Nutr Res Pract.  2021 Dec;15(6):798-806. 10.4162/nrp.2021.15.6.798.

Dietary glucosinolates inhibit splenic inflammation in high fat/cholesterol diet-fed C57BL/6 mice

• Gu H
• Gwon MH
• Kim SM
• Yun JM

• KMID: 2522658
• DOI: http://doi.org/10.4162/nrp.2021.15.6.798

Abstract

BACKGROUND/OBJECTIVES
Obesity is associated with chronic inflammation. The spleen is the largest organ of the lymphatic system and has an important role in immunity. Obesity-induced inflammatory responses are triggered by Toll-like receptor (TLR)-myeloid differentiation primary response 88 (MyD88) pathway signaling. Phenethyl isothiocyanate (PEITC) and 3,3′-diindolylmethane (DIM), major dietary glucosinolates present in cruciferous vegetables, have been reported to produce anti-inflammatory effects on various diseases. However, the effects of PEITC and DIM on the obesity-induced inflammatory response in the spleen are unclear. The purpose of this study was to examine the antiinflammatory effects of PEITC and DIM on the spleen and their mechanism in high fat/ cholesterol diet (HFCD)-fed C57BL/6 mice.
MATERIALS/METHODS
We established an animal model of HFCD-induced obesity using C57BL/6 mice. The mice were divided into six groups: normal diet with AIN-93G diet (CON), high fat diet (60% calories from fat) with 1% cholesterol (HFCD), HFCD with PEITC 30 mg/kg/ day or 75 mg/kg/day (HFCD+P30, HFCD+P75), and HFCD with DIM 1.5 mg/kg/day or 7.5 mg/kg/ day (HFCD+D1.5, HFCD+D7.5). Enzyme-linked immunosorbent assay was used to evaluate proinflammatory cytokine secretion. Western blot and quantitative polymerase chain reaction were used to analyze protein and mRNA levels of nuclear factor kappa B (NF-κB) p65, interleukin 6 (IL-6), cyclooxygenase 2 (COX-2), TLR2, TLR4, and MyD88 in spleen tissue.
RESULTS
Serum IL-6 levels were significantly higher in the HFCD group than in groups fed a HFCD with PEITC or DIM. Levels of NF-κB p65 protein and TLR2/4, MyD88, NF-κB p65, IL-6, and COX-2 mRNA were significantly higher in the HFCD group than in the CON group and were reduced by the PEITC and DIM supplements.
CONCLUSIONS
PEITC- and DIM-supplemented diets improved splenic inflammation by modulating the TLR2/4-MyD88 pathway in HFCD-fed mice. We suggest that dietary glucosinolates may at least partially improve obesity-induced inflammation of the spleen.

Keyword

Glucosinolates; spleen; obesity; inflammation; Toll-like receptors

Figure

• 

  Fig. 1 Effects of PEITC and DIM supplementation on spleen weight and images of representative spleen tissues from mice fed a high fat/cholesterol diet. (A) Summary of spleen weight in each group. (B) Representative macroscopic images of dissected spleens from each group. There was no significant difference in spleen weights. Data presented as means ± SD (n = 10); different letters indicate significant differences (P < 0.05), as determined by applying Duncan's multiple range test. CON: AIN-93G diet, HFCD: 60% calories from fat+1% cholesterol, HFCD+P30: HFCD+30 mg/kg/day of PEITC, HFCD+P75: HFCD+75 mg/kg/day of PEITC, HFCD+D1.5: HFCD+1.5 mg/kg/day of DIM, HFCD+D7.5: HFCD+7.5 mg/kg/day of DIM.PEITC, phenethyl isothiocyanate; HFCD, high fat/cholesterol diet; DIM, 3,3′-diindolylmethane.

• 

  Fig. 2 PEITC and DIM supplements suppress IL-6 secretion, IL-6 mRNA, COX-2 mRNA, and NF-κB p65 mRNA expressions and NF-κB p65 gene expression in serum and spleen of high fat/cholesterol diet-fed mice. (A) Pro-inflammatory IL-6 secretion was determined by enzyme-linked immunosorbent assay. Total RNA was subjected to qPCR analysis, as described in the materials and methods section. (B) Results of qPCR analysis of IL-6, COX-2, and NF-κB p65 gene expression in high fat/cholesterol diet-fed mice. The transcript mRNA levels of IL-6, COX-2, and NF-κB p65 were normalized to that of β-actin. (C) Lysates were prepared and underwent western blotting with NF-κB p65 antibody, as described in the materials and methods section. Increased NF-κB p65 expression was confirmed via western blot analysis. Values were analyzed using the 2-∆∆CT method. Significance was determined by comparison with β-actin normalized 2-∆∆CT values. Data presented as means ± SD (n = 3), and different letters indicate significant differences (P < 0.05, a > b), as determined by applying Duncan's multiple range test. CON: AIN-93G diet, HFCD: 60% calories from fat + 1% cholesterol, HFCD+P30: HFCD+30 mg/kg/day of PEITC, HFCD+P75: HFCD+75 mg/kg/day of PEITC, HFCD+D1.5: HFCD+1.5 mg/kg/day of DIM, HFCD+D7.5: HFCD+7.5 mg/kg/day of DIM.IL-6, interleukin 6; COX-2, cyclooxygenase 2; PEITC, phenethyl isothiocyanate; DIM, 3,3′-diindolylmethane; HFCD, high fat/cholesterol diet; NF-κB, nuclear factor kappa B; qPCR, quantitative polymerase chain reaction.

• 

  Fig. 3 PEITC and DIM suppress (A) TLR2, (B) TLR4, and (C) MyD88 mRNA expressions in the spleen of high fat/cholesterol diet-fed mice. Results of qPCR analysis of TLR2, TLR4, and MyD88 gene expression in high fat/cholesterol diet-fed mice. Total RNA was subjected to qPCR analysis, as described in the materials and methods section. The transcript mRNA levels of TLR2, TLR4, and MyD88 were normalized to that of β-actin. Values were analyzed using the 2-∆∆CT method. Significance was determined by comparison with β-actin normalized 2-∆∆CT values. Data presented as means ± SD (n = 3), and different letters indicate significant differences (P < 0.05, a > b > c), as determined by applying Duncan's multiple range test. CON: AIN-93G diet, HFCD: 60% calories from fat+1% cholesterol, HFCD+P30: HFCD+30 mg/kg/day of PEITC, HFCD+P75: HFCD+75 mg/kg/day of PEITC, HFCD+D1.5: HFCD+1.5 mg/kg/day of DIM, HFCD+D7.5: HFCD+7.5 mg/kg/day of DIM.TLR, Toll-like receptor; HFCD, high fat/cholesterol diet; MyD88, myeloid differentiation primary response 88; DIM, 3,3′-diindolylmethane; PEITC, phenethyl isothiocyanate; qPCR, quantitative polymerase chain reaction.

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