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Possible mechanism of neutrophil death in Diabetes mellitus
Author(s) -
Kuwabara Wilson Mitsuo Tatagiba,
Curi Rui,
Alba-Loureiro Tatiana Carolina
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.869.7
Subject(s) - cytochrome c , apoptosis , dna fragmentation , depolarization , caspase , apoptosis inducing factor , membrane potential , mitochondrion , chemistry , blot , inner mitochondrial membrane , microbiology and biotechnology , programmed cell death , medicine , biology , endocrinology , biochemistry , gene
Diabetes mellitus was induced by streptozotocin (65 mg/Kg, i.v.) two weeks before. Neutrophils were obtained from the peritoneal cavity 4 hours after injection of 1% oyster glycogen solution. Cells were maintained in culture for 0, 60, 120 and 180 minutes with or without PMA stimulus (20nM). Neutrophils were then analyzed by flow cytometer for membrane integrity, DNA fragmentation and mithocondrial transmambrane potential. Caspases 1 and 3 activity was evaluated by spectrofluorimetry. The content of mithocondrial proteins released during cellular death (Cytochrome C, Smac/Diablo, Endonuclease G (Endo G), Apoptosis Inducing Factor (AIF), High temperature requirement protein A2 (HtrA2)/Omi) was evaluated by Western Blotting. Our study showed that neutrophils from the diabetic rats presented a decrease in membrane integrity (p<0,001), increase in DNA fragmentation (p<0,01) and an increase in the mitochondrial depolarization (p<0,0001), after 60 min of PMA stimulus when compared to non‐stimulated cells from the same group. However, at time 0, neutrophils from diabetic rats already presented a decrease in the mitochondrial transmembrane potential (p<0,001) when compared to cells from the group control. Caspases 1 and 3 activity was not observed. It was not observed difference in the content of the mitochondrial proteins cytochrome C, Smac/Diablo and Endo G. However, content of AIF and HtrA2/Omi was increased 60 minutes after PMA stimulus. The death process in neutrophils from diabetic rats seems to be caspase 1 and 3 independent and dependent on alterations of mitochondrial transmembrane potential presented at time 0 without stimulus. FAPESP, CNPq and CAPES.