생쥐 대식세포에 대한 SNP의 Apoptosis 유발 효과 = Apoptotic Effect Induced by SNP in Murine Macrophage

다국어 초록 (Multilingual Abstract)

Endogenously generated Nitric oxide(NO) redox species by the activation of inducible NO synthase(iNOS) induce apoptotic cell death in macrophages. In this experiments to exclude possible interference with NOS induction, we examined the ability of NO generating compound, sodium nitroprusside(SNP) to induce apoptotic cell death of murine macrophages(J774A.1, RAW 264.7, and thioglycollate-induced peritoneal macrophage). The induction of apoptosis was sup-ported by the absence of significant trypan blue staining but the occurrence of biochemical and morphological apoptotic markers of the increased DNA fragmentation. For the quantitation of apoptosis we used relatively simple and effective method, diphenylarnine(DPA) colorimetric as-say which was confirmed by DNA ladder and TUNEL stain. Incubation of J774A.1 macrophages with increasing concentrations of SNP(0.5-3mM) for 8 hr led to concentration de-pendent apoptotic response, but with higher concentrations cell viability and DNA fragmentation started to decline. Apoptosis was rapid at 1mM and 2mM SNP in J774A.l macrophages, yielding specific DNA fragmentation after 4 and 3 hr with no further increase or slight decrease in fragmentation after 8 and 10 hr, respectively. As the concentration of SNP was increased, the time course of apoptosis was shortened. Increased apoptotic cells of late stage cause decreased cell viability and DNA fragmentation by the loss of cell membrane integrity and DNA de-gradation. As a results incubation with 1mM SNP for 5 hr is sufficient to induce apoptosis in J 774A.1 macrophages. Detailed kinetic studies revealed that there was no direct correleation between nitrite(NO2-) accumulation and death-eliciting potency. Nitrite , a final NO oxidation product, cannot correlated with various NO-related redox forms possibly involved in a apoptotic signaling. In different murine macrophage cell line, RAW264.7, apoptosis was also induced by the incubation of 1mM SNP for 5 hr and the response was not inhibited by the addition of 1mM NG-monomethyl-L-arginine(NMMA), establishing a definite link between exogenously applied NO and apoptotic cell death. In contrast to cell lines there was no induction of apoptosis in peritoneal macrophages and J774A.1 cultured with thioglycollate. Angry state of thioglycollate induced in-Rammed macrophages may suppress NO-induced apoptosis compared to fully activated ma-crophages which express a high level of iNOS, produce large quantities of NO, and perform self destruction. The balance between apoptosis-promoting and opposing signals may regulate the susceptibility of macrophages to NO-mediated apoptosis. Therefore under inflammatory conditions, there may be a tendency to maintain cytotoxic activities of macrophages against pathogens before elimination of activated macrophages by an apoptotic process after massive stimulation.