KR20200079382A - A composition for preventing or treating colitis comprising decursinol angelate - Google Patents

Abstract

The present invention relates to a composition for preventing or treating colitis, or for inhibiting a Type 17 helper T (Th17) cellular response, including decursinol angelate as an active ingredient, and to a method thereof. According to the present invention, it is shown that decursinol angelate is effective for inhibiting a Type 17 helper T cellular response. In addition, when using a DSS-induced colitis model, treatment with DA protects the large intestine from damages induced by DSS administration, alleviates serious conditions of the large intestine, including cut-down of the large intestine and weight loss, and significantly reduces Th17 cells accompanying with neutrophils in the large intestine tissue of a mouse suffering from colitis. Such results suggest that DA is a potential treatment agent for treating inflammation diseases, such as IBD, related with a Th17 cellular response.

Description

A composition for preventing or treating colitis comprising decursinol angelate

The present invention relates to a composition for preventing or treating colitis comprising decursinol angelate as an active ingredient.

Colitis is an inflammation of the large intestine, and includes inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and the like.

Inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD), which are typical diseases, have not been clearly identified, and severe chronic diarrhea and hemorrhage with abdominal pain It can cause diarrhea, hard to cure, and has the characteristic of repeating improvement and deterioration. Ulcerative colitis is a disease in which mucus (mirror) or ulcers are continuously formed on the mucous membrane of the large intestine. Bloody stools, mucus, diarrhea, and abdominal pain occur. In severe cases, systemic symptoms such as fever, weight loss, and anemia appear. .

In addition, ulcerative colitis can occur anywhere in the gastrointestinal tract. Crohn's disease is a disorder in which lesions such as ulcers occur non-continuously in any part of the digestive tract from the mouth to the anus. In addition to abdominal pain, diarrhea, and bloody stools, in severe cases, fever, lower blood pressure, weight loss, general malaise, anemia Back symptoms appear. Although ulcerative colitis and Crohn's disease are different in terms of lesions and inflammatory symptoms, they are similar in many ways, so the distinction between the two diseases is often unclear.

Conventionally, the incidence of ulcerative colitis and Crohn's disease has been known to be high in Westerners, but recently, the number of patients has rapidly increased in the East, including Korea, due to changes in lifestyle, such as eating habits. However, there is a reason why the cause is unclear, and a fundamental treatment is not established. For this reason, rather than aiming at complete treatment, there is a situation in which drugs that relieve symptoms and maintain these conditions for as long as possible are used.

As an agent for such symptomatic therapy, aminosalicylic acid, adrenal corticosteroids, immunosuppressants, etc. are mainly used, but various side effects have been reported. For example, salazosulfapyridine, which is frequently used as an amino salicylate, has been reported to cause side effects such as nausea, vomiting, anorexia, rash, headache, liver failure, leukocyte reduction, abnormal red blood cells, proteinuria, and diarrhea. In addition, the adrenal cortical steroid is generally used for oral administration of prednisolone, enema, suppository, and intravenous injection, but has strong side effects such as gastric ulcer or necrosis of the femoral head due to long-term use. However, because discontinuation of dosing causes symptoms to recur, these agents are compelled to continue to be used.

Accordingly, there is a need to develop a therapeutic agent for intestinal diseases such as ulcerative colitis and Crohn's disease, which is excellent in effect, yet safe and does not cause side effects.

Irritable bowel syndrome (IBS) is also a chronic abdominal disease whose cause is unclear, and currently, there are no underlying treatments for IBS, and symptomatic therapy aimed at alleviating the symptoms of each type. For example, anti-cholinergics that have antispasmodic effects that inhibit contraction of smooth muscles are used for lower IBS, constipation type IBS is difficult to control with a drug, and alternative type IBS is difficult to control with a drug, and basically, an agent for improving digestive tract movement function is used. have.

In order to treat such colitis, research has been actively conducted to develop a composition capable of treating colitis from natural products with few side effects. For example, Korean Registered Patent No. 1466308 discloses a composition for treating colitis comprising an extract of jimo as an active ingredient, and Korean Registered Patent 1526467 discloses a composition for treating colitis comprising an extract of cornus officinalis as an active ingredient. .

The present invention was devised by the necessity of the above, and an object of the present invention is to provide a novel composition for preventing or treating colitis.

