Effect of magnesium sulfate on the expression of NLRP3 inflammasoma in monocytes from pre-eclamptic pregnant women

Abstract

Hypertensive disorders of pregnancy are one of the main causes of maternal and perinatal mortality worldwide. Pre-eclampsia is a multisystem disease characterized by intense activation of innate immunity components such as the NLRP3 inflammasome complex, which is responsible for the production of high levels of Interleukin-1 (IL-1²) and IL-18 in both placenta and peripheral blood. As a clinical manifestation, pre-eclampsia can evolve into critical situations, directly related to the high rates of maternal and perinatal morbidity and mortality, which are hypertensive crisis, HELLP syndrome, and eclampsia. Eclampsia, defined as the manifestation of a generalized tonic-clonic seizure in a pregnant woman with pre-eclampsia, is considered one of the most serious complications of the disease. Evidence in the literature demonstrates that magnesium sulfate (MgSO4) is the medication of choice for the prevention and treatment of eclampsia, in addition to having an anti-inflammatory effect. Objective: Considering the therapeutic action of MgSO4 in the prevention and treatment of eclampsia, this project aims to: 1) evaluate the anti-inflammatory effect of MgSO4, administered intravenously, on the expression of NLRP3 inflammasome and pyroptosis by monocytes of pregnant women diagnosed with imminent eclampsia; 2) to evaluate the modulatory effect of MgSO4 in vitro on NLRP3 inflammasome expression and pyroptosis in monocytes from healthy women, after stimulation of these cells with monosodium urate (MSU). Subjects and methods: The study will include 16 pregnant women with isolated pre-eclampsia (PE), 16 with chronic arterial hypertension superimposed by pre-eclampsia (HAC+PE), both diagnosed with imminent eclampsia, and 16 healthy, non-pregnant women. In the first objective, the endogenous gene expression of the NLRP3 inflammasome by monocytes from pre-eclamptic pregnant women will be evaluated before and after treatment with MgSO4. After the inclusion of the patient in the study, four blood samples will be collected: before administration of the initial dose of MgSO4, after 12 and 24 hours of treatment with the maintenance dose, and 24 hours after delivery. The gene expression of the NLRP3 inflammasome and its components, caspase-1, IL-1², and IL-18, in addition to the cytokines tumor necrosis factor-alpha (TNF-±) and IL-10 will be determined by qPCR and by flow cytometry and, the plasma concentrations of caspase-1, IL-1², IL-18, TNF-±, and IL-10 will be detected by the ELISA technique. In the second objective, peripheral blood monocytes from healthy, non-pregnant women will be obtained and cultured in vitro in the presence or absence of MSU and MgSO4 for 4h to determine the gene expression of the NLRP3 inflammasome and its components by qPCR and by flow cytometry and, for 18 h for determination of caspase-1, IL-1², IL-18, TNF-± and IL-10 by ELISA. The determination of intracellular calcium levels in MgSO4 treated cells will be used to assess whether the effect of magnesium on the NLRP3 inflammasome involves an intracellular calcium-dependent mechanism. The occurrence of pyroptosis will be evaluated by flow cytometry and lactate dehydrogenase (LDH) cytotoxicity assays. The results will be analyzed by parametric or non-parametric tests, with a significance level of 5%. (AU)