Kohli V, Madden J F, Bentley R C, Clavien P A
Hepatobiliary and Liver Transplant Laboratory, Duke University Medical Center, Durham, North Carolina 27710, USA.
Gastroenterology. 1999 Jan;116(1):168-78. doi: 10.1016/s0016-5085(99)70241-6.
BACKGROUND & AIMS: Calpain proteases have been implicated in cell death by necrosis and more recently by apoptosis. Experiments were designed to determine the role of calpain proteases in ischemic rat liver injury by measurement of cytosolic calpain activity after different periods of ischemia-reperfusion and by evaluation of the effects of calpain inhibition on tissue injury and animal survival.
Calpain activity was measured in the cytosol using Suc-Leu-Leu-Val-Try-7 amino-4 methyl coumarin, a specific fluorogenic substrate, and Cbz-Leu-Leu-Tyr-CHN2, a specific inhibitor.
Calpain activity increased significantly with the duration of ischemia-reperfusion and was inhibited more than 80% by the inhibitor. Calpain inhibition resulted in a significant decrease in transaminase release and tissue necrosis and converted nonsurvival ischemic conditions to survival conditions. When the in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling assay for apoptosis was used, 35% +/- 6% of nonparenchymal cells and 16% +/- 3% of hepatocytes stained positively after 60 minutes of ischemia and 6 hours of reperfusion. In contrast, animals pretreated with the calpain inhibitor showed minimal evidence of apoptosis. This was further substantiated by gel electrophoresis assay for DNA fragmentation and by electron-microscopic evaluation.
These data suggest that calpain proteases play a pivotal role in warm ischemia-reperfusion injury of the rat liver through modulation of apoptosis and necrosis.
钙蛋白酶已被证明与坏死性细胞死亡有关，最近还与凋亡性细胞死亡有关。本实验旨在通过测量不同缺血再灌注时间后的细胞溶质钙蛋白酶活性，以及评估钙蛋白酶抑制对组织损伤和动物存活的影响，来确定钙蛋白酶在大鼠缺血性肝损伤中的作用。
使用特异性荧光底物Suc-Leu-Leu-Val-Try-7氨基-4甲基香豆素和特异性抑制剂Cbz-Leu-Leu-Tyr-CHN2测量细胞溶质中的钙蛋白酶活性。
钙蛋白酶活性随缺血再灌注时间的延长而显著增加，且被抑制剂抑制超过80%。钙蛋白酶抑制导致转氨酶释放和组织坏死显著减少，并将非存活的缺血状态转变为存活状态。当使用原位末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸-地高辛配基缺口末端标记法检测凋亡时，缺血60分钟和再灌注6小时后，35%±6%的非实质细胞和16%±3%的肝细胞呈阳性染色。相比之下，用钙蛋白酶抑制剂预处理的动物显示出最小的凋亡迹象。这通过DNA片段化的凝胶电泳分析和电子显微镜评估得到进一步证实。
这些数据表明，钙蛋白酶通过调节凋亡和坏死在大鼠肝脏的热缺血再灌注损伤中起关键作用。
