Identification of regulated proteins by resveratrol in glutamate-induced cortical injury of newborn rats

Abstract

Glutamate induces neuronal damage by generating oxidative stress and neurotoxicities. The neurological damage caused by glutamate is more severe during brain development in newborns than in adults. Resveratrol is naturally present in a variety of fruits and medicinal plants and exerts a neuroprotective effect against brain damage. The goal of this study was to evaluate the neuroprotective effects of resveratrol and to identify changed proteins in response to resveratrol treatment during glutamate-induced neonatal cortical damage. Sprague-Dawley rat pups (7 days old) were randomly divided into vehicle, resveratrol, glutamate, and glutamate and resveratrol groups. The animals were intraperitoneally injected with glutamate (10 mg/kg) and/or resveratrol (20 mg/kg) and their brain tissue was collected 4 hr after drug administration. Glutamate exposure caused severe histopathological changes, while resveratrol attenuated this damage. We identified regulated proteins by resveratrol in glutamate-induced cortical damaged tissue using two-dimensional gel electrophoresis and mass spectrometry. Among identified proteins, we focused on eukaryotic initiation factor 4A2, γ-enolase, protein phosphatase 2A subunit B, and isocitrate dehydrogenase. These proteins decreased in the glutamate-treated group, whereas the combination treatment of glutamate and resveratrol attenuated these protein reductions. These proteins are anti-oxidant proteins and anti-apoptotic proteins. These results suggest that glutamate induces brain cortical damage in newborns; resveratrol exerts a neuroprotective effect by controlling expression of various proteins with anti-oxidant and anti-apoptotic functions.

Keywords: cerebral cortex; glutamate; neonate; proteomics; resveratrol.

Fig. 1.

Representative photomicrographs of hematoxylin and eosin staining in the neonatal cerebral cortex of vehicle- (A), resveratrol- (B), glutamate- (C), and glutamate and resveratrol co-treated animals (D). Normal pyramidal cells with well-developed dendrites were observed in the vehicle animals, while swollen neuron with atypical shapes were observed in the glutamate-treated animals. These morphological changes were alleviated in the glutamate and resveratrol co-treated animals. Arrows present damaged neuron. Scale bar=40 μm

Fig. 2.

Two-dimensional electrophoresis analysis of proteins in neonatal cerebral cortex of vehicle- (A), resveratrol- (B), glutamate- (C), and glutamate and resveratrol co-treated animals (D). Isoelectric focusing was performed at pH 4–7 using IPG strips, followed by second-dimensional separation on 7.5–17.5% gradient SDS gels stained with silver. The square presents the protein spot with double intensity change between the glutamate- and the co-treated animals.

Fig. 3.

Image of eukaryotic initiation factor 4A2 (eIF4A2), γ-enolase, protein phosphatase 2A (PP2A) subunit B, and isocitrate dehydrogenase (ICDH) protein spots in neonatal cerebral cortex of vehicle-, resveratrol-, glutamate-, and glutamate and resveratrol co-treated animals. Arrows indicate the identified specific spots. Spot intensities were measured using PDQuest software. Protein levels were determined by the ratio of the intensity of each animals to the vehicle-treated aniamls. Data (n=4) and are shown as the mean ± S.E.M. ^#P<0.05.

Fig. 4.

Western blot analysis of eukaryotic initiation factor 4A2 (eIF4A2), γ-enolase, protein phosphatase 2A (PP2A) subunit B, and isocitrate dehydrogenase (ICDH) protein spots in neonatal cerebral cortex of vehicle-, resveratrol-, glutamate-, and glutamate and resveratrol co-treated animals. Each lane represents an individual experimental animal. Protein levels were determined by the ratio of the intensity of each protein to β-actin intensity. Data (n=5 per group) are presented as mean ± S.E.M. ^#P<0.05.

Fig. 5.

Reverse transcription-PCR of eukaryotic initiation factor 4A2 (eIF4A2), γ-enolase, protein phosphatase 2A (PP2A) subunit B, and isocitrate dehydrogenase (ICDH) protein spots in neonatal cerebral cortex of vehicle-, resveratrol-, glutamate-, and glutamate and resveratrol co-treated animals. Each lane represents an individual experimental animal. The band intensity of reverse transcription-PCR product was normalized to that of β-actin product. Data (n=5 per group) are presented as mean ± S.E.M. ^#P<0.05.

Fig. 6.

Schematic graphic of neuroprotection of resveratrol against glutamate toxicity in neonates.