時間分解蛍光イムノアッセイによるサイトカインの測定--心筋梗塞モデルラットの心筋におけるTNFαのup-regulation

摘要

TNF- alpha is involved in the pathogenesis of myocardial infarction (MI). Repeated angina attacks limit development of subsequent MI, which is known as ischemic preconditioning (IP). To reproduce MI, the coronary arteries of the rats were occluded. As measured by time-resolved fluoroimmunoassay, TNF- alpha level markedly increased with a peak at 1 hour in the membrane fraction. IP, conferred by three cycles of ischemia (3min)-reperfusion (5min), suppressed the Mi-induced TNF-alpha up-regulation. Real time PCR demonstrated up-regulation of TNF- alpha mRNA at 30 min of MI irrespective of IP. A transcription factor NF k B mediates pro-inflammatory or anti-apoptotic effects. The degradation of I k B, which indicates NF k B activation. In our samples, neither I k B degradation nor NF k B transcriptional activation was detected. IP abrogated the TNF- alpha up-regulation in MI, while p38MAP kinase inhibition blocked the TNF- alpha up-regulation. Immunoblot analyses using phospho-specific antibodies to MAP kinases shows the MI- induced activation of JNK, ERK and p38 MAP kinases. Only p38MAP kinase was inhibited partly by IP, though SB203580 inhibited the activation of the three MAP kinase enzymes. MI up-regulates TNF- alpha in the endothelium and cardiomyocyte partly through p38MAP kinase activation post-translationally, which is inhibited by IP.