Fermented Milk Containing Lactobacillus casei Strain Shirota Preserves the Diversity of the Gut Microbiota and Relieves Abdominal Dysfunction in Healthy Medical Students Exposed to Academic Stress.

UNLABELLED: Stress-induced abdominal dysfunction is an attractive target for probiotics. To investigate the effects of the probiotic Lactobacillus casei strain Shirota on abdominal dysfunction, a double-blind, placebo-controlled trial was conducted with healthy medical students undertaking an authorized nationwide examination for academic advancement. For 8 weeks, until the day before the examination, 23 and 24 subjects consumed an L. casei strain Shirota-fermented milk and a placebo milk daily, respectively. In addition to assessments of abdominal symptoms, psychophysical state, and salivary stress markers, gene expression changes in peripheral blood leukocytes and composition of the gut microbiota were analyzed using DNA microarray analysis and 16S rRNA gene amplicon sequence analysis, respectively, before and after the intervention. Stress-induced increases in a visual analog scale measuring feelings of stress, the total score of abdominal dysfunction, and the number of genes with changes in expression of more than 2-fold in leukocytes were significantly suppressed in the L. casei strain Shirota group compared with those in the placebo group. A significant increase in salivary cortisol levels before the examination was observed only in the placebo group. The administration of L. casei strain Shirota, but not placebo, significantly reduced gastrointestinal symptoms. Moreover, 16S rRNA gene amplicon sequencing demonstrated that the L. casei strain Shirota group had significantly higher numbers of species, a marker of the alpha-diversity index, in their gut microbiota and a significantly lower percentage of Bacteroidaceae than the placebo group. Our findings indicate that the daily consumption of probiotics, such as L. casei strain Shirota, preserves the diversity of the gut microbiota and may relieve stress-associated responses of abdominal dysfunction in healthy subjects exposed to stressful situations. IMPORTANCE: A novel clinical trial was conducted with healthy medical students under examination stress conditions. It was demonstrated that the daily consumption of lactic acid bacteria provided health benefits to prevent the onset of stress-associated abdominal symptoms and a good change of gut microbiota in healthy medical students.

Effects of Lacticaseibacillus paracasei Strain Shirota on Daytime Performance in Healthy Office Workers: A Double-Blind, Randomized, Crossover, Placebo-Controlled Trial.

Lacticaseibacillus paracasei strain Shirota (LcS) modulates psychological homeostasis via the gut-brain axis. To explore the possible efficacy of LcS for improving daytime performance, we conducted a double-blind, randomized, crossover, placebo-controlled study of 12 healthy office workers with sleep complaints. The participants received fermented milk containing viable LcS (daily intake of 1 × 1011 colony-forming units) and non-fermented placebo milk, each for a 4-week period. In the last week of each period, the participants underwent assessments of their subjective mood and measurements of physiological state indicators via an electroencephalogram (EEG) and heart rate variability in the morning and afternoon. The attention score in the afternoon as assessed by the visual analog scale was higher in the LcS intake period than in the placebo intake period (p = 0.041). Theta power on EEG measured at rest or during an auditory oddball task in the afternoon was significantly lower in the LcS period than in the placebo period (p = 0.025 and 0.009, respectively). The change rate of theta power was associated with the change in attention score. Treatment-associated changes were also observed in heart rate and the sympathetic nerve activity index. These results indicate that LcS has possible efficacy for improving daytime performance, supported by observations of the related physiological state indicators.

Effect of Lacticaseibacillus paracasei Strain Shirota on Improvement in Depressive Symptoms, and Its Association with Abundance of Actinobacteria in Gut Microbiota.

We previously reported lower counts of lactobacilli and Bifidobacterium in the gut microbiota of patients with major depressive disorder (MDD), compared with healthy controls. This prompted us to investigate the possible efficacy of a probiotic strain, Lacticaseibacillus paracasei strain Shirota (LcS; basonym, Lactobacillus casei strain Shirota; daily intake of 8.0 × 1010 colony-forming units), in alleviating depressive symptoms. A single-arm trial was conducted on 18 eligible patients with MDD or bipolar disorder (BD) (14 females and 4 males; 15 MDD and 3 BD), assessing changes in psychiatric symptoms, the gut microbiota, and biological markers for intestinal permeability and inflammation, over a 12-week intervention period. Depression severity, evaluated by the Hamilton Depression Rating Scale, was significantly alleviated after LcS treatment. The intervention-associated reduction of depressive symptoms was associated with the gut microbiota, and more pronounced when Bifidobacterium and the Atopobium clusters of the Actinobacteria phylum were maintained at higher counts. No significant changes were observed in the intestinal permeability or inflammation markers. Although it was difficult to estimate the extent of the effect of LcS treatment alone, the results indicated that it was beneficial to alleviate depressive symptoms, partly through its association with abundance of Actinobacteria in the gut microbiota.

