Avaliação dos efeitos da 1α,25-dihidroxivitamina D3 (VD3) sobre a neurotoxicidade induzida pela rotenona em células PC12: um modelo in vitro de doença de Parkinson

Abstract

Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic cells in the substantia nigra, pars compacta. The brains of PD patients also present neuroinflammation, oxidative stress and mitochondrial dysfunction. Among the neuroprotective agents currently tested, vitamin D (VD3) appears as a potentially compound in prevention of neurodegeneration and cell death. Thus, the aim of this study is evaluating the neuroprotective effect of VD3 in PC12 cells (in vitro model of Parkinson's disease) and in astrocytes (directly involved in neuroinflammation and in the pathophysiology of PD), after exposure to rotenone (ROT). Cell viability was assessed by MTT test and 7-AAD labeling in PC12 cells and astrocytes treated and untreated with VD3 (0.1; 0.5 and 1 ng / mL) and/or ROT (10 µg/mL or 5 µg/mL), and ROS production and cell death events measured by flow cytometry. Additionally, mitochondrial transmembrane potential, measures of glutathione and superoxide accumulation reduction and cell morphology were evaluated. In silico studies (molecular docking) and expression of tyrosine hydroxylase (TH) and NF-κB (Western blot) in PC12 cells were also performed. The results showed that VD3 improved the viability of cells previously treated and then exposed to ROT, preventing the occurrence of possible necrotic and apoptotic events. In addition, cells exposed to ROT showed increasement in ROS production and superoxide accumulation, which significantly decreased with previous treatment with VD3. Importantly, ROT decreased the mitochondrial transmembrane potential, which was significantly prevented by VD3, especially at a concentration of 1 ng/mL. Moreover, VD3 has been shown to interact strongly with TH, as observed by molecular docking experiments performed with PC12 cells. TH expression in these cells treated with ROT significantly decreased, whereas treatment with VD3 and subsequent exposure with ROT significantly increased. The expression of NF-κB increased in the treatment with ROT, while cells treated with VD3 at the concentrations evaluated and then exposed to ROT were able to protect against this inflammatory stimulus, reducing the expression of this transcription factor. Therefore, treatment with VD3 protects both cell lines from damage caused by ROT, decreasing oxidative stress, improving mitochondrial function, increasing TH expression (in PC12 cells) and causing a decrease in pro-inflammatory factors, such as NF-κB, suggesting its role cytoprotective in this PD model. A likely target appears to be the TH molecule, a rate-limiting step in dopamine synthesis. However, further investigation is needed regarding the participation and mechanism of action of VD3 in this cellular model of PD.