Evaluation of the beneficial effects of a GABA-based product containing Melissa officinalis on post-inflammatory irritable bowel syndrome: a preclinical study

Abstract

Introduction: Visceral pain represents the most common digestive issue, frequently resulting from long-term inflammation, such as inflammatory bowel diseases. The lack of effective drugs prompted search of new therapeutic approaches. In this regard, gamma-aminobutyric acid (GABA) and Melissa officinalis (Mo) appear as excellent candidates as they were recognized to have several positive effects on the digestive system. The aim of this research was to evaluate the effects of a compound containing GABA and Mo (GABA-Mo 5:1) in inflammation-induced intestinal damage and visceral pain.

Methods: Colitis was induced in rats by intrarectal 2,4-dinitrobenzenesulfonic acid (DNBS) administration. DNBS-treated animals received GABA-Mo (80 mg/kg BID), starting 3 days before DNBS administration, until 14 days after colitis induction (preventive protocol), or starting 7 days after DNBS until day 21 (curative protocol). Visceral pain was assessed by measuring the viscero-motor response (VMR) and the abdominal withdrawal reflex (AWR) to colorectal distension on day 7, 14 (both protocols) and 21 (curative protocol) after DNBS administration.

Results: In the preventive protocol, GABA-Mo reduced AWR at day 14 but had no effect on VMR. In the spinal cord, treatment with GABA-Mo significantly prevented microglia reactivity (Iba-1 positive cells). In the colon, the supplement significantly decreased malondialdehyde (MDA, index of oxidative stress) and IL-1β levels and counteracted the decreased expression of claudin-1. Moreover, GABA-Mo normalized the increased levels of plasma lipopolysaccharide binding protein (LBP, index of altered intestinal permeability). In the curative protocol, GABA-Mo significantly counteracted visceral hypersensitivity persistence in DNBS-treated animals (day 14 and 21). In the spinal cord, GABA-Mo significantly reduced GFAP positive cell density (astrocytes). Histological evaluations highlighted a mild but significant effect of GABA-Mo in promoting healing from DNBS-induced colon damage. Colonic MDA and myeloperoxidase (index of leukocyte infiltration) levels were reduced, while the decreased colonic claudin-1 expression was normalized. In addition, the increased levels of plasma LBP were normalized by GABA-Mo administration.

Discussion: In conclusion GABA-Mo, particularly in the curative protocol, was able to reduce visceral pain and intestinal inflammation, likely through a reinforcement of intestinal barrier integrity, thus representing a suitable approach for the management of abdominal pain, especially in the remission stages of colitis.

Keywords: GABA; Melissa officinalis; abdominal pain; colitis; irritable bowel syndrome.

FIGURE 1

Schematic representation of the preventive (A) and curative (B) protocol used in the study.

FIGURE 2

Effect of the preventive protocol of treatment with GABA-Mo on visceral pain induced by DNBS in rats. Animal nocifensive response associated with visceral pain was assessed by measuring the extent of the abdominal withdrawal response (AWR) to colorectal distension (CRD; 0.5–3 mL; A-B). Animal visceral sensitivity was assessed by measuring the extent of the abdominal contraction (EMG amplitude) related to visceromotor response (VMR) to colorectal distension (CRD; 0.5–3 mL; C-D). The test was performed 7 days (A,C) and 14 days (B,D) after colitis induction. The test was performed 7 days (A) and 14 days (B) after colitis induction. *P < 0.05, **P < 0.01 and ***P < 0.001 vs Control + vehicle treated animals. ^∧ P < 0.05 vs DNBS+ vehicle treated animals.

FIGURE 3

Effect of the curative protocol of treatment with GABA-Mo on visceral pain induced by DNBS in rats. Animal nocifensive response associated with visceral pain was assessed by measuring the extent of the abdominal withdrawal response (AWR) to colorectal distension (CRD; 0.5–3 mL; A-C). Animal visceral sensitivity was assessed by measuring the extent of the abdominal contraction (EMG amplitude) related to visceromotor response (VMR) to colorectal distension (CRD; 0.5–3 mL; D-E). The tests were performed 7 days [(A,D), pretest], 14 days (B,E) and 21 days (C,F) after colitis induction. **P < 0.01 and ***P < 0.001 vs. Control + vehicle treated animals. ^∧ P < 0.05, ^{∧ ∧} P < 0.01 and ^{∧ ∧ ∧} P < 0.001 vs. DNBS + vehicle treated animals.

