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Keywords:

  • Lactobacillus brevis;
  • heat shock protein;
  • barrier function;
  • proinflammatory cytokines;
  • p38MAPK;
  • TNF-α;
  • IL-1β;
  • IL-12

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Background:

Probiotics have been clinically administered to improve intestinal damage in some intestinal inflammations. However, probiotic treatments are not always effective for these intestinal disorders because live bacteria must colonize and maintain their activity under unfavorable conditions in the intestinal lumen when displaying their functions. This study investigated the physiological functions of a heat-killed body of a novel probiotic, Lactobacillus brevis SBC8803, on the protection of intestinal tissues, the regulation of cytokine production, the improvement of intestinal injury, and the survival rate of mice with dextran sodium sulfate (DSS)-induced colitis.

Methods:

Heat shock protein (Hsp) induction and mitogen-activated protein kinase (MAPK) phosphorylation in intestinal epithelia by heat-killed L. brevis SBC8803 were examined by Western blotting. The barrier function of intestinal epithelia was measured with [3H]-mannitol flux in the small intestine under oxidant stress. The effects of the bacteria on improving epithelial injury and cumulative survival rate were investigated with a DSS colitis model.

Results:

Heat-killed L. brevis SBC8803 induced Hsps, phosphorylated p38 MAPK, regulated the expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β and IL-12, and improved the barrier function of intestinal epithelia under oxidant stress. The induction of Hsp and the protective effect were negated by p38 MAPK inhibitor. These functions relieve intestinal impairments and improve the survival rate in mice with lethal colitis.

Conclusions:

The administration of heat-killed L. brevis SBC8803 helps to successfully maintain intestinal homeostasis, while also curing intestinal inflammation. A therapeutic strategy using heat-killed bacteria is expected to be beneficial for human health even in conditions unsuitable for live probiotics because the heat-killed body is able to exhibit its effects without the requirement of colonization. (Inflamm Bowel Dis 2011;)

The intestinal microflora is a unique ecological environment where microorganisms normally live in a balanced relationship with the host.1 The relationship appears to be associated with bidirectional interactions that influence the behavior of microflora as well as host responses essential for the maintenance of intestinal homeostasis. Human intestinal microflora is composed of 300–500 different species of bacteria2, 3 and the number of microbial cells within the gut lumen is about 10 times larger than that of eukaryotic cells in the human body.4 The constitutive interaction between the host and microbia provides health benefits to the human body, including the metabolism of nutrients, fortification of the mucosal barrier, xenobiotic metabolism, angiogenesis,1 and development of intestinal lymphoid tissue.5

Bacteria that provide specific health benefits when consumed as a food component or supplement are called probiotics.6 Probiotics are protective against Candida infections,7Cryptosporidium parvum,8 and Helicobacter pylori,9 and possess anticarcinogenic activity10 in several animal models. The mechanisms of the effects of probiotics are beginning to be explored. Potential mechanisms have been proposed including the upregulation of mucus production,11 improvement in epithelial barrier function,12 increase in IgA production,13 increased competition for adhesion sites on intestinal epithelia,14 the activation of cell signaling,15 and the production of antibiotic peptide bacteriocins.16 We also demonstrated the cytoprotective effect of Bacillus subtilis through the induction of heat shock proteins (Hsps) and activation of p38 mitogen-activated protein kinase (MAPK) and Akt pathway and identified the effective molecule derived from Bacillus subtilis as a competence and sporulation factor (CSF). Moreover, an epithelial cell membrane transporter, novel organic cation transporter isotype 2, has been demonstrated to transport the CSF and mediate the cytoprotective effects of the CSF in the mouse and human intestine.17 Accordingly, the mechanisms of probiotic activities are thought to be complex and dependent on the bacterial strains.

Probiotic treatment has been clinically administered to improve abdominal symptoms18 and intestinal damage in patients with inflammatory bowel disease (IBD),19–23 antibiotics-induced colitis,24, 25 and necrotizing enterocolitis.26, 27 However, these probiotics are not always effective for treating these intestinal disorders28, 29 because such live bacteria are required to colonize and maintain their activity under the various conditions of the lumen when displaying their beneficial functions for host health. Intestinal conditions in most patients with intestinal disorders are diverse due to the augmentation of pathogenic bacteria and/or the administration of drugs which may be harmful for probiotics. Therefore, probiotic treatment with live bacteria is not stably effective for each case of the intestinal disorder. Heat-killed probiotics can potentially solve this problem and achieve stable effects because these processed materials do not need to live and colonize in such unsuitable circumstances while exhibiting their physiological function.

