Altered vasoactive intestinal peptides expression in irritable bowel syndrome patients and rats with trinitrobenzene sulfonic acid-induced colitis

Abstract

Aim: To investigate the vasoactive intestinal peptides (VIP) expression in irritable bowel syndrome (IBS) and trinitrobenzene sulfonic acid (TNBS) induced colitis.

Methods: The VIP gene expression and protein plasma levels were measured in adult participants (45.8% male) who met Rome III criteria for IBS for longer than 6 mo and in a rat model of colitis as induced by TNBS. Plasma and colons were collected from naïve and inflamed rats. Markers assessing inflammation (i.e., weight changes and myeloperoxidase levels) were assessed on days 2, 7, 14 and 28 and compared to controls. Visceral hypersensitivity of the rats was assessed with colo-rectal distension and mechanical threshold testing on hind paws. IBS patients (n = 12) were age, gender, race, and BMI-matched with healthy controls (n = 12). Peripheral whole blood and plasma from fasting participants was collected and VIP plasma levels were assayed using a VIP peptide-enzyme immunoassay. Human gene expression of VIP was analyzed using a custom PCR array.

Results: TNBS induced colitis in the rats was confirmed with weight loss (13.7 ± 3.2 g) and increased myeloperoxidase activity. Visceral hypersensitivity to colo-rectal distension was increased in TNBS treated rats up to 21 d and resolved by day 28. Somatic hypersensitivity was also increased up to 14 d post TNBS induction of colitis. The expression of an inflammatory marker myeloperoxidase was significantly elevated in the intracellular granules of neutrophils in rat models following TNBS treatment compared to naïve rats. This confirmed the induction of inflammation in rats following TNBS treatment. VIP plasma concentration was significantly increased in rats following TNBS treatment as compared to naïve animals (P < 0.05). Likewise, the VIP gene expression from peripheral whole blood was significantly upregulated by 2.91-fold in IBS patients when compared to controls (P < 0.00001; 95%CI). VIP plasma protein was not significantly different when compared with controls (P = 0.193).

Conclusion: Alterations in VIP expression may play a role in IBS. Therefore, a better understanding of the physiology of VIP could lead to new therapeutics.

Trial registration: ClinicalTrials.gov NCT00824941.

Keywords: Gene expression; Irritable bowel syndrome; PCR arrays; Trinitrobenzene sulfonic acid; Vasoactive intestinal peptide.

Figure 1

Colitis induction following trinitrobenzene sulfonic acid administration. A: Weight loss after trinitrobenzene sulfonic acid (TNBS) enema. Graph shows the weight change in grams after TNBS treatment (n = 20 for control, n = 20 for Day 0, n = 20 for Day 2, n = 20 for Day 7, n = 12 for Day 14, n = 5 for Day 21); B: Visceral hypersensitivity after TNBS treatment. Graph representing colo-rectal distension (CRD) scores in mmHg before and after TNBS (n = 10 for 7 d, n = 15 for 14 d, n = 5 for 21 d, n = 8 for 28 d); C: somatic hypersensitivity after TNBS treatment. Graph representing the mechanical threshold testing on the hind paws, Von Frey. Data were shown as a percentage of control (naïve) threshold response (n = 21 for 7 d, n = 24 for 14 d, n = 12 for 21 d, n = 27 for 28 d); D: presence of inflammation following TNBS-induced colitis. Graph shows the average myeloperoxidase (MPO) activity (vehicle = ethanol, for all conditions, n = 3, each with 8 replicates). ^aP < 0.05, ^bP < 0.01 vs controls; ANOVA with Bonferroni’s and Dunnett’s posttests.

Figure 2

Vasoactive intestinal peptide co-localizes with the neuronal marker; Neurofilament. Vasoactive intestinal peptide (VIP) co-localizes with the neuronal marker; Neurofilament (NF). Top panel shows VIP positive cells (A), NF positive cells (B) and co-labeled cells in yellow with DAPI nuclear stain in blue (C) for control animals. Lower panel shows VIP positive cells (D), NF positive cells (E) and co-labeled cells in yellow with DAPI nuclear stain in blue (F) 14 d following TNBS treatment. TNBS: Trinitrobenzene sulfonic acid.

Figure 3

Vasoactive intestinal peptide in plasma after trinitrobenzene sulfonic acid treatment. Plasma concentration of vasoactive intestinal peptide (VIP) was assessed in naïve animals and after trinitrobenzene sulfonic acid (TNBS) treatment at the 2-h and 14-d time-points using a peptide enzyme immunoassay. n = 4 for all conditions, ^aP < 0.05 vs controls; ANOVA with Dunnett’s posttests.

Figure 4

Gene expression in irritable bowel syndrome participants vs healthy controls. Compares the expression of 88 genes and PCR controls for IBS participants (n = 12) vs healthy participants (n = 12). VIP fold regulation = 2.91. Boundary = 2. IBS: Irritable bowel syndrome; VIP: Vasoactive intestinal peptides.

Figure 5

Vasoactive intestinal peptide in human plasma. Vasoactive intestinal peptide (VIP) protein levels were not significantly different in IBS patients (n = 12, 0.193 ng/mL) when compared to controls (n = 11, 0.254 ng/mL), P = 0.173. IBS: Irritable bowel syndrome.