After cellular internalization, quercetin causes Nrf2 nuclear translocation, increases glutathione levels, and prevents neuronal death against an oxidative insult.
In this work we describe the protective effects of quercetin against H(2)O(2) in 24-h-pretreated neuronal cultures. We explored quercetin availability and subcellular fate through the use of HPLC-Diode Array Detection (DAD), epifluorescence, and confocal microscopy. We focused on quercetin modulation of thiol-redox systems by evaluating changes in mitochondrial thioredoxin Trx2, the levels of total glutathione (GSH), and the expression of the gamma-glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme of GSH synthesis, by the use of Western blot, HPLC, and real-time PCR techniques, respectively. We further explored the activation of the protective NF-E2-related factor 2 (Nrf2)-dependent signaling pathway by quercetin using immunocytochemistry techniques. Our results showed rapid quercetin internalization into neurons, reaching the nucleus after its addition to the culture. Quercetin pretreatment increased total GSH levels, but did not increase Trx2. Interestingly it caused Nrf2 nuclear translocation and significantly increased GCLC gene expression. At the moment of H(2)O(2) addition, intracellular quercetin or related metabolites were undetectable in the cultures although quercetin pretreatment prevented neuronal death from the oxidant exposure. Our findings suggest alternative mechanisms of quercetin neuroprotection beyond its long-established ROS scavenging properties, involving Nrf2-dependent modulation of the GSH redox system.
Arredondo
Florencia
F
Departamento de Neuroquímica, Instituto de Investigaciones Biológicas Clemente Estable, 11600 Montevideo, Uruguay. flor@iibce.edu.uy
Echeverry
Carolina
C
Abin-Carriquiry
Juan A
JA
Blasina
Fernanda
F
Antúnez
Karina
K
Jones
Dean P
DP
Go
Young-Mi
YM
Liang
Yong-Liang
YL
Dajas
Federico
F
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D000975:Antioxidants / Q000493:pharmacokinetics / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism; D019610:Cytoprotection / Q000187:drug effects*; D004353:Drug Evaluation, Preclinical; D005978:Glutathione / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000633:toxicity; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000493:pharmacokinetics / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects; D011794:Quercetin / Q000493:pharmacokinetics / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation / Q000187:drug effects
D000975:Antioxidants; D051267:NF-E2-Related Factor 2; D018696:Neuroprotective Agents; C495637:Nfe2l2 protein, rat; D011794:Quercetin; D006861:Hydrogen Peroxide; D005978:Glutathione