Banca de DEFESA: GRAZIELE CRISTINA FERREIRA

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
DISCENTE : GRAZIELE CRISTINA FERREIRA
DATA : 17/08/2021
HORA: 13:00
LOCAL: Modo remoto
TÍTULO:

EFFECT OF rBMTI-A (SERINE PROTEASE INHIBITOR) IN THE INFLAMMATORY PROCESS IN A549 PULMONARY CELLS

PÁGINAS: 126
GRANDE ÁREA: Ciências Biológicas
ÁREA: Bioquímica
SUBÁREA: Biologia Molecular
RESUMO:

  The serine protease inhibitor rBmTI-A is a recombinant protein based on an inhibitor from the tick Rhipicephalus microplus, belongs to Kunitz-BPTI family with two inhibitors domains and present activity on human neutrophil elastase (HNE), bovin trypsin, plasmin and human plasma kallikreins. In previous work, pulmonary emphysema was induced by porcine pancreatic elastase (PPE) in mice and the inhibitor was capable to prevent emphysema development when compared to the untreated control with reduction of the inflammatory response. In this work, we produced rBmTI-A, analyzed the inflammatory response induced by PPE in A549 human pulmonary cells and investigated the action of rBmTI-A in this process, furthermore, we analyzed the involvement of protease-activated receptor (PAR) in cytokine increase resulting from the action of PPE. The cells were incubated with rBmTI-A in different concentrations for two hours followed by addition of PPE (0.0125U/mL),controls were made only incubating the cellswith medium, PPE or inhibitor;after 16 hours, the supernatants were destined to inflammatory cytokines quantification by ELISA or Multiplex while the cells were submitted to cell viability assay by MTT or RNA extraction and then qPCR for analysis of inflammatory cytokine transcription. Similar procedure was accomplished with antagonist peptides PAR-1 and PAR-2 incubated for 30 minutes followed by addition of PPE (0.0125U/mL). Our results showed  that PPE at a concentration of 0.0125 U/mL did not alter the cellular viability and promoted an increase of relative expression of inflammatory cytokines. rBmTI-A at 8 nM promoted decrease of expressed cytokines in comparison with the induced PPE group with level of IL-8, IL-6, MCP-1 and TNF-α reduced in 56%, 20%, 45% and 64%, respectively. Concerning cytokines secretion in supernatant, rBmTI-A at 8 nM reduced 50% of IL-8 release and 75% of MCP-1 release. IL-10, on the other hand, was not detected neither by qPCR nor by Multiplextests. The PAR-1 and PAR-2 antagonist at 50µM were enough to reduce the genetic expression and secretion of IL-8 in PPE stimulated cells, presenting similar levels to the control. Through the results obtained in this work, we conclude that the treatment with rBmTI-A can decrease the inflammatory process induced by PPE in A549 cells and, additionally, we suggest that these cytokines increase, mainly IL-8, is connected to the action on PAR-1 and PAR-2 receptors.

MEMBROS DA BANCA:
Presidente - Interno ao Programa - 2605490 - SERGIO DAISHI SASAKI
Membro Titular - Examinador(a) Interno ao Programa - 1672728 - ANA CAROLINA SANTOS DE SOUZA GALVAO
Membro Titular - Examinador(a) Interno ao Programa - 3066269 - VINICIUS DE ANDRADE OLIVEIRA
Membro Titular - Examinador(a) Externo à Instituição - APARECIDA SADAE TANAKA - UNIFESP
Membro Titular - Examinador(a) Externo à Instituição - ERIKA SUZUKI DE TOLEDO
Membro Suplente - Examinador(a) Interno ao Programa - 1227329 - CESAR AUGUSTO JOAO RIBEIRO
Membro Suplente - Examinador(a) Interno ao Programa - 1675708 - DANIELE RIBEIRO DE ARAUJO
Membro Suplente - Examinador(a) Externo ao Programa - 1887027 - FERNANDO AUGUSTO DE OLIVEIRA RIBEIRO
Membro Suplente - Examinador(a) Externo à Instituição - BIANCA CARLA SILVA CAMPITELLI DE BARROS