Inflammatory diseases, comprising <i>Lactobacillus sakei </i>CVL-001 strain

Abstract

The present invention relates to a composition for preventing or treating inflammatory diseases, comprising a Lactobacillus sakei CVL-001 strain. The composition activates NOD2, which is an inflammation regulator, and exhibits the effects of inhibiting weight loss and improving clinical activity scores (disease activity index; DAI) in an animal model in which ulcerative colitis is induced, and thus the present invention can be effectively used in the prevention, treatment or alleviation of inflammatory diseases.

Claims

1. A method for alleviating or treating an inflammatory disease, the method comprising: administering to a subject in need thereof a composition comprising an effective amount of a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC 13816BP, wherein the strain is an inactivated form obtained by killing with heat, wherein the composition comprises the strain at a concentration of 510.sup.9 CFU/ml.

2. The method of claim 1, wherein the inflammatory disease is at least one selected from the group consisting of ulcerative colitis, ulcerative duodenitis, Crohn's disease, irritable bowel syndrome, intestinal Behcet's disease, hemorrhagic rectal ulcer, pouchitis, enteritis, ischemic colitis, extra-intestinal manifestations, dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, pharyngitis, tonsillitis, pneumonia, pancreatitis, gastritis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, and multiple sclerosis.

3. The method of claim 1, wherein the composition comprises a pharmaceutically acceptable carrier which is selected from the group comprising lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oils.

4. The method of claim 3, wherein the composition further comprises a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, and a preservative.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a graph comparing NOD2 activity levels by various types of lactic acid strains.

(2) FIG. 2 is a schematic diagram showing a schedule of constructing an ulcerative colitis mouse model through feeding of dextran sodium sulfate (DSS) to mice.

(3) FIG. 3 is a graph showing body weight changes by the feeding of Lactobacillus sakei CVL-001 strain in ulcerative colitis-induced mouse individuals.

(4) FIG. 4 is a graph showing changes in disease activity index (DAI) by the feeding of Lactobacillus sakei CVL-001 strain in ulcerative colitis-induced mouse individuals.

(5) FIG. 5 is a graph showing changes in colon length by the feeding of Lactobacillus sakei CVL-001 strain in ulcerative colitis-induced mouse individuals.

(6) FIG. 6A shows hematoxylin and eosin (H&E) staining images of colon tissues by the feeding of Lactobacillus sakei CVL-001 strain in ulcerative colitis-induced mouse individuals.

(7) FIG. 6B is a graph showing colon tissue changes by the feeding of Lactobacillus sakei CVL-001 strain in ulcerative colitis-induced mouse individuals, as determined by histologic scoring using H&E staining.

(8) FIG. 7 is a graph showing body weight changes by the feeding of the heat-treated Lactobacillus sakei CVL-001 strain and the culture in ulcerative colitis-induced mouse individuals.

(9) FIG. 8 is a graph showing DAI changes by the feeding of the heat-treated Lactobacillus sakei CVL-001 strain and the culture in ulcerative colitis-induced mouse individuals.

(10) FIG. 9 is a graph showing body weight changes by the feeding of heat-treated Lactobacillus sakei CVL-001 strain in ulcerative colitis-induced mouse individuals.

(11) FIG. 10 is a graph showing DAI changes by the feeding of heat-treated Lactobacillus sakei CVL-001 strain in ulcerative colitis-induced mouse individuals.

(12) FIG. 11 is a graph showing colon length changes by the feeding of heat-treated Lactobacillus sakei CVL-001 strain in ulcerative colitis-induced mouse individuals.

(13) FIG. 12A shows H&E staining images of colon tissues by the feeding of heat-treated Lactobacillus sakei CVL-001 strain in ulcerative colitis-induced mouse individuals.

(14) FIG. 12B is a graph showing colon tissue changes by the feeding of heat-treated Lactobacillus sakei CVL-001 strain in ulcerative colitis-induced mouse individuals, as determined by histologic scoring using H&E staining.

BEST MODE FOR CARRYING OUT THE INVENTION

(15) The present disclosure is directed to a pharmaceutical composition for preventing or treating an inflammatory disease, the pharmaceutical composition containing the Lactobacillus sakei CVL-001 strain deposited under Accession Number KCTC13816BP.

MODE FOR CARRYING OUT THE INVENTION

(16) Hereinafter, the present disclosure will be described in more detail by the following examples. However, these exemplary embodiments are used only for illustration, and the scope of the present disclosure is not limited by these exemplary embodiments.

