome by intraperitoneal injection into a control (vehicle) or 10 ug/200 ug of mitochondrial NOD/ShiLtJ mice.

The amount of saliva was measured by collecting saliva after administration of 0.1 mg of pilocarpine. As a result, mitochondria were administered a total of 5 times after the onset of the disease, and in the group treated with mitochondria at the 13th week after the onset of the disease, the salivary secretion ability (Figure 9A, measured by the flow rate, which is a saliva/weight value per minute) was higher than that of the control group (vehicle). It was confirmed that it increased significantly.

2. Confirmation of Th17/Treg cell regulation effect of muscle-derived mitochondria in Sjogren syndrome animal model

The present inventors performed flow cytometric analysis to identify Th17 cells (immunostaining with CD4 and IL-17 antibodies), B17 cells, and Th1 cells in the spleen tissue of the Sjogren syndrome mouse model. In addition, it was confirmed that the activity of IL-17 + CD4 + Th17 cells in the salivary gland tissue was reduced. As a result, it was confirmed that mitochondria decreased Th17 cells, B17 cells, and Th1 cells in spleen tissue and salivary gland tissue (FIG. 9B).

3. In the animal model of Sjogren syndrome, the effect of maintaining the cell morphology of the mitochondria derived from muscle and the invasion effect of inflammatory cells was confirmed

In order to confirm the effect of mitochondria for the treatment of Sjogren's syndrome, the present inventors investigated whether or not the morphology of cells is maintained in the lacrimal gland and the salivary gland of mice, which are animal models of Sjogren's syndrome, and the invasion of inflammatory cells. It was performed an experiment to confirm whether or not through the H & E staining method known in the art.

As a result, when the muscle-derived mitochondria were injected, it was confirmed that immune cell infiltration in the lacrimal gland and salivary gland tissue was reduced, and tissue destruction was significantly reduced compared to the control group (FIG. 9C).

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (10)

A pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases mediated by IL-17, comprising mitochondria isolated from muscles as an active ingredient. The composition of claim 1, wherein the mitochondria are isolated from muscle tissue or muscle cells. The composition of claim 1, wherein the mitochondria are isolated from tissues or cells derived from osteoarthritis patients. The composition of claim 1, wherein the immune disease or inflammatory disease is at least one disease selected from osteoarthritis, transplant rejection, Sjogren's syndrome and autoimmune diseases. The method of claim 4, wherein the osteoarthritis is
1) an increase in the number of cells expressing IL-1β, MMP13 and MCP1 in joint tissue;
2) COX4 is reduced in the joint tissue; and
3) Th17 cells in the spleen tissue having an increased condition, the composition. The composition of claim 4, wherein the transplant rejection reaction is at least one transplant rejection reaction selected from the group consisting of cells, blood, tissues and organs. The rejection reaction of claim 6, wherein the transplant rejection is at least one selected from the group consisting of bone marrow transplantation, heart transplantation, corneal transplantation, bowel transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney transplantation, and skin transplant rejection The composition that is. The composition of claim 4, wherein the graft rejection reaction is graft-versus-host disease (GVHD). The method of claim 4, wherein the autoimmune diseases of the nervous system are multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, ascending myelitis, and central myelitis. , Descending myelitis, transverse myelitis, myasthenia gravis, guillain-barre syndrome, autoimmuneuveitis, autoimmune encephalopathy, encephalopathy encephalomyelitis) and chronic inflammatory demyelinating polyneuropathy. The composition of claim 1, wherein the mitochondria decrease Th17 and increase Tregs.

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