Abolishment of TNBS-induced visceral hypersensitivity in mast cell deficient rats.

Mucosal mast cells are implicated in visceral hypersensitivity associated with irritable bowel syndrome (IBS). In this study, we investigated the role of mast cells in the development of visceral hypersensitivity by using mast cell deficient (Ws/Ws) rats and their control (W+/W+). In W+/W+ rats, an injection of 2,4,6-trinitrobenzene sulfonic acid (TNBS) into the proximal colon produced a significant decrease in pain threshold of the distal colon. Severe mucosal necrosis and inflammatory cell infiltration with concomitant increase in tissue myeloperoxidase activity were observed in the proximal colon that was directly insulted by TNBS, whereas neither necrosis nor increased myeloperoxidase activity occurred in the distal colon, indicating that TNBS-induced hypersensitivity is not caused by the local tissue damage or inflammation in the region of the gut where distention stimuli were applied. On the other hand, TNBS failed to elicit visceral hypersensitivity in Ws/Ws rats. This finding indicates that mast cells are essential for development of TNBS-induced visceral hypersensitivity in rats. Since the severity of TNBS-induced proximal colon injury and MPO activity was not affected by mast cell deficiency, it is unlikely that abolishment of visceral hypersensitivity in mast cell deficient rats was a result of altered development of the primary injury in the proximal colon. There was no difference between sham-operated Ws/Ws and W+/W+ rats in colonic pain threshold to distention stimuli, indicating that mast cells play no modulatory roles in normal colonic nociception. The present results support the view that mucosal mast cells play key roles in the pathogenesis of IBS.

Effect of a new alpha 2 delta ligand PD-217014 on visceral hypersensitivity induced by 2,4,6-trinitrobenzene sulfonic acid in rats.

PD-217014, a new GABA analog (1 alpha,3 alpha,5 alpha-3-aminomethyl-bicyclo[3.2.0]heptane-3-acetic acid), inhibited [(3)H]-gabapentin binding to alpha(2)delta subunit of voltage-gated calcium channels in a concentration-dependent manner (K(i) = 18 nmol/l). Oral treatment with PD-217014 significantly inhibited the visceral hypersensitivity induced by an intra-colonic injection of trinitrobenzene sulfonic acid in rats. The anti-hyperalgesic effect of PD-217014 increased in a dose-dependent manner, and reached a plateau level at 60 mg/kg p.o. The visceral analgesia produced by PD-217014 at 30 and 60 mg/kg p.o. correlated with blood concentrations within 4 h after dosing, and maximal efficacy was obtained 2 h after dosing when the maximal blood concentration was achieved at either dose. These results indicate that PD-217014 is a potent alpha(2)delta ligand and possesses visceral analgesic activity.

Colonic mast cell infiltration in rats with TNBS-induced visceral hypersensitivity.

Colonic mucosal mast cells are implicated in the pathogenesis of visceral hypersensitivity associated with irritable bowel syndromes. This study was designed to investigate the roles of mucosal mast cells in development of an experimental visceral hypersensitivity induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rats. TNBS, when injected into the proximal colon through laparotomy, produced a significant decrease in pain threshold of the distal colon to mechanical distention, indicating a visceral hypersensitivity. In the proximal colon that was directly insulted by TNBS, mucosal necrosis and extensive inflammatory cell infiltration were observed with concomitant increase in tissue myeloperoxide (MPO) activity. In the distal colon where distention stimuli were applied, the number of mucosal mast cells significantly increased following TNBS treatment, although neither mucosal injury nor increase in tissue MPO activity was observed. In an organ culture, spontaneous release of a mucosal mast cell-specific protease (RMCP-2) from the distal colon tissue of TNBS-treated rats was significantly larger than that of sham animals. Furthermore, TNBS-induced visceral hypersensitivity was significantly suppressed by subcutaneous pretreatment with a mast cell stabilizer doxantrazole in a dose-dependent manner. These findings suggest that prominent colonic mast cell infiltration associated with an enhanced spontaneous mediator release is responsible, at least partly, for development of visceral hypersensitivity induced by TNBS in rats.

