Quercetin Protects against Okadaic Acid-Induced Injury via MAPK and PI3K/Akt/GSK3β Signaling Pathways in HT22 Hippocampal Neurons

Abstract

Increasing evidence shows that oxidative stress and the hyperphosphorylation of tau protein play essential roles in the progression of Alzheimer's disease (AD). Quercetin is a major flavonoid that has anti-oxidant, anti-cancer and anti-inflammatory properties. We investigated the neuroprotective effects of quercetin to HT22 cells (a cell line from mouse hippocampal neurons). We found that Okadaic acid (OA) induced the hyperphosphorylation of tau protein at Ser199, Ser396, Thr205, and Thr231 and produced oxidative stress to the HT22 cells. The oxidative stress suppressed the cell viability and decreased the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), mitochondria membrane potential (MMP) and Glutathione peroxidase (GSH-Px). It up-regulated malondialdehyde (MDA) production and intracellular reactive oxygen species (ROS). In addition, phosphoinositide 3 kinase/protein kinase B/Glycogen synthase kinase3β (PI3K/Akt/GSK3β) and mitogen activated protein kinase (MAPK) were also involved in this process. We found that pre-treatment with quercetin can inhibited OA-induced the hyperphosphorylation of tau protein and oxidative stress. Moreover, pre-treatment with quercetin not only inhibited OA-induced apoptosis via the reduction of Bax, and up-regulation of cleaved caspase 3, but also via the inhibition of PI3K/Akt/GSK3β, MAPKs and activation of NF-κB p65. Our findings suggest the therapeutic potential of quercetin to treat AD.

Fig 1. Chemical structure of quercetin.

Fig 2. Effects of OA on the phosphorylation of tau protein.

Cell lysates were treated with various concentrations of OA (20-160nmol/L) for 12h. Protein levels of different phosphorylated tau protein, GSK3β and p38-MAPK were analyzed by western blotting, which were phosphorylated tau (A), phosphorylated GSK3β to pTyr216 and pSer9 (B) and phosphorylated p38-MAPK (C). Quantitative analysis of the blots showed in panel a-g. Data normalized by β-actin were analyzed by ANOVA with LSD’s post-hoc test (**P < 0.01 vs. control group).

Fig 3. Effects of quercetin on OA-induced neurotoxicity.

(A) Final concentration of DMSO in the mediumwas 0.5% (the control was also included). The quercetin did not affect the cell viability of HT22 cells after incubation for 24h with different concentrations(<100μmol/L); (B) Cells were pre-treated with different concentrations of quercetin for 12h before incubation of 80nmol/L OA for 12 h. The cell viability was measured by CCK-8 assay; (C) Morphology of HT22 cells was injured by OA but was reversed by quercetin (200x, 5, 10μM); (a) Control group; (b) 80nmol/L OA; (c) 5μmol/L quercetin + 80nmol/L OA; (d) 10μmol/L quercetin + 80nmol/L OA; Scale bar = 100μm. (D) Pre-treated with quercetin (5.0, 10.0μmol/L) for 12h decreased LDH release which was increased by OA at 80nmol/L. (n = 5, **P < 0.01 vs. control, ^#P < 0.05, ^##P < 0.01 vs. OA-control)

Fig 4. Effects of different concentrations of quercetin on OA induced ROS production in HT22 cells (200×).

Quercetin was added 12 h prior to OA induction and cells were incubated for another 12 h with OA. The ROS was quantified as previously described with the mean intensity of fluorescence. (A) control group; (B) 80nmol/L OA; (C) 5μmol/L quercetin + 80nmol/L OA; (D) 10μmol/L quercetin + 80nmol/L OA and (E) quantitative analysis of mean intensity of fluorescence (**P < 0.01 vs. blank control, ^##P < 0.01 vs. OA-control).

Fig 5. Quercetin reversed OA induced decrease of MMP in HT22 cells (200×).

Quercetin was added 12h before OA incubation. The MMP was quantified as the mean intensity of fluorescence. (A) Control group; (B) 80nmol/L OA; (C) 5μmol/L quercetin + 80nmol/L OA; (D) 10μmol/L quercetin + 80nmol/L OA; (E) quantitative analysis of the mean fluorescent intensity (**P < 0.01 vs. control, ^##P < 0.01 vs. OA-control).

