Patchouli alcohol improved diarrhea-predominant irritable bowel syndrome by regulating excitatory neurotransmission in the myenteric plexus of rats

Abstract

Background and Purpose: Irritable bowel syndrome (IBS) is usually associated with chronic gastrointestinal disorders. Its most common subtype is accompanied with diarrhea (IBS-D). The enteric nervous system (ENS) modulates major gastrointestinal motility and functions whose aberration may induce IBS-D. The enteric neurons are susceptible to long-term neurotransmitter level alterations. The patchouli alcohol (PA), extracted from Pogostemonis Herba, has been reported to regulate neurotransmitter release in the ENS, while its effectiveness against IBS-D and the underlying mechanism remain unknown. Experimental Approach: In this study, we established an IBS-D model in rats through chronic restraint stress. We administered the rats with 5, 10, and 20 mg/kg of PA for intestinal and visceral examinations. The longitudinal muscle myenteric plexus (LMMP) neurons were further immunohistochemically stained for quantitative, morphological, and neurotransmitters analyses. Key Results: We found that PA decreased visceral sensitivity, diarrhea symptoms and intestinal transit in the IBS-D rats. Meanwhile, 10 and 20 mg/kg of PA significantly reduced the proportion of excitatory LMMP neurons in the distal colon, decreased the number of acetylcholine (Ach)- and substance P (SP)-positive neurons in the distal colon and restored the levels of Ach and SP in the IBS-D rats. Conclusion and Implications: These findings indicated that PA modulated LMMP excitatory neuron activities, improved intestinal motility and alleviated IBS-induced diarrheal symptoms, suggesting the potential therapeutic efficacy of PA against IBS-D.

Keywords: colonic longitudinal muscle myenteric plexus; excitatory neurons; intestinal motility; irritable bowel syndrome with diarrhea; patchouli alcohol.

FIGURE 1

Evaluation of the IBS-D model. (A) Abdominal withdrawal reflex assessment in IBS-D rats. The AWR scores were used to assess visceral sensitivity in IBS-D rats at baseline, day 14 and day 28. (B) The defecation area evaluated diarrhea symptoms at baseline, day 7, day 14, day 21, and day 28 in different groups. (C) The intestinal transit rate revealed the changes in colon motility among model groups. Data are presented as mean ± SD (n = 6). **p < 0.01 vs. controls, *p < 0.05 vs. controls.

FIGURE 2

Effects of patchouli alcohol (PA) in IBS-D rats. (A) Abdominal withdrawal reflex assessment in different groups after PA. The AWR scores were used to assess visceral sensitivity at day 21 and day 28 after PA. (B) The defecation area evaluated diarrhea symptoms at day 7 and day 14 after PA. (C) The intestinal transit rate revealed the changes in colon motility among PA groups. Data are presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01, vs. controls; ^△ p < 0.05, ^△△ p < 0.01, vs. models.

FIGURE 3

Substance P (SP) was altered in IBS-D rats. SP proteins photographed by laser confocal microscopy. Immunofluorescence results of SP positive neurons in the proximal and distal colon of IBS-D rats at day 14, day 21, and day 28. HuC/D (Red), SP(Green). Scale Bar = 100 μm.

FIGURE 4

(A) SP-immunoreactive varicosities in the distal and proximal colon. (B) SP mRNA expression in model groups. RNA was isolated with TRIzol reagent and qRT-PCR was utilized to analyze the gene expression of SP. The GAPDH gene was defined as internal reference. Data were presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01, vs. controls.

FIGURE 5

Effects of patchouli alcohol (PA) on substance P (SP). SP proteins photographed by laser confocal microscopy. Immunofluorescence results of SP positive neurons in the proximal and distal colon of PA groups. HuC/D (Red), SP(Green). Scale Bar = 100 μm.

FIGURE 6

(A) SP-immunoreactive varicosities in the distal and proximal colon of PA groups. (B) SP mRNA expression in PA groups. RNA was isolated with TRIzol reagent and qRT-PCR was utilized to analyze the gene expression of SP. The GAPDH gene was used as internal reference. Data are presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01, vs. controls. ^△ p < 0.05, vs. models, ^△△ p < 0.01, vs. models.

FIGURE 7

Choline acetyltransferase (ChAT) is altered in IBS-D rats. ChAT proteins photographed by laser confocal microscopy. Immunofluorescence results of ChAT positive neurons in the proximal and distal colon of IBS-D rats at day 14, day 21, and day 28. HuC/D (Red), ChAT (Green). Scale Bar = 50 μm.

FIGURE 8

(A) The proportion of ChAT positive neurons in the distal and proximal colon. (B) ChAT mRNA expression in model groups. RNA was isolated with TRIzol reagent and qRT-PCR was used to analyze the gene expression of ChAT. The GAPDH gene was used as internal reference. Data are presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01, vs. controls.

FIGURE 9

Effects of patchouli alcohol (PA) on choline acetyltransferase (ChAT). ChAT proteins photographed by laser confocal microscopy. Immunofluorescence results of ChAT positive neurons in the proximal and distal colon of PA groups. HuC/D (Red), ChAT (Green) Scale Bar = 50 μm.

FIGURE 10

(A) The proportion of ChAT positive neurons in the distal and proximal colon of PA groups. (B) ChAT mRNA expression in PA groups. RNA was isolated with TRIzol reagent and qRT-PCR was utilized to analyze the gene expression of ChAT. The GAPDH gene was used as internal reference. Data are presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01, vs. controls. ^△ p < 0.05, vs. models, ^△△ p < 0.01, vs. models.

FIGURE 11

Effects of patchouli alcohol (PA) on substance P (SP) and choline acetyltransferase (ChAT) protein expression (SP and ChAT bands). SP and ChAT proteins were quantified by western blot. CP = proximal colon in control groups, CD = distal colon in control groups, MP = proximal colon in model groups, MD = distal colon in model groups.

FIGURE 12

Effects of patchouli alcohol (PA) on substance P (SP) and choline acetyltransferase (ChAT) protein expression. SP and ChAT proteins were quantified by western blot. β-actin was used as internal reference. (A) SP protein in the distal and proximal colon of PA groups. (B) ChAT protein in the distal and proximal colon of PA groups. Data are presented as mean ± SD (n = 6). *p < 0.05, vs. controls. ^△ p < 0.05, vs. models.