KR102177839B1 - Composition for treating chronic inflammatory bowel diseases or for improving bowel function - Google Patents

Abstract

The present invention relates to a composition for treating chronic inflammatory bowel disease or improving bowel function, and more particularly, a pharmaceutical composition for preventing or treating chronic inflammatory bowel disease, comprising copricin peptide CopA3 as an active ingredient, copricin peptide CopA3. It relates to a food composition for preventing or improving chronic inflammatory bowel disease and a food composition for improving intestinal function, including as an active ingredient.
The composition of the present invention promotes proliferation and apoptosis of intestinal epithelial cells to aid epithelial cell regeneration, and enhances barrier function by reinforcing close junctions between intestinal epithelial cells, thereby preventing various chronic inflammatory bowel diseases caused by increased intestinal mucosal permeability. Alternatively, it may be usefully used to develop therapeutic drugs, functional foods, and foods for improving intestinal function.

Description

Composition for treating chronic inflammatory bowel diseases or for improving bowel function}

The present invention relates to a composition for treating chronic inflammatory bowel disease or improving bowel function, and more particularly, a pharmaceutical composition for preventing or treating chronic inflammatory bowel disease, comprising copricin peptide CopA3 as an active ingredient, copricin peptide CopA3. It relates to a food composition for preventing or improving chronic inflammatory bowel disease and a food composition for improving intestinal function, including as an active ingredient.

Many mucosal surfaces, including the digestive tract, are surrounded by epithelial cells. Mucosal epithelial cells, in addition to their roles such as absorption of nutrients and secretion of waste products, act as a barrier that separates the boundary between the external and internal environment and blocks material exchange. The epithelial cells of the small intestine and large intestine exposed to food derived from the outside protect our body by blocking the invasion of pathogenic substances such as bacteria and viruses. In addition, the abnormal mucosa-immune system activation process by pathogens is also fundamentally blocked.

For the barrier function of epithelial cells, tight junctions formed between epithelial cells are of paramount importance. Close junction is a type of cell-to-cell bonding method, and is strong enough to appear as if the cell membranes of two cells are fused on an electron microscope. The barrier function is a function of blocking the passage of foreign substances between epithelial cells, and is important in both the blood vessels and the digestive tract. As a constituent molecule that forms this close junction, a membrane protein called occludin is representative, and proteins such as ZO protein and cingulin are also involved in the cytoplasm. The close junction structure and function are completed through the interaction of these proteins.

The barrier function of epithelial cells is damaged by various causes, and if abnormal mucosal permeation is caused, serious diseases may occur in various organs of the human body. Barrier function is particularly important in the digestive tract. It is known that mucosal permeability is highly increased in the large intestine of mice exposed to stress for a long time. In fact, increasing intestinal mucosal permeability in the small and large intestine causes serious changes in overall bowel function, including diarrhea. Even in patients with Crohn's disease who had no symptoms, intestinal permeability may increase rapidly from 1 year before recurrence. In addition, it was confirmed that celiac disease was also partially lost in close contact between intestinal epithelial cells in the early onset. Abnormally increased mucosal permeability changes are also found in patients with irritable growth syndrome. It has been reported that the close junction of intestinal epithelial cells is disrupted by abnormal activation of T cells located in the intestinal mucosa. Considering these reports, abnormal changes in intestinal mucosal permeability can be said to be the most important factor in the occurrence of enteritis.

According to recent studies, it is known that epidermal growth factor (EGF) promotes the growth of epithelial cells as well as strengthens the barrier function of epithelial cells. Attempts have been made to treat enteritis using the activity of EGF, and its therapeutic excellence has been confirmed. In other words, the barrier function enhanced by the administration of EGF blocks the penetration of pathogenic substances in the lumen into the body, and through this, the inflammatory reaction can be prevented. However, there is a limitation in drug development because EGF is expensive and the possibility of tumor occurrence that can be caused by an activity that increases strong epithelial cell growth. Therefore, unlike EGF drugs, there is a need to develop a variety of therapeutic agents for intestinal diseases caused by abnormal changes in intestinal mucosa permeability by discovering candidate substances capable of maximizing barrier function while showing appropriate growth promoting activity for epithelial cells.

Accordingly, the present inventors have shown that the copricin peptide CopA3 isolated from insects regulates the proliferation and death of intestinal epithelial cells to maintain a constant number of epithelial cells, and has the activity of inhibiting the occurrence of intestinal inflammation by strengthening the barrier function of intestinal epithelial cells. By confirming the present invention was completed.

Therefore, the object of the present invention

It is to provide a pharmaceutical composition for preventing or treating chronic inflammatory bowel disease comprising copricin peptide CopA3 as an active ingredient.

Another object of the present invention

It is to provide a food composition for preventing or improving chronic inflammatory bowel disease comprising copricin peptide CopA3 as an active ingredient.

