Agent for treating inflammatory bowel disease comprising lactic acid bacteria derived from phellodendron

Abstract

To provide an agent for treating inflammatory bowel disease.SOLUTION: The present invention provides an agent for treating inflammatory bowel disease containing Lactococcus lactis subsp. lactis L8 strain as an active ingredient, and a composition for treating inflammatory bowel disease containing Lactococcus lactis subsp. lactis L8 strain.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the use of the lactic acid bacteria Lactococcus lactis subsp. lactis L8 strain (hereinafter sometimes referred to as "L8" or "L8 strain") derived from Phellodendron amurense growing in the Shirakami Mountains for the treatment of inflammatory bowel disease. Furthermore, the present invention relates to a composition for the treatment of inflammatory bowel disease containing the Lactococcus lactis subsp. lactis L8 strain. [Background technology]

[0002] Inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease, are intractable inflammatory diseases of unknown etiology that cause chronic inflammation in the digestive tract. It has been shown that genetic predisposition and abnormalities in the intestinal immune system are involved in the pathology, but the detailed molecular mechanisms remain to be elucidated. In recent years, changes in the intestinal microbiota in patients with inflammatory bowel diseases have gradually become clear and are attracting attention. Compared to healthy individuals, patients with inflammatory bowel diseases have a reduced diversity of intestinal microbiota, with an increase in pathogenic bacteria such as Escherichia coli (E. coli), Fusobacterium, and Ruminococcus gnavus, and a decrease in beneficial bacteria such as Lachnospiraceae, Bifidobacterium, and Faecalibacterium prausnizii. This abnormal composition of the intestinal microbiota, i.e., dysbiosis, affects the metabolic pathways of amino acids, short-chain fatty acids, and bile acids. Such changes in the intestinal microbiota and metabolome impair intestinal homeostasis and are involved in the pathology of inflammatory bowel diseases.

[0003] Probiotics such as lactic acid bacteria are known to improve the intestinal environment and are effective in improving intestinal inflammation symptoms. Although there is still little medical evidence regarding their therapeutic effect on intestinal inflammation, some probiotics may be useful tools for the treatment of inflammatory bowel disease. For example, Bifidobacterium breve, Lactobacillus plantarum, Lactococcus lactis NCDO2118, and Lactobacillus paracasei have been reported to improve dextran sulfate sodium (DSS)-induced colitis, an animal model of ulcerative colitis.

[0004] Lactococcus lactis is a lactic acid bacterium that is often found in fermented foods such as dairy products. L8 is a lactic acid bacterium isolated from the plant Phellodendron amurense (Patent Document 1), but its functionality has not been elucidated. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2021 / 002449 publication Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a therapeutic agent for inflammatory bowel disease comprising L8, and a further object of the present invention is to provide a composition for treating inflammatory bowel disease comprising L8. [Means for solving the problem]

[0007] That is, the present invention includes the following embodiments.

[0008] (Embodiment 1) A treatment for inflammatory bowel disease that contains the Lactococcus lactis subsp. lactis L8 strain as an active ingredient.

[0009] (Embodiment 2) A composition for treating inflammatory bowel disease, comprising a Lactococcus lactis subsp. lactis L8 strain.

[0010] (Embodiment 3) The composition according to embodiment 2, which exerts a therapeutic effect on inflammatory bowel disease by suppressing TNF-α expression.

[0011] (Embodiment 4) The composition according to embodiment 3, which exerts a therapeutic effect against inflammatory bowel disease by changing the composition of the intestinal flora.

[0012] (Embodiment 5) A composition for suppressing TNF-α expression, comprising a Lactococcus lactis subsp. lactis L8 strain.

[0013] (Embodiment 6) A composition for changing the composition of the intestinal microbiota, comprising a Lactococcus lactis subsp. lactis L8 strain.

[0014] (Embodiment 7) The composition of embodiment 6, which alters the composition of the intestinal flora by increasing the proportion of Clostridiaceae.

[0015] (Embodiment 8) The composition of embodiment 6 or 7, which alters the composition of the intestinal flora by reducing the proportion of Rikenellaceae.

[0016] (Embodiment 9) 9. The composition according to any one of embodiments 2 to 8, which is a microcapsule.

