Protective effect of quercetin in primary neurons against Abeta(1-42): relevance to Alzheimer's disease

Abstract

Quercetin, a flavonoid found in various foodstuffs, has antioxidant properties and increases glutathione (GSH) levels and antioxidant enzyme function. Considerable attention has been focused on increasing the intracellular GSH levels in many diseases, including Alzheimer's disease (AD). Amyloid beta-peptide [Abeta(1-42)], elevated in AD brain, is associated with oxidative stress and neurotoxicity. We aimed to investigate the protective effects of quercetin on Abeta(1-42)-induced oxidative cell toxicity in cultured neurons in the present study. Decreased cell survival in neuronal cultures treated with Abeta(1-42) correlated with increased free radical production measured by dichlorofluorescein fluorescence and an increase in protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (protein-bound 4-hydroxy-2-nonenal). Pretreatment of primary hippocampal cultures with quercetin significantly attenuated Abeta(1-42)-induced cytotoxicity, protein oxidation, lipid peroxidation and apoptosis. A dose-response study suggested that quercetin showed protective effects against Abeta(1-42) toxicity by modulating oxidative stress at lower doses, but higher doses were not only non-neuroprotective but also toxic. These findings provide motivation to test the hypothesis that quercetin may provide a promising approach for the treatment of AD and other oxidative-stress-related neurodegenerative diseases.

Fig. 1

The chemical structure of quercetin dehydrate (2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one dihydrate), used in this study.

Fig. 2

(A) Shows representative blot for protein carbonyl, developed with a primary antibody against the hydrazone formed by reaction of carbonyl groups with DNPH. (B) Shows the increment to protein carbonyl formation in cultured neurons treated with Aβ (1-42) as compared to controls. The dose dependent effect of quercetin shows protection against protein carbonyls formation by Aβ (1-42) treatment. *p<0.01 and **p<0.001 compared to control and ^#p<0.01 and ^##p<0.001 compared to Aβ (1-42) (10 μM) treatment. The data are presented as mean ± SEM expressed as percentage of control (n=6).

Fig. 3

(A) Shows representative blot for 3-NT, developed with a primary antibody against 3-nitrotyrosine in proteins). (B) Shows the increment in 3-NT levels formation in cultured neurons treated with Aβ (1-42) as compared to controls. The dose dependent effect of quercetin shows protection against 3-NT formation by Aβ (1-42) treatment. *p<0.01 and **p<0.001 compared to control and ^#p<0.01 and ^##p<0.001 compared to Aβ (1-42) (10 μM) treatment. The data are presented as mean ± SEM expressed as percentage of control (n=6).

Fig. 4

(A) Shows representative blot for 4-HNE-bound proteins, developed with a primary antibody against the Michael adduct of HNE with proteins. (B) Shows the increment in 4-HNE formation in cultured neurons treated with Aβ (1-42) compared to the control. The dose dependent effect of quercetin shows protection against 4-HNE formation by Aβ (1-42) treatment. *p<0.01 and **p<0.001 compared to control and ^#p<0.01 and ^##p<0.001 compared to Aβ (1-42) (10 μM) treatment. The data are presented as mean ± SEM expressed as percentage of control (n=6).

Fig. 5

Shows the effect of varying concentrations of quercetin on cell viability that is reduced by Aβ (1–42) in primary cultured rat neurons. Quercetin was added to the culture 1 h prior to 10 μM Aβ (1–42) addition, and the cells were incubated for 24 h. Cell viability was assessed using the MTT reduction assay. The data are presented as mean±SEM expressed as percentage of control values. *p<0.01 and **p<0.001 compared to control and ^#p<0.01 and ^##p<0.001 compared to oxidant treatment.

Fig. 6

Quercetin protects against Aβ (1–42)-induced neuronal death, indexed by phase contrast microscopy. (A) Control; (B) neurons treated with Aβ (1–42); (C) neurons treated with Aβ (1–42)+5 μM quercetin; (D) neurons treated with Aβ (1–42)+10 μM quercetin; (E) neurons treated with Aβ (1–42)+20 μM quercetin; and (F) neurons treated with 40 μM quercetin only.