Another object of the present invention

It is to provide a food composition for improving intestinal function comprising the copricin peptide CopA3 as an active ingredient.

In order to achieve the above object, the present invention

It provides a pharmaceutical composition for preventing or treating chronic inflammatory bowel disease comprising copricin peptide CopA3 as an active ingredient.

In order to achieve another object of the present invention, the present invention

It provides a food composition for preventing or improving chronic inflammatory bowel disease comprising copricin peptide CopA3 as an active ingredient.

In order to achieve another object of the present invention, the present invention

It provides a food composition for improving intestinal function comprising coprisin peptide CopA3 as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention Coprisin Peptide CopA3 It provides a pharmaceutical composition for preventing or treating chronic inflammatory bowel disease comprising as an active ingredient.

In the present specification, the term "protein" is used interchangeably with "polypeptide" or "peptide", and, for example, refers to a polymer of amino acid residues as commonly found in proteins in a natural state.

One letter (three letters) of amino acids used herein means the following amino acids according to the standard abbreviation regulations in the field of biochemistry: A(Ala): alanine; C(Cys): cysteine; D(Asp): aspartic acid; E(Glu): glutamic acid; F(Phe): phenylalanine; G(Gly): glycine; H(His): histidine; I(IIe): isoleucine; K(Lys): lysine; L(Leu): leucine; M(Met): methionine; N(Asn): asparagine; O(Ply): pyrrolysine; P(Pro): proline; Q(Gin): glutamine; R(Arg): arginine; S(Ser): serine; T(Thr): threonine; U(Sec): selenocysteine; V(Val): valine; W(Trp): tryptophan; Y(Tyr): Tyrosine.

In the present invention, the "Cope lysine peptide CopA3 'is beetle Arabidopsis horns belong to the neck cow dung copper (Copris tripartitus) 43 amino acids (43mer) as consisting of defensin-like proteins of coprisin of α-helix peptide derived from the portion to be expressed in 9 It consists of four amino acids (9mer). Since the CopA3 peptide contains one cysteine (C) amino acid at the 3rd position of the amino acid sequence (LLCIALRKK; SEQ ID NO: 1), it tends to dimerize through a disulfide bond between the peptides. The CopA3 peptide for practicing the present invention is most preferably a dimer of 9mer CopA3 peptide, and is a D-peptide, a D-type (or D-form) optical isomer.

In one embodiment, the present inventors observed that the CopA3 peptide promotes the proliferation of HT29 cells, a human colon epithelial cell line. CopA3 peptide had the effect of promoting the proliferation of epithelial cells of the colon villi of mice, and was observed to increase apoptosis as well as cell proliferation.CopA3 controls the number of intestinal epithelial cells in the body to be constant while controlling the epithelial cells. Revealed that it has the effect of promoting the regeneration of

In addition, in another embodiment, it was confirmed that the CopA3 peptide has an activity of enhancing the close junction, which is important for the barrier function of intestinal epithelial cells. Intestinal mucosal permeability experiments of fluorescently labeled polymer (dextran) showed that the colonic mucosa permeability was significantly decreased in mice administered with CopA3.

Furthermore, the present inventors confirmed that the activity of CopA3 on intestinal epithelial cells is due to the fact that CopA3 promotes the degradation of p21, a cell proliferation-related signaling protein present in the cytoplasm. CopA3 peptide promotes ubiquitination of p21 by directly acting on the ubiquitin ligase of the ubiquitin/proteasome system responsible for proteolysis.

The activity of CopA3 on intestinal epithelial cells was also confirmed in a model of chronic inflammation induced by administering DSS to mice (DSS-induced mouse model of colitis). In the group treated with CopA3, the mortality rate and weight loss due to chronic inflammation induced by DSS significantly decreased, and the length of the large intestine (colon) observed in chronic enteritis induced by DSS, increased inflammatory cytokines, and mucosal damage. Symptoms also appeared to be greatly relieved overall. The model of inflammation used by the present inventors is a model representing the pathology of chronic inflammatory enteritis such as Crohn's disease or ulcerative colitis, and the present inventors confirmed in the animal model that CopA3 is also in the body through the activity of strengthening the barrier function of intestinal epithelial cells. It has been shown to have an excellent effect on chronic inflammatory enteritis and similar diseases.

Therefore, by using the activity of CopA3 on the intestinal epithelial cells identified by the present inventors, the function of the intestinal epithelial cells, in particular, to differentiate between the lumen environment of the digestive tract and the body environment, and to improve the barrier function to prevent indiscriminate transport or movement of substances or It can be seen that, by restoring the impaired barrier function, it is possible to prevent or treat chronic inflammatory diseases caused by weakened barrier function.

The chronic inflammatory bowel disease is a disease that occurs or intensifies due to a decrease in the barrier function of intestinal epithelial cells, and specifically, chronic enteritis, Crohn's disease, ulcerative colitis, Behcet's enteritis, irritable bowel syndrome, intestinal epithelial cell dysplasia, intestinal epithelial metaplasia. And it may be any one disease selected from the group consisting of secretory diarrhea.

Furthermore, as described above, the effect of the composition containing the CopA3 peptide according to the present invention as an active ingredient on chronic inflammatory bowel disease does not directly act on pathogens such as pathogenic bacteria or viruses introduced into the intestine. , It is due to the activity of enhancing the barrier function of the intestinal epithelial cells. Therefore, it is preferable that the chronic inflammatory bowel disease is not caused by bacterial infection, but is a non-bacterial chronic inflammatory bowel disease caused by toxic substances that cause epithelial damage such as stress or antibiotics.

In the present specification, the term'prevention' refers to any action that suppresses or delays the onset of a disease by administration of the composition of the present invention, and'treatment' refers to suppressing the occurrence or recurrence of a disease, alleviating symptoms, direct or indirect disease It refers to a decrease in pathological outcome, a decrease in the rate of disease progression, an improvement in a disease state, amelioration, amelioration or an improved prognosis.

The CopA3 peptide of the present invention may be derived from nature, and may be synthesized using a known method of synthesizing a polypeptide (genetic engineering method, chemical synthesis). Preparation of a peptide by a genetic engineering method can be carried out, for example, by preparing a nucleic acid encoding the polypeptide or a functional equivalent thereof according to a conventional method. The nucleic acid can be prepared by amplifying by PCR using an appropriate primer. Alternatively, a DNA sequence may be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer. The produced nucleic acid is operatively linked thereto to be inserted into a vector containing one or more expression control sequences (eg, promoters, enhancers, etc.) that control the expression of the nucleic acid to produce a recombinant expression vector. After transformation into a host cell, it is cultured under an appropriate medium and conditions to recover a substantially pure polypeptide expressed from the nucleic acid from the culture. The recovery can be performed using a method known in the art (eg, chromatography). In the above, "substally pure polypeptide" means that the polypeptide according to the present invention does not substantially contain any other proteins derived from host cells. The genetic engineering method for synthesizing the polypeptide of the present invention may be referred to the following literature: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., Second (1998) and Third (2000) Editions; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; And Hitzeman et al., J. Biol. Chem., 255:12073-12080, 1990.

In addition, the peptides according to the present invention can be prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press). , Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989). Peptides may also be synthesized using C-terminal amidation reaction or N-terminal acetylation reaction.

In addition, the peptide of the present invention is not only a polypeptide having a native amino acid sequence, but also an amino acid sequence variant thereof having the same activity against intestinal epithelial cells as the CopA3 peptide is included in the scope of the present invention. Variants of the polypeptide of the present invention are peptides having different sequences by deletion, insertion, non-conservative or conservative substitution, substitution of amino acid analogs, or combinations thereof in the amino acid sequence of the present invention. It means maintaining the effect of promoting proliferation and death and strengthening the close junction. Amino acid exchanges that do not totally alter the activity of a molecule are known in the art (H. Neuroth, R.L. Hill, The Proteins, Academic Press, New York, 1979).

In addition, the CopA3 peptide of the present invention is used for phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and An appropriate functional group may be added by acetylation or amidation.

In addition, the copricin peptide CopA3 included in the composition according to the present invention may be used by itself or in the form of a pharmaceutically acceptable salt. In the present invention,'pharmaceutically acceptable' means that it is physiologically acceptable, does not inhibit the action of the active ingredient when administered to humans, and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions. . As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is preferable, and an organic acid and an inorganic acid may be used as the free acid. The organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Includes glutamic acid and aspartic acid. In addition, the inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The pharmaceutical composition comprising the CopA3 peptide according to the present invention can be formulated in various ways according to the route of administration by a method known in the art together with a pharmaceutically acceptable carrier for the treatment of chronic inflammatory diseases. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

The pharmaceutical composition according to the present invention may be administered to a patient in an amount sufficient to promote proliferation and death of intestinal epithelial cells to facilitate regeneration, and to enhance the barrier function of intestinal epithelial cells by strengthening the close junction. For example, as a general daily dosage, it may be administered in the range of about 0.01 to 1000 mg/kg, and preferably, it may be administered in the range of about 1 to 100 mg/kg. The pharmaceutical composition of the present invention can be administered in one or several divided doses within a preferred dosage range. In addition, the dosage of the pharmaceutical composition according to the present invention may be appropriately selected by a person skilled in the art according to the route of administration, the subject of administration, age, sex, weight, individual difference and disease state.

The route of administration may be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration I can.

In the case of oral administration of the pharmaceutical composition of the present invention, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier for oral administration. It can be formulated in the form of such as. Examples of suitable carriers include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, gelatin, and polyvinylpyrrolidone may be included. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

In addition, when administered parenterally, the pharmaceutical composition of the present invention can be formulated according to a method known in the art in the form of injections, transdermal administrations and nasal inhalants together with a suitable parenteral carrier. In the case of such injections, they must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a mixture thereof and/or a solvent or dispersion medium containing vegetable oil. I can. More preferably, suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be additionally included. In addition, the injection may further include an isotonic agent such as sugar or sodium chloride in most cases.

In the case of transdermal administration, ointments, creams, lotions, gels, external solutions, pasta, liniment, air rolls, and the like are included. In the above, "transdermal administration" means that the active ingredient in an effective amount contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin. For example, the pharmaceutical composition of the present invention may be prepared in an injectable formulation and administered by lightly pricking the skin with a 30 gauge thin injection needle or applying it directly to the skin. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania, a prescription generally known for pharmaceutical chemistry.

In the case of inhalation administration, the compounds used according to the invention can be used in pressurized packs or with suitable propellants, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered from a nebulizer in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

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).

Pharmaceutical compositions according to the present invention include one or more buffers (e.g., saline or PBS), carbohythrate (e.g., glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g. For example, EDTA or glutathione), adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents and/or preservatives may further be included.

In addition, the pharmaceutical compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

In addition, the pharmaceutical composition of the present invention may be administered alone, or may be administered in combination with a known compound that has a therapeutic effect on chronic inflammatory bowel disease, or enhances the barrier function of intestinal epithelial cells.

In addition, the present invention Coprisin Peptide CopA3 It provides a food composition for preventing or improving chronic inflammatory bowel disease comprising as an active ingredient.

The present inventors provide a food composition for preventing or improving chronic inflammatory bowel disease by using the effect of enhancing the barrier function of the intestinal epithelial cells through the promotion of intestinal epithelial cell proliferation and death of the CopA3 peptide identified by the present inventors, and strengthening the epithelial cell adhesion junction.

The chronic inflammatory bowel disease is a disease that occurs or intensifies due to a decrease in the barrier function of intestinal epithelial cells, and specifically, any one selected from the group consisting of chronic enteritis, Crohn's disease, ulcerative colitis, Behcet's enteritis and irritable bowel syndrome. It could be a disease. The CopA3 peptide according to the present invention helps the regeneration of intestinal epithelial cells and strengthens the barrier function, so while maintaining a healthier population of intestinal epithelial cells, it prevents unnecessary substances and pathogens from entering the intestine, thereby preventing damage and inflammation of intestinal cells. It can be expected to reduce the effect of preventing the above-described chronic inflammatory bowel disease or improving symptoms.

The composition for food according to the present invention includes all forms such as functional food, nutritional supplement, health food, and food additive. These types can be prepared in various forms according to conventional methods known in the art. In order to use the food composition of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.

For example, as a health food, the food composition of the present invention itself may be prepared in the form of tea, juice and drinks to be consumed, or granulated, encapsulated, and powdered to be consumed. In addition, the food composition of the present invention can be prepared in the form of a composition by mixing with known substances or active ingredients known to be effective in suppressing the production or accumulation of fat in the body.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods thereof (eg, canned fruit, bottled, jam, marmalade, etc.), fish, meat and processed foods thereof (eg, ham, sausage corn beef, etc.). ), breads and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, Retort foods, frozen foods, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the food composition of the present invention.

The preferred content of the food composition according to the present invention is not limited thereto, but is preferably 0.01 to 50% by weight of the finally prepared food.

In addition, the present invention Coprisin Peptide CopA3 It provides a food composition for improving intestinal function comprising as an active ingredient.

As identified by the present inventors, the CopA3 peptide promotes the proliferation and death of intestinal epithelial cells, stabilizes the population of epithelial cells, helps regeneration, and enhances the barrier function of the intestinal epithelial cells by enhancing the adhesion of epithelial cells. This activity of CopA3 effectively blocks pathogens and toxic substances introduced from the outside while maintaining healthy intestinal epithelial cell population, and promotes the transport of selective substances such as nutrients that must be absorbed in the body, resulting in a safe maintenance of the intestinal cellular environment. It can be seen that the bowel function can be performed smoothly.

More specifically, the intestinal function may be maintenance of a numerical balance of intestinal epithelial cells or a barrier function of epithelial cells.

Preparation and examples of the food composition for improving intestinal function according to the present invention are as described in the food composition for preventing or improving chronic inflammatory bowel disease of the present specification.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating chronic inflammatory bowel disease, comprising a copricin peptide CopA3 as an active ingredient, a food composition for preventing or improving chronic inflammatory bowel disease, and a food composition for improving intestinal function. CopA3 peptide accelerates the decomposition of p21 protein to promote proliferation and apoptosis of intestinal epithelial cells so that the epithelial cell regeneration cycle proceeds smoothly, and has the effect of enhancing barrier function by reinforcing close junctions between intestinal epithelial cells.

1 shows the results of a cell proliferation rate measurement experiment using BrdU to confirm the growth promoting activity of human colon epithelial cells of CopA3 peptide.
1A is a cell proliferation rate measured after treating HT29 cells with various concentrations of CopA3 for 48 hours. * Means p <0.01. 1B is a cell proliferation rate measured after treating HT29 cells with culture medium ('Con'), EGF (100ng/ml), and CopA3 (20 μg/ml) for 48 or 72 hours, respectively. * Means p <0.005, # means p <0.001. All data are the results of three independent experiments expressed as mean ± standard error (mean ± SE).

2 shows experimental results confirming the effect of CopA3 peptide on proliferation and apoptosis of intestinal epithelial cells in the body.
The top panel ('Ki-67 staining') shows male C57BL/6 mice with CopA3 (2 μg/ml) added ('CopA3') or no ('con') drinking water for 5 days and separated colon ( colon), stained with Ki-67 antibody (blue, DAPI; green, Ki-67). Middle panel ('TUNEL') are TUNEL-stained dead cells observed in the tissue (arrow, TUNEL; blue, nucleus). The lower panel ('E-cadherin') is colon tissue stained with E-cadherin antibody, an epithelial cell marker (red).

Figure 3 shows the mucosal macromolecular permeability measured in the intestine of a control group ('con'), a mouse (n=8/group) administered with CopA3 (2 μg/ml) or P4 (2 μg/ml) for 5 days. Show the results. Data are expressed as the mean ± standard error (mean ± SE) of the results of three independent experiments, and * means p <0.005.

4 shows the results showing changes in p21 protein level and cell cycle distribution by treatment with CopA3 peptide in HT29 cells cultured in a plasma-free medium.
4A is a Western blot result confirming the levels of p21, p27, and β-actin in HT29 cell lysates cultured for 0, 24, 48, and 72 hours in plasma-free medium (15% SDS). 4B shows p21 in HT29 cell lysates cultured for 0, 24, 48, 72 hours in a plasma-free medium containing ('+') or not ('-') CopA3 (20 μg/ml), This is a Western blot result confirming the level of p27 and β-actin. 4C is a plasma-containing medium ('+serum'), a plasma-free medium ('-serum'), or a plasma-free medium ('-serum + CopA3') containing CopA3 (20 μg/ml) for 48 hours. This is the result of PI staining and flow cytometry (FACS) to confirm the cell cycle distribution of HT29 cells cultured during the period. 'S' indicates the S (synthesis) period of cell division, and the number under the'S' indicates the percentage of cells in the S stage. 4D is an adenovirus overexpressing p21 (bottom panel,'Adeno-p21 virus') or an adenovirus overexpressing control GFP (top panel,'Adeno-GFP virus') after infection for 24 hours, culture This is a result of analyzing the cell cycle distribution of HT29 cells cultured for 48 hours in medium, plasma-free medium, or plasma-free medium containing CopA3.

FIG. 5 shows the results of experiments of co-immunoprecipitation and ubiquitination reactions of p21 to confirm the change in the ubiquitination activity of ubiquitin ligase against p21 protein by CopA3. Immunoprecipitation and Western blot were performed using HT29 cell line lysate and anti-p21 antibody.

6 shows the results of HPLC analysis to confirm the intracellular location of the CopA3 peptide. mAU is an abbreviation of milli absorption units and represents the unit of detection of substances using HPLC. CopA3 peptide is present in the cytosol fraction, and the structure of the peptide is the same without any change after cell influx.

7 shows experimental results confirming the therapeutic efficacy of CopA3 in a DSS-induced enteritis model in mice. Drinking water with or without 2% DSS and CopA3 (2g/ml) or P4 (2g/ml) was supplied to C57BL/6 mice (n=30/group) for 16 days, and nothing was included in the control. Drinking water that was not used was supplied.
7A shows the weight change measured daily as an average amount of change (% Weight change) from the initial body weight. Data are expressed as mean±standard error (mean±SE), and the symbols in the drawings are as follows; Closed square, control; Open circle, DSS and CopA3 treated group; Group treated with open triangle, DSS and P4; Closed lozenge, treated with DSS only. 7B shows the survival rate. 7C shows the test results of measuring the length of the colon of the mouse measured after the end of the experiment. Data are expressed as the mean±standard error (mean±SE) of three independent experiments, and * indicates p <0.001. 7D and E show the concentrations of TNF-α and IL-6 measured in mouse colon tissue, respectively. * Denotes p <0.005. 7F is an optical micrograph of a mouse colon stained with hematoxylin & eosin (magnification 200×). G in FIG. 7 shows histological scores indicating the degree of epithelial cell damage and neutrophil infiltration, and * indicates that p <0.005 and # is p <0.001 compared to the group treated with only DSS. H in FIG. 8 shows mucosal macromolecular permeability measured by fluorescence intensity (n=8/group). * Denotes p <0.005.