Lactobacillus brevis SBC8803 is a type of plant bacteria identified from malt fermentation. L. brevis SBC8803 can ameliorate ethanol-induced liver injury and fatty liver by suppressing the upregulation of tumor necrosis factor alpha (TNF-α) through the inhibition of gut-derived endotoxin migration into the liver, suggesting the physiological effect of L. brevis SBC8803 in the enhancement of the intestinal barrier function.30

The present study proposed that heat-killed body of L. brevis SBC8803 induced Hsps, activated the p38 MAPK pathway, regulated the secretes of proinflammatory cytokines, protected intestinal tissues from oxidant stress, and improved both the intestinal injury and survival rate of mice with dextran sulfate sodium (DSS)-induced colitis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Bacteria Preparation

L. brevis SBC8803 and -8013 stored in Frontier Laboratories of Value Creation (Sapporo Breweries, Japan) were incubated in Lactobacilli-MRS broth (Difco, Detroit, MI) at 37°C for 24 hours. Bifidobacterium longum and Streptococcus faecalis stored in our laboratory were also incubated in MRS broth. These bacteria were centrifuged to separate the bacterial body from its supernatant. The pellet was rinsed with phosphate-buffered saline (PBS) three times, heat-killed at 121°C for 20 minutes, freeze-dried, and used for the following experiments.

Cell Culture

Human colonic epithelial Caco2bbe cells purchased from the ATCC (American Type Culture Collection, Manassas, VA) were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin, and 10 μg/mL transferrin (all from Invitrogen/GIBCO, Grand Island, NY) in a humidified atmosphere of 5% CO2. The cells were plated on 6- or 12-well plates at a density of 105 cells/cm2 and then were allowed to differentiate for 10–14 days before. Heat-killed bacteria (SBC8803 and -8013, B. longum and S. faecalis) and/or 30 μM of SB203580, 50 μM of PD98059, 20 μM of SP600125, or 50 μM of LY294002 (Sigma-Aldrich, St. Louis, MO) was incubated with differentiated Caco2bbe cells for 24 hours to examine the Hsps induction with Western blotting.

Mice

The studies were approved by the Institutional Animal Care and Use Committee of the Asahikawa Medical College. C57Bl/6 mice were purchased from Sankyo Labo Service (Tokyo, Japan). Small and large intestines with or without treatments were removed, rinsed with ice-cold saline, and the epithelium was gently sheared off with glass slides for protein determination.

Western Blots

After washing Caco-2bbe cells or mouse intestinal mucosa with PBS, proteins were extracted from these samples with 200 μL lysis buffer (1% vol/vol Triton X-100, 20 mM Tris, pH 8, 50 mM NaCl, 5 mM EDTA, 0.2% wt/vol BSA, and complete protease cocktail; Roche Molecular Biochemicals, Indianapolis, IN) and analyzed by Western blotting. Five to 20 μg of each sample was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%–12%) and immediately transferred to a polyvinylidene difluoride (PVDF) membrane using 1× transfer buffer (25 mM Tris pH 8.8, 192 mM glycine with 15% [vol/vol] methanol). PVDF membranes were incubated in PBS with 0.05% (vol/vol) Tween 20 (T-PBS) containing 5% (wt/vol) milk for 1 hour at room temperature to block nonspecific binding. The blots were incubated overnight at 4°C with antihuman Hsp27, Hsp 70, Hsc70 antibody (Stressgen, Victoria, British Columbia, Canada), antihuman p38 MAPK and phosph-p38 MAPK, antihuman extracellular signal-regulated kinase (ERK) and phosphor-ERK, antihuman c-Jun N-terminal kinase (JNK) and phosphor-JNK, antihuman phosphatidylinositol 3-kinase (Akt), and phosphor Akt and antihuman β-actin (Cell Signaling, Beverly, MA) as the primary antibody. The blots were washed five times for 10 minutes each in T-PBS at room temperature, incubated for 60 minutes in species-appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA) in T-PBS, washed four times in T-PBS, once in PBS, and developed using the Super-Signal West Pico enhanced chemiluminescence system (Pierce Chemical, Rockford, IL).

Ex Vivo Intestinal Loop Studies

C57Bl/6 mice (6 weeks old, male) were sacrificed and the small intestine was removed beginning at the ligament of Treitz. The first 18 cm were divided into three 6-cm lengths, each end ligated with silk suture, and the loops filled with RPMI 1640 medium, with or without concentrations of heat-killed L. brevis SBC8803. Loops were filled to moderate distention, about 1 mL per loop. Loops were placed in the organ culture dishes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) which were filled with 25 mL media as above. Loops were incubated for 30 minutes or 2 hours at 37°C in a 5% CO2 incubator. Mucosa were scraped off with glass slides and processed for protein analysis as described above. The segments were filled with RPMI 1640 medium with 1 mM mannitol and 1 mCi/mL [3H]-mannitol, and with or without 0.3 mM freshly prepared monochloramine to measure permeability effects. Loops were placed into the organ culture dish in 25 mL of RPMI 1640. Samples were taken at 5, 20, and 35 minutes to determine the flux of mannitol from lumen to the medium outside the bathing loops. The flux data at 15 and 30 minutes were calculated by subtracting that at 5 minutes from that at 20 and 35 minutes.

