Epigallocatechin-3-gallate confers protection against corticosterone-induced neuron injuries via restoring extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3 kinase/protein kinase B signaling pathways

Abstract

Extensive studies suggested epigallocatechin-3-gallate (EGCG) has significant neuroprotection against multiple central neural injuries, but the underlying mechanisms still remain poorly elucidated. Here we provide evidence to support the possible involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol-3 kinase/ protein kinase B (PI3K/AKT) pathways in EGCG-mediated protection against corticosterone-induced neuron injuries. As an essential stress hormone, corticosterone could induce obvious neurotoxicity in primary hippocampal neurons. Pre-treatment with EGCG ameliorated the corticosterone-induced neuronal injuries; however, it was blocked by pharmacological inhibitors for ERK1/2 (U0126) and PI3K/AKT (LY294002). Furthermore, the results confirmed that EGCG restored the corticosterone-induced decrease of ERK1/2 and PI3K/AKT phosphorylation, and attenuated the corticosterone-induced reduction of peroxisome proliferators-activated receptor-γ coactivator-1α (PGC-1α) expression and ATP production. Taken together, these findings indicated that EGCG has significant neuroprotection against corticosterone-induced neuron injuries partly via restoring the ERK1/2 and PI3K/AKT signaling pathways as well as the PGC-1α-mediated ATP production.

Fig 1. The effect of CORT and EGCG on cell viability of primary hippocampal neuron cultures.

Primary cultured hippocampal neurons were treated with various concentrations of CORT for 24 h and cell viability was assessed by MTT (A). Hippocampal neurons were exposed to different concentrations of EGCG for 24 h and cell viability was assessed by MTT (B). Hippocampal neurons were pretreated with different concentrations of EGCG for 30 min before subsequent stimulation with CORT for 24 h (C). ^a P<0.05 vs Control group; P<0.05 vs CORT group.

Fig 2. The changes of cell morphology of primary cultured hippocampal neurons.

Primary hippocampal neuron cultures were treated with CORT (10 μmol/L) for 24 h in the absence or presence of EGCG pretreatment (1μmol/L, at 30 min prior to CORT addition). A) Normal neurons, ×200; B) CORT treated neurons, ×200; C) EGCG co-incubation with CORT-treated neurons, ×200.

Fig 3. The effect of EGCG on CORT-induced morphological changes of hippocampal neurons by Hoechst 33342 staining.

Primary hippocampal neuron cultures were treated with CORT for 24 h in the absence or presence of EGCG pre-treatment (1 μmol/L, 2 h prior to CORT stimulation), ERK1/2 inhibitor (U0126, 10 μmol/L) or PI3K/AKT inhibitor (LY294002, 10 μmol/L) pre-treatment (30 min before CORT exposure). A) Normal control neurons; B) CORT treatment; C) EGCG and CORT co-treatment; D) EGCG+CORT+U0126 treatment; E) EGCG+CORT+ LY294002 treatment; F) The apoptotic index of hippocampal neurons. ^a P<0.05 vs Control group: ^b P<0.05 vs CORT group; ^c P<0.05 vs CORT+ EGCG group.

Fig 4. Corticosterone induces dynamic changes of ERK1/2 phosphorylation in primary hippocampal neuron cultures.

Primary hippocampal neuron cultures were stimulated with CORT for indicated time period, and the cellular levels of total and phosphorylated ERK1/2 were determined by western blot analysis with reference to a house-keeping protein (β-actin) as loading control. The specific signals were visualized with “ChemiDoc XRS” digital imaging system, and a representative western blot was shown (A). The relative protein levels were expressed as the relative band density of the corresponding protein (B, C). ^a P<0.05 vs normal control group: ^b P<0.05 vs CORT treatment 2 h group.

Fig 5. The effect of EGCG on CORT-induced alteration of ERK1/2 phosphorylation in primary hippocampal neuron cultures.

Primary hippocampal neuron cultures were pre-treated with EGCG at indicated concentrations for 2 h before stimulating with CORT for 1 h (A) and 24 h (D), respectively. The cellular levels of total and phosphorylated ERK1/2 were determined by western blot analysis with reference to a house-keeping protein (β-actin) as loading control. The relative protein levels were expressed as the relative band density of the corresponding protein (B, C, E and F). ^a P<0.05 vs normal control group: ^b P<0.05 vs CORT treatment group.

Fig 6. The effect of EGCG on CORT-induced decrease of AKT phosphorylation in primary hippocampal neuron cultures.

Primary hippocampal neuron cultures were stimulated with CORT for 0, 0.5, 1, 6 and 24 h, respectively in the absence of EGCG (A), or in the presence of a pre-treatment with EGCG at indicated concentration for 2 h before CORT stimulation for 24 h (B). The cellular levels of total and phosphorylated AKT were determined by western blot analysis with reference to a house-keeping protein (β-actin) as loading control. AKT and The relative protein levels were expressed as the relative band density of the corresponding protein (C, D). ^a P<0.05 vs normal control group: ^b P<0.05 vs CORT treatment group.

Fig 7. The effect of CORT and EGCG on ERK1/2 and AKT mRNA in primary hippocampal neurons.

Primary hippocampal neuron cultures were pre-treated with EGCG for 2 h at indicated concentrations before stimulation with CORT for 24 h. The total RNA was isolated to measure the expression of ERK1/2 and AKT mRNA by RT-PCR, and expressed as mean ± SD of GAPDH mRNA levels. A) The expression of ERK1/2 mRNA; B) The expression of AKT mRNA. ^a P<0.05 vs normal control group: ^b P<0.05 vs CORT treatment group.

Fig 8. Effect of EGCG on CORT-induced changes of ATP and PGC-1α expressions in primary hippocampal neurons.

Primary hippocampal neuron cultures were stimulated with CORT for indicated time period in the absence of presence of EGCG pretreatment (for 2 h) at indicated concentrations. The cultured hippocampal neurons were harvested and lysed to measure cellular ATP content, as well as PGC-1α protein and mRNA expression. The PGC-1α protein content was measured by western blot analysis, whereas the PGC-1α mRNA levels were determined by RT-PCR and expressed as mean ± SD of GAPDH mRNA level. A) The production of ATP; B, C) Examination of PGC-1α by western blot; D) The expression of PGC-1α mRNA; E, F) The relative expression of PGC-1α. ^a P<0.05 vs normal control group: ^b P<0.05 vs CORT treatment group.