Potential Involvement of Nrf2 in the Enhanced Antioxidant and Cytoprotective Effects of Quercetin and Kaempferol Following Their Exposure to a Nitrous Acid-Containing Pseudo-Gastric Fluid

Exposure of quercetin (Que) or kaempferol (Kae) to a nitrous-acid containing pseudo-gastric fluid (PGF) leads to their oxidative conversion into their respective hydroxylated-benzoyl-benzofuranones (BZFs), Que-BZF and Kae-BZF. This study evaluated the implications that the referred conversion could have on the antioxidant properties, cellular metabolic activity, and membrane integrity of such fluids when added to Caco-2 cells exposed to the ROS-generator, indomethacin. The addition of PGF containing either Que-BZF or Kae-BZF to the cells concentration-dependently (10–100 nM) prevented indomethacin-induced oxidative stress (dichlorodihydrofluorescein oxidation), mitochondrial-dependent metabolic dysfunction (MTT reduction), and plasma membrane disruption (LDH leakage). To achieve a level of intracellular antioxidant protection (80%), metabolic preservation (100%), and membrane integrity protection (100%) comparable to that induced by 10 nM of Que-BZF or Kae-BZF in the PGF, concentrations of quercetin or kaempferol of 10 µM, that is, three-orders of magnitude higher, were required. At 100 nM, the BZFs contained in the PGF promoted Nrf2 nuclear translocation and induced a near five-fold increase in the levels of the antioxidant enzymes HO-1 and NQO1, regardless of indomethacin treatment. Therefore, the present in vitro chemical simulation and cellular model of intestinal epithelium study demonstrates that the oxidative conversion that quercetin and kaempferol undergo following their incubation in a nitrous acid-containing PGF results in a substantial amplification of their original antioxidant and cytoprotective capabilities and suggests that the latter effects arise from the extremely potent ability of the hydroxylated-benzoyl-benzofuranones to activate the Nrf2-Keap1 pathway.