DSS-induced Colitis In Vivo

DSS with a molecular weight of 25,000 (Tokyo Chemical Industry, Japan) was dissolved in tap water to obtain a 2%–4% DSS solution. C57Bl/6 mice (18–25 g, 6 weeks old, male) were allowed free access to 3% DSS solution as drinking water for 5 days to develop a mouse model of DSS-induced colitis to determine the effect of heat-killed L. brevis SBC8803 on the treatment of acute colitis.31 The mice were transanally administered 0.1% of heat-killed L. brevis SBC8803 or PBS every day from day 1 and then were sacrificed on day 7 or day 14, or from day 4 and then were sacrificed on day 8 after the initial administration of DSS. A 10-mm piece of their colon was taken for protein determination and a 5-mm piece was fixed in 10% buffered formalin, sectioned at 4 μm, and stained with hematoxylin and eosin for light microscopic examination. To examine mRNA expressions of cytokines by real-time polymerase chain reaction (PCR), the mice were allowed free access to 2% DSS solution as drinking water and anally administrated 0% or 0.1% heat-killed L. brevis for 10 days and then a 10-mm piece of their colon was taken for RNA extraction.

In another set of experiments the weight and cumulative survival rate of the mice that were allowed free access to 4% DSS solution as drinking water and transanally administered either heat-killed L. brevis SBC8803 or PBS every day was investigated.32

Histological Analysis

The histological activity was assessed according to Berg's score described below.33 The grades of the intestinal inflammation were assessed in three representative parts of the colon in each mouse because of multifocal and variable severities of the intestinal lesions. A score from 0 to 4 was based on the following criteria: (grade 0) no change from normal tissue; (grade 1) one or a few multifocal mononuclear cell infiltrates in the lamina propria accompanied by minimal epithelial hyperplasia and slight to no depletion of mucus from goblet cells; (grade 2) the lesions tended to involve more of the intestine than grade 1 lesions, or were more frequent. Typical changes included mild inflammatory cell infiltrates in the lamina propria composed primarily of mononuclear cells with a few neutrophils. Small epithelial erosions were occasionally present and inflammation rarely involved the submucosa; (grade 3) lesions involved a large area of mucosa or were more frequent than grade 2 lesions. Inflammation was moderate and often involved the submucosa but was rarely transmural. Inflammatory cells were a mixture of mononuclear cells as well as neutrophils, and crypt abscesses were sometimes observed. Ulcers were occasionally observed; (grade 4) such lesions usually involved most of the intestinal section and were more severe than grade 3 lesions. Inflammation was severe, including mononuclear cells and neutrophils, and it was sometimes transmural. Crypt abscesses and ulcers were present.

Real-time PCR

RNA was harvested from large intestine of the mice administered 2% DSS as a drinking water using Trizol. RNA was reverse-transcribed using random primers and Superscript II RT (Invitrogen). mRNA of TNF-α, IL-1β, IL-6, IL-10, IL-12, and IL-17 was amplified using the iCycler iQ real-time PCR System (Bio-Rad Laboratories, Hercules, CA) using specific primers (sense, 5′-aagcctgtagcccacgtcgta-3′, antisense, 5′-ggcaccactagttggttgtctttg-3′ for the analysis of TNF-α, sense, 5′-tccaggatgaggacatgagcac-3′, antisense, 5′-gaacgtcacacaccagcaggtta-3′ for the analysis of IL-1β, sense, 5′-aagccagagctgtgcagatgagta-3′, antisense, 5′-tgtcctgcagccactggttc-3′ for the analysis of IL-6, sense, 5′-gaccagctggacaacatactgctaa-3′, antisense, 5′-gataaggcttggcaacccaagtaa-3′ for the analysis of IL-10, sense, 5′-tgtcttagccagtcccgaaacc-3′, antisense, 5′-tcttcatgatcgatgtcttcagcag-3′ for the analysis of IL-12, sense, 5′-tcacgagcgctccatctca-3′, antisense, 5′-ttctgtgggtagcggttctcatc-3′ for the analysis of IL-17c, sense, 5′-atgaagtgcacccgtgaaacag-3′, antisense, 5′-ctcagaatggcaagtcccaaca-3′ for the analysis of IL-17f) in triplicate. The averaged mRNA expression of these cytokines was normalized to β2-microgloblin expression (β2-microgloblin specific primers; sense, 5′-catccgtaaagacctctatgccaac-3′, antisense, 5′-atggagccaccgatccaca-3′).

Barrier Function Test in Caco2bbe Cells

Caco2bbe cells were grown in a transwell plate (Corning, Horsham, PA) for 14 days to form an orderly monolayer and then they were incubated with heat-killed L. brevis SBC8803 or PBS for 2 hours. When examining the association of p38 MAPK, ERK, JNK, and the Akt pathway with the protective effect of heat-killed L. brevis SBC8803 and 30 μM of SB203580, 50 μM of PD98059, 20 μM of SP600125, or 50 μM of LY294002 was added with heat-killed L. brevis SBC8803. The apical side was filled with RPMI 1640 medium with 1 mM mannitol and 1 mCi/mL [3H]-mannitol, and with or without 0.3 mM freshly prepared monochloramine to determine the permeability effects. Samples were taken at 5, 65, and 125 minutes to determine the flux of mannitol from the apical side to the basal side. The flux data at 60 and 120 minutes were calculated by subtracting the data obtained at 5 minutes from that obtained at 65 and 125 minutes.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Hsp 27/25 and 70 Induction and MAPK Phosphorylation by Heat-killed L. brevis SBC8803