[0017] (Embodiment 10) The therapeutic agent according to embodiment 1, or the composition according to any one of embodiments 2 to 9, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease. Effect of the Invention

[0018] The present invention makes it possible to provide a therapeutic agent for inflammatory bowel disease, and a composition for treating inflammatory bowel disease, which contain as an active ingredient the L8 strain of lactic acid bacteria derived from Phellodendron amurense that inhabits the Shirakami Mountains.The present invention also makes it possible to provide a composition that contains the L8 strain and suppresses TNF-α expression in the intestine, or a composition that contains the L8 strain and can change the composition of the intestinal bacterial flora. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will be described in detail below.

[0020] The present invention relates to a therapeutic agent for inflammatory bowel disease, which contains Lactococcus lactis subsp. lactis L8 strain as an active ingredient.

[0021] The L8 strain was deposited domestically on June 19, 2019 under the identification label "Lactococcus lactis l8" at the National Institute of Technology and Evaluation, Patent Microorganism Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) under the accession number NITE P-02970. After that, an application was made for the transfer of the strain to an international deposit under the Budapest Treaty, and an international deposit accession number (NITE BP-02970) was granted on June 23, 2020. Therefore, the strain can be obtained from each depository institution. Alternatively, it can be obtained by the method described in Patent Document 1.

[0022] The L8 strain of the present invention may be in the form of either live or killed cells, and may be in the form of a liquid or powder. In consideration of storage, transportation, etc. of the L8 strain, it is convenient and preferable to use heat-killed cells.

[0023] The present invention also relates to a composition for treating inflammatory bowel disease, containing Lactococcus lactis subsp. lactis L8 strain. The form of the composition is not particularly limited, and may be, for example, a tablet, capsule, microcapsule, jelly, paste, powder, granule, liquid, etc. Among them, from the viewpoint of exerting the effect of the L8 strain without being affected by digestive fluids, tablets, capsules, or microcapsules with an enteric coating that is not affected by gastric acid are preferred.

[0024] The composition of the present invention may contain additives such as excipients, disintegrants, binders, or lubricants that do not interfere with the action of the L8 strain and are medicamentously acceptable. Examples of such excipients include glucose, lactose, corn starch, sorbitol, etc.; examples of disintegrants include starch, sodium alginate, gelatin powder, calcium carbonate, calcium citrate, dextrin, etc.; examples of binders include dimethylcellulose, polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum arabic, gelatin, hydroxypropylcellulose, polyvinylpyrrolidone, etc.; examples of lubricants include talc, magnesium stearate, polyethylene glycol, hardened vegetable oil, etc.

[0025] In addition to the above excipients, the composition of the present invention may further contain minerals, vitamins, flavonoids, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, sweeteners, colorants, fragrances, stabilizers, preservatives, sustained-release regulators, surfactants, solubilizers, or humectants.

[0026] The therapeutic agent and composition of the present invention have a therapeutic effect on inflammatory bowel disease by suppressing the expression of TNF-α in the intestine and / or by changing the composition of the intestinal flora. In particular, the composition of the intestinal flora can be changed by increasing the ratio of Clostridiaceae and / or decreasing the ratio of Rikenellaceae.

[0027] Examples of inflammatory bowel diseases include ulcerative colitis, Crohn's disease, infectious enteritis, intestinal Behcet's disease, drug-induced enteritis, radiation-induced enteritis, obstructive enteritis, and ischemic enteritis.

[0028] In the composition of the present invention, the content of the L8 strain is not particularly limited, but can be 0.01 to 99% by weight, preferably 0.5 to 50% by weight, and more preferably 1 to 30% by weight, calculated as the weight of the lactic acid bacteria body relative to the weight of the total composition.

[0029] The therapeutic agent and composition of the present invention can be produced in various forms by conventionally known methods using the L8 strain and the above-mentioned excipients, additives, and other components. In addition, the therapeutic agents and compositions of the present invention can be used as foods, supplements, beverages, medicines, or feeds. EXAMPLES

[0030] The present invention will be described below with reference to specific embodiments, but it will be understood that the present invention is not limited to these embodiments, and that various changes and modifications therein can be made by those skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

[0031] Preparation of compositions containing L8 The L8 strain, provided by the Faculty of Agriculture and Life Sciences, Hirosaki University, was cultivated, heat-treated, and mixed with starch as a substrate to produce a powder. The resulting L8-containing powder (L8 and starch) and starch were used in the reference examples. The obtained L8-containing powder (L8 and starch) and starch were microencapsulated with an enteric coating by a known method and used in the examples.

