Antioxidants protect overexpression of Nurr1 in stressed dopaminergic cells

The nuclear receptor NR4A2 (Nurr1) is induced by stress and injury in brain; Nurr1 is a transcription factor necessary for the maintenance of the midbrain dopaminergic neurons because it regulates the dopaminergic synthesis and it has a key role in the protection against oxidative and inflammatory stress associated to neurodegeneration. Permethrin (PERM), a member of the family of synthetic pyrethroids, can induce dopaminergic damage. In particular, impairment of Nurr1 and tyrosine hydroxylase (TH) gene expression has been observed in animal models. The aim of this study was to evaluate in vitro, through PC12 dopaminergic cells used as paradigm of neurodegeneration in neurobiological and neurochemical studies, if the overexpression of Nurr1 induced by PERM can be counterbalanced by antioxidants. The expression of two of the main transcription factors regulating the genes involved in pro-inflammatory and anti-inflammatory responses Nrf2 and NF-kB respectively are also analyzed. Results show that the incubation of PC12 cells with 1mM PERM for 72 hours, leads to over expression of Nurr1 gene. This effect occurs with both corn oil and EVO used to solubilize the toxicant even if the cell viability and the Nrf2 expression were increased by the presence of EVO respect to corn oil. RT-PCR of Nurr1 showed that its up-regulation was significantly reduced in the presence of antioxidants glutathione (GSH), tocotrienols (TOC) and Electrolyzed reduced water (ERW), but the effect was more pronounced with the addition of ERW. Since ERW resulted from RT-PCR analysis the most efficient among the compounds used, western blotting was performed on PC12 cells treated with PERM or with PERM plus ERW. Nurr1 protein expression increased by PERM exposure was down-regulated in the presence of ERW while non-significant changes were observed for TH. In conclusion, the protective effect of antioxidants in this in vitro model suggests that PERM toxicity could be linked to the redox system imbalance. Even if EVO is not able to counterbalance Nurr1 expression impairment, improved cell viability and anti-inflammatory properties suggest a potential beneficial effect of this component of Mediterranean diet.