(17) Throughout the present specification, the % used to express the concentration of a specific material, unless otherwise particularly stated, refers to (wt/wt) % for solid/solid, (wt/vol) % for solid/liquid, and (vol/vol) % for liquid/liquid.

Example 1: NOD2 Activity Level by Lactobacillus sakei CVL-001 Strain

(18) Existing literature has reported that the protective effect of lactic acid bacteria on inflammatory bowel disease results from the anti-inflammatory effect of NOD2, which is an innate immune receptor protein. Therefore, it was investigated whether several types of lactic acid bacteria derived from kimchi had an ability to stimulate NOD2 in HEK293T cells.

(19) Specifically, HEK293 cells were treated with 1 MOI of several types of lactic acid bacteria, which had been transduced to generate luciferase by transcription of the luciferase gene through the stimulation of NOD2. For a control with respect to cell activity, HEK293 cells were treated with 10 ng/ml muramyl dipeptide (MDP) capable of stimulating NOD2 and then cultured in a cell incubator for 24 hours. Thereafter, the NOD2 activity was determined by luciferase assay on the luciferase generated from cell rupture. MDP, which is a component of the peptidoglycan constituting the cell wall of bacteria, is a ligand that directly acts on NOD2, and this was used as a control.

(20) TABLE-US-00001 TABLE 1 Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus sakei Strain MDP curvatus plantrum paracasei brevis graminis CVL-001 NOD2 1.00 40.70 4.11 4.01 1.39 0.83 3.64 23.63 Activity

(21) As can be confirmed from Table 1 and FIG. 1, the Lactobacillus sakei CVL-001 strain showed the highest level in NOD2 activity, and therefore, the Lactobacillus sakei CVL-001 strain was used for tests since the strain was predicted to exhibit a protective effect on inflammatory bowel disease.

Example 2: Culture of Lactobacillus sakei CVL-001

(22) The Lactobacillus sakei CVL-001 strain was inoculated (by streaking) on MRS agar medium, a selective medium for lactic acid bacteria, and cultured in a 30 C. bacterial incubator for 24 hours. Thereafter, single colonies were extracted and suspended in MRS broth, and the MRS broth thus obtained were pre-cultured for 12 to 16 hours in a bacterial incubator under conditions of 30 C. and 150 rpm.

(23) To obtain fresh bacteria, the pre-cultured bacteria were subjected to main culturing in fresh MRS broth for 2 to 3 hours by using a bacterial incubator under conditions of 30 C. and 150 rpm. The main-cultured bacteria were centrifuged at 3000 rpm for 15 minutes using a centrifuge, and the centrifuged bacteria were washed with phosphate buffer saline (PBS), followed by quantification through absorbance measurement. The quantified bacteria were used, along with PBS as a solvent, by the administration to mice.

Example 3: Ulcerative Colitis Mouse Model Construction (1)

(24) It is known that the feeding of dextran sodium sulfate (DSS) to mice usually causes inflammatory bowel disease, similar to ulcerative colitis in humans, followed by changes in body weight, clinical symptoms, colon length, and the like. Therefore, an ulcerative colitis mouse model was constructed by DSS feeding as shown in FIG. 2.

(25) Specifically, 54 C57BL/6J mice aged 7 weeks were grouped into six. To reduce the variation between groups, the mice were divided as follows such that the average body weight was constant. Group 1: water feeding (non-disease)+PBS Group 2: water feeding+10.sup.9 CFU of Lactobacillus sakei CVL-001 (high concentration of lactic acid bacteria) Group 3: DSS feeding (disease)+PBS Group 4: DSS feeding+10.sup.7 CFU of Lactobacillus sakei CVL-001 (low concentration of lactic acid bacteria) Group 5: DSS feeding+10.sup.8 CFU of Lactobacillus sakei CVL-001 (medium concentration of lactic acid bacteria) Group 6: DSS feeding+10.sup.9 CFU of Lactobacillus sakei CVL-001 (high concentration of lactic acid bacteria)

(26) The number of mice in each group was 9. The mice were acclimatized for 7 days, and then orally administered the Lactobacillus sakei CVL-001 strain at 200 l per individual once a day for 10 days. The mice were fed with water ad libitum, obtained by dissolving DSS in sterile tap water to 2%, for six days from the 10th day, and then, the recovery pattern was observed while the mice were fed with only sterile tap water ad libitum. The lactic acid bacteria were continuously administered until the test ended.

