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).
∗ P<0.05 versus the control group and VNS group; △P<0.05 versus the TNBS group.
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<0.05 versus the control group and VNS group; △P<0.05 versus the TNBS group.
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<0.05 versus the control group; △P<0.05 versus the TNBS group.
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).
* P<0.05 versus the control group; △P<0.05 versus the TNBS group.
Figure 5.
A–D. Effect of TNBS and VNS on LFnm(A), HFnm(B), LF/HF(C) and TP(D) on experimental colitis.
* P<0.05 versus control group and VNS group; # P<0.05 versus TNBS group; △P<0.05 versus VNS group.
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<0.05 versus the control group; Δ P<0.05 versus the TNBS group.
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<0.05 versus the control group; Δ P<0.05 versus the TNBS group.
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.