Riluzole-triggered GSH synthesis via activation of glutamate transporters to antagonize methylmercury-induced oxidative stress in rat cerebral cortex

Abstract

Objective: This study was to evaluate the effect of riluzole on methylmercury- (MeHg-) induced oxidative stress, through promotion of glutathione (GSH) synthesis by activating of glutamate transporters (GluTs) in rat cerebral cortex.

Methods: Eighty rats were randomly assigned to four groups, control group, riluzole alone group, MeHg alone group, and riluzole + MeHg group. The neurotoxicity of MeHg was observed by measuring mercury (Hg) absorption, pathological changes, and cell apoptosis of cortex. Oxidative stress was evaluated via determining reactive oxygen species (ROS), 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDAs), carbonyl, sulfydryl, and GSH in cortex. Glutamate (Glu) transport was studied by measuring Glu, glutamine (Gln), mRNA, and protein of glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1).

Result: (1) MeHg induced Hg accumulation, pathological injury, and apoptosis of cortex; (2) MeHg increased ROS, 8-OHdG, MDA, and carbonyl, and inhibited sulfydryl and GSH; (3) MeHg elevated Glu, decreased Gln, and downregulated GLAST and GLT-1 mRNA expression and protein levels; (4) riluzole antagonized MeHg-induced downregulation of GLAST and GLT-1 function and expression, GSH depletion, oxidative stress, pathological injury, and apoptosis obviously.

Conclusion: Data indicate that MeHg administration induced oxidative stress in cortex and that riluzole could antagonize this situation through elevation of GSH synthesis by activating of GluTs.

Figure 1

Light microphotographs showed that the morphological changes of cerebral cortex after MeHg exposure and/or riluzole pretreatment. Photographs of control (a), riluzole alone (b), MeHg alone (c), and riluzole + MeHg (d) were in this figure. The sections were stained with HE staining. Magnification × 40.

Figure 2

Alterations of hepatic apoptosis after MeHg exposure and/or riluzole pretreatment were shown in this figure. Scatter plots for control (a), riluzole alone (b), MeHg alone (c), and riluzole + MeHg (d) were in this figure. Data are mean ± SD for four animals in each group. **P < 0.01 denotes statistical significance compared with control group; ^## P < 0.01 denotes statistical significance compared with MeHg alone group.

Figure 3

The changes of intracellular ROS levels in cerebral cortex after MeHg exposure and/or riluzole pretreatment were shown in this figure. Data are mean ± SD for four animals in each group. **P < 0.01 denotes statistical significance compared with control group; ^## P < 0.01 denotes statistical significance compared with MeHg alone group.

Figure 4

The figure showed the immunohistochemistry assay of 8-OHdG in cerebral cortex after MeHg exposure and riluzole pretreatment. Light microphotographs were for control (a), riluzole alone (b), MeHg alone (c), and riluzole + MeHg (d) were observed respectively, the sections were stained with SABC, and the magnification was set at ×40. Data are mean ± SD for six animals in each group. The effects of MeHg exposure and riluzole pretreatment on 8-OHdG threshold area (e) and integral optical density (f) were also shown in this figure. **P < 0.01 denotes statistical significance compared with control group; ^## P < 0.01 denotes statistical significance compared with MeHg alone group.

Figure 5

The alteration of GLAST mRNA (a) and GLT-1 mRNA (b) in cerebral cortex after MeHg exposure and riluzole pretreatment was shown in this figure. Data are mean ± S.D. for four animals in every group and each RNA preparation was run three times by real time-PCR, *P < 0.05, **P < 0.01 denote statistical significance compared with control group; ^# P < 0.05, ^## P < 0.01 denote statistical significance compared with MeHg alone group.

Figure 6

The alteration of GLAST protein (a) and GLT-1 protein (b) in cerebral cortex after MeHg exposure and riluzole pretreatment was shown in this figure. Data are mean ± S.D. for four animals in every group and each RNA or protein preparation was run three times by real time-PCR or western blotting, *P < 0.05, **P < 0.01 denote statistical significance compared with control group; ^# P < 0.05, ^## P < 0.01 denote statistical significance compared with MeHg alone group.