Preeclampsia (PE) is a pregnancy-specific syndrome characterized by hypertension and proteinuria identified after the 20th week of gestation. The development of this disease is due to immune maladaptation in maternal-fetal interface, determining low uteroplacental blood flow, resulting in placental hypoxia / ischemia, oxidative stress and fetal growth restriction. The oxidative stress generated from injury caused by placental hypoxia and reperfusion induces an inflammatory response with increased production of mediators of endothelial cell dysfunction, such as proinflammatory cytokines and lipid mediators. This systemic inflammatory response becomes exacerbated, affecting the functional state of the cells of innate immunity. Endogenous products, called "danger signals" or damage-associated molecular patterns (DAMPS) can be released during tissue injury, resulting from sterile inflammation. These products are represented by molecules such as reactive oxygen species (ROS), proteins released from dead cells and extracellular matrix derivatives that interact with pattern recognition receptors (PRR) called NOD-like or NLR. These receptors consist of cytosolic proteins that recognize endogenous products and recruit other proteins, forming signaling complexes that promote inflammation. These complexes are called inflammasomes and their main function is to generate the production of IL-1b, responsible for the intense inflammatory response. Autophagy is a lysosomal degradation pathway that removes protein aggregates and damaged organelles maintaining the cellular integrity which may be compromised in pregnant women with PE, due to the presence of placental lesions caused by hypoxia / ischemia. Considering that the generation of inflammasome is related to intense inflammatory response and that autophagy can modulate the inflammatory process, contributing to the maintenance of intracellular homeostasis, this project aims to determine the presence of inflammasome NLRP3 and the occurrence of autophagy in placenta of pregnant women with PE. Forty pregnant women including 20 normotensive and 20 women with PE will be studied. Fragments of placenta containing the maternal and fetal sides will be obtained immediately after delivery. Each fragment will be cut into four equal parts, one being placed in formalin buffer for immunohistochemical analysis; another part will be frozen in liquid nitrogen for later preparation of placental homogenate and analysis by Western blot and enzyme linked immunoassay (ELISA). The third part will be used for technical real-time quantitative polymerase chain reaction (RT-qPCR) and the fourth fragment will be fixed in 2.5% glutaraldehyde for evaluating of the presence of autophagy by transmission electron microscopy. The occurrence of autophagy will also be determined by the expression of proteins LC3, Beclin-1 and mTOR by Western blot, immunohistochemistry and by RT-qPCR technique. For inflammasome determination the gene expression of NLRP3, caspase-1, IL-1², IL-18 and TNF-± will be evaluated by RT-qPCR. Protein concentration of caspase 1 and IL-1 ², IL-18, TNF ±-and HMGB1 will be quantified by Western blot and ELISA. The expression of NLRP3, caspase 1, IL-1 ², IL-18, TNF-± and HMGB-1 will also be determined by immunohistochemistry in placental sections.
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