In Vitro Insights into the Dietary Role of Glucoraphanin and Its Metabolite Sulforaphane in Celiac Disease

Abstract

Sulforaphane is considered the bioactive metabolite of glucoraphanin after dietary consumption of broccoli sprouts. Although both molecules pass through the gut lumen to the large intestine in stable form, their biological impact on the first intestinal tract is poorly described. In celiac patients, the function of the small intestine is affected by celiac disease (CD), whose severe outcomes are controlled by gluten-free dietary protocols. Nevertheless, pathological signs of inflammation and oxidative stress may persist. The aim of this study was to compare the biological activity of sulforaphane with its precursor glucoraphanin in a cellular model of gliadin-induced inflammation. Human intestinal epithelial cells (CaCo-2) were stimulated with a pro-inflammatory combination of cytokines (IFN-γ, IL-1β) and in-vitro-digested gliadin, while oxidative stress was induced by H₂O₂. LC-MS/MS analysis confirmed that sulforaphane from broccoli sprouts was stable after simulated gastrointestinal digestion. It inhibited the release of all chemokines selected as inflammatory read-outs, with a more potent effect against MCP-1 (IC₅₀ = 7.81 µM). On the contrary, glucoraphanin (50 µM) was inactive. The molecules were unable to counteract the oxidative damage to DNA (γ-H2AX) and catalase levels; however, the activity of NF-κB and Nrf-2 was modulated by both molecules. The impact on epithelial permeability (TEER) was also evaluated in a Transwell^® model. In the context of a pro-inflammatory combination including gliadin, TEER values were recovered by neither sulforaphane nor glucoraphanin. Conversely, in the context of co-culture with activated macrophages (THP-1), sulforaphane inhibited the release of MCP-1 (IC₅₀ = 20.60 µM) and IL-1β (IC₅₀ = 1.50 µM) only, but both molecules restored epithelial integrity at 50 µM. Our work suggests that glucoraphanin should not merely be considered as just an inert precursor at the small intestine level, thus suggesting a potential interest in the framework of CD. Its biological activity might imply, at least in part, molecular mechanisms different from sulforaphane.

Keywords: celiac disease; glucoraphanin; glucosinolates; gut barrier; gut inflammation; sulforaphane.

Figure A1

Cell viability measured by MTT assay. CaCo-2 cells were treated with sulforaphane (SFN, horizontal lines) or glucoraphanin (GFN, dots), in addition to the pro-inflammatory combination of IL-1β, IFN-γ (10 ng/mL), and digested gliadin (Ga) (black bar), for 24 h. Data from independent experiments (n = 3) were reported as mean absorbance (% ± SEM) vs. stimulus, to which was arbitrarily attributed the value of 100%.

Figure A2

Gene expression of catalase gene (hCAT) measured by rt-PCR in CaCo-2 cells. Cells were treated with sulforaphane (SFN, horizontal lines) or glucoraphanin (GFN, dots), in addition to the pro-oxidant stimulus H₂O₂ (1 mM) (black bar), for 6 h. Data from independent experiments (n = 3) were reported as fold change (% ± SEM) vs. untreated control (white bar), to which was arbitrarily attributed the value of 1.

Figure 1

Effect of sulforaphane and glucoraphanin on the release of chemokines by CaCo-2 cells. The release of CXCL-10 (A), IL-8 (B), and MCP-1 (C) was measured by ELISA assay. Cells were treated with sulforaphane (SFN, horizontal lines) or glucoraphanin (GFN, dots), in addition to the pro-inflammatory combination of IL-1β, IFN-γ (10 ng/mL), and digested gliadin (Ga, 1 mg/mL) (black bar), for 24 h. Apigenin 20 μM was used as reference inhibitor of CXCL-10 (−30%), IL-8 (−16%), and MCP-1 (−30%). Data from independent experiments (n = 3) were reported as mean of release (% ± SEM) vs. stimulus, to which was arbitrarily attributed the value of 100%. *** p < 0.001 vs. stimulus.