Example 4: Protective Effect of Lactobacillus sakei CVL-001 Strain on Ulcerative Colitis-Induced Mice

(27) 4-1. Changes in Body Weight and Disease Activity Index

(28) The ulcerative colitis-induced mice of Example 3 were administered the Lactobacillus sakei CVL-001 strain and measured for the body weight change (unit: %) and the change in disease activity index (DAI) indicating the extent of colitis. The results showed that the mice were recovered in proportion to the concentration of administration.

(29) TABLE-US-00002 TABLE 2 DSS + DSS + DSS + L. sakei L. sakei L. sakei L. sakei (10.sup.9 DSS + (10.sup.7 (10.sup.8 (10.sup.9 Day PBS CFU) PBS CFU) CFU) CFU) 0 1 1.02 0.10 1.26 0.14 1.73 0.65 2 1.38 0.69 2.65 0.38 1.72 1.61 3 3.57 0.94 2.52 1.84 0.13 3.94 4 5.53 3.55 3.85 3.02 3.28 4.84 5 4.16 4.27 1.37 1.10 0.82 1.87 6 4.78 3.36 3.08 4.81 4.38 2.51 7 5.62 3.02 11.61 11.23 10.78 8.80 8 5.75 3.60 16.07 15.90 16.16 12.81 9 6.10 4.05 19.49 18.69 19.09 15.99 10 5.60 4.50 22.14 20.37 19.91 16.54 11 7.15 5.06 22.26 18.55 17.26 15.32 12 7.88 4.81 20.38 13.83 12.03 9.59 13 6.17 3.32 16.74 11.00 7.66 6.41 14 8.21 4.03 15.24 9.29 5.17 2.69

(30) As can be confirmed in FIG. 3 and Table 2, the feeding of DSS to the mice caused diarrhea and bloody stools, resulting in weight loss of individuals. However, the body weight of the individuals was recovered in a concentration-dependent manner in the groups fed with the lactic acid bacteria. Especially, the degree of weight loss by DSS was relatively reduced and the recovery rate was also significantly increased in the high concentration administration group (10.sup.9 CFU/mice) compared with the other groups.

(31) TABLE-US-00003 TABLE 3 DSS + DSS + DSS + L. sakei L. sakei L. sakei L. sakei (10.sup.9 DSS + (10.sup.7 (10.sup.8 (10.sup.9 Day PBS CFU) PBS CFU) CFU) CFU) 0 1 0.00 0.00 0.00 0.00 0.00 0.00 2 0.00 0.00 0.00 0.00 0.00 0.00 3 0.00 0.00 0.00 0.00 0.00 0.00 4 0.00 0.00 1.17 1.61 1.50 0.78 5 0.00 0.00 2.56 3.89 2.89 3.11 6 0.00 0.00 7.22 8.33 7.00 6.56 7 0.00 0.00 6.89 5.72 5.33 5.11 8 0.00 0.00 6.89 5.83 6.17 5.56 9 0.00 0.00 6.56 6.17 6.50 5.89 10 0.00 0.00 6.72 5.56 5.89 5.00 11 0.00 0.00 5.50 4.67 4.67 4.61 12 0.00 0.00 5.72 5.22 5.11 4.44 13 0.00 0.00 5.17 4.17 4.00 3.39 14 0.00 0.00 3.39 2.33 2.00 1.22

(32) As can be confirmed in FIG. 4 and Table 3, the disease activity index of the mouse individuals, which was determined by observing the change of body weight, the state of stools, and the extent of bloody stool, were recovered.

(33) Therefore, the protective effect of the Lactobacillus sakei CVL-001 strain on inflammatory bowel disease could be confirmed.

(34) 4-2. Changes in Colon Length and Histologic Score Index Recovery

(35) Previous studies have reported that the colon length was shortened in groups fed with DSS when mouse organs were observed through autopsy. In a case where DSS causes damage to the colonic epithelium and disrupts the integrity of the epithelium, the in-vivo system, for recovery, induces an action to improve the integrity of the epithelium through various recovery mechanisms, such as fibrosis. Such an action shortens the overall colon length, and thus, the extent of damage to the colonic epithelium can be evaluated from the shortening of the colon length.

(36) The groups administered the lactic acid bacteria were measured for colon length and investigated for the change in histologic scoring index by examination of the epithelium integrity and inflammatory cell invasion.

(37) TABLE-US-00004 TABLE 4 DSS + DSS + DSS + L. sakei L. sakei L. sakei L. sakei (10.sup.9 DSS + (10.sup.7 (10.sup.8 (10.sup.9 Day PBS CFU) PBS CFU) CFU) CFU) Colon 77.22 77.00 54.22 62.00 60.56 64.67 length (mm)

(38) As can be confirmed in Table 4 and FIG. 5, the colon length was recovered in a concentration-dependent manner, according to the amount of administration of Lactobacillus sakei CVL-001 strain, in the groups having the disease caused by the feeding of DSS.