Hsps are essential for the maintenance of intestinal homeostasis,34–36 rendering colonic epithelial cells less susceptible to injury and stress.37 Therefore, the effects of heat-killed L. brevis SBC8803 on inducing Hsp27 expression in Caco2bbe were assessed. As shown in Figure 1A,B, 0.01% or 0.1% of heat-killed L. brevis SBC8803 induced Hsp27 in Caco2bbe cells, whereas 1.0% of the bacteria did not, thus suggesting the appropriate concentration of the heat-killed L. brevis SBC8803 required for protecting the intestinal epithelia to be around 0.01%–0.1%. Mouse small intestinal segments were filled with heat-killed L. brevis SBC8803 for 2 hours to elucidate Hsp induction by L. brevis SBC8803 ex vivo. Then Hsp25 and 70 induction was examined with Western blotting. Hsp25 (Fig. 1C,D) and 70 (Fig. 1E,F) were induced by either 0.01% or 0.1% of heat-killed L. brevis SBC8803 in mice intestine. MRS broth and the heat-killed bodies of the B. longum, another strain of L. brevis (SBC8013) possessed no activity for inducing Hsp27/25 or 70. S. faecalis as well as L. brevis SBC8803 induced Hsps in vitro and ex vivo, thus suggesting that L. brevis SBC8803 has a strong but not specific effect on the induction of Hsps (Fig. 1G–J). The Hsp25 induction rates (percent of control) of 0.01% and 0.1% of heat-killed L. brevis SBC8803 were 315 and 338, respectively. Similarly, the Hsp70 induction rates of 0.01% and 0.1% of the heat-killed L. brevis SBC8803 were 133 and 170, respectively. The phosphorylation of key signaling molecules p38 MAPK, ERK, JNK, and Akt were assessed by Western blotting to examine the physiological function on the signaling pathways by L. brevis SBC8803. As shown in Figure 2, p38 MAPK was phosphorylated by 0.1% of heat-killed L. brevis SBC8803 within 30 minutes in the mouse small intestine, while no phosphorylation of ERK, JNK, and Akt were identified. These data indicated that the bacterial body functioned as a strong inducer for Hsp27/25 and 70 and activator for p38 MAPK.

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Figure 1. Heat-killed L. brevis SBC8803 induced Hsps in Caco2bbe cells and mice small intestine. Caco2bbe cells were incubated with 0, 0.01, 0.1, 1% (wt/vol) of heat-killed L. brevis SBC8803 for 16 hours. Protein was extracted from the treated cells and Hsp27 expression was examined with Western blotting. Hsp27 expression in Caco2bbe cells treated with 0.01 and 0.1% of heat-killed L. brevis SBC8803 was increased while that with 1% of heat-killed L. brevis SBC8803 did not (A,B). To elucidate the effect of heat-killed L. brevis SBC8803 on Hsp induction, mice small intestine were filled with 0, 0.01, 0.1% of heat-killed L. brevis SBC8803 for 2 hours. Proteins were extracted from the mucosa shaved from the small intestine and Hsp25 and 70 expression were examined with Western blotting. Both Hsp25 and 70 expression were significantly higher in heat-killed L. brevis SBC8803-treated intestines than that in controls (C–F). Conversely, MRS broth itself, and Bifidobacterium longum, L. brevis (SBC8013) did not induce Hsp25 and 70 either in vitro (G,H) or ex vivo (I,J). Streptococcus faecalis as well as L. brevis SBC8803 induced Hsps in vitro (G,H) and ex vivo (I,J), thus suggesting that L. brevis SBC8803 has a strong, but not specific effect on the induction of Hsps induced Hsp25/27 and 70. Image shown is representative of four separate experiments (A,C,E,G,I). Densitometry was performed using NIH ImageJ, setting the control to 100% for each analysis (B,D,F,H,J). *P < 0.05 in comparison to the control based on an analysis of variance.

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Figure 2. p38 MAPK phosphorylation was increased in mice small intestine treated with 0.1% heat-killed L. brevis SBC8803. Mice small intestine were filled with 0.1% of heat-killed L. brevis SBC8803 for 2 hours, and then the phosphorylation of key signaling molecules, p38 MAPK, ERK, JNK, and Akt were assessed with Western blotting. p38 MAPK was phosphorylated by 0.1% of heat-killed L. brevis SBC8803 within 30 minutes in mouse small intestine while no phosphorylation of ERK, JNK, and Akt were identified (A,B). Image shown is representative of four separate experiments (A). Densitometry was performed using NIH ImageJ, setting the control to 100% for each analysis (B). *P < 0.05 in comparison to the control based on an analysis of variance.

