JP2003073286A - Agent for prevention and treatment of inflammatory bowel disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent for the prevention and treatment of inflammatory bowel disease containing bacteria of the genus Lactobacillus or a polysaccharide fraction derived from the bacterial cell as an active component, an IL-6 formation suppressing agent containing the treating agent as an active component and an agent for suppressing IL-6/STAT3 phosphorylation response containing the same. SOLUTION: The above problems are solved based on the findings that lactobacillus or a polysaccharide fraction derived from the bacterial cell has excellent effect for the prevention and treatment of inflammatory bowel disease and the mechanism of the action is the suppression of IL-6/STAT3 phosphorylation response based on the IL-6 formation suppressing action.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a safe and effective preventive and / or therapeutic agent for inflammatory bowel disease.

[0002]

BACKGROUND OF THE INVENTION Inflammatory bowel disease (IBD) has been increasing in number in recent years. This inflammatory bowel disease is divided into ulcerative colitis (UC) and Crohn's disease (CD).

The pathological condition of these inflammatory bowel diseases is the formation of sores and ulcers in the mucous membrane of the intestinal tract, and symptoms such as weight loss, diarrhea and bloody stool appear. The cause of this onset is said to be abnormal cytokine production response associated with disruption of the intestinal mucosal immune response system, but the details have not been clarified. In this way, inflammatory bowel disease is completely different from gastric ulcer and duodenal ulcer both in its pathological condition and cause of onset.

Therefore, it is desired to elucidate the cause of inflammatory bowel disease and to develop a safe and effective preventive / therapeutic agent.

[0005]

The inventors of the present invention have studied the cause of inflammatory bowel disease using animal models of ulcerative large intestine and animal models of Crohn's disease. The production was remarkably increased, and it was found that IL-6 is one of the causes of the onset.
IL-6 forms a complex with the soluble IL-6 receptor (IL-6R) secreted by monocytes / macrophages during the inflammatory response. Complexed IL-6 / IL-6R
Binds to a gp130 molecule that is expressed in almost all cells including non-lymphoid cells, and thus transmits an IL-6 signal to a cell line that does not express IL-6R (trans-signaling), and an inflammatory response. Are known to induce. Therefore, the present inventor also examined the IL-6 / STAT-3 phosphorylation response in an animal model of inflammatory bowel disease, and found that this was also significantly enhanced in inflammatory bowel disease. Therefore, the present inventor studied to find a therapeutic agent for inflammatory bowel disease focusing on highly safe edible fungi, and, surprisingly, Lactobacillus bacteria have an IL-6 production inhibitory effect, Results IL-6 /
The inventors have found that the STAT3 phosphorylation response is suppressed, and that they have a preventive and therapeutic effect on inflammatory bowel disease, and have completed the present invention. It was also found that these effects are particularly excellent in the polysaccharide fraction derived from bacterial cells of Lactobacillus bacteria.

That is, the present invention provides a prophylactic / therapeutic agent for inflammatory bowel disease, which comprises a lactobacillus bacterium or a polysaccharide fraction thereof as an active ingredient. Further, the present invention is
It is intended to provide an IL-6 production inhibitor containing, as an active ingredient, a Lactobacillus bacterium or a polysaccharide fraction derived from the bacterium.
Furthermore, the present invention provides an IL-6 / STAT3 phosphorylation response inhibitor containing, as an active ingredient, a Lactobacillus bacterium or a polysaccharide fraction thereof.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of Lactobacillus bacteria used in the present invention include Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus gasseri,
Lactobacillus reuteri and the like can be mentioned, among which Lactobacillus casei and Lactobacillus gasseri are preferred. As Lactobacillus casei, Lactobacillus casei YIT9029, (FERM BP
-1366), YIT9018 (FERM BP-66)
5) are mentioned. As Lactobacillus gasseri, Lactobacillus gasseri YIT0168 (J
CM 5813; Lactobacillus acidophilus in the old classification).

In the present invention, the bacterium of the genus Lactobacillus is
Either live cells or dead cells may be used, but it is preferable to use live cells.