Another object of the present invention is to provide a novel composition for inhibiting type 17 helper T (Th17) cell response and a method therefor.

In order to achieve the above object, the present invention provides a composition for preventing or treating colitis comprising decursinol angelate as an active ingredient.

In one embodiment of the present invention, the colitis includes inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis (UC), and Crohn's disease; CD) is preferably selected from the group consisting of, but is not limited thereto.

In addition, the present invention provides a composition for inhibiting a type 17 helper T (Th17) cell response comprising decursinol angelate as an active ingredient.

In addition, the present invention provides a method of inhibiting a type 17 helper T (Th17) cell response in a subject by treating decursinol angelate to the subject.

In one embodiment of the present invention, the object is preferably an animal excluding human, but is not limited thereto.

In addition, the present invention provides a composition for inhibiting the secretion of pro-inflammatory cytokines containing decursinol angelate as an active ingredient.

In one embodiment of the present invention, the pro-inflammatory cytokine is preferably TNF-alpha or IFN-gamma, but is not limited thereto.

In addition, the present invention provides a method of inhibiting the secretion of pro-inflammatory cytokines in a subject by treating decursinol angelate to the subject.

In addition, the present invention provides a composition for inhibiting IL-17 expression in CD4 T cells comprising decursinol angelate as an active ingredient.

In addition, the present invention provides a method of inhibiting IL-17 expression in a cell by treating decursinol angelate on CD4 T cells in vitro.

In the present invention, the colitis refers to a condition in which the colon is inflamed due to bacterial infection or pathological fermentation of the intestinal contents, and is a concept including infectious colitis and non-infectious colitis. Specific types of colitis that can be prevented or treated through the pharmaceutical composition according to the present invention include inflammatory bowel disease or irritable colitis syndrome, but are not limited thereto.

The inflammatory bowel disease includes, for example, ulcerative colitis, or Crohn's disease. In addition, colitis that can be prevented or treated through the pharmaceutical composition according to the present invention includes both acute colitis and chronic colitis. The acute colitis refers to inflammation of the colon or colon that comes acutely, and the mucous membrane is damaged due to the inflammation, and the symptoms of mucous diarrhea or acupuncture are mainly seen. Acute colitis in the present invention includes acute gastroenteritis and acute ulcerative colitis, as well as general acute infectious colitis.

When the composition is provided in the form of a pharmaceutical composition, it may further contain one or more pharmaceutically acceptable carriers, excipients or diluents in addition to the compounds of the present invention. "Pharmaceutically acceptable" as used herein is a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not normally cause an allergic or similar reaction such as gastrointestinal disorders, dizziness, or the like. Says

Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration.

Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration to the peptide preparation. In addition, the carrier for parenteral administration may include water, a suitable oil, saline, aqueous glucose and glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and formulations can be referenced as described in the following documents (Remington's Pharmaceutical Sciences, 19th ed, Mack Publishing Company, Easton, PA, 1995).

The composition of the present invention can be administered in any way to mammals, including humans. For example, it can be administered orally or parenterally. The parenteral administration method is not limited to this, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration. Can be

The pharmaceutical composition of the present invention may be formulated as a formulation for oral administration or parenteral administration according to the administration route as described above.

In the case of preparations for oral administration, the compositions of the present invention may be formulated using methods known in the art as powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Can. For example, oral preparations can obtain tablets or dragees by combining the active ingredient with a solid excipient, then grinding it and adding a suitable adjuvant to the granule mixture. Examples of suitable excipients are sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including cellulose starch, wheat starch, rice starch and potato starch, cellulose, etc. Fillers such as cellulose, gelatin, polyvinylpyrrolidone and the like may be included, including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose. In addition, if necessary, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant.

Furthermore, the pharmaceutical composition of the present invention may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

For formulations for parenteral administration, injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants can be formulated by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed, Mack Publishing Company, Easton, PA, 1995, a prescription generally known to all pharmaceutical chemistries.

The total effective amount of the composition of the present invention can be administered to a patient in a single dose, and can be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. The pharmaceutical composition of the present invention can vary the content of the active ingredient according to the degree of disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.001 μg to 10,000 mg per kg of patient body weight per day, most preferably 0.1 μg to 1,000 mg per kg of body weight per patient. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the formulation method, the route of administration and the number of treatments, as well as the patient's age, weight, health status, sex, disease severity, diet and excretion rate, etc. Considering this, one of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention.

