Effects of Corticosterone on Beta-Amyloid-Induced Cell Death in SH-SY5Y Cells

Abstract

Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuronal cell death and memory impairment. Corticosterone (CORT) is a glucocorticoid hormone produced by the hypothalamic-pituitary-adrenal axis in response to a stressful condition. Excessive stress and high CORT levels are known to cause neurotoxicity and aggravate various diseases, whereas mild stress and low CORT levels exert beneficial actions under pathophysiological conditions. However, the effects of mild stress on AD have not been clearly elucidated yet. In this study, the effects of low (3 and 30 nM) CORT concentration on Aβ_25-35-induced neurotoxicity in SH-SY5Y cells and underlying molecular mechanisms have been investigated. Cytotoxicity caused by Aβ_25-35 was significantly inhibited by the low concentration of CORT treatment in the cells. Furthermore, CORT pretreatment significantly reduced Aβ_25-35-mediated pro-apoptotic signals, such as increased Bim/Bcl-2 ratio and caspase-3 cleavage. Moreover, low concentration of CORT treatment inhibited the Aβ_25-35-induced cyclooxygenase-2 and pro-inflammatory cytokine expressions, including tumor necrosis factor-α and interleukin-1β. Aβ_25-35 resulted in intracellular accumulation of reactive oxygen species and lipid peroxidation, which were effectively reduced by the low CORT concentration. As a molecular mechanism, low CORT concentration activated the nuclear factor-erythroid 2-related factor 2, a redox-sensitive transcription factor mediating cellular defense and upregulating the expression of antioxidant enzymes, such as NAD(P)H:quinone oxidoreductase, glutamylcysteine synthetase, and manganese superoxide dismutase. These findings suggest that low CORT concentration exerts protective actions against Aβ_25-35-induced neurotoxicity and might be used to treat and/or prevent AD.

Keywords: Alzheimer’s disease; Beta-amyloid; Corticosterone; Inflammation; Neurotoxicity; Oxidative stress.

Fig. 1

Protective effect of CORT on Aβ_25-35-induced cytotoxicity in SH-SY5Y cells. The cells were incubated with media containing 3- and 30 nM CORT for 72 h and treated with a 10 μM Aβ_25-35 for an additional 24 h. Cell viability was determined using the MTT reduction assay. Data are presented as mean ± SD of three independent experiments, each performed in triplicate. **p<0.01 and ^##p<0.01 indicate statistically significant differences from vehicle-treated control group and Aβ_25-35 alone group, respectively.

Fig. 2

Effect of CORT on Aβ_25-35-induced apoptotic signals in SH-SY5Y cells. The cells were pretreated with 3- and 30 nM CORT for 72 h and then incubated with 10 μM Aβ_25-35 for 24 h. Expression of Bim, Bcl-2 (A), PARP, and cleaved caspase-3 (B) was determined by western blotting. Actin levels were measured as loading controls. Quantitative data were shown as fold induction in the right panel. Data are mean ± SD of three independent experiments. *p<0.05 or **p<0.01 vs vehicle-treated control group and ^#p<0.05 or ^##p<0.01 vs Aβ_25-35 alone group with statistical significance.

Fig. 3

Effect of CORT on Aβ_25-35-induced expression of COX-2 and cytokines in SH-SY5Y cells. The cells were pretreated with CORT (3- and 30 nM) for 72 h in the absence or presence of 10 μM Aβ_25-35 for 24 h. (A) The COX-2 protein expression was determined by western blot analysis. The relative ratio of COX-2 to Actin was represented in the right panel. (B) TNF-α and IL-1β expressions were analyzed by western blot analysis. Quantitative data for TNF-α/Actin and IL-1β/Actin protein levels were presented in the right panel. Data are presented as mean ± SD from three independent experiments. **p<0.01 vs vehicle-treated control group and ^#p<0.05 or ^##p<0.01 vs Aβ_25-35 alone group with statistical significance.

Fig. 4

Effect of CORT on Aβ_25-35-induced intracellular accumulation of ROS and lipid peroxidation in SH-SY5Y cells. (A) The intracellular ROS levels were determined by DCF-DA fluorescence staining. (B) The 4-HNE was measured by western blotting. Actin levels were measured as loading controls. Quantitative data were shown as fold induction in the right panel. Data are presented as mean ± SD of three independent experiments. **p<0.01 vs vehicle-treated control group and ^##p<0.01 vs Aβ_25-35 alone group with statistical significance.

Fig. 5

CORT-induced activation of Nrf2 through phosphorylation and upregulation of antioxidant enzymes. SH-SY5Y cells were pretreated with 3- and 30 nM CORT for 72 h and were incubated for another 24 h in the absence or presence of 10 μM Aβ_25-35. (A) Nrf2 phosphorylation was evaluated by western blot analysis. Quantitative data for the relative ratio of Nrf2 to actin were indicated in the upper right panel. (B) The protein expression of antioxidant enzymes including GCS, NQO1, and MnSOD was determined by western blot analysis. The relative ratio of aforementioned proteins to Actin was shown in the lower right panel. Data are presented as mean ± SD from three independent experiments. ^#p<0.05 or ^##p<0.01 vs Aβ_25-35 alone group with statistical significance.

Fig. 6

CORT-enhanced REST and BDNF protein levels in SH-SY5Y cells. The cells were pretreated with CORT (3- and 30 nM) for 72 h and were then incubated with or without 10 μM Aβ_25-35 for another 24 h. REST and BDNF expressions were analyzed by western blot analysis. Quantitative data and the relative ratio between REST/Actin and BDNF/Actin were represented in the lower panel. Data are presented as mean ± SD from three independent experiments. ^##p<0.01 vs Aβ_25-35 alone group with statistical significance.