Investigação dos efeitos da apigenina conjugada a ciclodextrinas em modelo in vitro de neuroinflamação em células da medula espinhal

Abstract

INTRODUCTION: Spinal cord injury is a serious damage to the central nervous system associated with high rates of morbidity and mortality, physical dependence, and financial burdens. Inflammation is the main characteristic of secondary injury that provides a cascade of cellular and molecular events that increase the area of injury, aggravating neurological deficits and tissue recovery. The literature presents different models of studies in the central nervous system in which apigenin has demonstrated beneficial effects. However, there is little research on the neuroprotective role of apigenin in spinal cord injury, and in addition, the low solubility of this flavonoid in water may be a limiting factor for its biological activities. Cyclodextrins are cyclic oligosaccharides whose chemical structure allows the incorporation of lipophilic substances in their cavity, giving these molecules greater stability and bioavailability. OBJECTIVE: To evaluate the anti-inflammatory and neuroprotective effect of flavonoid apigenin and conjugates of apigenin with β-cyclodextrins in an in vitro model of neuroinflammation in spinal cord cells. MATERIALS AND METHODS: In this work β-cyclodextrin (β-CD) and three other derivatives were used. The cytotoxicity of apigenin (API) and conjugates of apigenin with β-cyclodextrins (API-cd’s) was evaluated by MTT assay in PC-12 cells and spinal cord cells in a series of concentrations (0.1-100μM) for 24h. After its characterization, the primary culture of the spinal cord was submitted to inflammatory damage by LPS, and subsequently treated with apigenin and the best conjugates of apigenin. The investigation of changes in markers associated with neuroinflammation was performed by immunofluorescence. The nitric oxide production was analyzed with the supernatants of the experimental groups. The analysis of cell morphology was performed by phase contrast microscopy, Rosenfeld staining and immunofluorescence. Cell migration was evaluated by the wound healing assay and immunofluorescence. RESULTS AND DISCUSSION: The API and API-cd’s did not promote cytotoxic action in PC-12 cells at the concentrations tested. It was observed that three conjugates increased the viability of PC-12 cells, mainly at the concentration of 10μM. The API and API-cd’s did not induce cytotoxicity in primary culture of the spinal cord at the concentrations tested. The primary culture of spinal cord of neonate rats presents morphological diversity of astrocytes, microglia, and neurons, as well as cell growth clusters. Spinal cord cells were subjected to inflammatory damage by LPS at 5 μg/mL for 12 hours. The API and API-cd 52 decreased the expression of IBA-1 and CD-68-positive cells after LPS-induced damage. Two API-cd’s significantly reduced nitric oxide levels. Two API-cd’s significantly reduced the number of S100β-positive cells. CONCLUSIONS: Apigenin and apigenin conjugates appear to protect spinal cord cells in an in vitro model of neuroinflammation.