Cathepsin B inactivation attenuates the apoptotic injury induced by ischemia/reperfusion of mouse liver.
BACKGROUND
A major mechanism underlying warm ischemia/reperfusion (I/R) injury during liver transplantation is the activation of the caspase chain, which leads to apoptosis. Recently, it was demonstrated that the release of cathepsin B, a cysteine protease, from the cytosol in liver injury induces mitochondrial release of cytochrome c and the activation of caspase-3 and -9, thereby leading to apoptosis. The aim of this study was to ascertain if cathepsin B inactivation attenuates the apoptotic injury due to I/R in mouse liver.


METHODS
A model of segmental (70%) hepatic ischemia was used. Eighteen mice were anesthetized and randomly divided into three groups: (1) CONTROL GROUP: sham operation (laparotomy); (2) Ischemic group: midline laparotomy followed by occlusion of all structures in the portal triad to the left and median lobes for 60 min (ischemic period); (3) STUDY GROUP: like group 2, but with intraperitoneal administration of a pharmacological inhibitor of cathepsin B (4 mg/100 g) 30 min before induction of ischemia. Serum liver enzyme levels were measured by biochemical analysis, and intrahepatic caspase-3 activity was measured by fluorometric assay; apoptotic cells were identified by morphological criteria, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorometric assay, and immunohistochemistry for caspase-3.


RESULTS
Showed that at 6 h of reperfusion, there was a statistically significant reduction in liver enzyme levels in the animals pretreated with cathepsin B inhibitor (p<0.05). On fluorometric assay, caspase-3 activity was significantly decreased in group 3 compared to group 2 (p<0.0001). The reduction in postischemic apoptotic hepatic injury in the cathepsin B inhibitor -treated group was confirmed morphologically, by the significantly fewer apoptotic hepatocyte cells detected (p<0.05); immunohistochemically, by the significantly weaker activation of caspase-3 compared to the ischemic group (p<0.05); and by the TUNEL assay (p<0.05).


CONCLUSIONS
The administration of cathepsin B inhibitor before induction of ischemia can attenuate postischemic hepatocyte apoptosis and thereby minimize liver damage. Apoptotic hepatic injury seems to be mediated through caspase-3 activity. These findings have important implications for the potential use of cathepsin B inhibitors in I/R injury during liver transplantation.
Ben-Ari
Z
Z
Liver institute and Department of Medicine D, Rabin Medical Center, Beilinson Campus, Petah Tiqva, Israel. zbenari@clalit.org.il
Mor
E
E
Azarov
D
D
Sulkes
J
J
Tor
R
R
Cheporko
Y
Y
Hochhauser
E
E
Pappo
O
O
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism*; D004789:Enzyme Activation; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008099:Liver / Q000201:enzymology* / Q000473:pathology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015427:Reperfusion Injury / Q000201:enzymology* / Q000473:pathology*
D053148:Caspase 3; D002401:Cathepsin B