Quercetin Metabolites Up-Regulate the Antioxidant Response in Osteoblasts Isolated From Fetal Rat Calvaria

Oxidative stress contributes to osteoporosis by suppressing differentiation of osteoblasts, suggesting the osteoblast antioxidant response may be a viable strategy for osteoporosis prevention. Quercetin, an antioxidant flavonol, up-regulates the antioxidant response in many cell types, but studies are needed to understand the effects of quercetin plasma metabolites on the osteoblast antioxidant response. The first specific aim was to examine antioxidant response genes and proteins in osteoblasts exposed to plasma quercetin metabolites. The second specific aim was to identify potential signaling pathways in the osteoblast antioxidant response that mediate the effect of quercetin, specifically Nrf2, ERK1/2, and NFκB p65. Osteoblasts isolated from fetal rat calvaria were treated with doses up to 20 μM of three different quercetin metabolites found in blood plasma after consumption of quercetin-rich foods or supplements: quercetin aglycone (QRC), isorhamnetin (ISO), or quercetin 3-O-glucuronide (Q3G). Alternatively, some cells received a 2:1:1 mixture of all three metabolites (10 μM Q3G: 5 μM ISO: 5 μM QRC) to evaluate synergistic effects. Antioxidant response genes and proteins known to be up-regulated by quercetin were analyzed along with Nrf2, ERK1/2, and NFκB proteins. Both QRC and ISO, but not Q3G, up-regulated heme oxygenase-1 (HO-1) and γ-glutamate cysteine ligase catalytic subunit (GCLC) at the mRNA and protein level. Synergistic effects of metabolites were not observed. Up-regulation of HO-1 and GCLC was associated with suppression of phosphorylated ERK1/2 and NFκB, but no alterations in Nrf2 protein levels were observed. This study shows that the antioxidant response of osteoblasts is differentially stimulated by quercetin metabolites.