Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis
Ali Canbay, Maria Eugenia Guicciardi, Hajime Higuchi, Ariel Feldstein, Steven F. Bronk, Robert Rydzewski, Makiko Taniai, Gregory J. Gores
Ali Canbay, Maria Eugenia Guicciardi, Hajime Higuchi, Ariel Feldstein, Steven F. Bronk, Robert Rydzewski, Makiko Taniai, Gregory J. Gores
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Article Hepatology

Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis

Abstract

Although a lysosomal, cathepsin B–dependent (Ctsb-dependent) pathway of apoptosis has been described, the contribution of this pathway to tissue damage remains unclear. Our aim was to ascertain if Ctsb inactivation attenuates liver injury, inflammation, and fibrogenesis after bile duct ligation (BDL). In 3-day BDL mice, hepatocyte apoptosis, mitochondrial cytochrome c release, and serum alanine aminotransferase (ALT) values were reduced in Ctsb–/– versus Ctsb+/+ animals. Likewise, R-3032 (a Ctsb inhibitor) also reduced these parameters in BDL WT mice. Both genetic and pharmacologic inhibition of Ctsb in the BDL mouse reduced (a) hepatic inflammation, as assessed by transcripts for CXC chemokines and neutrophil infiltration, and (b) fibrogenesis, as assessed by transcripts for stellate cell activation and sirius red staining for hepatic collagen deposition. These differences could not be ascribed to alterations in cholestasis. These findings support a prominent role for the lysosomal pathway of apoptosis in tissue injury and link apoptosis to inflammation and fibrogenesis. Ctsb inhibition may be therapeutic in liver diseases.

Authors

Ali Canbay, Maria Eugenia Guicciardi, Hajime Higuchi, Ariel Feldstein, Steven F. Bronk, Robert Rydzewski, Makiko Taniai, Gregory J. Gores

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Liver injury and cytochrome c release are reduced in Ctsb–/– and R-3032–...
Liver injury and cytochrome c release are reduced in Ctsb–/– and R-3032–treated Ctsb+/+ mice. Ctsb+/+ and Ctsb–/– mice were BDL for 3 days as described in Methods. (a) Fixed liver specimens were analyzed by TUNEL assay to identify apoptotic hepatocytes (arrows). (b) The number of TUNEL-positive cells was significantly higher in Ctsb+/+ BDL mice than in sham-operated control or Ctsb–/– and R-3032–treated Ctsb+/+ BDL mice (P < 0.0001, n = 4 for each experimental group). (c) Cytosolic and mitochondrial fractions were prepared as described in Methods. Aliquots of 50 μg of cytosolic protein were subjected to SDS-PAGE and immunoblotted for cytochrome c. Release of cytochrome c in the cytosol was higher in Ctsb+/+ BDL livers compared with Ctsb–/– and R-3032–treated Ctsb+/+ BDL mice. A representative immunoblot from one animal from each group is shown, together with densitometric analysis of multiple immunoblots (n = 3 animals for each group). (d) Serum ALT values are significantly greater in Ctsb+/+ than in Ctsb–/– (P < 0.005, n = 4 for each experimental group) and R-3032–treated Ctsb+/+ mice 3 days after BDL (P < 0.0001, n = 4 for each experimental group). U/l, units per liter.