A Nonpolar Blueberry Fraction Blunts NADPH Oxidase Activation in Neuronal Cells Exposed to Tumor Necrosis Factor-α

Figure 6

Nonpolar blueberry fraction inhibits the association of p67^phox to the plasma membrane by modulating lipid raft platforms. SH-SY5Y cells were grown on polylysine-coated glass cover slips and following serum starvation (overnight), incubated with nonpolar (NP_BB) or polar (PO_BB) blueberry fractions (5 μg/mL each, 1 h) prior to insult (200 ng/mL TNFα or 400 ng/mL PMA, resp., 30 min). Live cultures were stained for lipid rafts (Alexa Fluor 555 lipid raft labeling kit), then fixed with 4% paraformaldehyde, immunostained against p67^phox, and mounted with PVA-DABCO. Images were acquired (Zeiss LSM510 confocal microscope) under rhodamine illumination (lipid rafts) and fluorescein illumination (p67^phox). Image overlay demonstrated colocalization of p67^phox and lipid rafts (yellow, arrow heads). SH-SY5Y cells under control conditions displayed very little colocalization of p67^phox with lipid rafts and the punctate staining is indicative of small dimension lipid raft domains (a). SH-SY5Y cells treated with NP_BB revealed a lipid raft-p67^phox colocalization indistinguishable from controls (b). In contrast, SH-SY5Y cells treated with PO_BB exhibited increased colocalization of p67^phox and lipid rafts (c). As expected, TNFα exposure induced the formation of large, virtually continuous lipid raft macrodomains and almost complete colocalization with p67^phox (d). Preincubation of SH-SY5Y cells with NP_BB potently interfered with the formation of lipid raft macrodomains with virtually no p67^phox colocalization upon exposure to TNFα (e) and the punctate, discontinuous staining patterns strongly resembled control conditions. Treatment of SH-SY5Ywith PO_BB prior to TNFα insult had no effect on lipid raft-p67^phox colocalization or lipid raft macrodomain formation (f). Similarly, SH-SY5Y cells responded to PMA exposure with the formation of lipid raft macrodomains and substantial p67^phox colocalization although to a lesser extent compared to TNFα (g). Treatment of SH-SY5Y cells with NP_BB prior to PMA insult diminished the formation of lipid raft macrodomains and p67^phox colocalization albeit less potently compared to a TNFα insult (h). PO_BB preincubation of SH-SY5Y was ineffective (i) (scale bar = 20 μm).