The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration and method of administration as long as it shows the effects of the present invention.

On the other hand, the term "prevention" used in the present invention means any action to suppress or delay the disease by administration of a composition comprising the mixture of the present invention. In addition, the term "treatment" as used in the present invention, by administration of a composition comprising a mixture of the present invention means any action that the symptoms of the disease is improved or cured.

Hereinafter, the present invention will be described.

The present invention identifies decursinol angelate (hereinafter referred to as'DA') as an immunomodulatory component of Angelica giga s and confirms the inhibitory effect on its type 17 helper T (Th17) cell response Did. Ex vivo helper T cell culture experiments showed that DA did not affect the survival and proliferation of CD4 T cells and interfered with IL-17 production and differentiation of Th17 cells. Using the DSS-induced colitis model, we determined the ability of DA to treat inflammatory bowel disease (IBD). DA treatment attenuated the severity of the large intestine, including protection from bowel tissue damage induced by DSS administration and shortening of the large intestine and reducing weight loss.

Th17 cells accompanying neutrophils to the colon tissues of colitis mice were significantly reduced by DA treatment. Overall, the results of the present invention suggest that DA may be a potential therapeutic agent to treat inflammatory diseases associated with Th17 cell responses such as IBD.

Hereinafter, the present invention will be described in detail.

As an anti-inflammatory component of Angelica Decursinol angelate cd4 T cell Infectious Inhibits cytokine production

The present inventors tested the effect of Angelica chemical components on ex vivo T cell activation using class II-MHC restricted and ovalbumin specific OT-II T cells.

Treatment with several compounds strongly attenuated the secretion of pro-inflammatory cytokines such as TNF-alpha and IFN-gamma from antigen-stimulated OT-II T cells measured by cytokine bead array (CBA) (Figure 1A & B).

Among the tested compounds, DA (decursinol angelate) was able to significantly inhibit IL-17 production of CD4 T cells (FIG. 1C ). IL-17 is produced primarily by Th17 cells and is associated with inflammatory and autoimmune diseases, including inflammatory bowel disease. Therefore, we further determined the effect of DA on Th17-related inflammatory diseases such as IBD and Th17 immune response.

Decursinol angelate IL-17 expression of CD4 T cells and Th17 Inhibits cell differentiation

To assess the immunomodulatory activity of the DA against the Th subset, we cultivated CD4 T cells in the presence of DA under Th1, Th2, and Th17 cell polarizing conditions. Flow cytometry analysis showed that DA treatment caused a dose-dependent decrease in intracellular expression of IL-17 in Th17-polarized cells (FIG. 2A ).

DA also inhibited expression in Th1 cells of IFN-gamma, but did not affect the expression of IL-4 in Th2 cells (FIGS. 7A & 7B ).

CBA results using the culture supernatant confirmed that DA inhibits IL-17 secretion significantly from activated Th17 cells (FIG. 2B ).

We also observed that IL-17A mRNA expression was reduced in DA-treated Th17 cells, suggesting that DA inhibits the induction of IL-17 at the transcription level (Figure 2C).

Although DA attenuated the induction of IFN-gamma and TNF-alpha in antigen-stimulated OT-II T cells and Th1 cells (Figures 1 and 2), DA did not affect the production of these cytokines from Th17 culture. Did (Figure 2B).

Rorc mRNA level (coding RORgt) Although significantly weakened in Th17 cells by DA treatment, the level of Tbx21 (encoding T-bet) was not affected (FIG. 2D ).

These results suggest that DA negatively regulates the programming of Th17 cells, leading to impaired production of IL-17.

When the inventors measured the anti-inflammatory cytokine IL-10 secreted from Th17 culture, the inventors did not observe the increased production of IL-10 by DA treatment (Fig. 2B). Even under Treg polarizing conditions, DA had no effect on the induction of FoxP3 ⁺ Treg cells (FIG. 7C ).

These results suggest that DA's anti-inflammatory activity against Th17 cells is not due to secondary effects on Treg cells' immune regulation.

