Lactobacillus acidophilus NCFM affects colonic mucosal opioid receptor expression in patients with functional abdominal pain - a randomised clinical study

Abstract

Background: In a recent double-blinded clinical trial, the probiotic combination of Lactobacillus acidophilus NCFM (L-NCFM) and B-LBi07 reduced bloating symptoms in patients with functional bowel disorders; an effect more evident in those who reported abdominal pain. In mice, L-NCFM but not B-LBi07 induced colonic mu-opioid receptor (MOR) and cannabinoid receptor 2 (CB2) expression, and reduced visceral sensitivity.

Aims: To determine if L-NCFM was the active component in the clinical trial and to investigate the mechanism of action in humans with mild to moderate abdominal pain.

Methods: Caucasian women (n = 20) 18-70 years with mild to moderate abdominal pain were enrolled in a double-blind, two-armed, single-centre study. Patients were given either L-NCFM alone or in combination with B-LBi07 for 21 days at a total dose of 2 × 10(10) CFU b.d. Colonic biopsies were collected during unsedated, unprepped flexible sigmoidoscopy before and at the end of probiotic consumption. mRNA and immunostaining were then performed on these biopsies. Patients kept symptom diaries for the 7 days prior to starting probiotic therapy and for the last 7 days of therapy.

Results: L-NCFM alone, but not with B-LBi07, induced colonic MOR mRNA and protein expression, as well as downstream signalling, as measured by enterocyte STAT3-phosphorylation. In contrast, CB2 expression was decreased. Both treatment groups trended towards improvement in symptoms, but the study was insufficiently powered to draw meaningful conclusions.

Conclusions: Lactobacillus acidophilus NCFM modulates mu-opioid receptor expression and activity, while the combination of L-NCFM and B-LBi07 does not. This study provides a possible mechanism for action by which probiotics modulates pain sensation in humans (Clinical Trial Number: NCT01064661).

Conflict of interest statement

COMPETING INTERESTS/DISCLOSURE

J.G is a paid technical consultant for Protagonist Therapeutics (Milpitas, CA).

The other authors declare that they have no competing interests.

© 2014 John Wiley & Sons Ltd.

Figures

Figure 1. Study design

A randomized, double blind pilot study with a 7 days run-in phase and a 30 day single or dual probiotic intervention phase. Patients completed a 7-day symptom diary during the run-in period and during the last 7 days of the intervention period. Mucosal biopsies were taken from unprepped colon pre and post probiotic interventions.

Figure 2. Probiotic therapy in human patients induces increased levels of hMOR mRNA in human colonic mucosa, driven by L-NCFM

A&B) Comparison by real-time PCR of expression levels pre- and post- treatment with one or both probiotics. For analysis, results from both the single- and dual-probiotic arms were pooled, and were performed without tracking per-patient changes. A) Real-time PCR shows an increase in the ΔCt and thus expression of hMOR mRNA in patients with abdominal pain receiving either one or both probiotics; -21.44 vs -17.65, P=0.0349, N=14/group. B) Real-time PCR shows a decrease in the ΔCt and thus expression of hCB2 mRNA in patients receiving either one or both probiotics; 1.287 vs. -2.396, P=0.0190, N=13/group.C) Per-patient increase in hMOR mRNA expression after treatment with one or both probiotics showed a 39.9-fold induction in patients given L-NCFM alone (P =0.0313). No significant induction was seen in patients given both L-NCFM and B-LBi07. D) Per-patient decrease in CB2 mRNA expression after treatment with L-NCFM alone showed a 4.15-fold decreased in the L-NCFM group (P=0.032). No change in expression was seen in patients given both probiotics.

Figure 3. Increase in levels of protein expression in human colonic mucosa by L-NCFM

A) Immunofluorescence of colonic mucosa samples of patients given L-NCFM alone showing increased MOR expression of colonic-mucosa. Representative of 4 independent experiments. White arrow heads point to colonic-mucosa-associated MOR. Scale bars equal 50 μm. B) Bar graph shows change in positive staining enterocytes after treatment with one or both probiotics; only the L-NCFM showed a statistically significant induction; 2.17-fold, P=0.0231, n=4/group.

Figure 4. Increase in post-receptor downstream signaling in human colonic mucosa by L-NCFM

Immunostaining is for pSTAT3 (Y705), with staining indicated by white arrow heads. Representative of 4 patients. Scale bars are 100 μm. Mean pre- to post-intervention increase in staining was 18 more cells after treatment (19.8 to 39.4 post-treatment, P=0.0469, n=4).