Examples of the polysaccharide fraction derived from the bacterium of Lactobacillus include, for example, JP-A-4-5236 or J. Bio
The fraction containing a polysaccharide-polypeptide glycan complex (hereinafter sometimes referred to as PS-PG) obtained by the method described in chem., 108, 568-571 (1990) is preferable. This fraction can be obtained, for example, as follows. First, the cells are suspended in an isotonic solution and treated with a cell wall lysing enzyme such as N-acetylmuramidice. Prior to this treatment, the microbial cells may be crushed by ultrasonic treatment or a French press. Solid cytoplasm is removed by centrifugation from the bacterial cell suspension after enzyme treatment, the supernatant is treated with nucleolytic enzyme, and then trypsin and pronase to decompose protein, and finally dialyzed with distilled water. Then, the low molecular weight fraction is removed, and the polysaccharide fraction is collected and, if necessary, lyophilized.

The Lactobacillus bacterium or the polysaccharide fraction derived from the bacterium suppresses IL-6 production in the intestinal mucosa of inflammatory bowel disease model and inflammatory bowel disease cases, and as a result, IL-6 / S.
It also significantly suppresses the TAT-3 phosphorylation response. further,
It has a strong preventive and therapeutic effect on inflammatory bowel disease model. Therefore, the Lactobacillus bacterium or its polysaccharide-derived polysaccharide fraction is useful as a prophylactic / therapeutic agent for inflammatory bowel disease, that is, ulcerative colitis and Crohn's disease.

Further, the polysaccharide fraction derived from Lactobacillus bacteria has a remarkably excellent inhibitory effect on IL-6 production as compared with the bacteria of Lactobacillus bacteria, and is a main active ingredient in the bacteria. Probability is high. Furthermore, the inhibitory effect on IL-6 production of the polysaccharide fraction derived from Lactobacillus bacterial cells is extremely stronger than the action of the polysaccharide fraction derived from Bifidobacterium bacterial cells. Particularly, the polysaccharide fraction derived from Lactobacillus casei is highly effective, and further, Lactobacillus casei YIT9029 or YIT9018 (FERM B
Those derived from P-665) are preferable.

The inflammatory bowel disease prophylactic / therapeutic agent of the present invention comprises a bacterial cell of Lactobacillus bacterium or a polysaccharide fraction derived from the bacterial cell, and its safety is established. However, if necessary, an excipient, a binder, a disintegrant, a lubricant, a coating agent, an emulsifier, a dispersant, a solvent, a stabilizer, etc. are appropriately added to the cells or fractions, and tablets , Granules, powders, powders, capsules and the like may be used. The daily dose for adults is 1 x live bacteria
It is preferable to administer a microbial cell or a polysaccharide fraction derived from a microbial cell in an amount corresponding to 10 ⁶ or more, particularly 1 × 10 ⁶ to 1 × 10 ¹³ cells, but the amount can be appropriately increased or decreased depending on the symptoms. The prophylactic / therapeutic agent for inflammatory bowel disease of the present invention can be orally administered as it is, or it can be added to any food or drink and taken daily.

[0013]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. Example 1 [1] Material and method

(A) Animal: SPF Balb / c mouse (female, 6 weeks old) and SPF SAMP1 / Yit strain (male, 10 and 20 weeks old) were used for the experiment.

(B) Enteritis model Ulcerative colitis (UC) model: DSS colitis model mouse (Gastroenterology (1990) 107, p1643) was used. Crohn's disease (CD) model: SAMP1 / Yit strain (Gut (1998) 43, p78, J.Clin. Inuest. (2001) 142, p24
3) was used.

(C) Detection of phosphorylated STAT in intestinal mucosa of IBD model and IBD cases Human IBD intestinal mucosa obtained from each model mouse and surgical material was added with dephosphorylating enzyme inhibitor and proteolytic enzyme inhibitor lysis Homogenized with buffer (J. Exp.
Med. (2001) 193, p471). The non-lesional part of the excised intestine of colorectal cancer was used as a control. The amount of protein in the supernatant obtained by centrifugation at 20,000 g for 10 minutes was measured. 8 μl of 10 μg protein
After electrophoresis on a% SDS gel, it was transferred onto a nylon membrane. The nylon membrane was blocked overnight with a 3% skim milk / PBS solution. The next day, reaction was performed with a specific antibody of each phosphorylated STAT molecule, and the reaction product was detected by a chemiluminescence system.

