Caspase inhibition protects from liver injury following ischemia and reperfusion in rats.
Normothermic ischemia and reperfusion of the liver results in microcirculatory failure followed by necrosis and cell death. Recently, another type of cell death, apoptosis or programmed cell death, was found to be activated during the early phase of reperfusion after liver ischemia. Caspases are cysteine proteinases specifically involved in the initiation and execution phases of apoptosis. The aim of this study was to demonstrate that inhibition of apoptosis by a specific inhibitor of caspases might protect the liver against ischemia/reperfusion injury. Rats were divided into three groups: group 1, control, PBS administration; group 2, Z-Asp-cmk (Z-Asp-2,6-dichlorobenzoyl-oxymethylketone) treatment; group 3, sham-operated control animals. Z-Asp-cmk (0.5 mg Z-Asp-cmk dissolved in 300 microl PBS solution containing 1% DMSO) was injected intravenously, 2 min prior to induction of 120 min ischemia. Survival rates were compared and serum activities of aspartate aminotransferases and alanine aminotransferases were assessed in the blood collected from the suprahepatic vena cava. Histology of the liver was assessed 6 h after the end of ischemia. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling method (TUNEL method) and by electrophoresis for analysis of DNA fragmentation. Caspase activity was determined by measuring hydrolysis of the CPP32-like substrate Ac-DEVD-pNA and absorption of paranitroaniline. Z-Asp-cmk treatment significantly increased 7-day survival (95%) compared with that in nontreated rats (30%, P < 0.001). Serum activities of aminotransferases and the extent of liver congestion and necrosis were significantly (P < 0.001) decreased after treatment with Z-Asp-cmk. TUNEL-positive cells were detected 3-6 h after reperfusion in the control group. In Z-Asp-cmk pretreated rats, a dramatic decrease in the number of TUNEL-positive cells was observed. Analysis of DNA fragmentation of freshly isolated hepatocytes confirmed these results. Caspase activity was increased 3-6 h after reperfusion in the control group, but significantly (P < 0.001) decreased after treatment with Z-Asp-cmk. These findings demonstrate that liver injury following ischemia and reperfusion can be prevented by inhibition of caspases. Caspase inhibitors may have important implications for therapy in liver disease and after liver transplantation.
Cursio
R
R
Laboratoire de Recherches Chirurgicales, Université de Nice, France.
Gugenheim
J
J
Ricci
J E
JE
Crenesse
D
D
Rostagno
P
P
Maulon
L
L
Saint-Paul
M C
MC
Ferrua
B
B
Mouiel
J
J
Auberger
P
P
D000410:Alanine Transaminase / Q000097:blood; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001219:Aspartate Aminotransferases / Q000097:blood; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects; D007511:Ischemia; D008099:Liver / Q000098:blood supply / Q000166:cytology / Q000187:drug effects*; D008297:Male; D051381:Rats; D011917:Rats, Inbred Lew; D015427:Reperfusion Injury / Q000517:prevention & control*
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; C421328:Z-Asp-2,6-dichlorobenzoyl-oxymethylketone; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase