Identification of changed proteins by retinoic acid in cerebral ischemic damage: a proteomic study

Abstract

Ischemic stroke is a severe neurodegenerative disease with a high mortality rate. Retinoic acid is a representative metabolite of vitamin A. It has many beneficial effects including anti-inflammatory, anti-apoptotic, and neuroprotective effects. The purpose of this study is to identify specific proteins that are regulated by retinoic acid in ischemic stroke. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia. Retinoic acid (5 mg/kg) or vehicle was injected intraperitoneally into male rats for four days prior to MCAO operation. Neurobehavioral tests were performed 24 hr after MCAO and the cerebral cortex was collected for proteomic study. Retinoic acid alleviates neurobehavioral deficits and histopathological changes caused by MCAO. Furthermore, we identified various proteins that were altered by retinoic acid in MCAO damage. Among these identified proteins, adenosylhomocysteinase, isocitrate dehydrogenase [NAD⁺] subunit α, glycerol-3-phosphate dehydrogenase, Rab GDP dissociation inhibitor β, and apolipoprotein A1 were down-regulated in MCAO animals with vehicle treatment, whereas retinoic acid treatment alleviated these reductions. However, heat shock protein 60 was up-regulated in MCAO animals with vehicle, while retinoic acid treatment attenuated this increase. The changes in these expressions were confirmed by reverse transcription-PCR. These proteins regulate cell metabolism and mediate stress responses. Our results demonstrated that retinoic acid attenuates the neuronal damage by MCAO and regulates the various protein expressions that are involved in the survival of cells. Thus, we can suggest that retinoic acid exerts neuroprotective effects on focal cerebral ischemia by modulation of specific proteins.

Keywords: cerebral ischemia; neuroprotection; proteomics; retinoic acid.

Fig. 1.

Neurological deficit scoring test (A), vibrissae-evoked forelimb placing test (B), adhesive-removal somatosensory test (C), and hematoxylin and eosin staining (D) in vehicle + middle cerebral artery occlusion (MCAO), retinoic acid (RA) + MCAO, vehicle + sham, and RA + sham animals. RA improved neurological behavioral defects caused by MCAO and alleviated histopathological changes. Arrows indicate shrunken nuclei and open arrows indicate vacuoles cytoplasm. Scale bar=100 μm. Data (n=15 per group) are represented as the mean ± S.E.M. **P<0.01 vs. vehicle + MCAO animals.

Fig. 2.

Image of two-dimensional electrophoresis analysis in vehicle + middle cerebral artery occlusion (MCAO), retinoic acid (RA) + MCAO, vehicle + sham, and RA + sham animals (n=5 per group). Isoelectric focusing was performed at pH 4–7 IPG strips and electrophoresed on 7.5–17.5% gradient SDS gels. Squares indicate the significantly changed protein spots between vehicle + MCAO and RA + MCAO animals.

Fig. 3.

Magnified protein spots (A) of adenosylhomocysteinase, isocitrate dehydrogenase [NAD⁺] subunit α, cytosolic glycerol-3-phosphate dehydrogenase, Rab GDP dissociation inhibitor β, apolipoprotein A1, and heat shock protein 60 in the cerebral cortex from vehicle + middle cerebral artery occlusion (MCAO), retinoic acid (RA) + MCAO, vehicle + sham, and RA + sham animals. Each square indicates the protein spots. The band intensity of protein spot (B) is expressed as a ratio of that of vehicle + sham. Data (n=5 per group) are represented as the mean ± S.E.M. *P<0.05, **P<0.01 vs. vehicle + MCAO animals.

Fig. 4.

Reverse transcription-PCR products (A) of adenosylhomocysteinase, isocitrate dehydrogenase [NAD⁺] subunit α, cytosolic glycerol-3-phosphate dehydrogenase, Rab GDP dissociation inhibitor β, apolipoprotein A1, and heat shock protein 60 in the cerebral cortex from vehicle + middle cerebral artery occlusion (MCAO), retinoic acid (RA) + MCAO, vehicle + sham, and RA + sham animals. The band intensity of PCR product (B) is expressed as a ratio of β-actin product intensity. Data (n=5 per group) are represented as the mean ± S.E.M. *P<0.05, **P<0.01 vs. vehicle + MCAO animals.