ABSTRACT
Objective:To investigate the structural alterations of mitochondria and its role in neutrophil death induced by ONO-AE-248.Methods:Human neutrophils were cultured in vitro with ONO-AE-248(5?10-5 mol/L)and medium alone. Transmission electron microscopy(TEM) was used to detect the structural alterations of mitochondria and the level of mitochondria membrane potential by flow cytometry using mitocapture dying.Results:ONO-AE-248 resulted in extremely swollen mitochondria within 6 hours. Meanwhile, a rapid loss of mitochondrial membrane potential of neutrophils occurred, especially in 3 hours and 6 hours. There were obviously differences between spontaneous apoptosis and non-apoptosis programmed cell death induced by ONO-AE-248.Conclusion:The experiment results suggest that changes of mitochondrial structure and function be typically morphological, physiological and biochemical features in this unique form of neutrophil death, and that the mitochondrial pathway might play a more important role in ONO-AE-248-induced death of neutrophils.
ABSTRACT
Objective:To investigate the roles of apoptosis-associated speck-like protein containing CARD(ASC),one of differentially expressed proteins in neutrophil death induced by ONO-AE-248.Methods:Human neutrophils were cultured in vitro with or without ONO-AE-248(5?10-5 mol/L).The expression of ASC mRNA was detected by RT-PCR.PMN proteins were extracted at 12 h and then separated by 2D electrophoresis.20 differentially expressed proteins were selected and determined by PDQest 2-DE software.Results:ONO-AE-248 decreased the expression of ASC mRNA strikingly in 2 hours;ASC was one of the important differentially expressed proteins between spontaneous apoptosis and non-apoptosis programmed cell death induced by ONO-AE-248 in 12 hour and ASC protein point was weaker in ONO-AE-248 group.Conclusion:ASC might act as double switch that leads to apoptosis of neutrophils in the high expression and removes the forbiddance of pathway of ONO-AE-248 signals in the low expression,resulting in non-apoptosis programmed cell death of neutrophils.
ABSTRACT
Objective:To investigate the role of apoptosis-associated speck-like protein(ASC) in non-apoptotic,non-necrotic cell death of neutrophils induced by ONO-AE-248.Methods:The morphological changes of neutrophil nuclei and DNA fragmentation of the cells were examined by confocal microscopy and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL).Neutrophils were stimulated with medium alone,LPS,TNF-? or ONO-AE248 respectively.Then expression of ASC was detected by Western blot.Results:ONO-AE-248-induced neutrophil death was negative in TUNEL assay.Western blot showed that the expression of ASC protein was down-regulated by ONO-AE-248.Conclusion:The DNA degradation of the neutrophils when stimulated by ONO-AE-248 is probably different from that of the typical apoptosis.ASC might be a crosslink point between the ONO-AE-248-induced neutrophil death and spontaneous apoptosis of neutrophils.