Efeito citoprotetor de nanopartículas poliméricas contendo resveratrol em modelo in vitro de doença de Parkinson induzida por rotenona: envolvimento da via Nrf2

Abstract

Parkinson's disease (PD) is a neurodegenerative condition characterized by the loss of dopaminergic neurons in the substantia nigra, resulting in motor impairment, whose prevalence is expected to increase in the coming decades. Considering the limitations of current clinical treatment (levodopa), new therapeutic strategies are needed. Resveratrol (RSV) has antioxidant and anti-inflammatory properties, but its low bioavailability limits its clinical application, making nanoencapsulation a promising alternative. The aim of this study was to evaluate the in vitro protective effect of resveratrol polymeric nanoparticles (NP RSV) and the involvement of the Keap1/Nrf2/ARE pathway in a mimetic model of PD induced by rotenone (ROT). For in vitro assays, PC12 neuronal cells and astrocytes were pretreated with NP RSV, RSV, and dopamine (DA) for 1 hour, followed by exposure to ROT for 24 hours. Cell viability was assessed by the MTT assay. The pattern of cell death, reactive oxygen species production, and mitochondrial transmembrane potential (ΔΨm) were evaluated by flow cytometry assays. Cellular morphological changes were assessed by optical microscopy. In silico tests were performed to investigate the theoretical interaction of RSV with the Kelch and Brick-to-Brick (BTB) domains of the Keap1 protein. Gene expression of Nrf2 and HMOX, elements of the Keap1/Nrf2/ARE pathway, was performed by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). The results showed that ROT induced a reduction in cell viability, increased oxidative stress, depolarization of ΔΨm, and cell death by apoptosis; however, pretreatment with NP RSV was able to significantly protect the cells, preserving cell viability, reducing the formation of reactive oxygen species, maintaining mitochondrial function, and decreasing apoptosis. The protective effects of NP RSV were observed at concentrations of 1.56 µM, 0.78 µM, and 0.39 µM in PC12 cells and 6.25 µM, 3.12 µM, and 1.56 µM in astrocytes; while RSV showed a protective effect at concentrations of 12.5 µM, 6.25 µM, and 3.12 µM in both cell lines. DA (400 µM-100 µM) showed limited neuroprotective effects. Morphological analyses corroborated these findings, showing cellular changes and reduced cell density after exposure to ROT and maintenance of these characteristics when pretreated with NP RSV. In silico assays indicated a stable interaction between RSV and Keap1 protein, sustained by hydrogen bonds. Regarding gene expression, ROT promoted increased expression of NRF2 and HMOX-1, while pretreatment with NP RSV modulated this response. Together, the data indicate that NP RSVs exert a neuroprotective effect, acting in an integrated manner on the main mechanisms involved in the pathophysiology of PD, with emphasis on the modulation of the Keap1/Nrf2/ARE pathway. Thus, the present study contributes to the understanding of the molecular mechanisms associated with neuroprotection mediated by nanostructured systems, presenting scientific relevance.