Figure 2

Effect of sulforaphane and glucoraphanin on DNA damage caused by H₂O₂ in CaCo-2 cells. DNA double-strand break was revealed by immunofluorescence staining of phospho-γ-H2AX (ser 139). Cells were treated with sulforaphane (SFN 25 μM) or glucoraphanin (GFN 25 μM) for 1 h, in addition to the pro-oxidant stimulus H₂O₂ (1 mM). Resveratrol 20 μM (Resv.) was used as reference antioxidant compound. Representative images from independent experiments (n = 3) were reported (60× objective magnification, scale bar equivalent to 50 μm).

Figure 3

Effect of sulforaphane and glucoraphanin on Nrf-2 driven transcription and catalase activity in CaCo-2 cells. The activity of Nrf-2 was measured after plasmid transfection by luciferase assay (A), while catalase activity was measured by enzymatic assay (B). Cells were treated with sulforaphane (SFN, horizontal lines) or glucoraphanin (GFN, dots) for 6 h in addition to the pro-oxidant stimulus H₂O₂ 1 mM. Resveratrol 20 μM was used as reference inducer of Nrf-2 (+220%). Data from independent experiments (n = 3) were reported as mean activity (% ± SEM) vs. stimulus (black bar), to which was arbitrarily attributed the value of 100%. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. stimulus.

Figure 4

Effect of sulforaphane and glucoraphanin on NF-κB driven transcription in CaCo-2 cells. The activity of NF-κB was measured after plasmid transfection by luciferase assay. Cells were treated with sulforaphane (SFN, horizontal lines) or glucoraphanin (GFN, dots) for 6 h in addition to the pro-inflammatory combination of IL-1β, IFN-γ (10 ng/mL) and digested gliadin (Ga, 1 mg/mL) (A), or pro-oxidant stimulus H₂O₂ 1 mM (B). Apigenin 20 μM and resveratrol 20 μM were used as reference inhibitors (−97% and −90%, respectively). Data from independent experiments (n = 3) were reported as mean luminescence (% ± SEM) vs. stimulus (black bar), to which was arbitrarily attributed the value of 100%. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. stimulus.

Figure 5

Effect of sulforaphane and glucoraphanin on CaCo-2 epithelial barrier (Transwell^® model). Cells were treated with sulforaphane (SFN, horizontal lines) or glucoraphanin (GFN, dots) for 24 h, in addition to the pro-inflammatory combination of IL-1β, IFN-γ (10 ng/mL), and digested gliadin (Ga, 1 mg/mL): (A) The release of MCP-1 was measured by ELISA assay. Data from independent experiments (n = 3) were reported as mean release (%) ± SEM vs. stimulus (black bar), to which was arbitrarily attributed the value of 100%. (B) Epithelial integrity was measured as normalized TEER variation (ΔΩ = Ωt24h − Ωt0). Data from independent experiments (n = 4) were reported as normalized ΔΩ ± SEM vs. stimulus, to which was arbitrarily attributed the value of 0. Sodium butyrate 2 mM was used as reference inhibitor of MCP-1 release (−40%) and trophic factor for the epithelial barrier (+40 Ω). *** p < 0.001 vs. stimulus.

Figure 6

Effect of sulforaphane and glucoraphanin on the epithelial barrier in the co-culture of CaCo-2 and THP-1 macrophages (Transwell^® model). Cells were treated with sulforaphane (SFN, horizontal lines) or glucoraphanin (GFN, dots) for 24 h, in addition to the pro-inflammatory combination of LPS (100 ng/mL) and IFN-γ (10 ng/mL): (A,B) The release of MCP-1 and IL-1β was measured by ELISA assay on media collected from co-culture or THP-1, respectively. Data from independent experiments (n = 3) were reported as mean release (%) ± SEM vs. stimulus (black bar), to which was arbitrarily attributed the value of 100%. (C) Epithelial integrity was measured as normalized TEER variation (ΔΩ = Ωt24h − Ωt0). Data from independent experiments (n = 4) were reported as normalized ΔΩ ± SEM vs. stimulus, to which was arbitrarily attributed the value of 0. Sodium butyrate 2 mM was used as reference inhibitor of MCP-1 release (−17%) and trophic factor for the epithelial barrier (+220 Ω). Apigenin 20 μM was used as reference inhibitor of IL-1β (−72%). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. stimulus.