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Blood-brain Barrier Disruption in Diabetic Mice Is Linked to Nrf2 Signaling Deficits: Role of ABCB10?

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Blood-brain Barrier Disruption in Diabetic Mice Is Linked to Nrf2 Signaling Deficits: Role of ABCB10?

Ravi K Sajja et al. Neurosci Lett.

Abstract

Blood-brain barrier (BBB) damage is a critical neurovascular complication of diabetes mellitus that adversely affects the CNS health and function. Previously, we showed the protective role of NF-E2 related factor-2 (Nrf2), a redox sensitive transcription factor, in regulation of BBB integrity. Given the pathogenic role of mitochondrial oxidative stress in diabetes-related microvascular complications, we focused on assessing: 1) the impact of diabetes on brain Nrf2 in correlation with BBB permeability and 2) Nrf2-dependent regulation of the mitochondrial transporter ABCB10, an essential player in mitochondrial function and redox balance at BBB endothelium. Using live animal fluorescence imaging, we demonstrated a strong increase in BBB permeability to 70kDa dextran in db/db diabetic mice that correlated with significant down-regulation of brain Nrf2 protein. Further, Nrf2 gene silencing in human BBB endothelial cells markedly suppressed ABCB10 protein, while Nrf2 activation by sulforaphane up-regulated ABCB10 expression. Interestingly, ABCB10 knockdown resulted in a strong-induction of Nrf2 driven anti-oxidant responses as evidenced by increased expression of Nrf2 and its downstream targets. Nrf2 or ABCB10 silencing elevated endothelial-monocyte adhesion suggesting an activated inflammatory cascade. Thus, our results demonstrate a novel mechanism of ABCB10 regulation driven by Nrf2. In summary, Nrf2 dysregulation and ABCB10 suppression could likely mediate endothelial oxidative/inflammatory stress and BBB disruption in diabetic subjects.

Keywords: ABCB10; In vivo imaging; Leukocyte; Mitochondria; Oxidative stress; db/db mice.

Conflict of interest statement

5. Conflict of Interest

The authors disclose that no conflicts of interest are perceived in publishing this study.

Figures

Fig. 1
Fig. 1. Increased BBB permeability in diabetic mouse brain correlates with Nrf2 down-regulation
Live animal florescent imaging of diabetic (db/db) and control (db/+) mouse brains at 10 (A) and 30min (B) following tail vein injection of RITC dextran (n=5 mice/group). Fluorescence intensity was measured across identical regions of interest and expressed in terms of total radiance. (C) Nrf2 expression were analyzed by western blotting in brain homogenates extracted from diabetic and control mice (n=5 mice/group).
Fig. 2
Fig. 2. Nrf2 regulates mitochondrial ABCB10 expression in BBB endothelial cells
(A) Nrf2 knockdown by siRNA in hCMEC/D3 cells leads to subsequent down-regulation of NQO1 and ABCB10, as determined by western blots. (B) Treatment with SFN (5μM) for 24h significantly enhanced Nrf2 and ABCB10 protein expression in hCMEC/D3 cells, possibly through increased phosphorylation of AKT (Ser 473). Representative western blots were shown. N=3 biological replicates and two independent experiments. *** p < 0.001 and * p < 0.05, vs. control.
Fig. 3
Fig. 3. ABCB10 gene silencing induces oxidative stress response in BBB endothelial cells
(A) Knockdown efficiency of ABCB10 and its effects on the protein expression of Nrf2 and its downstream targets heme oxygenase 1 (HO-1) and (NQO1) in hCMEC/D3 cells were analyzed by western blotting. (B1) Immunofluorescence analyses ABCB10 expression in hCMEC/D3 cells following exposure to low and high glucose conditions and (B2) total cellular levels of ROS in hCMEC/D3 cells. (C) Nrf2 or ABCB10 gene silencing in hCMEC/D3 cells by siRNA significantly enhance THP-1 monocyte adhesion to endothelial cells.. Immunofluorescence images were obtained at 40X magnitude and scale size was 100μm. *** p < 0.001, ** p<0.01 and * p < 0.05, vs. control; # p<0.05 vs 15 mM D-Glucose

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