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Nitroxide Radical-Containing Redox Nanoparticles Protect Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine
Monika Pichla1, Łukasz Pulaski2,3, Katarzyna Dominika Kania3
1Department of Analytical Biochemistry, Institute of Food Technology and Nutrition, College of Natural Sciences, Rzeszow University, Zelwerowicza Street 4, 35-601 Rzeszow, Poland.
Abstract:
Parkinson's disease (PD) patients can benefit from antioxidant supplementation, and new efficient antioxidants are needed. The aim of this study was to evaluate the protective effect of selected nitroxide-containing redox nanoparticles (NRNPs) in a cellular model of PD. Antioxidant properties of NRNPs were studied in cell-free systems by protection of dihydrorhodamine 123 against oxidation by 3-morpholino-sydnonimine and protection of fluorescein against bleaching by 2,2-azobis(2-amidinopropane) hydrochloride and sodium hypochlorite. Model blood-brain barrier penetration was studied using hCMEC/D3 cells. Human neuroblastoma SH-SY5Y cells, exposed to 6-hydroxydopamine (6-OHDA), were used as an in vitro model of PD. Cells were preexposed to NRNPs or free nitroxides (TEMPO or 4-amino-TEMPO) for 2 h and treated with 6-OHDA for 1 h and 24 h. The reactive oxygen species (ROS) level was estimated with dihydroethidine 123 and Fluorimetric Mitochondrial Superoxide Activity Assay Kit. Glutathione level (GSH) was measured with ortho-phtalaldehyde, ATP by luminometry, changes in mitochondrial membrane potential with JC-1, and mitochondrial mass with 10-Nonyl-Acridine Orange. NRNP1, TEMPO, and 4-amino-TEMPO (25-150 μM) protected SH-SY5Y cells from 6-OHDA-induced viability loss; the protection was much higher for NRNP1 than for free nitroxides. NRNP1 were better antioxidants in vitro and permeated better the model BBB than free nitroxides. Exposure to 6-OHDA decreased the GSH level after 1 h and increased it considerably after 24 h (apparently a compensatory overresponse); NRNPs and free nitroxides prevented this increase. NRNP1 and free nitroxides prevented the decrease in ATP level after 1 h and increased it after 24 h. 6-OHDA increased the intracellular ROS level and mitochondrial superoxide level. Studied antioxidants mostly decreased ROS and superoxide levels. 6-OHDA decreased the mitochondrial potential and mitochondrial mass; both effects were prevented by NRNP1 and nitroxides. These results suggest that the mitochondria are the main site of 6-OHDA-induced cellular damage and demonstrate a protective effect of NRNP1 in a cellular model of PD.