Disturbance in the Mucosa-Associated Commensal Bacteria Is Associated with the Exacerbation of Chronic Colitis by Repeated Psychological Stress; Is That the New Target of Probiotics?

Psychological stress can exacerbate inflammatory bowel disease. However, the mechanisms underlying how psychological stress affects gut inflammation remain unclear. Here, we focused on the relationship between changes in the microbial community of mucosa-associated commensal bacteria (MACB) and mucosal immune responses induced by chronic psychological stress in a murine model of ulcerative colitis. Furthermore, we examined the effect of probiotic treatment on exacerbated colitis and MACB composition changes induced by chronic psychological stress. Repeated water avoidance stress (rWAS) in B6-Tcra-/- mice severely exacerbated colitis, which was evaluated by both colorectal tissue weight and histological score of colitis. rWAS treatment increased mRNA expression of UCN2 and IFN-γ in large intestinal lamina propria mononuclear cells (LI-LPMC). Interestingly, exacerbated colitis was associated with changes in the microbial community of MACB, specifically loss of bacterial species diversity and an increase in the component ratio of Clostridium, revealed by 16S rRNA gene amplicon analysis. Finally, the oral administration of a probiotic Lactobacillus strain was protective against the exacerbation of colitis and was associated with a change in the bacterial community of MACB in rWAS-exposed Tcra-/- mice. Taken together, these results suggested that loss of species diversity in MACB might play a key role in exacerbated colitis induced by chronic psychological stress. In addition, probiotic treatment may be used as a tool to preserve the diversity of bacterial species in MACB and alleviate gut inflammation induced by psychological stress.

Chronic Psychological Stress Disrupted the Composition of the Murine Colonic Microbiota and Accelerated a Murine Model of Inflammatory Bowel Disease.

The effect of psychological stress on the gastrointestinal microbiota is widely recognized. Chronic psychological stress may be associated with increased disease activity in inflammatory bowel disease, but the relationships among psychological stress, the gastrointestinal microbiota, and the severity of colitis is not yet fully understood. Here, we examined the impact of 12-week repeated water-avoidance stress on the microbiota of two inbred strains of T cell receptor alpha chain gene knockout mouse (background, BALB/c and C57BL/6) by means of next-generation sequencing of bacterial 16S rRNA genes. In both mouse strains, knockout of the T cell receptor alpha chain gene caused a loss of gastrointestinal microbial diversity and stability. Chronic exposure to repeated water-avoidance stress markedly altered the composition of the colonic microbiota of C57BL/6 mice, but not of BALB/c mice. In C57BL/6 mice, the relative abundance of genus Clostridium, some members of which produce the toxin phospholipase C, was increased, which was weakly positively associated with colitis severity, suggesting that expansion of specific populations of indigenous pathogens may be involved in the exacerbation of colitis. However, we also found that colitis was not exacerbated in mice with a relatively diverse microbiota even if their colonic microbiota contained an expanded phospholipase C-producing Clostridium population. Exposure to chronic stress also altered the concentration of free immunoglobulin A in colonic contents, which may be related to both the loss of bacterial diversity in the colonic microbiota and the severity of the colitis exacerbation. Together, these results suggest that long-term exposure to psychological stress induces dysbiosis in the immunodeficient mouse in a strain-specific manner and also that alteration of microbial diversity, which may be related to an altered pattern of immunoglobulin secretion in the gastrointestinal tract, might play a crucial role in the development of chronic stress-induced colitis.

Intragastric injection of Lactobacillus casei strain Shirota suppressed spleen sympathetic activation by central corticotrophin-releasing factor or peripheral 2-deoxy-d-glucose in anesthetized rats.

