Shuganyin decoction improves the intestinal barrier function in a rat model of irritable bowel syndrome induced by water-avoidance stress

Abstract

Background: To determine the effect of Shuganyin decoction (SGD) on the intestinal barrier function in an irritable bowel syndrome (IBS) rat model induced by water-avoidance stress.

Methods: Forty male Wistar rats were divided into control, water-avoidance stress (WAS) group, WAS plus Shuganyin decoction (SGD) group and WAS plus dicetel (Dicetel) group. IBS was induced in rats by subjecting them to water-avoidance stress for 7 days. On day 4 of the WAS protocol, the rats were treated for 7 consecutive days (days 4-11) with SGD, dicetel or a negative control (saline). The number of feces granules, histopathological changes of the intestine and mast cell (MC) morphometry were determined. Intestinal permeability was approximated by measuring the absorption of FITC-dextran 4400 (FD-4) from the lumen into the bloodstream in vivo and in vitro experiments. Also, the expression of protease active receptor-2 (PAR-2) and tumor necrosis factor-α (TNF-α) was estimated using immunohistochemical staining and ELISA, respectively. Tight junction (TJ) protein abundance was measured following a quantitative immunofluorescent analysis of intestinal sections and western blotting.

Results: In vivo, WAS elicited a significantly increase in the transfer of FD-4 from the intestine to blood about threefold in 30 min compared with control group. After treated with SGD, the intestinal permeability to FD-4 of WAS-induced rats was significantly attenuated (P < 0.05). In vitro, the permeability coefficient (Papp) values were measured for FD-4 absorption across the excised intestine. WAS was shown to increase the intestinal permeability to (4.695 ± 0.3629) × 10^-7 cm/s in 120 min, which was 2.6-fold higher than the control group. Rats treated with SGD showed a significant decrease in Papp values of FD-4 as compared to WAS group (P < 0.05). Furthermore, by immunofluorescent detection we found that WAS elicited the irregular distribution of TJ proteins. Using the quantitative analysis software of the medical image, the average optical density and protein abundance of TJ proteins was shown to be lower in the WAS group as compared to control group, (P < 0.05). SGD could attenuate this response and improve TJ distribution (P < 0.05). Western blot analysis confirmed that TJ protein abundance was significantly decreased in WAS group and that they could be returned to control levels following an SGD treatment. WAS also induced an increase in number of MCs, their area and diameter as compared to controls. These observations were attenuated with an SGD or dicetel treatment. Similarly, the expression of PAR-2 and TNF-α exceeded control values in the WAS group and were shown to be successfully attenuated with an SGD treatment.

Conclusion: WAS-induced IBS rat model exhibited intestinal barrier dysfunction, which was manifested as tight junction damage and structural rearrangements that increased the intestinal permeability. Under these conditions, MCs were activated and degranulated in the intestinal mucosa leading to the activation of PAR-2. Our data showed that SGD could inhibit the activation of MCs and down-regulate the expression of both PAR-2 and TNF-α. In turn, this was shown to improve the expression and structural arrangement of TJ proteins in the intestinal mucosa, thereby regulating the intestinal permeability. It was concluded that Shuganyin could protect the intestinal barrier.

Keywords: Intestinal barrier function; Mast cell; Protease activated preceptor-2; Rat model; Shuganyin decoction; Water-avoidance stress.

Fig. 1

Body weights of control and WAS rats both before and after intervention with SGD or Dicetel. a Body weight of rats in the control, WAS, SGD and Dicetel groups on day 1 (before drug administration). b Body weight of rats in the control, WAS, SGD and Dicetel groups on day 6 (after drug treatment). c Body weight of rats in the control, WAS, SGD and Dicetel groups on day 10 (after drug treatment). Control, control group; WAS, water avoidance stress model group; SGD, WAS model + Shuganyin decoction; Dicetel, WAS model + Dicetel treatment as a positive control. n = 10 per group