(D) Immunohistochemical detection of phosphorylated STAT3 molecule Frozen sections of ileal lesions of SAMP1 / Yit strain were prepared. Immunostaining was performed according to a conventional method using a specific antibody (Biocource) against the phosphorylated STAT3 molecule. After immunostaining, they were nuclear-stained with hematoxylin and observed under a microscope.

(E) IL in intestinal mucosa of IBD mouse
-6 Examination of productivity A Balb / c mouse and SPF after DSS colitis induction
Colonic lamina propria cells (LPLs) were isolated from Balb / c mice by a known method (ENBO J. (1998) 17, p100.
6). 2.0 × 10 ⁶ LPLs were stimulated with immobilized TCR β chain antibody (H57-597: 10 μg / mL) / liquid phased CD28 antibody (2 μg / mL). The culture supernatant after 48 hours of culture was collected, and the amount of IL-6 produced was detected by the ELISA method.
In addition, after separating the lamina propria cells from the ileum part of SAMP1 / Yit strain, RNA was extracted according to a conventional method. The expression of IL-6 mRNA was examined by the RT-PCR method using G3PDH and IL-6 specific primers.

(F) Lactobacillus casei YIT9
Verification of suppression of enteritis by strain 029 Heat-killed YIT9029 strain-containing feed (0.05
%) Administration group (L group) and control group (normal feed: C group) DS
The mice were given 2 weeks before the induction of S colitis. Chronic DSS colitis was induced according to a conventional method, and the state of enteritis was evaluated by each item shown in G.

(G) Evaluation of enteritis (a) Mouse mortality (b) Disease activity index
(Clinitis clinical score, LabInvest (1993) vol 69 p 238-24
9) (C) FACS analysis (D) Cytokine production

(H) Preparation of NK cells from spleen cells and N
Measurement of K activity After preparing spleen cells, NK cells were isolated by the MACS method using a mouse NK cell-specific antibody. According to the usual method,
The cytotoxic activity was measured by targeting Yac-1 cells.

(I) Statistical analysis Statistical processing was performed by t-test.

[2] Results (1) Phosphorylated STAT content of intestinal mucosa of IBD model mouse
Phosphorylation STAT intestinal mucosa expression UC and CD model mice element group
The expression of the molecule was detected by immunoblotting. The result is shown in Figure 1.
Shown in. In the UC model (DSS-induced colitis),
The phosphorylation reaction of STAT3 molecule was remarkable (Fig. 1).
A). Moreover, in the colitis non-induced mouse, STAT
No phosphorylation reaction of the molecular group was observed. On the other hand, CD
In the intestinal mucosa of a model mouse (SAMP1 / Yit strain), phosphorylation of STAT1, STAT3, and STAT6 molecules (indicated by PY in the figure) was observed in the intestinal mucosa at the site of inflammation in the active phase (10 weeks of age). , STAT3
The phosphorylation reaction was most prominent (Fig. 1B). Further, in the inactive period (20-week-old) inflammation, only the STAT3 phosphorylation reaction was observed. The phosphorylation reaction of STAT5 was observed regardless of the difference between the lesioned part and the non-lesioned part (FIG. 1B).

(2) Intestinal mucosa in human IBD cases
Expression of unoxidized STAT molecule group From the results of (1) above, it was found that the phosphorylation reaction of STAT3 molecule was enhanced in the intestinal mucosa of both CD and UC models as the pathological condition was expressed. Therefore, it was confirmed whether a similar reaction was enhanced in the intestinal mucosa of human IBD cases. The result is shown in FIG. In both UC (2 cases) and CD (1 case), the phosphorylation reaction of STAT3 molecule was remarkable as in the mouse model (FIG. 2). No phosphorylation of STAT3 molecule was observed in normal intestinal tissue (Nor in the figure) derived from the case of colorectal cancer (FIG. 2).

