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.
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- MATERIALS AND METHODS
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.