Received June 01, 2018; Accepted October 11, 2018; Published November 15, 2018
http://dx.doi.org/10.18081/2333-5106/018-336-348
You-lin Lee; Qiong Chung; Lei Zhang; Zhu Shen; Jie Chan
Abstract
The transcriptional regulation of the iNOS gene in different cells is very complicated and diverse, and includes at least five transcription factors (ISRE, NF-κB, C/EBPβ, ATF2, and AP-1). Our hypothesis is that NFATc3 plays an important role in the expression of iNOS in macrophages during myocardial ischemia and reperfusion. To verify the hypothesis, the expression of NFATc3 and iNOS protein in the myocardium of rats with myocardial ischemia and reperfusion injury was observed and the changes in the nucleus were detected using western blot and immunohistochemistry. Finally, the effects of LPS and reagent-assisting reperfusion in RHRMC cells in the activation of NFATc3 and iNOS after induced by H2O2 were assessed. It showed that the protein level of NFATc3 in the nucleus of rats increased significantly (p < 0.05) at 4 h after reperfusion, peaked at 8 h in the rats with myocardial ischemia and reperfusion. It was sustained for 4 h in the sham group of the normal heart rat. The expression of NFATc3 in both the nucleus of myocardium and RHRMC cells accordingly resulted in significant enhancement at mRNA level. The effect of reperfusion occurred predominantly after myocardial ischemia and lasted for 2 h in the rat of Langendorff perfusion. The increment value of NFATc3 protein 1 h after reperfusion was approximately twice that of I/R group, and NFATc3 continued to rise till 4 h after reperfusion (p < 0.05). The protein level of iNOS in the myocardium leads NFATc3 by 4 h in the agents assisting reperfusion combined with H2O2, the peak of iNOS protein induced was attained at the same time as NFATc3. The level of iNOS increased faster than NFATc3 under these circumstances. Following reperfusion, a slight increase of iNOS was induced in the myocardial ischemia, the peak of iNOS protein induced was attained 10 h after reperfusion. The mRNA level of iNOS in the myocardium at 8 h after reperfusion was higher than that of 4 h (p < 0.05). The mRNA of iNOS in RHRMC cells induced by the reagent-assisting reperfusion was more than that induced by H2O2. The protein of iNOS peaked at 8 h (p < 0.05) and NFATc3 peaked at 4 h after reperfusion. The protein of iNOS peaked at 8 h and NFATc3 peaked at 4 h after reperfusion in I/R + LPS group (p < 0.05). The protein and the mRNA level of NFATc3 and iNOS were different in the myocardium after reperfusion. Also, NFATc3 and iNOS were detected in RHRMC cells implicated in both damaged and normal rat myocardial tissues.
Keywords: Myocardial ischemia and reperfusion; NFATc3; Langendorff; macrophage iNOS
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