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Heat-killed L. brevis SBC8803 Protects Mice Intestine from Oxidant Stress

The barrier function of the intestinal epithelia was evaluated with a transmural [3H]-mannitol flux study to determine the physiological effect of the heat-killed L. brevis SBC8803 in the protection of the mouse intestine against oxidant stress. As shown in Figure 3A, increased mucosal permeability in small intestinal loops caused by exposure to an oxidant (NH2Cl 0.3 mM) was significantly inhibited by 0.1% of heat-killed L. brevis SBC8803. This indicated that heat-killed L. brevis SBC8803 possessed the ability to augment the barrier function of intestinal epithelia in the presence of oxidant stress. In contrast, the MRS broth itself, B. longum, S. faecalis, and L. brevis (SBC8013) possess no protective effects for intestinal epithelia against oxidant stress (Fig. 3B).

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Figure 3. Increased mucosal permeability in small intestinal loops caused by exposure to oxidant was inhibited by 0.1% of heat-killed L. brevis SBC8803. Mice small intestine was removed beginning at the ligament of Treitz. The first 18 cm were divided into three 6-cm lengths, each end ligated with silk suture, and the loops filled with RPMI 1640 medium, with or without concentrations of heat-killed L. brevis SBC8803. Loops were filled to moderate distention, about 1 mL per loop. Loops were placed in the organ culture dishes for 2 hours at 37°C in a 5% CO2 incubator and washed three times with PBS, and then the segments were filled with RPMI 1640 medium with 1 mM mannitol and 1 mCi/mL [3H]mannitol and with or without 0.3 mM freshly prepared monochloramine. Loops were placed into an organ culture dish in 25 mL of RPMI 1640. Samples were taken at 5, 20, and 35 minutes to determine the flux of mannitol from the lumen to the medium outside the bathing loops. Increased mucosal permeability in small intestinal loops caused by exposure to oxidant (NH2Cl 0.3 mM) was significantly inhibited by 0.1% of heat-killed L. brevis SBC8803 at 15 and 30 minutes (n = 4) (A), but not by 0.1% of B. longum, S. faecalis, and L. brevis (SBC8013) (B). *P < 0.05 in comparison to the control based on an analysis of variance.

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Heat-killed L. brevis SBC8803 Improves Intestinal Injury in DSS-induced Colitis Mice

Mice were allowed free access to 3% DSS solution as drinking water (for 5 days) and transanally administered 0.1% of heat-killed L. brevis SBC8803 or PBS every day. Seven days after the initial DSS treatment, the length of the large intestine was significantly longer in the heat-killed L. brevis SBC8803-administered mice than that in the PBS-administered mice, while the weight of mice revealed no difference between the control mice and heat-killed L. brevis SBC8803-administered mice (Fig. 4A–C). The inflammation grade of the large intestine in mice treated with heat-killed L. brevis SBC8803 was significantly lower than that in PBS-treated mice (Fig. 5A,B), suggesting the preventive effect of heat-killed L. brevis SBC8803 for the intestinal damage in DSS-induced colitis. Moreover, on day 14 after the initial treatment, the efficacy of heat-killed L. brevis SBC8803 for improving body weight loss, intestinal shortening (Fig. 4D–F), and histological severity of intestinal inflammation (Fig. 5C,D) in the DSS-induced colitis mice was more clearly exhibited. In another set of experiments, on day 4 after the initial administration of DSS, while the body weight loss was not improved (Fig. 4G), the administration of heat-killed L. brevis SBC8803 relieved intestinal shortening (Fig. 4H,I) and ameliorated the histological grade of the intestinal inflammation (Fig. 5E,F) in DSS-induced colitis mice, thereby indicating the heat-killed L. brevis SBC8803 to possess a potential for treating intestinal impairment even after the development of inflammation.

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Figure 4. The transanal administration of 0.1% heat-killed L. brevis SBC8803 improved the shortening of the large intestine. Mice were allowed free access to 3% DSS solution as drinking water for 5 days and transanally administered 0.1% of heat-killed L. brevis SBC8803 or PBS every day. The weight of mice revealed no difference between the control mice and heat-killed L. brevis SBC8803 administered mice (n = 6) (A). In contrast, the length of large intestine was significantly longer in the mice administered heat-killed L. brevis SBC8803 than that in the PBS administered mice at 7 days after the initial DSS treatment (n = 6) (B,C). At 14 days after the initial treatment, the efficacy of heat-killed L. brevis SBC8803 for the improvement of weight loss (D) and intestinal shortening (E,F) in the DSS-induced colitis mice was enhanced. While the administration of heat-killed L. brevis SBC8803 from day 4 after the initial administration of DSS did not improve weight loss (G), it relieved the intestinal shortening (H,I). *P < 0.05 in comparison to the control based on an analysis of variance. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 5. The inflammation grade of large intestine in DSS-induced colitis mice administered with heat-killed L. brevis SBC8803 was significantly lower than that in PBS-treated mice. The histological findings are representative of four separate experiments. Severe infiltration of mononuclear cells and neutrophils and ulceration were seen in the large intestine of DSS-induced colitis mice treated with PBS, while only mild inflammatory cell infiltrates in the lamina propria composed of mononuclear cells and a few neutrophils were seen in those treated with heat-killed L. brevis SBC8803 (A). The inflammation grade of the large intestine in the mice treated with heat-killed L. brevis SBC8803 was significantly lower than that in PBS-treated mice (B). Moreover, at 14 days after the initial treatment heat-killed L. brevis SBC8803 dramatically improved the histological severity of intestinal inflammation in the DSS-induced colitis (C,D). In another set of experiments, the administration of heat-killed L. brevis SBC8803 from day 4 after the initial administration of DSS also ameliorated the histological grade of the intestinal inflammation in the DSS-induced colitis mice (E,F). The boxplot demonstrates the groups of numerical data through their five-number summaries: the smallest observation (the lower whisker), lower quartile (the bottom of the box), median (the band near the middle of the box), upper quartile (the top of the box), and largest observation (the upper whisker). Any data not included between the whiskers is plotted as an outlier with a dot (B,D,F). *P < 0.05 in comparison to the control based on an analysis of variance. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Heat-killed L. brevis SBC8803 Improved Survival Rate of Mice Treated with a Lethal Dose of DSS