[0032] (Example) Creation of animal models Thirty-six 6-week-old female C57BL6 / J mice were purchased from CLEA Japan. After one week of preliminary breeding, they were divided into four groups (vehicle / water group (n=6), vehicle / DSS group (n=12), L8 / water group (n=6), and L8 / DSS group (n=12). The first two groups were given starch, and the last two groups were given L8 plus distilled water, each for 12 consecutive days using an oral feeding tube (1x109 lactobacillus count / mouse / day). From day 4 to day 11, colitis was induced in the mice by allowing them to drink 3.5% DSS ad libitum (vehicle / DSS group, L8 / DSS group). On day 12, the mice were dissected and colonic tissues and fecal samples were collected. Histological evaluation, quantitative PCR, ELISA, and gut microbiota analysis were performed using the collected colonic tissues and fecal samples.

[0033] (Test 1) Verification of the effect of L8 on DSS colitis The body weight of mice in each group was measured from day 0 to day 12. In the group in which DSS was started, body weight gradually decreased from day 5. No significant difference in body weight change was observed between the vehicle / DSS group and the L8 / DSS group (Fig. 1a). Comparing the intestinal length of the colon, the vehicle / DSS group was significantly shorter than the vehicle / water group, but the L8 / DSS group had a significantly longer intestinal length than the vehicle / DSS group (Fig. 1b, Fig. 1c). Compared with the vehicle / water group, the vehicle / DSS group showed clear histological findings of inflammation, namely, loss of crypt structure, extensive ulcer formation, and marked inflammatory cell infiltration (Fig. 2c). These histological findings were only mild to moderate in the L8 / DSS group (Fig. 2c, Fig. 2d).

[0034] (Test 2) Histological evaluation Colon tissue was fixed in 10% formalin, embedded in paraffin, and sectioned (Figure 2a-d). H&E staining was performed and the degree of inflammation was scored as follows: Inflammation level (0: none, 1: mild, 2: moderate, 3: severe) Inflammatory cell infiltration (0: none, 1: mucosa, 2: submucosa, 3: deep to the muscular layer) Crypt damage (0: none, 1: 1 / 3 from the bottom, 2: 2 / 3 from the bottom, 3: only the surface epithelium remains, 4: crypt structure and epithelium lost) Range of enteritis (1: 1-25%, 2: 26-50%, 3: 51-75%, 4: 76-100%) These scores were summed to obtain a tissue score, and the results were compared between the vehicle / DSS group and the L8 / DSS group. The results are shown in Figure 2e.

[0035] (Test 3) Analysis of cytokine expression by quantitative PCR RNA was extracted from colon tissue and cDNA was prepared. Cytokine (TNF-α, IL-6, IL-17A, IL-10, and TGF-β) expression was analyzed by quantitative PCR. The results are shown in Figure 3a to Figure 3e. TNF-α mRNA was significantly higher in the DSS-treated group, and L8 reduced this expression level (Fig. 3a). Furthermore, L8 tended to reduce the expression of IL-6 and IL-17A, cytokines that aggravate intestinal inflammation (Fig. 3b, c). IL-10 and TGF-β expression was comparable among the four groups (Fig. 3d, e).

[0036] (Test 4) Analysis of cytokine expression by ELISA (Enzyme-linked immunosorbent assay) To examine cytokine expression at the protein level, colon tissue homogenates were used to analyze the levels of TNF-α in the tissues using a commercially available ELISA kit, and the results are shown in Figure 3f. The TNF-α concentration was significantly decreased in the L8 / DSS group compared with the vehicle / DSS group.

[0037] (Test 5) Analysis of intestinal flora 16S rRNA gene sequencing was performed using fecal samples collected from mice (n=5 for each group). First, DNA was extracted and the V3-V4 region of 16S ribosomal RNA (rRNA) was amplified by PCR. The resulting amplicons were sequenced using Illumina MiSeq and analyzed using QIIME2 to examine the ratio of constituent bacteria. The results are shown in Figure 4. The main family-level constituent bacteria were Lactobacillaceae and Bacteroidaceae in the non-inflammation group, whereas Enterobacteriaceae in the inflammation-induced group (Fig. 4a). The intestinal microbiota diversity was reduced in mice with inflammation (Fig. 4b). The Shannon index, an index of α diversity, tended to increase in the L8 / DSS group, but this was not significant (Fig. 4b). Compared with the vehicle / water group, the ratio of Bacteroides was reduced in the L8 / water group (Fig. 4c). Furthermore, compared with the vehicle / DSS group, the ratio of Clostridiaceae was higher and the ratio of Rikenellaceae was lower in the L8 / DSS group (Fig. 4d, Fig. 4e).