(3) Immune system of phosphorylated STAT3 molecule
Histologic localization of phosphorylated STAT3 molecules in the ileal lesions of SAMP1 / Yit strain was detected by immunohistochemistry. The result is shown in FIG. The phosphorylated STAT3 molecule was localized in the cell nucleus distributed in the lamina propria and submucosa (Fig. 3A, arrow). It was also observed in the epithelial cell nuclei at the tip of some intestinal villi (Fig. 3B, arrow).

(4) IL in the intestinal mucosa of IBD mouse
Examination of -6 productivity The IL-6 productivity of LPLs cells isolated from the UC model (DSS colitis) was significantly higher than that of normal mice (Fig. 4A). In addition, a CD model of the active stage of enteritis (SAM
PPLs isolated from mouse lesions (P1 / Yit strain)
IL-6 mRNA of was increased compared with the inactive phase (FIG. 4B).

(5) Lactobacillus casei YIT9
Examination on Enteritis Suppressing Ability of Strain 029 In order to verify whether suppression of IL-6 / STAT3 phosphorylation response which was enhanced in intestinal mucosa of mouse enteritis model and human IBD case leads to suppression of enteritis, it was heat killed. The YIT9029 strain was administered to DSS chronic colitis-induced mice and the state of enteritis was compared with that of the control group. Chronic DS
After induction of S colitis, about half of the mice died in the control group, but no mice died in the YIT9029 strain-administered group (FIG. 5). YIT9 was also used in the clinical score of enteritis using weight loss of mice, diarrhea and bloody stool as indicators
An improvement effect on enteritis was observed in the 029 strain group (FIG. 6). After dissecting the mice, the IL-6 production response in the large intestine LPLs of both groups was compared. As a result, YIT90
IL-6 production was suppressed in the 29 strain-administered group (FIG. 7). In addition, in the YIT9029 strain-administered group, an increase in LPLs cells (αβTCR T
Cells, B220 cells) were suppressed (Fig. 8). In the enteritis-induced mice, the NK cells in the spleen cells were remarkably reduced, and the NK cell level cytotoxic activity was also reduced (FIGS. 9 and 10). On the other hand, in the YIT9029 strain-administered group, the decrease of NK cells and the decrease of the cytotoxic activity at the cell level were suppressed (FIGS. 9 and 10).

Example 2 [1] Material and Method Animal : Balb / c (female, 8 weeks old) was used for the experiment.

Chronic colitis 4% (v / w) dextran sodium sulfate (DSS:
ICN) tap water was administered to mice for 1 week, and the administration was stopped for 1 week to perform 4 cycles to induce chronic colitis (Gastroenterology (1990) 102, p43).

Of colonic lamina propria cells (LI-LPLs)
Separation According to a conventional method, LI-LPLs were separated (Microb
ial Ecology in Health and Disease (2000) 12, p10
2).

IL-6 production response system of LI-LPLs Lipopolysaccharide derived from Escherichia coli (LPS; 10 μg /) was added to 2.0 × 10 ⁵ freshly isolated LI-LPLs cells.
(mL) was added and cultured for 48 hours. The amount of IL-6 in the culture supernatant was detected by the ELISA method.

Pairing LI-LPLs with the IL-6 production response system
Effect of lactic acid bacteria on heat-killed Lactobacillus casei YIT902
Nine strains and Lactobacillus gasseri YIT0168 strain were prepared, and various concentrations were added to the above culture system. Culture 4
The amount of IL-6 after 8 hours was examined by the ELISA method. The same examination was conducted for live bacteria (heat-killed untreated practical strains). In addition, the mouse macrophage cell line RAW2
A similar experiment was performed using 64.7 strain.

[2] Results L for the LI-LPLs culture system derived from mouse chronic colitis
IL-6 production response after PS stimulation L was added to the LI-LPLs culture system derived from chronic colitis-induced mouse.
When PS was added, an IL-6 production response was observed (Fig. 1
1).

To IL-6 production response system after LPS stimulation
Effect of heat-killed strain (YIT90)
29, YIT0168) was added to examine the IL-6 production response. As a result, in each strain, IL-
6 production was suppressed. 1 μg / mL-0.5 in each strain
Before and after mg / mL, the effect of suppressing IL-6 production was highest, and the suppressing rate was about 40% (Fig. 12).