The mice were treated with 4% DSS and anally administered heat-killed L. brevis SBC8803 every day to examine the effect of heat-killed L. brevis SBC8803 on survival rate of mice with severe colitis. As shown in Figure 6A, the weight of mice treated with heat-killed L. brevis SBC8803 was significantly heavier than that of PBS-treated mice. Whereas all PBS-treated mice died within 12 days, 80% of the mice anally treated with heat-killed L. brevis SBC8803 were still alive. The cumulative survival rate of the mice anally administered heat-killed L. brevis SBC8803 was significantly higher than that of PBS-treated mice (Fig. 6B).

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Figure 6. The cumulative survival rate of the mice treated with heat-killed L. brevis SBC8803 was significantly higher than that in PBS-administered mice. Mice were allowed free access to 4% DSS solution as drinking water and transanally administered 0.1% of heat-killed L. brevis SBC8803 or PBS every day. The weight of the heat-killed L. brevis SBC8803 treated mice was significantly higher than that in PBS-treated mice at days 9 and 10 (n = 6) (A). The cumulative survival rate of the mice treated with 0.1% heat-killed L. brevis SBC8803 was also high in comparison to that in PBS-treated mice (n = 6) (B). *P < 0.05 in comparison to the control based on an analysis of variance.

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Heat-killed L. brevis SBC8803 Decreases mRNA Expression of Proinflammatory Cytokines, TNF-α, IL-1β, and IL-12, Augmented in DSS-induced Colitis

The expression of inflammation-related cytokines including TNF-α, IL-1β, IL-6, IL-10, and IL-12 are upregulated in DSS-induced colitis.38, 39 Real-time PCR revealed a significant decrease of mRNA expression of TNF-α, IL-1β, and IL-12 in the mice treated with heat-killed L. brevis SBC8803 while that of IL-4, IL-6, IL-10, and IL-17c and -f were not changed, thus indicating that the inhibitory effect of heat-killed L. brevis SBC8803 on proinflammatory cytokines (Fig. 7). This suggests that heat-killed L. brevis SBC8803 settles the intestinal inflammation through the downregulation of proinflammatory cytokines as well as the augmentation of the intestinal barrier function.

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Figure 7. mRNA expression of TNF-α, IL-1β, and IL-12 in the mice with DSS-induced colitis were significantly decreased by the transanal administration of 0.1% heat-killed L. brevis SBC8803. RNA was harvested from the large intestine of the mice administered 2% DSS in drinking water at day 5. Real-time PCR revealed the significant decrease of mRNA expression of TNF-α, IL-1β, and IL-12 in the mice administered 0.1% heat-killed L. brevis SBC8803 while that of IL-4, IL-6, IL-10, and IL-17c and -f were not changed. mRNA expression was calculated by dividing threshold cycle of β2-microglobin into those of examined cytokines. *P < 0.05 in comparison to the control based on an analysis of variance.

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Hsp Induction and the Protective Effect of Heat-killed L. brevis SBC8803 in Caco2bbe Cells Was Eliminated by p38 MAPK Inhibitor

Whereas heat-killed L. brevis SBC8803 induced Hsp 27, SB203580 resulted in nearly complete reversal of the Hsp induction (Fig. 8A). Furthermore, the permeability of [3H]mannitol from the apical to basal side of Caco2bbe cells treated with heat-killed L. brevis SBC8803 was significantly lower than that with PBS. However, SB203580 negated the decrease in the permeability of [3H]mannitol by heat-killed L. brevis SBC8803 at both 60 and 120 minutes (Fig. 8B). Conversely, other inhibitors including PD98059 (Fig. 8C,D), SP600125 (Fig. 8E,F), and LY294002 (Fig. 8G,H) did not affect either the induction of Hsps or the enhancement of the barrier function (Fig. 8I) of the intestinal epithelia by L. brevis SBC8803, thus indicating that the p38 MAPK pathway is a major mediator of the protective effect of L. brevis SBC8803 on intestinal epithelia.

