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. 2018 Aug 23;8(1):12708.
doi: 10.1038/s41598-018-31137-7.

In Vitro Modulation of Redox and Metabolism Interplay at the Brain Vascular Endothelium: Genomic and Proteomic Profiles of Sulforaphane Activity

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Free PMC article

In Vitro Modulation of Redox and Metabolism Interplay at the Brain Vascular Endothelium: Genomic and Proteomic Profiles of Sulforaphane Activity

Ravi K Sajja et al. Sci Rep. .
Free PMC article

Abstract

Sulforaphane (SFN) has been shown to protect the brain vascular system and effectively reduce ischemic injuries and cognitive deficits. Given the robust cerebrovascular protection afforded by SFN, the objective of this study was to profile these effects in vitro using primary mouse brain microvascular endothelial cells and focusing on cellular redox, metabolism and detoxification functions. We used a mouse MitoChip array developed and validated at the FDA National Center for Toxicological Research (NCTR) to profile a host of genes encoded by nuclear and mt-DNA following SFN treatment (0-5 µM). Corresponding protein expression levels were assessed (ad hoc) by qRT-PCR, immunoblots and immunocytochemistry (ICC). Gene ontology clustering revealed that SFN treatment (24 h) significantly up-regulated ~50 key genes (>1.5 fold, adjusted p < 0.0001) and repressed 20 genes (<0.7 fold, adjusted p < 0.0001) belonging to oxidative stress, phase 1 & 2 drug metabolism enzymes (glutathione system), iron transporters, glycolysis, oxidative phosphorylation (OXPHOS), amino acid metabolism, lipid metabolism and mitochondrial biogenesis. Our results show that SFN stimulated the production of ATP by promoting the expression and activity of glucose transporter-1, and glycolysis. In addition, SFN upregulated anti-oxidative stress responses, redox signaling and phase 2 drug metabolism/detoxification functions, thus elucidating further the previously observed neurovascular protective effects of this compound.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
SFN impact on mitochondria-related gene expression. (A1) Cell viability was not affected by SFN (5 µM) as determined by MTT assay. (A2) SFN treatment upregulates the expression level of Nrf2 and its downstream target Nqo1 in mice brain microvascular endothelial cells. (B1B3) Volcano plots of gene expression in response to different concentration of SFN (B1) 2 µM vs. control; (B2) 5 µM vs control and (B3) 2 vs. 5 µM) as analyzed by MitoChip microarray in primary cells. Horizontal line represents p = 0.05. Genes with expression not significantly altered are indicated by solid black circles and genes with expression significantly altered are indicated by solid red circles. Significantly down-regulated genes are in the upper left quadrant and significantly up-regulated genes are in the upper right quadrant. N = 3–4 independent biological samples per condition repeated in triplicates. Blots of Nrf2, Nqo1 and β-actin were taken from different gels.
Figure 2
Figure 2
SFN modulate the redox-metabolic interplay of the brain vascular endothelium to promote ATP production. SFN promotes upregulation of Glut1 expression (A) and transport activity (B) as determined by 2-NBDG uptake normalized to total protein. (C) Key genes involved in the regulation of glycolysis were also upregulated including hexokinase 1 (Ht1) and pyruvate kinase 2 (Pkm2) as analyzed by MitoChip array (C1) and confirmed by RT-PCR (C2). The overall cellular effect was a substantial increase in ATP production (D). N = 3 independent biological samples per condition assayed in triplicates. “*”p < 0.05, “**”p < 0.01 compared to controls (fold changes). For the time-dependent study (24 vs. 48 h SFN treatment) “+” p < 0.05 vs. 24 h. Blots of Glut1 and β-actin were taken from different gels.
Figure 3
Figure 3
SFN promotes the expression of genes involved in oxidative stress responses and redox signaling. MitoCHIP array-based analysis of anti-oxidative gene expression changes (A1). Most prominent/relevant gene expression were validated ad hoc by RT-PCR (A2). SFN treatment up-regulated the expression of Abc transporters (B1) and most relevant gene expression changes were validated ad hoc by RT-PCR (B2). N = 4 biological replicates per condition (assayed in triplicates). *p < 0.05, **p < 0.005 and ***p < 0.001 vs. controls (fold changes).
Figure 4
Figure 4
SFN promotes the expression of the Slc40a1 iron importer in brain vascular endothelial cells. SFN promotes the transcription of the Slc40a1 iron exporter gene (fold change) as shown by MitoChip array (A1) and RT-PCR analysis (A2). Results were further confirmed through measurements of Slc40a1 protein expression by western blots (B1) and immunofluorescence (B2). Image scale = 100 µm at 40x magnification. N = 3–4 biological replicates per condition (assayed in triplicates). **p < 0.01, ***p < 0.001 and ****p < 0.0001, vs. controls. Blots of Slc40a1 and β-actin were taken from different gels.
Figure 5
Figure 5
SFN regulates the expression of phase 2 drug metabolism/detoxification genes of the glutathione system in a dose-dependent manner. (A1) MitoChip array data of GSTs enzymes involved in cellular detoxification that were affected by SFN. Changes in the gene expression levels of relevant GST enzymes (Gsta1 and Gstm1) were validated ad hoc by RT-PCR (A2). Results demonstrated a dose and time-dependent effect of SFN treatment over upregulation of these enzymes. N = 4 samples per condition and assayed in triplicates. *p < 0.05, **p < 0.005 and ***p < 0.001 compared to controls (fold changes).

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