Resumo:
Brain ischemia is characterized by the interruption of blood supply, reducing the supply of oxygen and nutrients to the nervous tissue. As a consequence, energy deficit, oxidative stress and inflammation occur, which lead to cell death. Glutamatergic excitotoxicity is one of the pathophysiological mechanisms present in chronic and acute neurodegenerative diseases, such as ischemic stroke. Excess glutamate promotes apoptosis in neurons, increasing the entry of Ca2+ into the cell, resulting in DNA damage and cell death. On the other hand, astrocytes actively control excess glutamate to prevent neuronal death. Our previous studies demonstrated improvement in the function of neural cells treated with compounds present in the dichloromethane extract of Amburana cearensis seeds (EDAC) against oxygen and glucose deprivation (OGD) and stress due to excess glutamate. However, more studies need to be carried out to better understand the mechanisms and characterize coumarin, the main compound in EDAC, as a pharmacologically active agent. It is known that cerebral ischemia can lead to the activation of cascades such as PI3K/AKT and MAPK, increasing the expression of proteins such as AKT and ERK1/2, and also modulating the expression of glutamine synthetase (GS), a marker of astrogliosis, which converts glutamate into glutamine, a non-toxic amino acid. Therefore, this study aims to investigate the mechanism of action of EDAC and isolated coumarin in the regulation of MAPK and AKT pathway proteins in vitro model of ischemic stroke. Thus, PC12 cell lines were subjected to OGD or glutamate (20 mM) and/or concomitantly treated with EDAC (500 μg/mL) and coumarin (500 μm/mL) for 24 hours. Subsequently, cell viability was assessed by propidium iodide or MTT. Furthermore, the expression of AKT and MAPK pathway proteins was investigated by Western Blot, and the expression of caspase 3 by immunofluorescence. EDAC and coumarin were able to protect PC12 cells in OGD condition and EDAC was able to protect cells against glutamate toxicity (20 mM). Both were able to regulate the expression of AKT, ERK1/2 and phosphorylated ERK and negatively regulate caspase 3. Our results demonstrate that EDAC and coumarin have a potential pharmacological effect in acute models of ischemia and clarify the mechanisms of action related to the effect neuroprotective compound from A. cearensis seeds in a study model of brain ischemia.