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Figure 8. Hsp induction and protective effect of heat-killed L. brevis SBC8803 in Caco2bbe cells was eliminated by p38 MAPK inhibitor. A p38 MAPK inhibitor, SB203580 (30 μM), resulted in a nearly complete reversal of the Hsp induction by heat-killed L. brevis SBC8803 (n = 6) (A,B). Conversely, either PD98059 (50 μM) (C,D), SP600125 (20 μM) (E,F), or LY294002 (50 μM) (G,H) exerted no effects on inducing Hsps. The reduced permeability of [3H]mannitol from the apical to basal side of Caco2bbe cells treated with heat-killed L. brevis SBC8803 was negated by SB203580 (n = 6), but not by PD98059 (50 μM), SP600125 (20 μM), or LY294002 (50 μM) (n = 6) (I). Image shown is representative of four separate experiments (A,C,E,G). Densitometry was performed using NIH ImageJ, setting the control to 100% for each analysis (B,D,F,H). *P < 0.05 in comparison to the control based on an analysis of variance.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The current study demonstrated that L. brevis SBC8803 induced Hsps, activated p38 MAPK, regulated the production of proinflammatory cytokines, and improved the barrier function of intestinal epithelia in the presence of oxidant stress. Furthermore, heat-killed L. brevis SBC8803 reduced the intestinal impairment of the mice with acute colitis even after inflammation developed and improved the survival rate of the mice with lethal colitis. This suggests that heat-killed L. brevis SBC8803 possess an ability to maintain the intestinal homeostasis as well as to cure intestinal disorders. It is noteworthy that the heat-killed probiotic requires no colonization in the intestinal lumen when exhibiting their physiological functions, while live probiotics need to colonize the intestinal lumen and maintain their activities. Therefore, the heat-killed bodies of L. brevis SBC8803 are expected to be effective, even if the patient condition (including the intestinal environment) is not suitable for the colonization of live probiotics. This proposes a novel concept of probiotic treatment to acquire stable effects for maintaining intestinal homeostasis and curing intestinal disorders in diverse conditions of the intestinal tract.

Some probiotics including Lactobacillus rhamnosus GG (LGG),11, 13–15, 40, 41Bifidobacterium,42 and B. subtilis17 protect the intestinal epithelia from oxidant stress or pathogenic bacteria; however, the mechanisms of probiotic effects seem to vary. The current study proposed that heat-killed L. brevis SBC8803 activated p38 MAPK, but not JNK, ERK, and Akt, and induced Hsp in intestinal epithelia. Furthermore, the induction of Hsp and the protective effect of oxidant stress by heat-killed L. brevis SBC8803 was negated when p38 inhibitor, SB203580, was added. This demonstrates that these effects of heat-killed L. brevis SBC8803 are initiated through some type of signal transduction pathway involving p38 MAPK, which is a key kinase associated with both the stress response and adaptive immunity.43 LGG also stimulated the phosphorylation of p38 MAPK and induced Hsps,15 thus suggesting that Lactobacillus strains commonly activate the p38 MAPK pathway and protect the intestinal epithelia via the induction of Hsps. Otherwise, some possible mechanisms of intestinal cytoprotection by probiotics have been proposed, including the upregulation of mucus production,11 increase in IgA production,13 increased competition for adhesion sites on intestinal epithelia,14 and the production of bacteriocins which inhibit the growth of pathogenic bacteria as anti-biotics.16 Recent studies have shown that VSL#3 prevents Salmonella-induced impairment of intestinal barrier function and induces mucin gene expression.42 LGG or its soluble factors improves the intestinal barrier function impaired by pathogenic bacteria via the activation of protein kinase C,40 or the protection of tight junction proteins.41 These studies therefore indicate that the mechanisms of intestinal protection by probiotics are diverse and dependent on the bacterial strains. The characteristic function and its mechanism of each strain should be adequately grasped when choosing the appropriate strain for an individual case or disease.