Hereinafter, the present invention will be described in detail.

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

< Example 1>

CopA3 Captain of Epithelial cell line Proliferation promoting activity

Recently, the present inventors have reported that the CopA3 peptide isolated from Arabidopsis dung beetles can promote the growth of nerve cells (Republic of Korea Patent Registration 10-1336023). Based on this, we investigated whether CopA3 affects the growth of human colon epithelial cells.

To this end, HT29 cells, a human colon epithelial cell line, were treated with CopA3 at different concentrations (1, 5, 10, 20, 50 μg/ml) for 48 hours, and then BrdU cell proliferation assay was performed to measure the change in cell growth rate.

As a result, it was confirmed that the growth rate of colon epithelial cells increased by about 20% or more at a CopA3 concentration of 20 μg/ml or more ( FIG . 1A ). Next, the effect of CopA3 on promoting colon epithelial cell growth was compared with EGF. As shown in B of FIG. 1 , it was confirmed that the degree of promotion of growth of human colon epithelial cells by CopA3 (20 μg/ml) was similar to the EGF effect of 100 ng/ml concentration.

< Example 2>

CopA3 Activity to promote colonic epithelial cell proliferation and apoptosis

In order to confirm whether CopA3 exhibits the activity of promoting colonic epithelial cell proliferation even in vivo, mice were used as an animal model in the body ( in vivo ) It was confirmed the change in the growth rate of epithelial cells in the colon.

For 5 days, colon tissue was removed from mice supplied through drinking water with or without CopA3 (2 μg/ml) peptide, and the tissue was stained with Ki-67 antibody to measure changes in cell proliferation. Ki-67 is a nuclear protein essential for cell growth and is known as a protein involved in ribosomal RNA transcription. In general, it is known that cells that actively divide and grow rapidly in small and large intestine tissues (eg, stem cells) exist only in the crypt site of the villi.

It was observed that the epithelial cells stained with Ki-67 significantly increased in the crypts in the colon of mice supplied with drinking water containing CopA3 peptide compared to the control group (con) that consumed only drinking water not containing CopA3 ( Fig. 2, upper part, Ki-67 staining panel). This indicates that the D-type (enantiomer) CopA3 peptide synthesized so as not to be biodegraded in the gastrointestinal tract for this experiment reaches the large intestine while maintaining the activity of increasing the growth of epithelial cells without being degraded in the intestine, thereby exhibiting the effect of promoting epithelial cell growth. To show.

Further, as a result of confirming the intestinal apoptosis pattern by TUNEL staining, it was found that apical region of the mouse villi treated with CopA3 increased apoptosis compared to the control group (middle of FIG. 2 , TUNEL panel). This shows that the cell growth and apoptosis process are balanced and the cell number is properly maintained. If the cell growth rate is greater than the apoptosis rate, the possibility of tumorigenicity may be a problem, but since CopA3 promotes not only cell growth but also apoptosis, it can be seen that the possibility of tumor formation by CopA3 is very low. Cells in the apical region of the villous during apoptosis were cells expressing E-cadherin, and were reconfirmed that they were epithelial cells, not leukocytes or cells constituting general connective tissue (bottom of FIG. 2 , E-cadherin panel).

< Example 3>

CopA3 Strengthening activity of intestinal epithelial cells in the body

In addition to promoting the growth of human colon epithelial cells, the effect of CopA3 peptide on the close junction important to epithelial cell function was confirmed.

To this end, mice fed with CopA3 (2 μg/ml) for 5 days were anesthetized by intraperitoneal injection of Avertin (250 mg/kg), and both kidneys were tied with silk so that the fluorescent probe did not escape into the urine. The ileum was tied at both ends of the ileum for about 3 cm to form a pocket, and then 0.3 ml of physiological saline containing a fluorescent dextran (25 mg/ml) having a molecular weight of 4000 kDa was injected with an insulin syringe. During the operation, the mice stably maintained anesthesia in a warm state to maintain body temperature, and the abdomen was covered with aluminum foil to block light. After 3 hours, 0.5 ml of blood was collected from the heart of the mouse and centrifuged at 5000 rpm for 10 minutes to separate plasma. After mixing plasma and PBS at a volume ratio of 1:2, fluorescence intensity was measured with a GloMax 20/20 (Promega, USA) fluorescence analyzer.