[0038] (Test 6) Effect of heat-killed L8 bacteria on cytokine production in macrophages C57BL6 / J female mice were intraperitoneally administered 2 mL of 4% thioglycollate. Three days later, the peritoneal cavity was washed with 10 mL of phosphate-buffered saline (PBS) to collect free cells in the peritoneal cavity. The cells were seeded on a culture plate, and one hour later, the plate was washed to remove unadhered cells, yielding peritoneal macrophages.

[0039] Peritoneal macrophages prepared as described above were cultured at 10 5 ~10 7 The cells were stimulated with 100 ng / mL of heat-killed L8 bacteria and E. coli-derived LPS (lipopolysaccharide) (100 ng / mL). After 16 hours, the culture supernatant was collected and the amount of IL-12 and IL-10 produced was analyzed by ELISA. Among the cytokines produced by macrophages, IL-12 acts on T cells and activates the immune response. On the other hand, IL-10 is known as an anti-inflammatory cytokine and has the effect of suppressing excessive immune responses. Neither cytokine was produced by L8 administration alone. When co-stimulated with LPS, IL-12 was produced at high concentrations (10 7 IL-10 production was significantly increased at low concentrations (10 5The IL-10 production was significantly increased even in the case of heat-killed L8 (cells / mL) (Fig. 6a, b). Therefore, the results of this study indicate that L8 can affect the immune response of macrophages even in the case of heat-killed L8, and that the effect may differ depending on the concentration. In addition, the enhancement of IL-10 production under low-concentration stimulation of L8 suggests that it may be effective against intestinal inflammation through its anti-inflammatory effect.

[0040] (Reference example) Creation of animal models Ten 6-week-old C57BL6 / J female mice were purchased from CLEA Japan. After one week of preliminary breeding, they were divided into two groups (vehicle / DSS group (n=5) and L8 / DSS group (n=5). Starch was administered to the vehicle / DSS group, and non-microencapsulated L8 dry powder was dissolved in distilled water and administered to the L8 / DSS group using an oral administration sonde every day for 15 days (1x10 lactic acid bacteria count). 9 The mice were allowed to drink 3.5% DSS ad libitum from day 3 to day 10, and enteritis was induced in the mice. The mice were weighed for each group from day 0 to day 15. On day 12, the mice were dissected and the colon tissues were collected. Histological evaluation was performed using the collected colon tissues as in the examples. The results are shown in FIG. 5.

[0041] Body weight loss was greater in the L8 / DSS group than in the vehicle / DSS group (Figure 5a). Comparing the length of the colon, the L8 / DSS group was significantly longer than the vehicle / DSS group (Figure 5b). Colon tissue was almost equivalent in both groups (Figures 5c-d).

[0042] (result) The above test showed that heat-killed L8 bacteria improved intestinal mucosal damage in mice with DSS colitis. Compared to the vehicle / DSS group, inflammatory cell infiltration was milder in the L8 / DSS group. As reflected by histological findings, TNF-α expression was elevated in mice with colitis, but administration of L8 significantly reduced this level. TNF-α is a multifunctional cytokine that is also involved in the pathology of chronic colitis. Since anti-TNF-α antibody preparations are highly effective in IBD patients, this molecule is considered to be an important therapeutic target. Therefore, the results of this test suggest that L8 may also be effective against the pathology of inflammatory bowel disease.