Fig. 2

Control and WAS group defecation before and after treatment with SGD or Dicetel. A The number of feces from control, WAS, SGD and Dicetel groups recorded on day 3 (before drug treatment). B The number of feces from control, WAS, SGD and Dicetel groups recorded on day 6 (after drug treatment). C The number of feces from control, WAS, SGD and Dicetel groups recorded on day 10 (after drug treatment). Data were expressed as the mean ± SD. ^aP < 0.01 compared to the control group; ^bP < 0.01 compared to the WAS group; ^cP > 0.05 compared to the SGD group. n = 10 per group

Fig. 3

WAS-induced alterations in rat intestine permeability estimated by the rate of FD-4 transfer across the intestinal epithelium. A, B In vitro Papp values for FD-4 absorption across excised ileum. C In vivo detection of FD-4 in the blood stream 120 min after infusion into the lumen of the small intestine. Data were expressed as the mean ± SD. ^aP < 0.05 compared to the control group; ^bP < 0.05 compared to the WAS group; ^cP > 0.05 compared to the SGD group. n = 10 per group

Fig. 4

Immunohistological detection of ZO-1, occludin and F-actin protein levels in intestinal mucosa. A Immunohistological detection ZO-1 protein in the intestinal mucosa of animals from control group (a), WAS group (b), SGD group (c) and Dicetel group (d), (×400); average optical density levels of ZO-1 protein in the different groups (right panel). B Immunohistological detection of occludin protein levels in intestinal mucosa of animals from control group (a), WAS group (b), SGD group (c) and Dicetel group (d), (×400); average optical density levels of occludin in different groups (right panel). C Immunohistological detection of F-actin protein level in intestinal mucosa of animals from control group (a), WAS group (b), SGD group (c) and Dicetel group (d), (×400); average optical density levels of F-actin in different groups (right panel). Data were expressed as the mean ± SD. ^aP < 0.05 compared to the control group; ^bP < 0.05 compared to the WAS group; ^cP > 0.05 compared to the SGD group. n = 10 per group

Fig. 5

Western blot analysis of ZO-1, occludin and F-actin protein level in intestinal mucosa homogenates. A Western blot of ZO-1, occludin and F-actin proteins in intestinal mucosa homogenates from the control group, WAS group, SGD group and Dicetel group; B–D quantification the protein levels of ZO-1 (B), occludin (C) and F-actin (D) in the different experimental groups. ^aP < 0.05 compared to the control group; ^bP < 0.05 compared to the WAS group; ^cP > 0.05 compared to the SGD group. n = 3–5 per group

Fig. 6

Morphology of mast cells in intestinal mucosa. A Toluidine blue staining revealing mast cells in the mucosa of animals from control group (a), WAS group (b), SGD group (c) and Dicetel group (d), (×200). B–D Quantification of mast cell characteristics in the mucosa and sub mucosa of colon. ^aP < 0.05 compared to the control group; ^bP < 0.05 compared to the WAS group; ^cP > 0.05 compared to the SGD group. n = 10 per group

Fig. 7

The immunohistochemical of PAR-2 in colon tissue. A Immunohistochemical staining revealing PAR-2 in the mucosa of rats from control group (a), WAS group (b), SGD group (c) and Dicetel group (d), (×200). B IHC of PAR-2 in different groups. Data were expressed as the mean ± SD. ^aP < 0.05 compared to the control group; ^bP < 0.05 compared to the WAS group; ^cP > 0.05 compared to the SGD group. n = 10 per group

Fig. 8

The expression of TNF-α in colon tissue. The expression of TNF-α in different groups. Data were expressed as the mean ± SD. ^aP < 0.05 compared to the control group; ^bP < 0.05 compared to the WAS group; ^cP > 0.05 compared to the SGD group. n = 10 per group

Fig. 9

The morphological of liver, kidney and lung tissues stained with H&E after treated with SGD. H&E staining revealing in the liver tissue (a), kidney tissue (b), lung tissue (c) (×200)

Fig. 10

The biochemical parameters of rats after treated with SGD. a The expression of ALT before and after treated with SGD; b The expression of AST before and after treated with SGD; c The expression of BUN before and after treated with SGD; d The expression of Cr before and after treated with SGD. Data were expressed as the mean ± SD. n = 10 per group