As can be seen in Figure 3 , unlike the control (con) supplied with only water not containing CopA3, the fluorescence intensity in the ileum of the mouse (CopA3) supplied with drinking water containing CopA3 was significantly reduced, resulting in a markedly reduced mucosal transmittance. Appeared to decrease. As another negative control, the P4 peptide (consisting of 9 amino acids, NH ₂ -RLLLAIGRG-COOH; SEQ ID NO: 2) isolated from the white spotted flower radish ( Protaetia brevitarsis ), which is known to have no growth promoting effect on colon epithelial cells, was used. Using the same experiment was carried out. In the case of the P4 peptide (2 μg/ml), it was confirmed that it had no effect on the mucosal permeability of the large intestine of mice, even though it was consumed as drinking water for 5 days. This shows that CopA3 peptide has the effect of enhancing the barrier function of epithelial cells by strengthening the close junction between intestinal mucosal epithelial cells.

This result also shows that it can be developed as a food composition for improving intestinal function containing CopA3 as an active ingredient. In other words, when CopA3 is treated, the intestinal epithelial cell population is kept healthy and can effectively block pathogens and toxic substances introduced from the outside, and also maintains the intestinal environment safely by promoting the selective transport of nutrients, such as nutrients, that must be absorbed in the body. It can be seen that it not only helps the intestines function smoothly.

< Example 4>

CopA3 On by p21 Decreased protein levels

Through the results of the previous examples, it was confirmed that the CopA3 peptide promotes the growth and apoptosis of human colon epithelial cells, thereby stably maintaining the epithelial cell population and strengthening the close junction of epithelial cells. The mechanism of molecular signaling in human colon epithelial cells regulated by the related CopA3 peptide was investigated.

To this end, human colon epithelial cells were cultured in a plasma-removed medium (plasma-free medium) to inhibit cell growth, and it was confirmed whether or not cell growth was restored again after treatment with CopA3 peptide. In addition, a signal molecule whose level changes in cells after treatment with CopA3 peptide was identified.

In human colon epithelial cells cultured in plasma-free medium, the level of p21, which is well known as a protein that inhibits cell growth, was significantly increased ( FIG. 4A ). In contrast, in the cells treated with CopA3, the p21 protein level was significantly reduced compared to the control (-) ( FIG . 4B ). The decrease in p21 was more evident as the CopA3 treatment time increased.

The colonic epithelial cells cultured in plasma-free medium decreased the number of cells in the DNA replication process during cell division compared to cells cultured in normal medium, but it was observed to be significantly recovered by treatment with CopA3 ( FIG. 4C ). .

When the p21 protein was artificially overexpressed in human colon epithelial cells by using the P21 adenovirus expression system, the p21 protein level did not decrease even if CopA3 was treated, and the cell growth promoting effect was not exhibited ( FIG . 4D ). The above results suggest that the process of promoting human colon epithelial cell growth by CopA3 peptide is the result of the reduction of p21 protein. Considering that p21 is a potent cell growth inhibitory factor, it can be understood that the mechanism of promoting human colon epithelial cell growth by CopA3 is through p21 inhibition.

< Example 5>

CopA3 of p21 Ubiquitination Promoting activity

We tried to find out the mechanism by which the CopA3 peptide inhibits the p21 protein.

To this end, p21 protein was isolated from the HT29 cell line by immunoprecipitation. The isolated p21 was provided as a substrate to the ubiquitin ligase enzyme involved in the process of proteolysis, and it was confirmed that ubiquitination occurred.

As can be seen in Figure 5 , it was observed that the p21 protein was ubiquitinated by ubiquitin ligase in the presence of ATP. On the other hand, when the CopA3 peptide was added to the ubiquitination reaction, the ubiquitination reaction of p21 was more remarkably increased. This means that CopA3 directly affected the activity of the ubiquitin ligase enzyme. As a result of conducting the same experiment using P4 peptide isolated from white spotted radish as a negative control, P4 peptide did not affect the ubiquitinization activity of ubiquitin ligase against p21 at all. The above results show that CopA3 decreases the p21 protein level through the ubiquitin/proteasome system, and in particular, CopA3 peptide directly increases the activity of the ubiquitin ligase enzyme, thereby maximizing the ubiquitination reaction to p21.

< Example 6>

CopA3 The intracellular location of

It has been reported that the CopA3 peptide is highly reactive to various cells, but the exact intracellular action site of the CopA3 peptide has never been identified. To clarify this, attempts such as fluorescent labeling or biotin conjugation to CopA3 peptide were attempted, but there was a problem in that the peptide was crystallized. Accordingly, the present inventors treated the colon epithelial cells with CopA3, and then obtained a cell fraction and analyzed the fractional extract by HPLC to confirm the intracellular location of CopA3.

To this end, HT29 cells were treated with CopA3 peptide (20 μg/ml) for 2 hours, then the cells were disrupted and centrifuged to obtain a cell extract. In order to measure the amount of CopA3 remaining in the culture medium without entering the cells, the culture medium was also recovered. Even in the control experimental conditions, both the culture medium and the cell extract were separated and analyzed by HPLC ( FIG. 6 ).

