Daikenchuto, a traditional Japanese herbal medicine, ameliorates postoperative ileus by anti-inflammatory action through nicotinic acetylcholine receptors

Abstract

Background: Daikenchuto (DKT), a gastrointestinal prokinetic Japanese herbal medicine, is prescribed for patients with postoperative ileus (POI) and adhesive bowel obstruction following abdominal surgery. Several mechanisms for the amelioration of POI by DKT have been suggested; however, it has remained unclear whether DKT shows anti-inflammatory effects in POI. In the present study, we investigated the effects of DKT in a mouse POI model and attempted to clarify the detailed mechanisms of action.

Method: Intestinal manipulation (IM) was applied to the distal ileum of mice. DKT was administered orally to the animals 4 times before and after IM. Gastrointestinal transit in vivo, leukocyte infiltration, cytokine mRNA expression and gastrointestinal motility were analyzed. We also investigated the effects of the α7nAChR antagonist methyllycaconitine citrate (MLA) on the DKT-mediated ameliorative action against POI, and we studied the effects of DKT on inflammatory activity in α7nAChR knockout mice.

Results: DKT treatment led to recovery of the delayed intestinal transit induced by IM. DKT significantly inhibited the infiltration of neutrophils and CD68-positive macrophages, and inhibited mRNA expressions of TNF-α and MCP-1. MLA significantly reduced the anti-inflammatory action of DKT, and the amelioration of macrophage infiltration by DKT was partially suppressed in α7nAChR knockout mice.

Conclusions: In conclusion, in addition to the gastrointestinal prokinetic action, DKT serves as a novel therapeutic agent for POI characterized by its anti-inflammatory potency. The DKT-induced anti-inflammatory activity may be partly mediated by activation of α7nAChR.

Fig. 1

HPLC profile of DKT. DKT analyzed by HPLC (ACQUITY UPLC; Nihon Waters K.K, Tokyo, Japan) under following conditions: column, COSMOSIL C18-MS-II (3.0 × 50 mm); mobile phase, H₂O:CH₃Hn (9: 1 → 1:1, linear gradient, for 95 min); flow rate; 1.0 ml/min; oven temperature, 30 °C; injection volume, 10 μl

Fig. 2

Ameliorative action of DKT on gastrointestinal transit in mouse POI model. Detailed procedures are described in “Materials and methods. ” a Shows distribution of PR in the normal group (left panel), at 24 h after IM + Vehicle (middle panel) and after IM + DKT (95 mg/kg, right panel). Columns indicate mean ± SEM of n = 4/group. b, c Show geometric center and gastric emptying rate, as calculated from a. Bars indicate mean ± SEM. ##; significantly different from normal at P < 0.01. * and ** are significantly different from IM + Vehicle at P < 0.05 and P < 0.01, respectively

Fig. 3

Effect of DKT on excretion and cumulative excretion of ¹³CO₂, as calculated by ¹³C-acetate breath test in POI model mice. a, b Showed excretion and cumulative excretion curves for ¹³CO₂, respectively. Detailed procedures are described in “Materials and methods”. Open diamonds, closed squares, open triangles and open circles indicate normal, 24 h after IM, IM with 15 mg/kg DKT and IM with 95 mg/kg DKT groups, respectively. ## are significantly difference from normal (P < 0.01). Symbols indicate mean ± SEM of 5–7 experiments. ** are significantly different between IM group and IM + DKT group (19 and 95 mg/kg), respectively

Fig. 4

Ameliorative effects of DKT on MPO-stained neutrophil infiltration in IM. a Indicates representative results for whole mount immunohistochemical staining of myenteric plexus region to detect MPO-positive neutrophils. b Shows quantification of neutrophil cells in n = 5/normal and IM + DKT (95 mg/kg) group, n = 7/IM + Vehicle and IM + DKT (19 mg/kg) group. Columns indicate mean ± SEM. ## and *: significantly different from normal and IM + Vehicle at P < 0.01 and P < 0.05, respectively

Fig. 5

Ameliorative effects of DKT on CD68-positive macrophage infiltration in IM. a Indicates representative results for whole mount immunohistochemical staining of myenteric plexus region to detect CD68-positive macrophages. b Shows quantification of macrophage cells in n = 5/normal and IM + DKT (95 mg/kg) group, n = 7/IM + Vehicle and IM + DKT (19 mg/kg) group, independent experiments. Columns indicate mean ± SEM. ## and *: significantly different from normal and IM + Vehicle at P < 0.01 and P < 0.05, respectively. Position of myenteric plexus region was confirmed by staining with protein gene product 9.5, which detects myenteric nerve plexus. Red signals indicate PGP9.5-positive myenteric nerves. Green signals indicate CD68-positive macrophages

Fig. 6

Effects of DKT on mRNA expression of inflammatory mediators in inflamed muscle layer of small intestine in mouse POI model. a Showed typical results of RT-PCR. b–f Showed quantitative results of mRNA expression of TNF-α, IL-6, IL-1β, MCP-1 and iNOS. Bars indicate mean ± SEM from n = 4/group. # and ##; significantly different from normal at P < 0.05 and P < 0.01, respectively. *: significantly different from IM + Vehicle at P < 0.05

Fig. 7

Ameliorative effects of DKT and negative effects of MLA on MPO activity and MPO-positive neutrophil infiltration in mouse POI model. a, c Show representative images of MPO-positive neutrophil infiltration into myenteric plexus region and quantification results from n = 4/normal and IM + Vehicle group, n = 5/IM + DKT + MLA group. b Shows results for MPO activity. Columns indicate mean ± SEM from n = 4/group. ##: significantly different from normal at P < 0.01. * and ** are significantly different from IM at P < 0.05, P < 0.01.ψ: significantly different between IM + DKT and IM + DKT + MLA at P < 0.05

Fig. 8

Ameliorative effects of DKT and negative effects of MLA on CD68-positive macrophage infiltration in mouse POI model. a, b Show representative images of CD68-positive macrophage infiltration into the myenteric plexus region and quantification results. Columns indicate mean ± SEM from n = 4/normal, IM + Vehicle and IM + DKT group, n = 5/IM + DKT + MLA group. ## and *: significantly different from normal and IM at P < 0.01, P < 0.05, respectively

Fig. 9

Ameliorative effects of DKT on MPO- and CD68-positive neutrophils and macrophage infiltration in wild-type and α7nAChR KO mice. a, b Show the effects of DKT on MPO-positive neutrophil and CD68-positive macrophage infiltration induced by IM, respectively. Columns represent mean ± SEM from n = 7/wild-type mice, n = 8/normal and IM + Vehicle group in α7nAChR KO mice, n = 9/IM + DKT group in α7nAChR KO mice. Wild-type mice are C57BL/6J mice and α7nAChR KO mice are of C57BL/6J background. ##: significantly different from normal at P < 0.01. * and **: significantly different between IM and IM + DKT at P < 0.05 and P < 0.01, respectively. ψψ: significantly different between wild-type mice and α7nAChR KO mice at P < 0.01