Pregnancies complicated by preeclampsia (PE) are associated with placental ischemia/hypoxia, oxidative stress, and elevated levels of proinflammatory cytokines. These physiological changes can trigger cell death due to apoptosis, an essential process for the maintenance of homeostasis in human tissues, including the placenta and considered an intermediate event in the generation of pregnancy syndromes, such as PE. The triggering of PE may be dependent on the deficiency of regulatory factors capable of modulating this inflammatory response such as vitamin D. This vitamin has effects on the innate and adaptive immune system, generating a more tolerogenic immune status, particularly for its regulatory activities on the inflammatory response. The onset of apoptosis in placental tissues may be involved in the exacerbated systemic inflammatory state of PE. This project aims to evaluate the immunomodulatory action of vitamin D on apoptosis in BeWo trophoblastic cell line stimulated with monosodium urate (MSU), hydrogen peroxide (H2O2) and tumor necrosis factor (TNF-alpha). BeWo cells will be cultured in the presence or absence of MSU, H2O2 and TNF-alpha and treated with vitamin D for 4 and 24 hours. After this period, the presence of annexin V that binds to externalized phosphatidylserine in the early stages of apoptosis and 7AAD, which is a DNA intercalator, will be evaluated and will mark cells in late apoptosis and/or necrosis. BeWo cells will be submitted for evaluation of the gene and protein expression of IL-1beta, TNF-alpha, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and vitamin D receptor (VDR) by qPCR and ELISA. This project is expected to determine whether vitamin D has a protective effect during the inflammatory process and on apoptosis in BeWo cells against the above-mentioned stimuli.
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