For the survival and proliferation of CD4 T cells decursinol angelate effect

DA This anti-inflammatory effect seems to be due to attenuating cytotoxicity and/or the expansion of activated CD4 T cells. However, DA did not affect the survival rate of CD4 T cells to 40 mM concentration (FIG. 3A).

The results of Annexin-V staining indicated that DA did not induce Epoptosis of T cells (FIG. 3B ).

Using the EdU uptake assay, we measured the effect of DA on cell cycling activated CD4 T cells in Th17 culture. 3C, the rate of EdU ⁺ cells did not decrease by DA treatment, suggesting that DA negatively regulates cytokine production and differentiation of Th17 cells independent of CD4 T cell survival and proliferation.

Decursinol angelate dextran sulphate sodium-induced colitis Improve

To explore whether DA has therapeutic potential for treating IBD, we used a murine ulcerative colitis (UC) model.

Mice given DSS in drinking water for 7 days showed disease symptoms including diarrhea and UC symptoms related to weight loss (FIGS. 4A-C ).

When mice were treated with DA every 2 days, the weight loss and colitis symptoms triggered by the administration of DSS were markedly improved (FIGS. 4A-C).

We also observed that the length of the large intestine and the size of the appendix were longer and larger in DA-treated mice than in vehicle-treated mice (FIG. 4D ).

DA treatment significantly inhibited colitis-induced upregulation of plasma IL-6, an indicator of systemic inflammation (FIG. 4E ).

Histological evaluation of colon sections showed that DSS administration caused histopathology of colitis including destruction of epithelial structures, cellular filtration, damaged crypt structures and muscle thickening (FIG. 4F ).

In contrast, DA treatment protected mice from DSS-induced histopathological changes and immune cell penetration in the colon (Figure 4F). These results provide evidence that DA may improve the symptoms and severity of DSS-induced colitis in mice.

Decursinol angelate In the large intestine Neutrophil Recruitment and Th17 Inhibits DSS-induced activation of cells

To evaluate the inhibitory effect of DA on the Th17 response in vivo , the present inventors took the epithelial and lymph nodes of the large intestine from DSS-administered mice to isolate and characterize immune cells.

IL-17-producing Th17 cells increased significantly in the mucosal stromal layer (lamina propria), mesenteric lymph nodes and colon transepithelium by induction of colitis, but IFN-gamma-producing Th1 cells were upregulated only in the mucosal genital layer ( Figure 5A). Interestingly, DA treatment significantly reduced the induction of Th17 cells in colitis mice (Figure 5A).

In contrast, DA did not inhibit DSS-triggered Th1 responding cells in lymph nodes and colon tissues. Consistent with in vitro data (FIG. 2 ), these results confirm the immunomodulatory potential for the function and activation of the Th17 response of DA.

The inventors observed a large colitis induced increase of CD11b ⁺ Gr-1 ⁺ cells in colon tissues (FIG. 5B ).

Consistent with reduced induction of Th17 cells, DA treatment significantly attenuated recruitment of the CD11b ^hi Gr-1 ^hi neutrophil group in the colon tissue of colitis mice (FIG. 5B ).

Anti-inflammatory Treg cells in lymph nodes and colon tissues for DSS administration also increased. Although DA did not affect IL-10 production and FoxP3 induction of Treg cells in vitro, DA treatment significantly reduced the incidence of Treg cells in the colon tissue of colitis mice (FIG. 7 ).

Overall, the data of the present invention suggests that DA improves DSS-induced colitis through the regulation of Th17 cells and this contributes to the recruitment of neutrophils in colon tissues.

As can be seen through the present invention, the inhibitory effect of decursinol angelate on type 17 helper T (Th17) cell response was confirmed, and using a DSS-induced colitis model, DA Treatment attenuated the severity of the large intestine, including protection from bowel tissue damage induced by DSS administration, shortening of the colon, and reduction of weight loss. Th17 cells accompanying neutrophils to the colon tissue of colitis mice were markedly treated by DA treatment. These results suggest that DA may be a potential treatment for inflammatory diseases associated with Th17 cell responses such as IBD.

