The role of astrocyte-dependent, Nrf2-ARE induction in flavonoid-mediated protection for primary mouse cortical cultures

Oxidative stress is proposed to have an early role in the development and progression of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Activation of the nuclear factor E2-related factor 2/antioxidant response element (Nrf2/ARE) pathway leads to increased expression of many protective antioxidant and phase II enzymes, including reduced-nicotinamide adenine dinucleotide phosphate quinone oxidoreductase (NQO1). Numerous compounds, including some phenolic antioxidants, are ARE inducers that convey neuroprotection against oxidative stress. Here, we report that kaempferol, quercetin-3-glucoside (Q3G) and quercetin significantly increased NQO1-ARE gene transcription in an ARE promoter-directed luciferase assay. In addition, kaempferol and Q3G significantly increased NQO1 enzyme activity, at 5 μM and 10 μM respectively. Pretreatment with Q3G or kaempferol provided significant protection to primary cortical cultures against a toxic concentration of H2O2 (25 μM), at 7.5-30 μM and 20 μM respectively. Overexpression of a dominant-negative version of Nrf2 (Ad-DN-Nrf2) in C57Bl6 cortical cultures abolished kaempferol-mediated protection, while an enhanced green fluorescent protein adenovirus control (Ad-EGFP) did not. Adenovirus infection occurs only in astrocytes; thus the Nrf2/ARE pathway in astrocytes contributes to kaempferol-mediated protection against oxidative stress-induced cortical culture death. Q3G-mediated protection at 7.5-10 μM is abolished by preventing ARE gene expression with dominant negative Nrf2. However, a significant loss in Q3G-mediated protection after Ad-DN-Nrf2 infection is only observed at 20 μM. This suggests that the Nrf2/ARE pathway contributes to the antioxidant efficiency of some flavonoids and confers protection to primary cortical cell cultures against oxidative stress.