(39) As for histologic scoring, the colon tissue was extracted from the mice, fixed with formalin, and subjected to hematoxylin and eosin (H&E) staining, to prepare a sample, and then the sample was observed to determine the histologic scoring index.

(40) TABLE-US-00005 TABLE 5 DSS + DSS + DSS + L. sakei L. sakei L. sakei L. sakei (10.sup.9 DSS + (10.sup.7 (10.sup.8 (10.sup.9 Day PBS CFU) PBS CFU) CFU) CFU) Histologic 4.40 3.05 2.51 2.21 scoring index

(41) As can be confirmed in Table 5 and FIGS. 6A and 6B, the histologic scoring index was recovered, considering the integrity of the epithelium and the reduction in inflammatory cell invasion in a concentration-dependent manner, in the groups administered the Lactobacillus sakei CVL-001 strain.

Example 5: Ulcerative Colitis Mouse Model Construction (2)

(42) To determine what characteristics of the strain exhibit a protective effect on inflammatory bowel disease, heat-treated strains having only the characteristics of strain walls were prepared by heat killing (H.K.) of the Lactobacillus sakei CVL-001 strain through heat treatment. In addition, test groups were established by preparing the heat-treated strain from a culture containing strain metabolites and the culture excluding the strain while constructing the ulcerative colitis mouse model in the same manner as in Example 3. Group 1: water feeding (non-disease)+PBS Group 2: DSS feeding (disease)+PBS Group 3: DSS feeding+10.sup.9 CFU Lactobacillus sakei CVL-001 strain (H.K.) Group 4: DSS feeding+Lactobacillus sakei CVL-001 culture

(43) The mice were acclimatized for 7 days, and then orally administered the heat-treated Lactobacillus sakei CVL-001 strain and the culture at 200 l per mouse once a day for 10 days. The number of mice for each group was five or six. The rest of the procedure was carried out in the same manner as in Example 3.

Example 6: Protective Effects of Heat-Treated Lactobacillus sakei CVL-001 Strain and Culture on Ulcerative Colitis-Induced Mice

(44) 6-1. Changes in Body Weight and Disease Activity Index

(45) The ulcerative colitis-induced mice in Example 5 were administered the heat-treated Lactobacillus sakei CVL-001 strain and the culture and then measured for the weight change (unit: %) and DAI change, and as a result, the recovery of the mice was observed in only the group using the heat-treated strain.

(46) TABLE-US-00006 TABLE 6 DSS + L. sakei DSS + L. sakei Day PBS DSS + PBS (10.sup.9 CFU) sup 0 1 0.00 0.00 0.00 0.00 2 0.74 0.21 1.20 0.51 3 2.73 1.36 0.93 1.55 4 3.17 1.46 1.20 1.19 5 1.04 2.01 2.36 4.57 6 6.59 4.23 3.91 7.72 7 7.98 7.70 4.94 10.34 8 2.75 13.89 7.61 17.90 9 4.11 16.96 8.49 22.77 10 7.37 19.23 7.05 24.57 11 9.57 20.84 4.16 25.50 12 7.29 19.69 2.28 22.02

(47) As can be confirmed in FIG. 7 and Table 6, the weight of the individual was recovered in the group fed with the lactic acid bacteria killed by heat treatment. Especially, the degree of weight loss by DSS was relatively reduced and the recovery rate was also significantly increased in the high-concentration administration group (10.sup.9 CFU/mouse) compared with the other groups. The group treated with the culture showed no protective effect.

(48) TABLE-US-00007 TABLE 7 DSS + L. sakei DSS + L. sakei Day PBS DSS + PBS (10.sup.9 CFU) sup 0 1 0.00 0.00 0.00 0.00 2 0.00 0.00 0.00 0.00 3 0.00 0.00 0.00 0.00 4 0.00 2.90 2.50 2.83 5 0.00 6.40 3.42 6.25 6 0.00 6.80 3.75 9.67 7 0.00 7.80 3.50 7.33 8 0.00 5.50 3.42 6.17 9 0.00 5.20 3.00 5.83 10 0.00 5.00 2.08 5.50 11 0.00 3.80 1.42 4.83 12 0.00 2.10 0.67 3.50

(49) As can be confirmed in FIG. 8 and Table 7, the recovery of the mice was observed when the mice were administered the lactic acid bacteria killed by heat treatment and measured for the DAI change. The group treated with the culture showed no protective effect.