1 is a figure showing that the immunomodulatory component of Angelica is Decursinol angelate.
(A) Lymph node cells isolated from OT-II mice were treated with Angelica chemical components and activated in the presence of IL-2 with 0.2 μg/ml ova peptide. Five days after OT-II T cell activation, the culture supernatant was analyzed by CBA assay. Data represent mean (± SD) cytokine levels from three independent experiments: *, p<0.05; **, p<0.01.
Figure 2 is a diagram showing that DA inhibits Th17 differentiation and function,
Purified CD4 ⁺ T cells were activated and cultured for 5 days under Th17 polarizing conditions in the presence of DA.
(A) Cells were treated with 50 ng/ml PMA and 1 μg/ml ionomycin for 6 hours and analyzed by intracellular cytokine staining (ICS). Representative FACS profiles are shown at the living CD4 T cell gate: Inset numbers represent the mean (± SD) percent of IL-7 ⁺ cells obtained from three independent experiments.
(B) Th17-polarized T cells were re-stimulated with anti-CD3e and anti-CD28 for 24 hours, and the culture supernatant was analyzed by CBA (C&D). CD4 T cells were cultured for 5 days under Th17 polarizing conditions, and qRT-PCR was performed. The concentrations of the described mRNAs were normalized to that of Actb and the mean (± SD) of relative levels from three replicates was shown: p <0.05; **, p<0.01.
Figure 3 is a diagram showing the effect on the survival and proliferation of DA CD T cells. The purified CD4 T cells were activated with anti-CD3 and anti-CD28 for 3 days in the presence or absence of DA. Cell viability (A) was determined with the Ez-Cytox kit, and Epoptosis (B) was evaluated by Annexin-V staining. Data represent mean (± SD) obtained from three independent experiments.
(C) CD4 T cells were cultured as in A, pulsed with EdU for 12 hours and analyzed by flow cytometry. Representative histograms from living CD4 T cell gates, and the mean (± SD) of EdU ⁺ cells averaged over three independent experiments.
Figure 4 is a diagram showing that DA improves DSS-induced colitis in mice,
(A) Schematic illustration of the experimental process.
(B & C) Body weight and colitis severity were monitored for 9 days.
Mean (± SEM) percent of body weight (B) and disease activity index (DAI) (C) at day 7 for n=6; *, p <0.05; **, p <0.01.
(D) On day 9, mice were euthanized and the colon was separated and photographed. Colon length was measured.
(E) IL-6 levels in plasma on day 9 were analyzed by CBA. (F) Formalin-immobilized colon was sectioned and stained with hematoxylin and eosin (H&E). Representative mean H&E stained sections and histological score mean (±SD): n=3; *, p <0.05.
FIG. 5 is a diagram showing that DA weakens activation of Th17 cells and recruitment of neutrophils in the colon of DSS-administered mice.
(A) Mice were administered with DSS-water as shown in FIG. 4, and leukocytes were separated from mesenteric lymph nodes (MLN), colon transepithelium (colon:TE), and mucosal eigenlayer (colon:LP). Cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for 5 hours and analyzed by ICS. Representative FACS profiles with inset numbers indicating the incidence of IL-17 ⁺ and/or IFN-g ⁺ cells in the living CD4 T cell gate. The mean (±SD) percent of IL-17A ⁺ cells was calculated from three independent experiments.
(B) Permeation of the granulocyte group from colon transepithelium and lamina was analyzed by flow cytometry. CD11b ^hi Gr-1 ^hi from three independent experiments Average (± SD) incidence of neutrophils and representative FACS profile in living leukocyte gates.
Figure 6 shows the effect of DA on Th1, Th2, and Treg cell differentiation:
Purified CD4 ⁺ T cells were cultured in the presence or absence of DA under Th1 (a), Th2 (b) and Treg (c) polarizing conditions for 5 days.
(a & b) Cells were re-stimulated with PMA and ionomycin for 6 hours and analyzed by intracellular cytokine staining. Representative FACS profiles in living CD4 T cell gates with inset numbers indicating the mean (± SD) prevalence of IFN-g ⁺ and IL-4 ⁺ cells.
(c) Treg cells were cultured for 5 days and FoxP3 expression was measured by flow cytometry. Representative FACS profile at a live CD4 T cell gate with an inset number indicating the mean (± SD) prevalence of GITR ⁺ FoxP3 ⁺ cells.
7 is a diagram showing that DA treatment in the colon of a colitis mouse negatively regulates the induction of Treg cells.
Lymphocytes were separated from mesenteric lymph nodes (MLN, a), colon transepithelium (colon:TE, b) and mucosal eigenlayer (colon:LP, c) and stained with FoxP3 staining kit. Representative FACS profile in TCR ⁺ CD4 ⁺ gated cells with an inset number indicating the incidence of GITR ⁺ FoxP3 ⁺ cells.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Dextran sulphate sodium (Mr ~40000 kD) used in the present invention was purchased from Sigma-Aldrich (St. Louis, USA), and the chemical compound of Angelica, including decursinol angelate, was provided by Professor Yongho Ho of Konkuk University. Fluorophore attached antibodies to IFN-g (XMG1.2), CD11b (M1/70) and Gr-1 (RB6-8C5); Neutralizing antibodies to IFN-g (XMG1.2), IL-12 (C17.8) and IL-4 (11B11); Antibodies to CD3e (145-2C11) and CD28 (37.51) and recombinant human IL-2 were purchased from BD biosciences (CA, USA). FITC attached IL-17A antibody (eBio17B7) was purchased from Thermo Fisher Scientific (MA, USA). Recombinant IL-6, IL-12, IL-4, IFN-g, human TGF-b, and human IL-23 were purchased from PeproTech (NJ, USA).

