Emilia Zgórzyńska1, Barbara Dziedzic2, Anna Gorzkiewicz2, Dawid Stulczewski2, Katarzyna Bielawska3, Kuan-Pin Su4, Anna Walczewska2. 1. Department of Cell-to-Cell Communication, Medical University of Lodz, Łódź, Poland. Electronic address: emilia.zgorzynska@umed.lodz.pl. 2. Department of Cell-to-Cell Communication, Medical University of Lodz, Łódź, Poland. 3. Department of Analytical Chemistry, Medical University of Bialystok, Białystok, Poland. 4. Department of Psychiatry and Mind-Body Interface Laboratory, China Medical University, Taichung, Taiwan.
Abstract
BACKGROUND: Neuronal tolerance to hypoxia and nutrient defficiency highly depends on GSH levels and antioxidant enzyme activity in astrocytes. Omega-3 polyunsaturated fatty acids (ω-3PUFA) enhance antioxidant defence in different cells. The aim of present study was to investigate if ω-3PUFA improve antioxidant status in astrocytes. METHODS: Rat primary astrocytes were incubated for 24h with DHA and EPA (30μM), then lysed, fractioned and fatty acids were determined by gas chromatography. GSH and protein thiols were assayed by enzymatic methods. Glutamate cysteine ligase (GCL), glutathione synthetase (GS), glutathione peroxidase 4 (GPx4) and Nrf2 protein expression was validated by Western blot. Intracellular ROS level using H2DCF-DA, and Nrf2 activation by ELISA were measured. RESULTS: Incubation of cells with DHA doubled DHA, not EPA content in the membranes, and incubation with EPA increased both fatty acids content compared to control. However, both ω-3PUFAs reduced ROS generation in dose-dependent manner in basal condition and in H2O2-treated cells, and significantly increased GSH, GCL and GPx4 levels. The thiols level was higher only in DHA-treated cells. DHA and EPA activated Nrf2 in a dose-dependent manner but p38MAPK-Nrf2 activation was found only in DHA-enriched astrocytes. CONCLUSION: Both ω-3PUFA improved the antioxidant defense in astrocytes via an Nrf2-dependent mechanism, however, upstream pathways of Nrf2 activation may depend on proportion of DHA to EPA incorporated into membrane phospholipids. These results suggest that enrichment of astrocytes with ω-3PUFA may better protect neurons during harmful conditions.
BACKGROUND: Neuronal tolerance to hypoxia and nutrient defficiency highly depends on GSH levels and antioxidant enzyme activity in astrocytes. Omega-3 polyunsaturated fatty acids (ω-3PUFA) enhance antioxidant defence in different cells. The aim of present study was to investigate if ω-3PUFA improve antioxidant status in astrocytes. METHODS:Rat primary astrocytes were incubated for 24h with DHA and EPA (30μM), then lysed, fractioned and fatty acids were determined by gas chromatography. GSH and protein thiols were assayed by enzymatic methods. Glutamate cysteine ligase (GCL), glutathione synthetase (GS), glutathione peroxidase 4 (GPx4) and Nrf2 protein expression was validated by Western blot. Intracellular ROS level using H2DCF-DA, and Nrf2 activation by ELISA were measured. RESULTS: Incubation of cells with DHA doubled DHA, not EPA content in the membranes, and incubation with EPA increased both fatty acids content compared to control. However, both ω-3PUFAs reduced ROS generation in dose-dependent manner in basal condition and in H2O2-treated cells, and significantly increased GSH, GCL and GPx4 levels. The thiols level was higher only in DHA-treated cells. DHA and EPA activated Nrf2 in a dose-dependent manner but p38MAPK-Nrf2 activation was found only in DHA-enriched astrocytes. CONCLUSION: Both ω-3PUFA improved the antioxidant defense in astrocytes via an Nrf2-dependent mechanism, however, upstream pathways of Nrf2 activation may depend on proportion of DHA to EPA incorporated into membrane phospholipids. These results suggest that enrichment of astrocytes with ω-3PUFA may better protect neurons during harmful conditions.
Authors: Grace Y Sun; Runting Li; Bo Yang; Kevin L Fritsche; David Q Beversdorf; Dennis B Lubahn; Xue Geng; James C Lee; C Michael Greenlief Journal: Int J Mol Sci Date: 2019-02-21 Impact factor: 5.923
Authors: Maria E Fernandez; Jackelyn M Kembro; Maria L Ballesteros; Jorge M Caliva; Raul H Marin; Maria C Labaque Journal: PLoS One Date: 2019-05-09 Impact factor: 3.240