(50) For the improvement of reproducibility and reliability, tests for weight and disease activity index were again carried out for only the heat-treated Lactobacillus sakei CVL-001 strain low concentration (10.sup.7 CFU) and high concentration (10.sup.9 CFU) groups, with the exclusion of the culture treatment group, in the test groups established in Example 5.

(51) The mice were acclimatized for 7 days, and then orally administered the heat-treated Lactobacillus sakei CVL-001 strain at 200 l per mouse once a day for 10 days. The number of mice for each group was nine. The rest of the procedure was carried out in the same manner as in Example 3.

(52) TABLE-US-00008 TABLE 8 DSS + L. sakei DSS + L. sakei Day PBS DSS + PBS (10.sup.7 CFU; HK) (10.sup.9 CFU; HK) 0 1 0.43 0.38 0.45 0.36 2 0.01 0.26 0.60 0.19 3 0.59 0.78 0.42 0.51 4 0.75 0.56 0.48 0.96 5 0.80 3.59 3.95 2.79 6 1.59 10.97 8.68 4.84 7 1.93 17.28 13.05 7.36 8 1.55 21.54 14.59 7.43 9 2.17 24.35 14.96 4.92 10 1.71 25.42 12.66 0.45 11 5.14 24.08 8.21 2.48

(53) As can be confirmed in FIG. 9 and Table 8, the weight of the individuals was recovered in a concentration-dependent manner in the groups fed with the lactic acid bacteria killed by heat treatment. Especially, the degree of weight loss by DSS was relatively reduced and the recovery rate was also significantly increased in the high-concentration administration group (10.sup.9 CFU/mouse) compared with the other groups.

(54) TABLE-US-00009 TABLE 9 DSS + L. sakei DSS + L. sakei Day PBS DSS + PBS (10.sup.7 CFU; HK) (10.sup.9 CFU; HK) 0 1 0.00 0.00 0.00 0.00 2 0.00 0.00 0.00 0.00 3 0.00 0.00 0.00 0.00 4 0.00 1.94 2.39 2.06 5 0.00 3.83 4.06 2.56 6 0.00 7.67 6.22 4.89 7 0.00 7.83 5.44 4.28 8 0.00 6.78 4.44 3.11 9 0.00 5.67 3.67 3.17 10 0.00 5.28 3.50 1.39 11 0.00 5.11 3.06 0.56

(55) As can be confirmed in FIG. 10 and Table 9, the mice were recovered in a concentration-dependent manner when the mice were administered the lactic acid bacteria killed by heat treatment and measured for the DAI change. Especially, the clinical symptoms by DSS were relatively reduced and the recovery rate was also significantly increased in the high-concentration administration group (10.sup.9 CFU/mouse) compared with the other groups.

(56) 6-2. Changes in Colon Length and Histologic Scoring Index Recovery

(57) The groups administered the lactic acid bacteria were measured for colon length and investigated the change in histologic scoring index by examination of the epithelium integrity and inflammatory cell invasion.

(58) TABLE-US-00010 TABLE 10 DSS + L. sakei DSS + L. sakei day PBS DSS + PBS (10.sup.7 CFU; HK) (10.sup.9 CFU; HK) Colon length 78.44 51.56 59.11 70.33 (mm)

(59) As can be confirmed in Table 10 and FIG. 11, the colon length was recovered in a concentration-dependent manner in the groups administered the Lactobacillus sakei CVL-001 strain among the groups having the disease caused by the feeding of DSS.

(60) TABLE-US-00011 TABLE 11 DSS + L. sakei DSS + L. sakei Day PBS DSS + PBS (10.sup.7 CFU; HK) (10.sup.9 CFU; HK) Histologic scoring 0.00 4.88 3.70 1.60 index

(61) As can be confirmed from Table 11 and FIGS. 12A and 12B, the histologic scoring index was recovered in a concentration-dependent manner, considering the integrity of the intestinal epithelium and the reduction in inflammatory cell invasion, in the groups administered the Lactobacillus sakei CVL-001 strain.

INDUSTRIAL APPLICABILITY

(62) The present disclosure relates to a composition containing Lactobacillus sakei CVL-001 strain for preventing or treating an inflammatory disease and, more specifically, to a composition capable of exhibiting activity to prevent, treat, or alleviate an inflammatory disease by using Lactobacillus sakei CVL-001 strain.