Involvement of MAPK/NF-κB signaling in the activation of the cholinergic anti-inflammatory pathway in experimental colitis by chronic vagus nerve stimulation

Abstract

Background: Autonomic nervous system dysfunction is implicated in the etiopathogenesis of inflammatory bowel diseases (IBD). Therapies that increase cardiovagal activity, such as Mind-Body interventions, are currently confirmed to be effective in clinical trials in IBD. However, a poor understanding of pathophysiological mechanisms limits the popularization of therapies in clinical practice. The aim of the present study was to explore the mechanisms of these therapies against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats using a chronic vagus nerve stimulation model in vivo, as well as the lipopolysaccharide (LPS)-induced inflammatory response in human epithelial colorectal adenocarcinoma cells (Caco-2) by acetylcholine in vitro.

Methods and results: Colitis was induced in rats with rectal instillation of TNBS, and the effect of chronic VNS (0.25 mA, 20 Hz, 500 ms) on colonic inflammation was evaluated. Inflammatory responses were assessed by disease activity index (DAI), histological scores, myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOS), TNF-α and IL-6 production. The expression of Mitogen-activated protein kinases (MAPK) family members, IκB-α, and nuclear NF-κB p65 were studied by immunoblotting. Heart rate variability (HRV) analysis was also applied to assess the sympathetic-vagal balance. DAI, histological scores, MPO activity, iNOS, TNF-α and IL-6 levels were significantly decreased by chronic VNS. Moreover, both VNS and acetylcholine reduced the phosphorylation of MAPKs and prevented the nuclear translocation of NF-κB p65. Methyllycaconitine (MLA) only reversed the inhibitory effect on p-ERK and intranuclear NF-κB p65 expression by ACh in vitro, no significant change was observed in the expression of p-p38 MAPK or p-JNK by MLA.

Conclusion: Vagal activity modification contributes to the beneficial effects of the cholinergic anti-inflammatory pathway in IBD-related inflamed colonic mucosa based on the activation of MAPKs and nuclear translocation of NF-κB. Our work may provide key pathophysiological mechanistic evidence for novel therapeutic strategies that increase the cardiovagal activity in IBD patients.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. A-B. Time course of the disease activity index (DAI) (mean± SEM) scores and body weight (mean±SEM) in the control group (n=8), VNS group (n=8), TNBS group (n=10) or TNBS+VNS group (n=10).

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Figure 2. Chronic VNS reduces the severity of TNBS-induced colitis in rats.

(A) Effects of administration of VNS after colonic instillation with TNBS; CMDI scores were quantified and expressed as the mean±SEM. (B) Representative macroscopic appearance of colonic mucosa across groups. * P

Figure 3. A–E. Chronic VNS ameliorates TNBS-induced colitis histologically.

Photomicrographs (magnification ×100 & ×200) are representative of H&E stained slides of colonic tissues. A: Normal colonic mucosa of the SD rats. B: Mucosa of normal SD rats after VNS administration. C: Severe inflammation is present on the mucosa of TNBS-treated rats with inflammatory cell infiltration, ulcerations and goblet cell depletion (arrow). D: Treatment of TNBS-rats with chronic VNS for 6 days markedly decreased the inflammatory cell infiltration in the mucosa, and the arrow indicates the remaining goblet cells. E: The mean ± SEM of the histologic inflammatory scores are calculated for each group, as described in the methods section. * P

Figure 4. Colonic mucosal ACh level in the control group (n=8), VNS group (n=8), TNBS group (n=10) or TNBS+VNS group (n=10).

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Figure 5. A–D. Effect of TNBS and VNS on LFnm(A), HFnm(B), LF/HF(C) and TP(D) on experimental colitis.

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Figure 6. Chronic VNS inhibits the activation of NF-κB p65 on TNBS-induced colitis.

Photomicrographs (magnification ×400) are representative of immunohistochemically stained slides with NF-κB p65 anti-body in colon mucosa. (A) and (B) show normal colon mucosa, and colon mucosa with TNBS (C) shows markedly increased NF-κB p65 nuclear-positive cells (arrow). Colon mucosa from the TNBS model treated with VNS (D) shows much less translocation of NF-κB p65. Western blot was also performed with NF-κB p65 anti-body (E), and densitometric analysis was normalized to Histone H3. The results are expressed as the mean ± SEM (n = 3). The data shown are representative of three independent experiments. * P

Figure 7. Acetylcholine inhibits LPS-induced TNF-α expression and NF-κB translocation in Caco-2 cells.

Caco-2 cells (5×105 cells/well) were pretreated with increasing concentrations of Acetylcholine (0.1-10 µM) with or without methyllycaconitine (10 µM) for 60 min and then incubated with or without LPS (10 µg/ml) for 24 h. Cells were then lysed, proteins from the whole cell were extracted, and the nuclear extract cells were analyzed by immunoblotting with anti-TNF-α antibody (A) and anti-NF-κB p65 antibody (B). Densitometric analysis was normalized to β-actin and Histone H3, respectively, and the results are expressed as the mean ± SEM (n = 3). The data shown are representative of three independent experiments. * P<0.05 versus the control group; # P<0.05 versus the LPS group.

Figure 8. Chronic VNS inhibition of IκB-α (A) degradation and p-ERK1/2 (B), p-JNK (C), and p-p38 (D) activation in colon tissue from TNBS-induced colitis rats.

Densitometric analysis was normalized to the control (ERK1/2, JNK, p38 and β-actin, respectively), and the results are expressed as the mean ± SEM (n = 3). The data shown are representative of three independent experiments. * P

Figure 9. Acetylcholine inhibits LPS-induced activation of p-ERK1/2 (A), p-JNK (B) and p-p38 MAPK (C) in Caco-2 cells.

Caco-2 cells (5×105 cells/well) were pretreated with 10 µM Acetylcholine with or without methyllycaconitine (10 µM) for 60 min and then incubated with or without LPS(10 µg/ml) for 24 h. Cells were then lysed, and the proteins were analyzed by western blot. Densitometric analysis was normalized to the control (ERK1/2, JNK, and p38, respectively), and results are expressed as the mean±SEM (n = 3). The data shown are representative of three independent experiments. * P<0.05 versus the control group; # P<0.05 versus the LPS group.