The same experiment was performed using RAW241 cells, which is a mouse macrophage-derived cell line. RAW
IL-6 production response was observed when LPS was added to the cell culture system (FIG. 13). When heat-killed Lactobacillus casei YIT9029 strain was added to this assay system, IL-6 production was suppressed in inverse proportion to the amount of bacterial cells added, as in the case of using LI-LPLs cells derived from chronic colitis. (FIG. 13).

The ability to suppress IL-6 production was compared between live cells (non-heat-treated cells) and heat-killed cells. LI-L derived from chronic colitis
Inhibition of IL-6 production in PLs cells was also observed in viable bacteria, and the activity was the same as that when heat-killed cells were used (Fig. 14).

Production Example 1 (Preparation of PS-PG) Lactobacillus casei YIT9029 cells were treated with ILS.
The cells were cultured in the medium until the stationary phase, harvested by centrifugation, and washed with deionized water. The washed cells were suspended in deionized water, heated at 100 ° C. for 30 minutes, collected by centrifugation, and then
The cells were washed with deionized water and freeze-dried. The cells (60 g) thus obtained were added to 3 liters of 5 mM Tris-malate.
Suspend in buffer (pH 6.5, containing ₂ mM CaCl ₂ ) and
0 mg (10 mg x 5) of N-acetylmuramidase (Seikagaku Corporation, 10 mg) was added, and the mixture was shaken at 42 ° C for 40 hours, then 8
Centrifuged at 500 rpm for 1 hour. After centrifugation, the supernatant was collected, and Benzon nuclease (Kanto Kagaku, Grade I: 10000) was added to the supernatant.
U), 1 vial was added, and the mixture was shaken at 37 ° C. for 20 hours to give pronase (Roche Diagnostics, 50 mg /
(5 mL filtered) was added, and the reaction was carried out at 37 ° C. for 20 hours. Centrifuge this (8500 rpm, 60 minutes)
Then, the supernatant was collected and concentrated with an ultrafiltration membrane having a cutoff of 300,000, and the inner solution was collected, dialyzed and freeze-dried. The dried product was subjected to gel filtration treatment with Sephacryl S-300, dialyzed and freeze-dried to obtain a PS-PG fraction.

Example 3 Lactobacillus casei on IL-6 producing ability in peripheral blood mononuclear cells (PMNC) derived from patients with ulcerative colitis
YIT9029 strain and a polysaccharide fraction derived from the strain (Production Example 1
The effect of PS-PG) obtained in step 1 was examined. That is, 1.5 × 10 ⁵ PMNC cells collected from a patient with ulcerative colitis
Individuals were cultured for 48 hours in the presence of LPS. Y in this culture system
The IT9029 strain bacterial cell or its polysaccharide fraction was added at various concentrations. The amount of IL-6 in the culture supernatant was detected by the ELISA method.

As a result, as shown in FIG. 15, YIT9
The strain 029 strain exhibited an IL-6 production inhibitory action, but the bacterial cell-derived polysaccharide fraction (PS-PG) exhibited a stronger IL-6 production inhibitory action than the bacteria.

Example 4 Lactobacillus casei-derived polysaccharide fraction and bifido against IL-6 production response system when peripheral blood mononuclear cells (PMNC) derived from a patient with ulcerative colorectal disease are stimulated with lipopolysaccharide The action of the polysaccharide fraction derived from Bifidobacterium was examined. That is, lipopolysaccharide (LPS: 10 μg / mL) was added to 1.5 × 10 ⁵ PMNC cells collected from a patient with ulcerative colitis and cultured for 48 hours.
Lactobacillus casei YIT9029 was added to this culture system.
A polysaccharide fraction derived from the strain (the same fraction as in Example 3) or a polysaccharide fraction derived from the Bifidobacterium bifidum YIT4007 strain (fractionated in the same manner as the YIT9029 strain of Example 3) was added at various concentrations. .

As a result, as shown in FIG. 16, the polysaccharide fraction derived from Lactobacillus casei showed an extremely strong IL-6 production inhibitory action as compared with the polysaccharide fraction derived from Bifidobacterium bifidum. It was

[0042]

The lactic acid bacterium or the polysaccharide fraction derived from the bacterium has a high prophylactic and therapeutic effect on inflammatory bowel disease, and its action mechanism is IL.
This is due to the IL-6 production inhibitory action based on the inhibition of -6 / STAT3 phosphorylation response.

