Fondevila Constantino, Shen Xiu-Da, Tsuchihashi Seiichiro, Uchida Yoichiro, Freitas Maria Cecilia, Ke Bibo, Busuttil Ronald W, Kupiec-Weglinski Jerzy W
Dumont-University of California at Los Angeles Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, CA 90095, USA.
Liver Transpl. 2008 Aug;14(8):1133-41. doi: 10.1002/lt.21496.
Activation of the complement cascade represents an important event during ischemia/reperfusion injury (IRI). This work was designed to investigate the role of the membrane attack complex (MAC; C5b-9) in the pathogenesis of hepatic IRI. Livers from B&W/Stahl/rC6(+) and C6(-) rats were harvested, stored for 24 hours at 4 degrees C, and then transplanted [orthotopic liver transplantation (OLT)] to syngeneic recipients. There were 4 experimental groups: (1) C6(+)-->C6(+), (2) C6(+)-->C6(-), (3) C6(-)-->C6(+), and (4) C6(-)-->C6(-). At day +1, C6(-) OLTs showed decreased vascular congestion/necrosis, contrasting with extensive necrosis in C6(+) livers, that was independent of the recipient C6 status (Suzuki score: 7.2 +/- 0.9, 7.3 +/- 1.3, 4.5 +/- 0.6, and 4.8 +/- 0.4 for groups 1-4, respectively, P < 0.05). The liver function improved in recipients of C6(-) grafts (serum glutamic oxaloacetic transaminase: 2573 +/- 488, 1808 +/- 302, 1170 +/- 111, and 1188 +/- 184 in groups 1-4, respectively, P < 0.05). Intragraft macrophage infiltration (ED-1 immunostaining) and neutrophil infiltration (myeloperoxidase activity) were reduced in C6(-) grafts versus C6(+) grafts (P = 0.001); these data were confirmed by esterase staining (naphthol). The expression of proinflammatory interferon-gamma, interleukin-1beta, and tumor necrosis factor messenger RNA/protein was also reduced in C6(-) OLTs in comparison with C6(+) OLTs. Western blot-assisted expression of proapoptotic caspase-3 was decreased in C6(-) OLTs versus C6(+) OLTs (P = 0.006), whereas antiapoptotic Bcl-2/Bag-1 was enhanced in C6(-) OLTs compared with C6(+) OLTs (P = 0.001). Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining of apoptotic cells was enhanced (P < 0.05) in C6(+) OLTs compared with C6(-) OLTs. Thus, the terminal products of the complement system are essential in the mechanism of hepatic IRI. This is the first report using a clinically relevant liver cold ischemia model to show that local MAC inhibition attenuates IRI cascade in OLT recipients.
补体级联反应的激活是缺血/再灌注损伤（IRI）过程中的一个重要事件。本研究旨在探讨膜攻击复合物（MAC；C5b-9）在肝IRI发病机制中的作用。采集B&W/Stahl/rC6(+)和C6(-)大鼠的肝脏，在4℃下保存24小时，然后移植[原位肝移植（OLT）]给同基因受体。共有4个实验组：（1）C6(+)→C6(+)，（2）C6(+)→C6(-)，（3）C6(-)→C6(+)，和（4）C6(-)→C6(-)。在第1天，C6(-)OLT显示血管充血/坏死减少，与C6(+)肝脏中的广泛坏死形成对比，这与受体C6状态无关（Suzuki评分：第1-4组分别为7.2±0.9、7.3±1.3、4.5±0.6和4.8±0.4，P<0.05）。C6(-)移植物受体的肝功能得到改善（血清谷氨酸草酰乙酸转氨酶：第1-4组分别为2573±488、1808±302、1170±111和1188±184，P<0.05）。与C6(+)移植物相比，C6(-)移植物中的移植内巨噬细胞浸润（ED-1免疫染色）和中性粒细胞浸润（髓过氧化物酶活性）减少（P = 0.001）；这些数据通过酯酶染色（萘酚）得到证实。与C6(+)OLT相比，C6(-)OLT中促炎干扰素-γ、白细胞介素-1β和肿瘤坏死因子信使核糖核酸/蛋白的表达也降低。与C6(+)OLT相比，C6(-)OLT中促凋亡半胱天冬酶-3的蛋白质印迹辅助表达降低（P = 0.006），而与C6(+)OLT相比，C6(-)OLT中抗凋亡Bcl-2/Bag-1增强（P = 0.001）。与C6(-)OLT相比，C6(+)OLT中凋亡细胞的末端脱氧核苷酸转移酶介导的dUTP缺口末端标记染色增强（P<0.05）。因此，补体系统的终末产物在肝IRI机制中至关重要。这是首次使用临床相关的肝脏冷缺血模型报告表明局部MAC抑制可减轻OLT受体中的IRI级联反应。