Example One; Mouse and colitis model

OT-II transgenic mice were provided by Mark Boothby (Vanderbilt University). C57BL/6J mice were purchased from DBL (Seoul, Korea) and maintained in sterile conditions at the Hallym University Laboratory Animal Center.

All animal experiments were conducted in accordance with the Korea Food and Drug Administration guidelines. The experimental protocol was approved by the Hallym University Animal Experiment Ethics Committee.

7 to 8 week old mice were administered 2.5% DSS through drinking water for 7 days and the rest of the experiment was administered with distilled water. Control mice provided distilled water throughout the experiment. DA or vehicle at 4 mg/kg was injected intraperitoneally every 2 days as in Figure 4A. The body weight of the mice was recorded daily.

The disease activity index was scored as follows: weight loss ('0': no decrease; '1': decrease >1-5%; '2': decrease >5 -10%; '3': decrease >10-15 ; '4': reduction >15-20%); The presence of blood ('0': absent; '1': present) and stool hardening ('0':dandan;'1': loose; '2': diarrhea). The score was added to give a maximum score of 7 points.

Example 2: CD4 ⁺ T cell activation and helper T cell culture

Lymph node cells from OT-II mice were activated with 0.2 ug/ml OVA _{323 -339} peptide in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS and 1% penicillin-streptomycin and 5 days with 4 ng/ml IL-2 Cultured. CD4 ⁺ T cells were purified using CD4 (L3T4) microbeads (Miltenyi Biotech, Gladbach, Germany) from the spleen of C57BL/6J mice according to the manufacturer's instructions.

Purified T cells were activated with plate attached anti-CD3e (2.5 ug/ml) and soluble anti-CD28 (2.5 ug/ml). Cultures were 10 ng/ml IL-12 and 5 ug/ml anti-IL-4 for Th1 differentiation; 10 ng/ml IL-4, 5 ug/ml anti-IL-12 and 5 ug/ml for Th2 anti-IFN-g; And 5 ng/ml TGF-b, 10 ng/ml IL-6, 10 ng/ml IL-23, 5 ug/ml IL-12, 5 ug/ml IFN-g and 5 ug/ml anti IL for Th17 Supplemented with -4. Treg cells were supplemented with 5 ug/ml TGF-b and 4 ng/ml IL-2.

Example 3: Cell separation from the large intestine

The colon was washed 3 times with PBS and stirred for 30 minutes in the presence of 0.015% DTT and 1 μM EDTA in RPMI media at 37°C. The supernatant contained epithelial cells (IEPs). The remaining tissue was sliced with scissors and cultured by stirring in RPMI medium containing 1.5 ug/ml collagenase and 0.5 ug/ml dispase at 37 °C. The cell suspension containing LP (Lamina propria) cells was washed, passed through a 70 μm strainer, counted and analyzed by flow cytometry.