There were no peaks showing the detection of CopA3 peptide in both the control culture medium (standard buffer) and the control epithelial cell extract not treated with CopA3. In the cells treated with CopA3, a peak of CopA3 was observed around the retention time of 13.9min. Through quantitative analysis, CopA3 was detected in the cytosol as much as the amount of CopA3 decreased in the media, and it was found to exist in a dimerized form (~14.7 μg/ml). CopA3 was not detected at all in the nucleus and membrane fractions. This suggests the possibility that the CopA3 peptide rapidly enters and acts into human colon epithelial cells by binding to a receptor present in the cell membrane. Considering that the proteasome and ubiquitin ligase enzymes that degrade proteins are also present in the cytoplasm, the fact that CopA3 was identified in the cytoplasmic fraction corresponds to the function of promoting p21 protein degradation by CopA3 acting directly on the enzyme in the cytoplasm.

< Example 7>

In an animal model of chronic enteritis CopA3 The efficacy of treatment

It was intended to verify the efficacy of CopA3 in an animal model of chronic enteritis. A model of chronic animal enteritis was established by injecting dextran sulfate sodium (DSS). This model is the standard method commonly used by academics to study Crohn's disease and ulcerative colitis in humans.

Six-week-old male C57BL/6 mice were fed for 7 days through drinking water containing CopA3 (2 μg/ml). After that, drinking water containing 2% DSS was supplied for more than 10 days to induce chronic enteritis. As a negative control, the P4 peptide isolated from the white-spotted radish insect was applied in the same manner.

DSS treatment reduced the weight of the mice by more than 30% ( FIG. 7A ), and after 15 days, the entire mice died ( FIG . 7B ). However, in the group of mice treated with CopA3, the weight loss was weak ( FIG. 7A ), and the whole mice survived ( FIG. 7B , 100% survival). The decrease in colon length in the DSS-induced chronic enteritis animal model was also recovered to a normal level by treatment with CopA3 ( C in FIG. 7 , control: 7.8 cm; DSS: 5 cm; DSS+CopA3: 6.7 cm). And inflammatory cytokines that increase in chronic enteritis intestine ( Fig. 7 D, E , It was confirmed that TNF-α and IL-6) and mucosal damage ( FIG. 7F ) were significantly inhibited by CopA3 treatment. In histopathological evaluation, it was confirmed that CopA3 treatment recovered all of the overall colonic enteritis ( FIG . 7G ). Moreover, it was also confirmed that the mucosal permeability, which is strongly increased in the DSS-induced chronic enteritis model, is recovered by treatment with CopA3 ( FIG . 7H ). However, in the case of the P4 peptide used as a negative control, it was confirmed that there is no effect on the change in the large intestine ( FIG. 7 ). These results show that CopA3 has an excellent therapeutic effect on chronic inflammatory enteritis in humans such as Crohn's disease and ulcerative colitis.

As described above, the composition of the present invention promotes the proliferation and death of intestinal epithelial cells to promote the regeneration cycle of epithelial cells, and enhances the barrier function by strengthening the close junction between the intestinal epithelial cells. It can be usefully used to develop pharmaceuticals, functional foods, and foods for improving intestinal function for preventing or treating various chronic inflammatory bowel diseases.

<110> Daejin University Center for Educational Industrial Cooperation <120> Composition for treating chronic inflammatory bowel diseases or for improving bowel function <130> NP16-0065 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CopA3 peptide <400> 1 Leu Leu Cys Ile Ala Leu Arg Lys Lys 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> P4 peptide <400> 2 Arg Leu Leu Leu Ala Ile Gly Arg Gly 1 5

Claims (7)

A pharmaceutical composition for preventing or treating chronic inflammatory bowel disease, comprising copricin peptide CopA3 as an active ingredient.
The composition of claim 1, wherein the coprisin peptide CopA3 consists of an amino acid sequence represented by SEQ ID NO: 1.
According to claim 1, The chronic inflammatory bowel disease is any one disease selected from the group consisting of chronic enteritis, Crohn's disease, ulcerative colitis, Behcet's enteritis, irritable bowel syndrome, intestinal epithelial cell dysplasia, intestinal epithelial metaplasia, and secretory diarrhea. Composition, characterized in that.
The composition of claim 1, wherein the chronic inflammatory bowel disease is non-bacterial.
Food composition for preventing or improving chronic inflammatory bowel disease comprising copricin peptide CopA3 as an active ingredient.
Food composition for improving intestinal function comprising copricin peptide CopA3 as an active ingredient.
The composition according to claim 6, wherein the intestinal function is maintaining a numerical balance of epithelial cells or a barrier function of epithelial cells.

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