A Nonpolar Blueberry Fraction Blunts NADPH Oxidase Activation in Neuronal Cells Exposed to Tumor Necrosis Factor-α
Figure 1
A nonpolar blueberry fraction inhibits oxidative stress in neuroblastoma cells exposed to TNFα and PMA. SH-SY5Y human neuroblastoma cells were loaded with 50 μM H2DCFDA (1 h) in the presence or absence of pharmacological inhibitors, nonpolar blueberry fractions (NPBB), or polar blueberry fractions (POBB). Following exposure to 200 ng/mL TNFα or 400 ng/mL PMA (1 h), maximum DCF-fluorescence intensities were measured in whole cell lysates. For the quantification of ROS formation, all measurements were adjusted to total protein and normalized to the average maximum DCF-fluorescence intensity under control conditions. (a) TNFα stimulated a significant increase in relative maximum DCF-fluorescence in SH-SY5Y cells (filled bar) indicative of ROS formation compared to basal ROS levels in controls (open bar). Preincubation with crude blueberry extract (5 μg/mL) negated ROS formation upon exposure to TNFα without affecting basal ROS levels (grey bars). (b) Fractionation of crude blueberry extracts according to increasing polarity produced nonpolar colorless fractions and polar, strongly colored fractions enriched in polyphenolics (insert). Preincubation of SH-SY5Y cells with 5 μg/mL NPBB completely suppressed ROS formation upon exposure to TNFα to control levels (open bar) whereas preincubation with 5 μg/mL POBB was ineffective. TNFα-stimulated ROS formation was also negated when inhibiting NOX (10 μM DPI, 1 mM AEBSF), scavenging ROS (1 mM NAC), or blocking ceramide production (13.8 μM GW4869) (gray bars). (c) PMA exposure of SH-SY5Y cells also induced significant ROS formation (filled bar), which was blocked with 5 μg/mL NPBB, 10 μM DPI, 1 mM AEBSF, or 1 mM NAC compared to control (open bar). PMA-evoked ROS formation was not disrupted by preincubation with 5 μg/mL POBB. All data represent the mean of at least four independent experiments ± standard deviations (SD), and statistical significance was determined at * (ANOVA and Tukey’s post hoc analysis).
