Alleviation of Irritable Bowel Syndrome-Like Symptoms and Control of Gut and Brain Responses with Oral Administration of Dolichos lablab L. in a Mouse Model

Abstract

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder manifesting as unexplained abdominal pain and bowel habit changes. The pathogenesis of post-infectious IBS is associated with gut⁻brain axis dysfunction, including low-grade colonic inflammation and anxiety-related long-term brain changes. This study analyzed the efficacy of a standardized extract of Dolichos lablab L. extract (DL), a bean species, in an IBS mouse model resembling post-infectious, diarrhea-dominant IBS. Using a zymosan-induced animal IBS model, we found that oral administration of DL significantly attenuated zymosan-induced increases in colonic macroscopic scores and minimized weight loss without affecting food intake. In the DL-treated mice, the mast cell count and tumor necrosis factor-α level in the colon markedly decreased, similar to results in sulfasalazine-treated mice and in mice with lipopolysaccharide-stimulated bone marrow-derived mast cells. The number of visceral pain-related behaviors was much lower in the DL-treated mice. Anxiety-like behaviors significantly improved, comparable to that after treatment with amitriptyline. The c-Fos expression level in the prefrontal cortex was significantly reduced. Our data suggest that DL could be beneficial for treating IBS by acting on the gut and brain.

Keywords: Dolichos lablab L.; anxiety; colonic inflammation; gut–brain axis; irritable bowel syndrome; mouse model; pain; zymosan-induced IBS.

Figure 1

HPLC chromatogram for determining DL. (A) HPLC-UV chromatogram of trigonelline (1, a) and DL (b) at 264 nm. (B) HPLC fluorescence chromatogram of arginine (3, c) and aspartic acid (2, d) as standard components, and DL (e). HPLC, high-performance liquid chromatography; DL, D. lablab L. extracts.

Figure 1

HPLC chromatogram for determining DL. (A) HPLC-UV chromatogram of trigonelline (1, a) and DL (b) at 264 nm. (B) HPLC fluorescence chromatogram of arginine (3, c) and aspartic acid (2, d) as standard components, and DL (e). HPLC, high-performance liquid chromatography; DL, D. lablab L. extracts.

Figure 2

Animal experimental scheme. DL, Dolichos lablab L. extract; AMT, amitriptyline; SSZ, sulfasalazine; EPM, elevated plus maze; OFT, open field test.

Figure 3

Effects of DL on the body weight and food intake of zymosan-induced irritable bowel syndrome (IBS) mice. (A) Body weight and (B) food intake were measured on the indicated days. Data are presented as a mean ± standard deviation (n = 6, one-way ANOVA; ### p < 0.001 vs. naïve; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). DL, D. lablab L. extracts; AMT, amitriptyline; SSZ, sulfasalazine.

Figure 4

Effects of DL on colonic inflammation. Formalin-fixed colons were stained with (A) H&E and toluidine blue. Cells in the colon were visualized under a visible-light microscope at a magnification of 200×. (B) The mucosa thickness were measured and (C) infiltrated inflammatory cells and (D) mast cells were counted by ImageJ software. (E) TNF-α expression levels and colon histology in zymosan-induced IBS mice. The level of TNF-α mRNA in the colon on days 4 and 14 were determined by reverse transcription polymerase chain reaction (RT-PCR). (F) Bone marrow-derived mast cells (BMMCs) were treated with various concentrations of DL for 1 h followed by stimulation with LPS for 6 h. Levels of TNF-α were determined by enzyme-linked immunosorbent assay (ELISA). The images represent one of three or six independent experiments. Data are presented as a mean ± SD (n = 6, one-way ANOVA; ## p < 0.01, ### p < 0.001 vs. naïve; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). H&E, hematoxylin and eosin; DL, D. lablab L. extracts; AMT, amitriptyline; SSZ, sulfasalazine; TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharides; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Effects of DL on visceral pain-related behaviors in zymosan-induced IBS mice. (A) Day 1, (B) day 7, and (C) day 14 according to the methods described in the Materials and Methods. Data are presented as a mean ± standard deviation (n = 6, one-way ANOVA; ## p < 0.01 vs. naïve; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). DL, D. lablab L. extracts; AMT, amitriptyline; SSZ, sulfasalazine.

Figure 6

Effects of DL on anxiety-related behaviors in the EPM test and OFT. (A) The number of entries, (B) percentage of relative stay in open arms, and (C) travel distance in the OFT were measured on days 1, 7, and 14. Data are presented as a mean ± standard deviation (n = 8, one-way analysis of variance; # p < 0.05, ### p < 0.001 vs. naïve; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). DL, D. lablab L. extracts; AMT, amitriptyline; SSZ, sulfasalazine; EPM, elevated plus maze test; OFT, open field test.

Figure 7

Effects of DL on c-Fos protein expression in the prefrontal cortex of zymosan-induced IBS mice. (A) IF staining was performed to determine c-Fos expression in the prefrontal cortex (1.78 mm before the bregma). The frozen sections were stained with c-Fos antibodies (green) and evaluated by confocal microscopy. The dashed line indicates the location of the prefrontal cortex. The protein level of c-Fos was quantified by ImageJ software. (B) Prefrontal cortex lysates were analyzed by Western blotting using c-Fos antibodies. Band density was analyzed by ImageJ software. β-actin was used as the loading control. Data are presented as a mean ± standard deviation (n = 4, one-way ANOVA; ## p < 0.01 vs. naïve; *** p < 0.001 vs. control). DL, D. lablab L. extracts; AMT, amitriptyline; SSZ, sulfasalazine; IF, immunofluorescence; DAPI, 4′,6-diamidino-2-phenylindole.