Intragastric (IG) administration of probiotic strain Lactobacillus casei Shirota (LcS) decreases the sympathetic nerve outflow of anesthetized rats in a tissue-specific manner. In the present study, we examined the effects of IG administration of LcS on sympathetic activation induced by an intracerebroventricular (ICV) injection of corticotrophin-releasing factor (CRF) and an intravenous (IV) injection of 2-deoxy-d-glucose (2DG) or interleukin (IL)-1ß in urethane-anesthetized rats. The IG administration of LcS differently affected the stimulatory responses of sympathetic nerve outflow to CRF. LcS suppressed the increase in splenic sympathetic nerve activity (Spleen-SNA), induced by central CRF, in a dose-dependent manner; however, it did not alter adrenal sympathetic nervous activity (ASNA). In contrast, LcS did not affect spleen-SNA and ASNA following an IV injection of IL-1ß. On the other hand, IG administration of LcS suppressed the activation of ASNA following an IV injection of 2DG. These findings suggest that the suppression of central CRF-induced sympathetic activation by LcS is tissue-specific. Moreover, it can suppress the 2DG-induced sympathetic activation. Furthermore, we found that stomach-specific vagotomy attenuates the suppressive effect of LcS on CRF-mediated spleen-SNA activation. Thus, the present study suggests that LcS administered to the stomach may act on the afferent vagal nerve and send afferent signals to the brain to regulate efferent SNA induced by sympathetic stimulators.

Injection of Lactobacillus casei strain Shirota affects autonomic nerve activities in a tissue-specific manner, and regulates glucose and lipid metabolism in rats.

AIMS/INTRODUCTION: Previously, it was observed that long-term ingestion of a probiotic strain Lactobacillus casei Shirota (LcS) ameliorates insulin resistance and glucose intolerance in rats fed a high-fat diet. In the present study, we examined its possible role in the autonomic nervous system during LcS-induced modulations in glucose and lipid metabolism or cardiovascular functions. MATERIALS AND METHODS: The present study examined the effects of intragastric (IG) LcS injection on autonomic nerve tones in anesthetized rats by electrophysiological method. RESULTS: We found that an IG injection of LcS suppressed neural activity of sympathetic nerves supplying the white adipose tissue of urethane-anesthetized rats in a dose-dependent manner, whereas sympathetic nerve outflow to brown adipose tissue was not affected by the IG LcS injection. Furthermore, the IG LcS injection reduced efferent sympathetic nerve outflow to the adrenal gland and liver, but did not alter gastric vagal nerve activity, renal sympathetic nerve activity, as well as mean arterial pressure. To test the involvement of afferent vagal nerves and the abdominal organs, we examined the adrenal sympathetic response to an LcS injection in rats with ablated afferent vagal nerves, and found that the adrenal sympathetic nerve response to LcS was inhibited in vagotomized rats. In addition, we found that oral ingestion of LcS attenuated the hyperglycemic response to glucose loading and blood glycerol levels in conscious rats. CONCLUSIONS: Our data suggest that LcS might affect tissue-specific autonomic nerves through the afferent vagal nerve pathway to modulate glucose and lipid metabolism.

Involvement of parasympathetic pelvic efferent pathway in psychological stress-induced defecation.

AIM: To investigate the role of the pelvic nerve pathway in stress-induced acceleration of colorectal transit and defecation in rats. METHODS: Surgical transection of rectal nerves (rectal branches of the pelvic nerve), vagotomy (Vag) or adrenalectomy (Adx) were performed bilaterally in rats. Number of fecal pellet output of these rats was measured during 1-h water avoidance stress (WAS). To evaluate the colonic transit, rats were given phenol red through the catheter indwelled in the proximal colon and subjected to WAS. After WAS session, entire colon and rectum were isolated and distribution of phenol red was measured. Distal colonic and rectal transit was evaluated using glass bead. Rats were inserted the glass bead into the distal colon and evacuation rate of the bead was measured. Neural activation was assessed by immunohistochemical staining of c-Fos and PGP9.5 in colonic whole-mount preparations of longitudinal muscle myenteric plexus (LMMP). RESULTS: In the sham-operated rats (sham op), WAS significantly increased defecation and accelerated colorectal transit with marked elevation of plasma corticosterone level. Compared with sham-operated rats, increase in the excretion of fecal pellets during WAS was significantly reduced by rectal nerve transection (RNT) (sham op: 6.9 ± 0.8 vs RNT: 4.3 ± 0.6, P < 0.05) or Vag (sham op: 6.4 ± 0.8 vs Vag: 3.7 ± 1.1, P < 0.05), although corticosterone level remained elevated. Adx-rats significantly increased the defecation despite the lower corticosterone level. Distribution pattern of phenol red showed RNT inhibited distal colonic and rectal transit accelerated by WAS, while Vag inhibited proximal colonic transit. Suppression of distal colonic and rectal transit by RNT was further confirmed by the bead evacuation rate (sham op: 80.0% vs RNT: 53.8%). WAS significantly increased the number of c-Fos-immunoreactive neural cells in the LMMP of the proximal and distal colon, whereas c-Fos expression was decreased by RNT in the distal colon (sham op: 9.0 ± 2.0 vs RNT: 4.4 ± 1.0, P < 0.05) and decreased by Vag in the proximal colon. CONCLUSION: Pelvic nerve conveys WAS stimuli from the brain to the distal colon, and directly activate the myenteric neurons, followed by the increase of its motility.

