Inhibitory Effect of Steamed Soybean Wastewater Against DSS-Induced Intestinal Inflammation in Mice

Abstract

This study was performed to examine the beneficial potential of steamed soybean wastewater (SSW), which is generated during the manufacture of fermented soybean products and usually discarded as a by-product. The SSW was found to contain considerable amounts of isoflavones and had concentration-dependent radical scavenging capabilities. Moreover, oral administration of SSW effectively prevented colonic damage induced by dextran sulfate sodium (DSS), based on improvement of morphological and histological features, reduction of oxidative stress indicators, suppression of proinflammatory cytokine production, downregulation of inflammatory marker expression in the colonic tissue, and inhibition of the inflammatory activation of macrophages. It suggests that SSW could prevent intestinal inflammation in humans, although its efficacy should be verified through careful study design in humans. These findings have implications for enhancement of the value-added of SSW and for reduction of wastewater treatment costs incurred by the food industry.

Keywords: anti-inflammation; colitis; isoflavones; oligosaccharides; steamed soybean wastewater.

Figure 1

Free radical scavenging activities of steamed soybean wastewater (SSW). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) (A) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (B) radical scavenging activities were determined at various concentrations (0–40 mg/mL) of 80% ethanol extract of SSW powder. Values on bars are presented as mean ± SEM (n = 3). Bars not sharing a common letter are significantly different (p < 0.05).

Figure 2

Oral supplementation of SSW prevented the loss of body weight and the shortening of large intestine in DSS-treated mice. Mice were fed SSW at a dose of 500 or 1000 mg/kg body weight (BW) for 3 weeks and treated with DSS for the last 9 days before sacrifice. SFSA, sulfasalazine, an anti-inflammatory drug to treat ulcerative colitis, was used as a positive control at a dose of 50 mg/kg BW. (A) The trend in average body weight over the experimental period. An asterisk indicates statistical significance compared with the control (p < 0.05). (B,C) The length of the large intestine dissected after sacrifice. (B) Representative photographs of the large intestine (from the cecum to the rectum). (C) Quantification of the large intestinal length. Values are presented as means ± SD (n = 7 mice per group). Bars not sharing a common letter are significantly different (p < 0.05). DSS, dextran sulfate sodium; SFSA, sulfasalazine; SSW, steamed soybean wastewater.

Figure 3

Oral supplementation of SSW protected the colon from DSS-induced histological changes in mice. (A) Representative images of the colon tissue sections stained with H&E (40× magnification). (B) Histological disease score of the colon tissue sections; larger numbers indicate comparatively greater damage. Values are presented as mean ± SD (n = 7 mice per group). Bars not sharing a common letter are significantly different (p < 0.05). DSS, dextran sulfate sodium; SFSA, sulfasalazine; SSW, steamed soybean wastewater.

Figure 4

Oral supplementation of SSW decreased the levels of oxidative markers. (A,B) The levels of 8-OHdG in the plasma (A) and malondialdehyde (MDA) in the liver homogenate (B) were quantified for each experimental group. Values are presented as means ± SD (n = 7 mice per group). Bars not sharing a common letter are significantly different (p < 0.05). DSS, dextran sulfate sodium; SFSA, sulfasalazine; SSW, steamed soybean wastewater.

Figure 5

Oral supplementation of SSW decreased the productions of pro-inflammatory cytokines. (A–C) The plasma levels of pro-inflammatory cytokines, IL-6 (A), TNF-α (B), and IL-1β (C), were quantified by ELISA. (D) The level of anti-inflammatory cytokine, IL-10, was examined in the colonic tissue homogenate. Values are presented as means ± SD (n = 6 mice per group). Bars not sharing a common letter are significantly different (p < 0.05). DSS, dextran sulfate sodium; SFSA, sulfasalazine; SSW, steamed soybean wastewater.

Figure 6

Oral administration of SSW decreased the expression of inflammatory regulators in the colon. (A) Of the cytoplasmic proteins isolated from the colon tissue samples, the relative expression levels of COX-2 and iNOS were analyzed by Western blotting. (B) The nuclear level of NF-κB was comparatively analyzed and quantified. Values are presented as means ± SD (n = 2 mice per group). Bars not sharing a common letter are significantly different (p < 0.05). DSS, dextran sulfate sodium; SFSA, sulfasalazine; SSW, steamed soybean wastewater; NS, not significant.

Figure 7

SSW decreased intracellular reactive oxygen species (ROS) level in RAW264.7 cells. (A) SSW at the concentrations of ≤800 µg/mL were not cytotoxic. (B) The ROS levels increased in tBHP-treated RAW264.7 cells were decreased by SSW treatment at 400 µg/mL or higher. Values are presented as means ± SEM (n = 3). Bars not sharing a common letter are significantly different (p < 0.05). ROS, reactive oxygen species; tBHP, tert-butyl hydroperoxide; SSW, steamed soybean wastewater.

Figure 8

SSW downregulated inflammatory mediators in lipopolysaccharide (LPS)-treated RAW264.7 cells. (A) The nitric oxide (NO) production from LPS-treated RAW264.7 cells was significantly decreased by SSW treatment at 800 µg/mL (B–D) The secretion levels of pro-inflammatory cytokines, IL-1β (B), IL-6 (C), and TNF-α (D), to the culture media were quantified by ELISA. (E,F) The cytoplasmic COX-2 and iNOS levels (E) and nuclear NF-κB level (F) were analyzed by Western blotting. The bars represent the mean ± SEM (n = 3). Bars not sharing a common letter are significantly different (p < 0.05). NO, nitric oxide; LPS, lipopolysaccharide; SSW: steamed soybean wastewater; iNOS, inducible nitric oxide synthase; NS, not significant.