[Brief description of drawings]

FIG. 1 Phosphorylated STAT of intestinal mucosa of IBD model mouse
It is a figure which shows the result of the western blotting which investigated the expression of the molecular group (A: DSS induction colitis, B: SAMP1 / Y.
it mouse).

FIG. 2 is a diagram showing the results of Western blotting examining the expression of phosphorylated STAT molecule groups in human UC (2 cases), human CD (1 case) and normal (Nor) intestinal tissue.

FIG. 3 shows the results of immunohistological detection of phosphorylated STAT3 molecules in ileal lesions of SAMP1 / Yit strain (A: lamina propria and submucosa, B: epithelial cells at the tip of intestinal villi). .

FIG. 4 shows the results of examination of IL-6 producing ability in the intestinal mucosa of IBD mice (A: DSS colitis, B: S).
AMP1 / Yit line).

FIG. 5 is a graph showing the life-prolonging effect of Lactobacillus casei YIT9029 strain on DSS colitis.

FIG. 6 shows the effect of Lactobacillus casei strain YIT9029 on the clinical score of enteritis in DSS colitis mice.

FIG. 7: Lactobacillus against DSS colitis mice
It is a figure which shows the IL-6 production inhibitory effect of casei YIT9029 strain.

FIG. 8 shows the effect of Lactobacillus casei strain YIT9029 on the increase of LPLs cells in DSS enteritis mice.

FIG. 9 shows the effect of Lactobacillus casei strain YIT9029 on the decrease in NK cell number in DSS colitis mice.

FIG. 10 shows the effect of Lactobacillus casei strain YIT9029 on reduction of NK cell level cytotoxic activity in DSS colitis mice.

FIG. 11 is a diagram showing an IL-6 production response after LPS stimulation to a LI-LPLs culture system derived from a chronic colitis mouse.

FIG. 12 is a diagram showing the influence of lactic acid bacteria on the IL-6 production response system after LPS stimulation.

FIG. 13 shows the IL-6 production response when LPS was added to the RAW264.7 cell culture system and the effect of lactic acid bacteria on this.

FIG. 14 shows the effects of heat-killed lactic acid bacteria and viable bacteria on the IL-6 production response when LPS was added to the RAW264.7 cell culture system.

FIG. 15 is a diagram showing the influence of lactic acid bacteria and bacterial cell-derived polysaccharide fractions on IL-6 producing ability in a PMNC cell culture system derived from a patient with ulcerative colitis.

FIG. 16 is a graph showing the influence of a lactic acid bacterium-derived polysaccharide fraction and a Bifidobacterium bacterium-derived polysaccharide fraction on the IL-6 production response when LPS was added to a PMNC cell culture system derived from a ulcerative colitis patient. is there.

Continued front page    (72) Inventor Keiichi Mitsuyama             67 Asahi-cho, Kurume-shi, Fukuoka Kurume University School of Medicine             Second internal medicine (72) Inventor Michio Sada             67 Asahi-cho, Kurume-shi, Fukuoka Kurume University School of Medicine             Second internal medicine F-term (reference) 4C087 AA01 AA02 BC56 BC57 CA09                       CA14 NA14 ZA66 ZB11 ZC02

Claims (4)

[Claims] 1. A prophylactic / therapeutic agent for inflammatory bowel disease, which comprises a polysaccharide fraction derived from a bacterium of the genus Lactobacillus or a bacterium thereof as an active ingredient. 2. The prophylactic / therapeutic agent for inflammatory bowel disease according to claim 1, wherein the Lactobacillus bacterium is Lactobacillus casei or Lactobacillus gasseri. 3. An IL-6 production inhibitor containing a lactobacillus bacterium or a polysaccharide fraction derived from the bacterium thereof as an active ingredient. 4. An inhibitor of IL-6 / STAT3 phosphorylation response containing a polysaccharide fraction derived from a bacterium belonging to the genus Lactobacillus or a bacterial cell thereof as an active ingredient.