Example 4; Flow cytometry

Cells were washed with FACS wash buffer (1% FBS in DPBS) and incubated for 30 minutes with fluorophore attached antibody. For intracellular cytokine staining (ICS), cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for 6 hours in the presence of Golgi-stop reagent (BD Biosciences). Cells were washed with FACS buffer and stained with PerCp-attached CD4 antibody. After immobilizing them with 4% paraformaldehyde and permeating 1% saponin, the cells were intracellular with FITC-attached anti-IL17A, PE-attached anti-IL-4, and APC-attached anti-IFN-g cytokines. Dyed. FoxP3 staining was performed with FoxP3 Staining Kit (Thermofisher Scientific) according to the manufacturer's instructions. Data were obtained with a FACS Canto II instrument (BD Biosciences) and analyzed with FlowJo V10 software.

Example 5: CBA ( Cytometric Bead Array)

Cytometric Bead Array was performed with a BD CBA Mouse Th1/Th2/Th17 Cytokine kit (BD biosciences) according to the manufacturer's instructions. Briefly, the cell supernatant was serially diluted with assay diluent and incubated with the mixture capture beads and PE detection reagent. After 2 hours, samples were analyzed by FACSCalibur (BD Biosciences).

Example 6: cells Survival rate And Apoptosis Assay

CD4 ⁺ T cells were activated with 2.5 ug/ml anti-CD3e and 2.5 ug/ml anti-CD28 and cultured in the presence or absence of DA for 3 days. The cell viability analysis was performed according to the manufacturer's instructions with the EZ-Cytox Enhanced Cell Viability Assay Kit (DoGenBio, Seoul, Korea). To detect apoptosis, cells were washed with Annexin V binding buffer (25 mM CaCl ₂ , 140 mM NaCl and 10 mM Tris -pH 7.4) and incubated with PE-attached Annexin V (BD Biosciences) for 30 minutes. FACS analysis was performed.

Example 7: EdU Uptake analysis:

CD4 ⁺ T cells were cultured under Th17 polarizing conditions and their EdU (5-ethynyl-2'-deoyuridine) uptake analysis was performed according to the manufacturer's instructions with a Click-iT Plus EdU Flow Cytometry Assay Kit (ThermoFisher). In summary, after pulsed with 10 μM Edu 12 hours before harvest, the cells were stained with a surface marker, immobilized and permeated with a Click it kit. To detect EdU uptake, cells were stained with Alexa Fluor 647-attached detection reagent and analyzed by flow cytometry.

Example 8: Histology

Clones were fixed with formalin, filled in paraffin, and sectioned into 5 μM sections. Tissue sections were stained with hematoxylin and eosin staining reagents and histological scores (0-6) were determined under a microscope. Presence of cell permeation ('0': none; '1': scattered inflammation foci; multiple inflammation foci, and '3': transmural cell penetration); crypt organization ('0': normal; '1': presence of elongated crypts, and '2': absence of crypts); And edema ('0': absent and '1': presence of edema) on the colon wall.

The experiment of the present invention was repeated at least twice or three times, and unless otherwise specified, the results of three or more independent experiments were expressed as the mean ± standard deviation. Statistical significance between samples was calculated by unpaired bilateral t-test (Instat; GraphPad, Inc). In the figure, * and ** represent P values less than 0.05 and 0.01, respectively.

Claims (11)

A composition for preventing or treating colitis comprising decursinol angelate as an active ingredient. The method of claim 1, wherein the colitis is inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), ulcerative colitis (UC) and Crohn's disease (CD). A composition characterized in that it is selected from the group consisting of. A composition for inhibiting a type 17 helper T (Th17) cell response comprising decursinol angelate as an active ingredient. A method of treating decursinol angelate on a subject to inhibit the type 17 helper T (Th17) cell response in the subject. The method of claim 4, wherein the subject is an animal other than a human. Decursinol angelate (decursinol angelate) composition for inhibiting the secretion of pro-inflammatory cytokines containing as an active ingredient. 7. The composition of claim 6, wherein the pro-inflammatory cytokine is TNF-alpha or IFN-gamma. A method of treating decursinol angelate on a subject to inhibit the secretion of pro-inflammatory cytokines from the subject. 9. The method of claim 8, wherein the subject is an animal other than a human. CD4 T containing decursinol angelate as an active ingredient A composition for inhibiting IL-17 expression in cells. Decursinol angelate (decursinol angelate) in vitro by treating CD4 T cells to inhibit IL-17 expression in the cells.

Images