Efeito neuroprotetor do extrato padronizado e cumarina de Amburana cearensis (cumaru) em modelo de Parkinson em células neuronais: avaliação da neurotoxicidade e expressão de NF-ΚB

Abstract

Parkinson's disease (PD) is the second most common neurodegenerative disease in the world, with projections indicating that more than 12 million people will be affected by it by 2040. PD is characterized by the progressive loss of motor and behavioral functions, associated with the loss of dopaminergic neurons in the substantia nigra and the presence of protein inclusions called Lewy bodies, which are related to neuroinflammation, mitochondrial dysfunction and oxidative stress. Given the limitations of current pharmacotherapy for PD, which is only symptomatic, research into new drugs is essential. Medicinal plants from the Caatinga, such as Amburana cearensis (cumaru) and their active ingredients, such as coumarin (CM), have shown promising effects for the control of neurotoxicity and neuroinflammation. In view of the above, the present study aims to investigate the neuroprotective effect of the dry extract of A. cearensis (ESAC) and CM, measured by cytotoxicity and the expression of the inflammatory transcription factor NF-κB, in a Parkinson's model in neuronal cells (PC-12 cell line). For this purpose, ESAC was characterized by HPLC (major active markers - CM = 35.16 ± 0.01 mg/g; vanillic acid = 2.02 ± 0.01 mg/g; amburoside A = 1.16 ± 0.01 mg/g). The effect of ESAC (5, 10, 25, 50, 100 and 200 µg/mL) and CM (5, 10, 25, 50 and 100 µg/mL) on neurotoxicity induced by 6-hydroxydopamine (6-OHDA) was evaluated by the MTT test, and the evaluation of the expression of the pro-inflammatory transcription factor (NF-κB p65) was performed by the western blotting technique. The results showed that ESAC (5 - 200 µg/mL) and CM (5 - 100 µg/mL) did not significantly alter cell viability when compared to the control group (DMSO 0.1%, vehicle, 100% viability). Pretreatment of PC-12 cells with ESAC (10 µg/mL) resulted in a reduction of up to 20% in neurotoxicity induced by 6-OHDA (25 µg/mL), compared to the 6-OHDA+DMSO 0.1% group (48.8 ± 5.0%). Similar results were observed for CM. Exposure of PC-12 cells to 6-OHDA increased the expression of NF-κB p65 compared to the untreated group (DMSO 0.1%). However, treatment with ESAC (10 and 25 µg/mL) inhibited the increase in NF-κB p65 expression induced by 6-OHDA by up to 29%, while treatment with CM (5 and 10 µg/mL) achieved a reduction of up to 39% in the expression of the NF-κB p65 pathway. The study allowed the use of the Parkinson's model in dopaminergic neuronal cells previously standardized by our laboratory, with unprecedented determination of the neuroprotective effect of ESAC and CM, which appears to be associated with the reduction of the inflammatory microenvironment via NF-κB p65.