Protein kinases expressed by interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are involved in the generation of electrical rhythmicity of intestinal muscle and in the transduction of neural inputs in the gut. Although the expression of receptors for neurotransmitters and hormones and some second messengers have been investigated in ICC, the protein kinases present in these cells have not been well documented. This study has demonstrated the immunohistochemical localisation of PKA, PKC gamma and PKC theta in ICC that were identified by the known ICC marker, c-Kit, in the guinea-pig gut. Other PKCs, PKC alpha, beta, delta, epsilon, eta, iota and lambda, and Ca(2+)-calmodulin-dependent protein kinase II were not localised in ICC. Double labelling studies were conducted on longitudinal muscle-myenteric plexus and external muscle-myenteric plexus preparations of the oesophagus, stomach (fundus, corpus and antrum), duodenum, distal ileum, caecum, proximal and distal colon, and rectum. The three protein kinases were detected in c-Kit-immunoreactive ICC at the level of the myenteric plexus (IC-MY), in the muscle (IC-IM) and at the level of the deep muscular plexus (IC-DMP) in the small intestine. PKA was found in over 90% of IC-IM in all regions examined, and in over 90% of IC-MY in the gastric body and antrum and throughout the small and large intestines. PKC gamma was in the majority of ICC in the gastric body and antrum and in the small intestine, but was largely absent from ICC in the oesophagus, proximal stomach and large intestine. PKC theta occurred in the majority of ICC in all regions except the rectum. The intensity of staining was greatest for PKA, with PKC gamma giving comparatively weak labelling of ICC. PKA was also detected in myenteric neurons, smooth muscle, macrophages and fibroblast-like cells. PKC gamma labelling occurred in large, multipolar neurons throughout the small and large intestine, as well as in lymph vessels and in capillaries. It is concluded that PKA, PKC gamma and PKC theta are all present in ICC, with the differences in their localisations suggesting specific roles for each in ICC function.

Comparison of the effects of phorbol dibutyrate and low-frequency stimulation of synaptic inputs on the excitability of myenteric AH neurons.

Low-frequency stimulation of synaptic inputs to after-hyperpolarising (AH) neurons in the guinea-pig small intestine causes sustained increases in excitability that far outlast the stimulus period. This excitation has been called sustained, slow, post-synaptic excitation (SSPE). Intracellular microelectrodes were used to record the effects of the protein kinase C (PKC) stimulant, phorbol dibutyrate (PDBu), and compare these with changes seen during the SSPE, in AH neurons of the small intestine of the guinea-pig. PDBu (1 nM-1 microM) increased excitability, depolarised the membrane and increased input resistance concentration dependently, mimicking the effects of low-frequency stimulation of pre-synaptic inputs. These changes developed slowly after the start of infusion and were only slowly reversible after wash out. PDBu suppressed a late after-hyperpolarising potential (AHP) that depends on Ca2+ entry via voltage-gated Ca2+ channels during the action potential. The effects of PDBu (10 nM) on the late AHP were indistinguishable from those observed during the SSPE. PDBu, at a concentration that inhibited the AHP, had no effect on the action potential half-width or the slope of its first repolarisation phase (the early phase of repolarisation is slowed by the Ca2+ influx of the action potential). Thus PDBu inhibited K+ channel opening underlying the late AHP, but did not suppress Ca2+ entry during the action potential. The hyperpolarisation-activated cation current (Ih) in intrinsic primary afferent neurons (IPANs) was not affected by PDBu. We conclude that PDBu mimics the sustained excitation caused by low-frequency stimulation of synaptic inputs to IPANs by closing IK channels responsible for the AHP or restricting their opening by Ca2+ and by reducing the current carried by K+ channels that are active at rest. IK channels, the opening of which results in the AHP, have consensus sites for PKC and are likely targets for phosphorylation during the SSPE.