Modulatory effect of progesterone, vitamin D and silibinin on the inflammasomes in monocytes from pegnant women with peeclampsia

Abstract

Preeclampsia (PE) is a specific syndrome of pregnancy, characterized by hypertension and proteinuria, identified after the 20th week of pregnancy. This pathology is associated with hyperuricemia, elevated serum levels of inflammatory cytokines, leukocyte activation and oxidative stress. Uric acid crystals (MSU) and hyalorunan (HA) may activate an intracellular complex called inflammasome, a multi-protein structure which is important for processing and release of inflammatory cytokines such as interleukin-1 beta (IL-1²) and IL-18. The imbalance between pro- and anti-inflammatory cytokines in PE could be dependent on the deficiency of regulatory factors capable of modulating inflammatory response such as vitamin D and progesterone, or this imbalance could be improved by administration of substances with anti-inflammatory properties such as silibinin. The aim of this study is to evaluate the modulatory effect of progesterone, vitamin D and silibinin on NLRP1 and NLRP3 inflammasomes in monocytes from pregnant women with PE and in THP-1 human monocytes cell line. Monocytes obtained from peripheral blood of 20 pregnant women with PE and THP-1 cells will be cultured in the presence or absence of MSU, HA, progesterone, vitamin D or silibinin for different periods: a) 30 min for determination of the NF-kB phosphorylation and analysis of TLR4, IºB and NF-kB expression by flow cytometry, b) 4 h for the gene expression of TLR4, MYD88, NFKB1, NLRP1, NLRP3, CASP1, IL1B, IL18, TNF and IL-10 by RT-qPCR and c) 18 h for the determination of the cytokines concentration by ELISA. Additionally, the culture of THP-1 cells, stimulated or not with MSU, HA, progesterone, vitamin D and silibinin, for 18h will be used to evaluate NLRP1, NLRP3 and caspase-1 proteins by Western blot. The results will be analyzed using non-parametric tests at 5% significance level.