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Signaling pathways involved in induction of neutrophil apoptosis by oleic and linoleic acids

Grant number: 15/07815-5
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): July 01, 2015
Effective date (End): November 30, 2016
Field of knowledge:Biological Sciences - Physiology
Principal Investigator:Adriana Cristina Levada-Pires
Grantee:Eliane Borges da Silva
Home Institution: Pró-Reitoria de Pós-Graduação e Pesquisa. Universidade Cruzeiro do Sul (UNICSUL). São Paulo , SP, Brazil
Associated research grant:10/50848-8 - Role of oxygen reactive species and ceramides in human neutrophil death induced by fatty acids, AP.JP

Abstract

Endoplasmic reticulum (ER) stress is a protective mechanism to prevent the formation of unfolded proteins, however ER prolonged can induces apoptosis of neutrophils and other cell types. Recent studies have demonstrated that fatty acids can induce ER in neurons, therefore the effect of these metabolites on ER of human neutrophils has not been established. Our group has shown that oleic and linoleic acid at a concentration of 250 uM induce apoptosis in human neutrophils after culture for 3 hours. The possible involvement of ER stress on apoptosis of neutrophils induced by fatty acids and signaling pathways involved in this process remain to be elucidated. Thus, this study will investigate the induction of ER stress by oleic and linoleic acids and the possible involvement of ER stress on apoptosis of human neutrophils induced by these fatty acids. Human neutrophils are pre-treated for a period of 3 hours (for 1, 2 and 3 hours), with oleic and linoleic acids into non-toxic (100 µM) and toxic (250 µM) concentration and then determining a stress induction the ER by oleic and linoleic acids through the determination of pathways and proteins involved in ER stress tests modulation(for probe ER-tracker, cytometry), gene expression of cleaved protein XBP-1 (PCR) and quantification of proteins involved in endoplasmic reticulum stress : PERK, pPERK, peIF2 alfa, IRE-1 and pIRE1 (by western blotting). In addition, it is determined the share of ER stress in the death of neutrophils induced by fatty acids through quantification of proteins involved in endoplasmic reticulum stress: CHOP, GRP78, Ero-1, JNK and ASK (by western blotting) and determining the production of ROS by neutrophils treated with acids (by using lucigenin chemiluminescence). Furthermore, the importance of endoplasmic reticulum stress-induced neutrophil apoptosis by the tested fatty acids is investigated by assessing cell viability and DNA fragmentation after culture of neutrophils in the endoplasmic reticulum stress 4PBA inhibitor prior to addition of fatty acids. The means of statistical comparisons will be performed by analysis of variance (ANOVA, one-way, non-parametric) followed by Tukey-Kramer multiple comparison test. The level of significance will be p <0.05. Will be used GraphPad Prism Software - version 4.03.