Fig 6. Effects of quercetin on OA induced hyperphosphorylation of tauprotein.

Protein levels of phosphorylated tau proteins and total tau protein (Tau-5) were measured by western blotting (A, B). Statistical analysis for protein levels was shown in panel a-f. (**P < 0.01 vs. control, ^#P < 0.05, ^##P < 0.01 vs. OA-control).

Fig 7. Effects of quercetin and OA induced protein levels of Akt and GSK3β.

Cell were treated with 80nmol/L OA for 12h after pre-incubation of quercetin for 12h. Theprotein levels of phosphorylated Akt (A) and the phosphorylated GSK3β at pTyr216 (B), pSer9 (B) and total GSK3β (B) were measured by Western blotting. Quantitative analysis for the protein levels was shown in panel a-d. (**P < 0.01 vs. control, ^#P < 0.05, ^##P < 0.01 vs. OA-control).

Fig 8. Effects of quercetin on OA induced elevation of cleaved caspase-3 and Bax in HT22 cells.

Cleaved caspase-3 (A) and Bax (B) were measured by western blot. Quantitative analysis of images was shown in panel a and b. Data were analyzed by ANOVA with LSD’s test. (*P < 0.05, **P < 0.01 vs. blank control, ^#P < 0.05, ^##P < 0.01 vs. OA-control)

Fig 9. Effects of quercetin and LY294002 on PI3K/Akt/GSK3β Signaling Pathway.

HT22 cells were pre-treated with 5μmol/L quercetin or 5μmol/L quercetin plus LY294002 (10μmol/L) for 12 h before exposure to 80nmol/L OA. The protein levels of total Akt, p-Akt (Ser473), total GSK3β, GSK3β (pY216) and PT205 were quantified. We found that pre-treatment of 5μmol/L quercetin decreased the level of GSK3β (pY216) and increased the levels of p-Akt (Ser473) and PT205. However, the effect of quercetin was blocked by 10μmol/L LY294002. Protein levels of phosphorylated GSK3β and Akt were quantified which were normalized by total GSK3β and total Akt respectively. Data were expressed as mean±S.E.M (**P < 0.01 vs. control; ^##P < 0.01 vs. OA-control; ⁺P < 0.05, ⁺⁺P < 0.01 vs. quercetin pre-treatment group).

Fig 10. Effects of quercetin and LiCl on PI3K/Akt/GSK3β signaling pathway.

HT22 cells were pre-treated with 5μmol/L quercetin or quercetin plus 10mmol/L LiCl for 12h before exposure to 80nmol/L OA. The protein levels of total GSK3β, GSK3β (pSer9), GSK3β (pTyr216) and PT205 were quantified. It revealed that pre-treatment with 5μmol/L quercetin or 10mmol/L LiCl decreased the level of GSK3β (pTyr216) and increased the levels of GSK3β (pSer9) and PT205. Data were expressed as mean±S.E.M (**P < 0.01 vs. control; ^##P < 0.01 vs. OA-control; ⁺P < 0.05, ⁺⁺P < 0.01 vs. quercetin pre-treatment group).

Fig 11. Effects of quercetin, LY294002 and LiCl on PS396.

OA (80nmol/L) induced increase of PS396, compared to control group and this effect was reversed by quercetin(5μmol/L) and LiCl (10mmol/L) (n = 3, **P < 0.01 vs. control group; ^##P < 0.01 vs. OA-control group; ⁺⁺P < 0.01 vs. Quercetin pre-treatment group).

Fig 12. Effects of quercetin on the MAPK pathway in HT22 cells.

Protein levels for phospho-ERK1/2/ERK1/2, phospho-JNK/JNK and phospho-p38/p38were shown in panel A-C for OA group and quercetin groups. Quantitative analysis of the bands were shown in panel a-c. Data were analyzed by one-way ANOVA with LSD’s post-hoc test and are mean±S.E.M (**P< 0.01 vs. control, ^##P < 0.01 vs. OA-control).

Fig 13. Effects of quercetin on NF-κB activity in OA-treated HT22 cells.

OA increased protein level of p-p65 NF-κB and this effect was reversed quercetin (5,10μmol/L) (A). Quantitative analysis was shown in the below panel (A). Data are presented as the mean ± S.E.M (**P < 0.01 vs. control, ^##P < 0.01 vs. OA-control).