Although the mechanisms of action of probiotics regarding protection of the intestinal epithelia, such as the activation of the cell signaling pathways and the enhancement of the barrier function, are becoming clear, it is unclear how probiotics are sensed by mammalian epithelia. Mice deficient in MyD88, which is an adaptor molecule essential for Toll-like receptor (TLR) signaling, have impaired survival after administration of DSS.44 This result was replicated in wildtype mice after eradication of their commensal bacteria by broad-spectrum antibiotics, thus suggesting that some commensal bacteria appear to maintain intestinal homeostasis through TLR stimulation. Otherwise, mice lacking multidrug resistance 1 gene (MDR1), which is a member of the ATP-binding cassette subfamily B acting as a transmembrane efflux pump for many drugs (digoxin, quinidine, Actinomycin D, e.g.), spontaneously develop enteritis and this intestinal inflammation disappeared with the administration of broad-spectrum antibiotics.45 We previously demonstrated that a bacterial quorum-sensing molecule secreted by B. subtilis was internalized into human intestinal epithelial cells through the transport of novel organic cation transporter isotype 2 (OCTN2) and stimulated production of an Hsp that can protect the intestinal epithelia from oxidant-induced injury.17 It is notable that mice with a polymorphism of either the MDR1 or OCTN2 gene are susceptible to IBD,46, 47 illustrating the strong association of the transport of bioactive molecules derived from probiotics with the etiology of IBD. Subsequently, two molecules were identified from conditioned media of LGG which prevent cytokine-induced apoptosis in human and mouse intestinal epithelial cells by regulating signaling pathways.48 The current study also showed that the heat-killed body which possibly contained bioactive molecules produced by L. brevis SBC8803 exhibited intestinal cytoprotection against oxidant stress. Soluble factors derived from probiotics may mediate their physiological function through an interaction with TLRs or transport by cell membrane transporters. While the effective molecule responsible for the activity of L. brevis has not yet been identified, our preliminary study confirmed that the conditioned media successfully induced Hsp 25 in Caco2bbe cells and protected the mouse small intestine from oxidant stress (data not shown). This suggests that some molecules secreted by B. subtilis mediate the effects of L. brevis on the intestinal cytoprotection in some manner. Conversely, lipopolysaccharide and lipo-taicooic acid, which are components of the bacterial wall, failed to induce Hsp 27 and 70, thus suggesting that the common components of the bacterial wall are not the molecules responsible for these effects. Further investigation analyzing the conditioned media of L. brevis will identify the effective molecules responsible for the effects and elucidate the mechanisms with regard to how the probiotics induce a protective effect for intestinal epithelia against oxidant stress or for improving intestinal injury due to inflammation.

The current study has also shown that heat-killed L. brevis SBC8803 decreased the expression of TNF-α, IL-1β, and IL-12, which are excessively expressed in association with intestinal inflammation induced by DSS. TNF-α and IL-1β are involved in local and systemic inflammation and are members of a group of cytokines that stimulate the acute phase reaction.49, 50 IL-12 is involved in the differentiation of naive T cells into Th0 cells which will further develop into either Th1 cells or Th2 cells. The excess production of these proinflammatory cytokines is thought to lead to an excessive inflammatory response. Indeed, anti-TNF-α monoclonal antibody is an efficient suppressor for such inflammatory reactions and clinically applied for the treatment of IBD. Therefore, heat-killed L. brevis SBC8803 may reduce intestinal inflammation through the regulation of proinflammation cytokines as well as T-cell differentiation. Some other probiotic strains including various Lactobacilli decrease the excess expression of proinflammatory cytokines such as TNF-α, IL-1β, IL-4, IL-8, or IL-17 in some manner.51–58 Whereas some bacteria such as Lactobacillus acidophilus NCFM, Bifidobacterium bifidum BI-98, and BI-504 are thought to reduce regulatory T cell through suppressing the production of IL-1758 which is a pivotal cytokine participating in autoimmune inflammation59 and T-cell-mediated colitis,60 the current study showed no influence of heat-killed L. brevis SBC8803 on mRNA expression of IL-17. This suggests that, in the process of improving the damage to the intestinal epithelia, the cytoprotective effect of L. brevis SBC8803 may be a dominant, rather than a suppressive effect on the excessively activated immune cells. Further studies analyzing the effects of L. brevis SBC8803 on immune cell lines and proinflammatory cytokine-deficient models will provide important clues which will help to elucidate the roles of the L. brevis SBC8803 for immune cells as well as the intestinal epithelia. Conversely, some bacterial strains induce antiinflammatory mediators. Lactobacillus johnsonii induces transforming growth factor beta (TGF-β) production in Caco-2 cells cocultured with leukocytes.61Bifidobacterium breve also induced TGF-β in the serum of infants.62 VSL#3 or its three individual component Bifidobacterium species induce antiinflammatory cytokine IL-10 form human dendritic cells.63 Regulatory T cells can also be stimulated by either Lactobacillus paracasei treatment or bacterial-induced dendritic cell maturation.64, 65 Accordingly, each probiotic appears to display antiinflammatory activity through suppressing various proinflammatory cytokines or stimulating antiinflammatory mediators or pathways which are essential for the aggravation or regulation of intestinal inflammation.

In summary, heat-killed L. brevis SBC8803 induced Hsps, activated the p38 MAPK pathway, regulated the production of pivotal proinflammatory cytokines, TNF-α, IL-1β, and IL-12, and improved the barrier function of the intestinal epithelia in the presence of oxidant stress. These physiological functions of heat-killed L. brevis SBC8803 reduced the intestinal impairments in the mice with acute colitis and improved the survival rate in the mice with lethal colitis. The administration of heat-killed L. brevis SBC8803 may therefore have the potential to both maintain intestinal homeostasis as well as cure intestinal disorders, even in conditions unsuitable for live probiotics, because heat-killed L. brevis SBC8803 can exhibit its physiological effects without the need for colonization.

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  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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