Facultad de Farmacia
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Browsing Facultad de Farmacia by Author "Arriagada Cancino, Solange Pilar"
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Item Efecto de la vitamina C en células endoteliales microvasculares de cerebro humano en modelo de isquemia-reperfusión in vitro.(Universidad de Concepción, 2023) Arriagada Cancino, Solange Pilar; Rivas Rocco, Coralia; Inostroza Pérez, EvelingStroke is the second leading cause of death in Chile, where 60% of cases are ischemic. In these cases, a reduction of nutrients and oxygen occurs in the affected area of the brain, triggering a redox imbalance with an increase in reactive oxygen species (ROS) levels that generate cellular damage and death. Among the affected cells are endothelial cells (EC), which are responsible for protecting the integrity of the blood-brain barrier (BBB). Therefore, their death facilitates the destruction and loss of BBB functionality. Antioxidants such as vitamin C (VitC) are available to regulate the redox imbalance. VitC exists in the body in its oxidized form (dehydroascorbic acid; DHA) and reduced form (ascorbic acid; AA). AA is a potent reductant capable of neutralizing free radicals. Both forms of VitC enter the cell through different transporters: DHA enters through GLUTs, and AA through SVCTs. There is evidence that SVCT2 transporters increase their expression in ischemic processes in mouse cerebral endothelial cells, but this has not yet been studied in human cerebral endothelial cells. In this study, we implemented an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC), which, upon differentiation, express BBB-like tight junction proteins (TJPs). We evaluated the TJPs through ZO-1 protein expression using immunofluorescence. We established an in vitro model of ischemia and reperfusion and evaluated its effects on cell viability through the MTT assay, the expression of HIF1-α through western blot, and the decrease in ZO-1 protein expression through immunofluorescence. Subsequently, we assessed the expression of VitC transporters after ischemia and reperfusion using RT-qPCR and immunofluorescence. Additionally, we analyzed the increased levels of ROS after ischemia and reperfusion and the effect of VitC treatment on these levels using flow cytometry. Our results demonstrate that the in vitro ischemia and reperfusion model is capable of generating endothelial damage in our BBB model. We also found a decrease in the expression of GLUT1 and SVCT2 transporters after ischemia and reperfusion, while SVCT1 showed increased expression. Furthermore, we observed elevated levels of ROS after ischemia and reperfusion, which decreased after 24 hours of reperfusion. There was a trend towards decreased ROS levels, and AA treatment generated lower ROS levels compared to the untreated condition. These results shed light on the important role of antioxidants in oxidative damage, particularly emphasizing the relevance of VitC and its transporters in the BBB under an ischemia and reperfusion state that resembles the conditions occurring in a stroke. We highlight the finding of increased SVCT1 transporter expression, which may play a crucial role in such pathologies. While there are still many aspects to be studied, the obtained results potentially open up therapeutic opportunities to mitigate the damage caused by stroke and other conditions that currently lack effective treatments.