[0043] Lactic acid bacteria probiotics are thought to suppress enteritis by suppressing the proliferation of pathogenic bacteria and enhancing the host's immune function. There is debate as to whether heat-killed bacteria, which are not live bacteria, directly change the composition of the intestinal flora, but it is gradually becoming clear that they can modify the composition of the intestinal flora when improving enteritis. It has been reported that mice administered Bifidobacterim bifidum B1628 increased Lactobacillus, and administration of Lactobacillus acidphilus PIN7 suppressed the increase in Proteobacteria. It is known that dysbiosis similar to that observed in patients with inflammatory bowel disease is observed in DSS enteritis mice, and in particular, an increase in E. coli, Bacteroides, and Helicobacter has been confirmed. In this study, the ratio of Enterobacteriaceae was significantly increased in DSS enteritis mice. L8 did not change the ratio of such harmful bacteria (Figure 4a). Therefore, it is thought that L8 itself does not cause enteritis. [Industrial Applicability]

[0044] The present invention provides a therapeutic agent and composition for inflammatory bowel diseases, such as ulcerative colitis or Crohn's disease, which contain Lactococcus lactis subsp. lactis L8 as an active ingredient. Furthermore, it is possible to suppress TNF-α expression in the intestine or change the composition of the intestinal microflora by using a composition containing Lactococcus lactis subsp. lactis L8. [Brief description of the drawings]

[0045] [Figure 1] Figure 1a is a graph showing the changes in body weight in the vehicle / water group, the L8 / water group, the vehicle / DSS group, and the L8 / DSS group. Figure 1b is a graph comparing the intestinal length of the colon in each group, and Figure 1c is a photograph of the colon in each group. **P<0.01, *P<0.05, ns no significant difference. [Diagram 2] Figures 2a to 2d are photographs showing histological findings of inflammation in the vehicle / water group, the L8 / water group, the vehicle / DSS group, and the L8 / DSS group. Figure 2e is a diagram showing histological findings of inflammation in the vehicle / DSS group compared with the vehicle / water group. *P<0.05. [Diagram 3] Figures 3a to 3e are graphs showing the analysis of cytokine (TNF-α, IL-6, IL-17A, IL-10, and TGF-β) expression by quantitative PCR. Figure 3f is a graph showing the analysis of cytokine expression by ELISA. ***P<0.001, **P<0.01, *P<0.05, ns not significant. [Figure 4]Figure 4a shows the ratio of constituent bacteria in fecal samples from the vehicle / water group, the L8 / water group, the vehicle / DSS group, and the L8 / DSS group. Figure 4b shows the analysis results of the Shannon index, an index of alpha diversity, in each group. Figure 4c shows the ratio of Bacteroides in the vehicle / water group and the L8 / water group, Figure 4d shows the ratio of Clostridiaceae in the vehicle / DSS group and the L8 / DSS group, and Figure 4e shows the ratio of Rikenellaceae in the vehicle / DSS group and the L8 / DSS group. **P<0.01, ns no significant difference. [Diagram 5] Figure 5a is a graph showing the changes in body weight in the vehicle / DSS group and the L8 / DSS group. Figure 5b is a graph comparing the intestinal length of the colon in each group. Figures 5c and 5d are photographs showing histological findings of inflammation in each group. *P<0.05. [Figure 6a] We investigated the effect of L8 heat-killed bacteria on IL-12 production by macrophages. IL-12 production was significantly increased at high concentrations (107 cells / mL). ns: not statistically significant, *p<0.01, **p<0.001 (common to Fig. 6a and Fig. 6b). [Figure 6b] We investigated the effect of L8 heat-killed bacteria on IL-10 production by macrophages. IL-10 production was significantly increased at a range of concentrations from low (105 cells / mL) to high (107 cells / mL). In particular, it was significantly increased at the low concentration (105 cells / mL).

Claims

1. A therapeutic agent for inflammatory bowel disease, comprising Lactococcus lactis subsp. lactis L8 strain as an active ingredient.

2. A composition for treating inflammatory bowel disease, comprising Lactococcus lactis subsp. lactis L8 strain.

3. The composition according to claim 2, which exerts a therapeutic effect on inflammatory bowel disease by suppressing TNF-α expression.

4. The composition according to claim 3, which exerts a therapeutic effect on inflammatory bowel disease by changing the composition of intestinal bacterial flora.

5. A composition for suppressing TNF-α expression, comprising Lactococcus lactis subsp. lactis L8 strain.

6. A composition for altering the composition of the intestinal microbiota, comprising Lactococcus lactis subsp. lactis L8 strain.

7. The composition of claim 6, which alters the composition of the intestinal flora by increasing the proportion of Clostridiaceae.

8. The composition according to claim 6 or 7, which alters the composition of the intestinal flora by reducing the proportion of Rikenellaceae.

9. The composition according to any one of claims 2 to 8, which is a microcapsule.

10. The therapeutic agent according to claim 1, or the composition according to any one of claims 2 to 9, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

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