Natural vitamin E α-tocotrienol protects against ischemic stroke by induction of multidrug resistance-associated protein 1

Abstract

Background and purpose: α-Tocotrienol (TCT) represents the most potent neuroprotective form of natural vitamin E that is Generally Recognized As Safe certified by the U.S. Food and Drug Administration. This work addresses a novel molecular mechanism by which α-TCT may be protective against stroke in vivo. Elevation of intracellular oxidized glutathione (GSSG) triggers neural cell death. Multidrug resistance-associated protein 1 (MRP1), a key mediator of intracellular oxidized glutathione efflux from neural cells, may therefore possess neuroprotective functions.

Methods: Stroke-dependent brain tissue damage was studied in MRP1-deficient mice and α-TCT-supplemented mice.

Results: Elevated MRP1 expression was observed in glutamate-challenged primary cortical neuronal cells and in stroke-affected brain tissue. MRP1-deficient mice displayed larger stroke-induced lesions, recognizing a protective role of MRP1. In vitro, protection against glutamate-induced neurotoxicity by α-TCT was attenuated under conditions of MRP1 knockdown; this suggests the role of MRP1 in α-TCT-dependent neuroprotection. In vivo studies demonstrated that oral supplementation of α-TCT protected against murine stroke. MRP1 expression was elevated in the stroke-affected cortical tissue of α-TCT-supplemented mice. Efforts to elucidate the underlying mechanism identified MRP1 as a target of microRNA (miR)-199a-5p. In α-TCT-supplemented mice, miR-199a-5p was downregulated in stroke-affected brain tissue.

Conclusions: This work recognizes MRP1 as a protective factor against stroke. Furthermore, findings of this study add a new dimension to the current understanding of the molecular bases of α-TCT neuroprotection in 2 ways: by identifying MRP1 as a α-TCT-sensitive target and by unveiling the general prospect that oral α-TCT may regulate miR expression in stroke-affected brain tissue.

Figures

Figure 1. MRP1 knockdown attenuated the neuroprotection of α-tocotrienol in primary cortical neurons

After 24h of seeding, primary cortical neurons were treated with glutamate (5mmol/L, 24h). Glutamate challenge induced the expression of MRP1 protein (A). After MRP1 siRNA transfection, MRP1 mRNA expression was significantly down-regulated (B). C, Glutamate-challenged primary neurons with MRP1 knockdown exhibited loss of functional MRP1 by retaining more calcein compared to corresponding control cells. D, α-TCT (1μmol/L) was added into cell culture medium 6h before glutamate treatment. After glutamate (5mmol/L, 24h) challenge, LDH leakage was measured. Neuroprotection of α-TCT was compromised under conditions of MRP1 knockdown. Bar=50μm, n=3, *P<0.05 compared with control; §P< 0.05 compared with control siRNA-transfected, glutamate-treated primary neurons.

Figure 2. MCAO-induced brain injury was exacerbated in MRP1 deficient mice

Transient focal cerebral ischemia was induced in MRP1 deficient (n=20) or FVB mice (n=15) by MCAO. A, MRP1 KO mice presented larger stroke-induced lesion compared to FVB mice after 48h reperfusion (FVB, n=8; MRP1 KO, n=12). B, Protein expression of MRP1 was increased in infarct hemisphere of FVB mice (n=3). C, MRP1 deficient mice contained significantly higher level of GSSG in stroke-affected hemisphere compared to that of FVB mice (n=4). *P<0.05 compared with corresponding contralateral hemisphere; †P<0.05 compared with FVB; §P<0.05 compared with infarct hemisphere of FVB.

Figure 3. MCAO-induced neurodegeneration was accentuated in MRP1 deficient mice

A, MRP1 expression was upregulated at the stroke affected cortex of FVB mice (red-MRP1 protein; blue-DAPI stained nuclei) B, The infarct hemisphere of MRP1 deficient mice presented abundant Fluoro-Jade signals in cortical region (green-Fluoro-Jade positive neurons; blue-DAPI stained nuclei). Bar=50μm, n=3, *P<0.05 compared with corresponding contralateral hemisphere; §P<0.05 compared with infarct hemisphere of FVB.

Figure 4. miR-199a-5p silences MRP1 in primary cortical neurons

After 72h of miR-199a-5p mimic delivery, miR-199a-5p expression was significantly upregulated (A) while miR-199a-5p hairpin inhibitor significantly downregulated miR-199a-5p (B). miR-199a-5p mimic/inhibitor negatively regulated protein (C-D) of MRP1 in primary cortical neurons. n=3, *P<0.05 compared with control.

Figure 5. Orally supplemented α-tocotrienol protected against stroke via MRP1 upregulation

C57BL/6 mice were orally gavaged with vitamin E-stripped corn oil (n=18) or 50mg α-TCT per kg body weight (n=23) for 13 weeks then MCAO was performed. A, α-TCT supplementation significantly reduced MCAO-induced brain injury (corn oil supplemented mice, n=9; α-TCT supplemented mice, n=10). miR-199a-5p expression was significantly down-regulated (B) while MRP1 mRNA (C) and protein (D) levels were significantly elevated in the stroke affected tissue of α-TCT fed mice (n=3). *P<0.05 compared with corresponding control; §P<0.05 compared with infarct hemisphere of corn oil fed mice.

Figure 6. Orally supplemented α-tocotrienol attenuated MCAO-induced neurodegeneration and lipid peroxidation

A, The abundance of MRP1 positive cells were significantly upregulated in the stroke affected cortex of α-TCT supplemented mice (red-MRP1 protein; blue-DAPI stained nuclei). B, Fluoro-Jade positive neurons were fewer in the infarct hemisphere of α-TCT fed group compared to those at the infarct site of corn oil fed group (green-Fluoro-Jade; blue-DAPI stained nuclei). C, The abundance of 4-HNE-positive cells was lower in infarct hemisphere of α-TCT supplemented mice. Bar(white)=50μm, Bar(black)=20μm. n=3, *P<0.05 compared with corresponding contralateral hemisphere; §P<0.05 compared with infarct hemisphere of corn oil fed mice.