Banca de DEFESA: MILENA DANIELA SOUZA SILVA

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : MILENA DANIELA SOUZA SILVA
DATE: 28/04/2022
TIME: 08:00
LOCAL: Sala Virtual Remota
TITLE:

ROLE OF PLA2S ISOLATED FROM Bothrops jararacussu VENOM OF NLRP3 INFLAMMASOME ACTIVATION IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS

KEY WORDS:

Snake venom, PLA2s, PBMCs, inflammassome NLRP3

PAGES: 77
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

Phospholipases A2 (PLA2s) are proteins found in abundance in the venom of snakes of the genus Bothrops and play an important role in the inflammatory reaction and leukocyte activation. Monocytes and lymphocytes are cells of the immune system characterized by mediating and presenting effective responses during an inflammatory response. The NLRP3 inflammasome is a multiprotein complex that is present in cells of the immune system and is activated by stimuli such as pathogen-associated molecular patterns (PAMPs) and damageassociated molecular patterns (DAMPs). The action of PLA2s against human peripheral blood mononuclear cells (PBMCs) is still not well understood. In this sense, the present study aimed to evaluate the role of PLA2s (BthTX-I Lys49) or (BthTX-II Asp49) from Bothorps jararacussu on NLRP3 inflammasome activation. Thus, viability tests were carried out from human PBMCs in a period of 4 hours by DNA labeling with 7AAD. The results showed that at concentrations of 5 and 10 μg/mL there was no cell death. The relative gene expression of NLRP3, Caspase1, ASC, IL-1β and GAPDH (internal control) gene were performed by RT-qPCR. Furthermore, the protein expression of these components (NLRP3, Caspase-1, ASC), gasdermin D (GSDMD) and β-actin (internal control) was verified by Western Blot. The results obtained showed significant amplification when compared to the GAPDH control of NLRP3, ASC, Caspase-1 and IL-6 in 1 hour and, of IL-1β, in 2 hours. Protein expression was performed after 4 hours of PBMC stimulation, and the results obtained showed significant expression of NLRP3 and GSDMD inflammasome complex proteins. Another method of proving the activation of the NLRP3 inflammasome was the counting of punctas formed by the NLRP3 sensor component by immunofluorescence. Then, to verify the cell death dependent on the activation of caspase-1, pyroptosis, LDH was measured in the supernatant of the PBMCs incubated with the different stimuli after 4 and 6 hours. The release of LDH occurred in 6 hours, as well as the expression of GSDMD, suggesting that the release of this molecule occurs due to the formation of pores in the membrane, a consequence of pyroptosis. Taken together, the data obtained so far show that the PLA2s, BthTX-I and BthTX-II, isolated from Bothrops jararacussu venom, activate the NLRP3 inflammasome complex culminating in the formation of membrane pores through GSDMD in human PBMCS that contribute to the inflammatory response observed in envenomation by this snake.

BANKING MEMBERS:
Externo à Instituição - FABIO HENRIQUE KWASNIEWSKI - UEL
Externa à Instituição - LUCIANE ALARCÃO DIAS MELICIO - UNESP
Presidente - 1726461 - JULIANA PAVAN ZULIANI TRENCH DE SOUZA