Triagem de potenciais neuroprotetores em modelo celular de toxicidade induzida por MPP+: Angiotensina- (1-7) como um candidato

Abstract

Introduction: Cellular models have been used to study a range of diseases. They are particularly useful for exploring isolated pathophysiological aspects, the genes and proteins involved, mechanisms of action of specific neurotoxins, for preliminary screening of compounds, and providing valuable information for validation in animal models. SH-SY5Y cells constitute the cell lineage most widely used as a model of neurons. This cell was established in 1970 from a metastatic bone tumor derived from neuroblastoma, an embryonic peripheral sympathetic nervous system tumor, and the most common extracranial solid tumor of childhood. Despite their tumor origin, these cells are widely used to study neurodegenerative diseases, such as Parkinson's disease (PD). These cells express several dopaminergic neuronal markers, such as tyrosine hydroxylase (TH), dopamine-β-hydroxylase, DA transporter (DAT), and DA receptor subtypes 2 and 3 (D2R and D3R). Furthermore, most of the genes and pathways involved in PD are found intact in these cells. Due to ample evidence for the involvement of oxidative stress in PD, SH-SY5Y cells are used in most studies to assess the neuroprotective effect of compounds against oxidative insult. The toxic insult with 1-Methyl-4-Phenylpyridinium (MPP+) is the most widely used and has the advantage of causing specific degeneration of dopaminergic neurons since it is actively captured only by DATs. It inhibits the complex I activity of the electron transport chain leading to ATP depletion and cell death. Currently, there are no proven neuroprotective therapies for PD. Current therapies used in patients with PD have only symptomatic action and do not reduce or prevent neuronal degeneration. Although several compounds have demonstrated neuroprotective activity in preclinical trials in recent years, most have failed to reproduce these results in clinical trials, or are poorly tolerated by patients because of their side effects. In the past few years, attention has been given to components of the renin-angiotensin system as potential neuroprotective targets, such as Angiotensin II inhibitors and Angiotensin- (1-7) (Ang- (1-7)). Recent studies suggest that Ang- (1-7) has a neuroprotective role, but the mechanisms involved are not yet fully understood. Objective: To evaluate the in vitro neuroprotective potential of Ang- (1-7) against the toxicity induced by MPP+ using human neuronal cells SH-SY5Y differentiated with retinoic acid as a model. Method: The neuroprotective effect was evaluated by viability assays (MTT and LDH), labeling cells with propidium iodide (IP), and a fluorimetric assay using the marker 2,7-dichlorofluorescein diacetate (DCF,-DA) and neurite outgrowth. Results: Pretreatment of cells with Ang- (1-7) for 24 hours protected against loss of cell viability, membrane integrity, oxidative stress, and neuritic degeneration caused by MPP+. The protective effect was completely abolished when the cells were treated with Ang- (1-7) + A-779. This demonstrates that in this model, the neuroprotective effect of Ang- (1-7) is dependent on the Mas receptor. Conclusions: The present study allows us to conclude that, in the proposed experimental model, treatment with Ang- (1-7) was able to protect SH-SY5Y cells against MPP+ damage. These findings support the idea that the Ang- (1-7) / MasR axis should be included as a potential pharmacological target against oxidative injury in more complex models of neuroprotection and PD.