Antioxidant effect of an alpha-MSH analogue in primary astrocytes cultures

DELIA RAMÍREZ; LILA CARNIGLIA; JULIETA SABA; DANIELA DURAND; CARLA CARUSO; MERCEDES LASAGA

Bilbao

Congreso; XII European Meeting on Glial Cells in Health and Disease; 2015

Inflammatory processes in the central nervous system (CNS) contribute to the development of neurodegenerative disorders and oxidative stress appears to be connected with the loss of neurons during the progression of these diseases. Astrocytes are the cell type with the most antioxidant capabilities within the CNS and they supply neurons with glutathione (GSH) precursors. Melanocortins are neuropeptides with proved anti-inflammatory and neuroprotective properties. Previously, we have shown that alpha-melanocyte-stimulating hormone (alpha-MSH) exerts an anti-inflammatory action through MC4 receptors in astrocytes. Considering the importance of developing new therapeutic strategies focused on the regulation of antioxidant mechanisms, we decided to evaluate melanocortins? antioxidant effect in cultured rat primary astrocytes using NDP-MSH, a synthetic analogue of alpha-MSH. Primary cultured rat astrocytes were incubated for 24 h with NDP 0.1 μM and intra and extracellular GSH levels were evaluated using monochlorobimane. NDP-MSH increased extracellular GSH levels by 21.2% (p less than 0.001) while it did not change intracellular GSH levels. Next, we determined the enzymatic activity of gamma-glutamate-cysteine ligase (gamma-GCL), the rate-limiting enzyme in GSH synthesis. Exposure of astrocytes to NDP-MSH increased gamma-GCL activity in a dose-dependent manner (p less than 0.01). However, treatment with NDP-MSH for 6 h did not modify gene expression of either catalytic (GCLc) or modifier (GCLm) subunits of gamma-GCL, assessed by real time-PCR. Nuclear factor erythroid 2-related factor 2 (Nrf2) is considered the primary cellular defense against cytotoxicity caused by oxidative stress, by modulating the expression of antioxidant enzymes like heme oxygenase 1 (HO-1). We observed nuclear translocation of Nrf2 by immunocytochemistry in astrocytes treated with NDP-MSH for 6 h. Moreover, HO-1 mRNA levels were also increased upon NDP-MSH treatment.Finally we tested superoxide dismutase (SOD) activity through the epinephrine auto-oxidation technique. NDP-MSH increased SOD activity by 52.7% (p less than 0.05). In summary, NDP-MSH might exert an antioxidant effect in primary cultured astrocytes by means of modulation of gamma-GCL and SOD activities, stimulation of GSH release and induction of Nrf2 nuclear translocation and HO-1 gene expression, thereby contributing to the neuroprotective properties of melanocortins.