Kin Hajime, Wang Ning-Ping, Halkos Michael E, Kerendi Faraz, Guyton Robert A, Zhao Zhi-Qing
Cardiothoracic Surgery Laboratory, Carlyle Fraser Heart Center, Emory Crawford Long Hospital, Emory University, Atlanta, Georgia 30308-2225, USA.
J Surg Res. 2006 Sep;135(1):170-8. doi: 10.1016/j.jss.2006.02.019. Epub 2006 Apr 17.
This study tested the hypothesis that depletion of neutrophils (PMNs) reduces myocardial apoptosis via reducing oxidant generation and inhibiting NFkappaB-mediated signaling pathways after ischemia/reperfusion.
Anesthetized rats were randomly divided into one of four groups:
30 min ischemia and 3 h of reperfusion; PMN depletion: anti-PMN serum was injected 6 h before ischemia; N-acetylcysteine (NAC): NAC was given twice before ischemia and at reperfusion. Sham: the ligature was placed without coronary occlusion. Apoptosis was detected by TUNEL staining and DNA fragmentation. PMN accumulation was studied by immunohistochemical staining. Levels of TNF-alpha, IL-6, and caspase-3 were detected by Elisa kits. Expression in NFkappaB, Bcl-2, and Bax was assessed by Western blotting analysis.
Relative to CONTROL, depletion of PMNs or NAC treatment reduced levels of plasma TNFalpha (567 +/- 130* and 231 +/- 72* versus 1994 +/- 447 pg/ml) and IL-6 (791 +/- 473* and 666 +/- 300* versus 3724 +/- 1233, pg/ml), accompanying a reduction in PMN accumulation (12 +/- 1* and 13 +/- 0.6* versus 20 +/- 1 mm2 myocardium) in ischemic myocardium. Both groups showed a reduction in expression of nuclear NFkappaB relative to CONTROL (62 +/- 9* and 67 +/- 8* versus 124 +/- 16 arb.u), consistent with reduced NFkappaB binding activity. The number of apoptotic cells (%) in area at risk myocardium was comparably reduced in anti-PMN and NAC groups relative to CONTROL (12 +/- 1* and 14 +/- 0.9* versus 20 +/- 1), consistent with reduced appearance of DNA ladders. Furthermore, activated caspase-3 was significantly reduced and Bcl-2 was increased relative to CONTROL. No difference in all parameters measured was detected during the course of experiment in the Sham group.
These data suggest that the oxidants generated from activated PMNs after ischemia/reperfusion trigger myocardial apoptosis, which is further supported by an anti-oxidant therapy with NAC, potentially mediated by enhanced NFkappaB-TNFalpha signaling pathway, activated caspase-3 and down-regulated Bcl-2. *P < 0.05 versus CONTROL.
本研究检验了以下假设，即中性粒细胞（PMN）耗竭通过减少缺血/再灌注后氧化剂生成及抑制NFκB介导的信号通路来减少心肌细胞凋亡。
将麻醉的大鼠随机分为四组之一：
缺血30分钟，再灌注3小时；PMN耗竭组：在缺血前6小时注射抗PMN血清；N-乙酰半胱氨酸（NAC）组：在缺血前及再灌注时两次给予NAC。假手术组：放置结扎线但不进行冠状动脉闭塞。通过TUNEL染色和DNA片段化检测细胞凋亡。通过免疫组织化学染色研究PMN聚集情况。使用酶联免疫吸附测定试剂盒检测TNF-α、IL-6和半胱天冬酶-3的水平。通过蛋白质免疫印迹分析评估NFκB、Bcl-2和Bax的表达。
相对于对照组，PMN耗竭或NAC处理降低了血浆TNFα水平（分别为567±130和231±72，而对照组为1994±447 pg/ml）和IL-6水平（分别为791±473和666±300，而对照组为3724±1233 pg/ml），同时缺血心肌中的PMN聚集减少（分别为12±1和13±0.6，而对照组为20±1 mm²心肌）。相对于对照组，两组的核NFκB表达均降低（分别为62±9和67±8，而对照组为124±16 arb.u），这与NFκB结合活性降低一致。相对于对照组，抗PMN组和NAC组中危险区域心肌的凋亡细胞数量（%）显著减少（分别为12±1和14±0.9，而对照组为20±1），这与DNA梯带的出现减少一致。此外，相对于对照组，活化的半胱天冬酶-3显著减少，Bcl-2增加。在假手术组的实验过程中，未检测到所有测量参数的差异。
这些数据表明，缺血/再灌注后活化的PMN产生的氧化剂触发心肌细胞凋亡，NAC抗氧化治疗进一步支持了这一点，这可能由增强的NFκB-TNFα信号通路、活化的半胱天冬酶-3和下调的Bcl-2介导。*与对照组相比，P<0.05。