FIGURE 4

Effect of DNBS treatment and preventive protocol with GABA-Mo on astrocytes and microglia activation in the spinal cord. The graphs show the quantification of GFAP immunoreactivity (A) and the density of GFAP-positive cells (B), with representative images showing the expression of GFAP (purple); (C), 14 days after DNBS injection. The figure reports the quantification of the percentage of reactive Iba-1-positive cells (D) and the density of Iba-1-positive cells (E), with representative images showing the expression of Iba-1 (green); (F), 14 days after DNBS injection. The quantitative analysis of immunofluorescence was performed by collecting independent fields from the dorsal horns of the spinal cord. *P < 0.05 vs. Control + vehicle group. ^∧ P < 0.05 vs. DNBS + vehicle group. Original magnification: ×20.

FIGURE 5

Effect of DNBS treatment and curative protocol with GABA-Mo on astrocytes and microglia activation in the spinal cord. The graphs show the quantification of GFAP immunoreactivity (A) and the density of GFAP-positive cells (B), with representative images showing the expression of GFAP (purple); (C), 21 days after DNBS injection. The figure reports the quantification of the percentage of reactive Iba-1-positive cells (D) and the density of Iba-1-positive cells (E), with representative images showing the expression of Iba-1 (green); (F), 14 days after DNBS injection. The quantitative analysis of immunofluorescence was performed by collecting independent fields from the dorsal horns of the spinal cord. *P < 0.05 and **P< 0.01 vs. Control + vehicle group. ^{∧ ∧} P< 0.01 vs. DNBS + vehicle group. Original magnification: ×20.

FIGURE 6

Effect of preventive protocol with GABA-Mo on colon damage induced by DNBS in rats. Representative pictures of haematoxylin–eosin-stained sections of full-thickness colon were reported (A); Original magnification ×10). The column graphs report the colon macroscopic (B) and microscopic (C) damage score; **P < 0.01 vs. Control + vehicle.

FIGURE 7

Effect of curative protocol with GABA-Mo on colon damage induced by DNBS in rats. Representative pictures of haematoxylin–eosin-stained sections of full-thickness colon were reported (A); Original magnification ×10). The column graphs report the colon macroscopic (B) and microscopic (C) damage score; **P < 0.01 vs. Control + vehicle. ^∧ P < 0.05 vs. DNBS + vehicle.

FIGURE 8

Colonic levels of MPO (A) and MDA (B) in the preventive protocol and MPO (C) and MDA (D) in the curative protocol in control or DNBS rats treated with vehicle or GABA-Mo. Each column represents the mean ± SEM from eight animals. *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. Control + vehicle group. ^∧ P < 0.05 and ^{∧ ∧} P < 0.01 vs. DNBS + vehicle group.

FIGURE 9

Preventive protocol. IL-1β levels in plasma (A) and colonic tissues (B); TNF levels in plasma (C) and colonic tissues (D), IL-10 levels in plasma (E) and colonic tissues (F) in control or DNBS rats treated with vehicle or GABA-Mo. Each column represents the mean ± SEM from eight animals. **P < 0.01 vs. Control + vehicle group. ^{∧ ∧} P < 0.01 vs. DNBS + vehicle group.

FIGURE 10

Curative protocol. IL-1β levels in plasma (A) and colonic tissues (B); TNF levels in plasma (C) and colonic tissues (D), IL-10 levels in plasma (E) and colonic tissues (F) in control or DNBS rats treated with vehicle or GABA-Mo. Each column represents the mean ± SEM from eight animals.

FIGURE 11

Calprotectin levels in the preventive protocol of treatment (A) and curative protocol of treatment (B) in feces from control or DNBS rats treated with vehicle or GABA-Mo. Each column represents the mean ± SEM from eight animals.

FIGURE 12

Preventive protocol. Representative blots (A and C) and densitometric analysis of the expression levels of S100β (B), occludin (D) and claudin-1 (E) in control or DNBS rats treated with vehicle or GABA-Mo. Each column represents the mean ± SEM from eight animals. *P < 0.05, **P < 0.01 vs. Control + vehicle group. ^∧ P < 0.05 vs. DNBS + vehicle group.

FIGURE 13

Curative protocol Representative blots (A and C) and densitometric analysis of the expression levels of S100β (B), occludin (D) and claudin-1 (E) in control or DNBS rats treated with vehicle or GABA-Mo. Each column represents the mean ± SEM from eight animals. *P < 0.05, **P < 0.01 vs. Control + vehicle group. ^∧ P < 0.05 vs. DNBS + vehicle group.

FIGURE 14

LBP levels in the preventive protocol of treatment (A) and curative protocol of treatment (B) in plasma from control or DNBS rats treated with vehicle or GABA-Mo. Each column represents the mean ± SEM from eight animals. *P < 0.05, **P < 0.01 vs. Control + vehicle group. ^∧ P < 0.05 vs. DNBS + vehicle group.