Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis (original) (raw)

Extrahepatic cholestasis by ligation of the common hepatic duct. The use and the care of the animals were reviewed and approved by the Institutional Animal Care and Use Committee at the Mayo Clinic. C57BL/6 Ctsb knockout (Ctsb–/–) mice and WT littermates (Ctsb+/+) 6 to 8 weeks of age were used for these studies. The generation and use of these animals has previously been described in detail (20). Common BDL was performed as previously described in detail (12). Sham-operated mice, used as controls, underwent a laparotomy with exposure, but no ligation of the common bile duct was performed.

Pharmacologic Ctsb inhibition. In selected experiments, mice were treated with the Ctsb inhibitor R-3032 (4.9 mg/ml in 30:70 polyethylene glycol 400/H2O at pH 7.5) intraperitoneally twice a day. The agent was obtained from Celera Genomics. The dose was based on pharmacokinetic data demonstrating a half-life of 4.2 hours, and preliminary data demonstrating that R-3032 extracellular concentrations of 10 μM were required to maximally inhibit TNF-α/actinomycin D–mediated apoptosis in isolated cultured murine hepatocytes (not shown). R-3032 is a reversible Ctsb inhibitor. The _K_I for Ctsb is 0.02 μM, whereas the _K_I for cathepsin K is 89 μM, cathepsin L is 12 μM, and cathepsin S is 3 μM. The _K_I for Ctsb is at least 2 logs lower than it is for these other lysosomal cathepsin proteases, and the drug does not inhibit caspases. Thus, the drug is a selective Ctsb inhibitor.

Histology and the TUNEL assay. The liver was diced into 5 × 5-mm sections, fixed in 4% paraformaldehyde for 48 hours, and then embedded in paraffin (Curtin Matheson Scientific Inc., Houston, Texas, USA). Tissue sections (4 μm) were prepared with a microtome (Reichert Scientific Instruments, Buffalo, New York, USA) and placed on glass slides. Hematoxylin and eosin staining was performed according to standard techniques. TUNEL assay was performed with a commercially available kit, according to the manufacturer’s instructions (In Situ Cell Death Detection Kit; Roche Diagnostics Corp., Indianapolis, Indiana, USA). Hepatocyte apoptosis in liver sections was quantitated by counting the number of TUNEL-positive cells in 30 random microscopic low-power fields (×630).

Determination of serum total bilirubin, bile acids, and alanine aminotransferase. Total serum bilirubin, total serum bile acid, and serum alanine aminotransferase (ALT) determinations were performed using commercially available assay kits according to the manufacturer’s instructions (Sigma-Aldrich Diagnostics kit no. 505, 550, and 450; Sigma-Aldrich, St. Louis, Missouri, USA).

Preparation of subcellular fractions. Cytosolic extracts (S-100) were prepared from mouse liver using the approach previously described (10). Protein concentration was measured with the Bradford assay (Bio-Rad Laboratories Inc., Hercules, California, USA).

Immunoblot analysis. Aliquots of S-100 cytosolic extracts containing 50 μg of protein were subjected to 15% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were first probed overnight at 4°C with a mouse anti–cytochrome c antibody (PharMingen, San Diego, California, USA), dilution 1:1,000, and then incubated with an HRP-conjugated goat anti-mouse IgG secondary antibody (BioSource International, Camarillo, California, USA) diluted 1:5,000 for 45 minutes at room temperature. Blots were developed by the enhanced chemiluminescence system (Amersham Life Sciences Inc., Arlington Heights, Illinois, USA), according to the manufacturer’s instructions.

Real-time PCR. Total RNA was obtained from whole liver with the Trizol Reagent (Invitrogen Corp., Carlsbad, California, USA). For each RNA sample, a 10-μg aliquot was reverse transcribed into cDNA using oligo-dT random primers and Moloney murine leukemia virus reverse transcriptase. Real-time PCR was performed with Taq polymerase (Invitrogen Corp.) and primers for α-smooth muscle actin (α-SMA), collagen α1(I) (COL1A1), TGF-β1, and tissue inhibitor of metalloproteinases (TIMP) as previously described (18). Primers for the experiment using inflammation were as follows: CXC chemokine ligand 1 (KC), forward 5′-TGGGATTCACCTCAAGAACA-3′, reverse 5′-TGGGGACACCTTAGCATC-3′ (yielding a 167-bp product); macrophage inflammatory protein–2 (MIP-2), forward 5′-CCACCAACCACCAGGCTAC-3′, reverse 5′-GCTTCAGGGTCAAGGGCAAA-3′ (221-bp); GAPDH, forward 5′-ACCCAGAAGACTGTGGATGG-3′, reverse 5′-CATCGAAGGTGGAAGAGTGG-3′ (340-bp). Both GAPDH and 18S primers (Ambion Inc., Austin Texas, USA) were used as controls for RNA integrity. All PCR products were confirmed by gel electrophoresis.

Real-time PCR was performed with the LightCycler (Roche Diagnostics Corp., Mannheim, Germany) and SYBR green as the fluorophore (Molecular Probes Inc., Eugene, Oregon, USA). The result was expressed as a ratio of product copies per milliliter to copies per milliliter of housekeeping gene 18S and GAPDH from the same RNA (respective cDNA) sample and PCR run.

Determination of liver fibrosis. Liver fibrosis was quantified with sirius red. Direct red 80 and Fast green FCF (color index 42053) were provided by Sigma-Aldrich. The liver was diced into 5 × 5-mm sections, immersion fixed in PBS containing 4% paraformaldehyde for 24 hours at 4°C, and embedded in paraffin. Ten-micrometer sections were mounted on glass slides. Sections were deparaffinized and the slides rehydrated as follows with a wash for each 5-minute step: Xylene (twice), 100% ethyl alcohol (EtOH), 95% EtOH, 70% EtOH, 30% EtOH, 1× PBS, and distilled water (twice). The sections were incubated for 2 hours in room temperature with an aqueous solution of saturated picric acid containing 0.1% Fast green FCF and 0.1% direct red 80. The sections were covered with aluminum foil during the incubation. Stained slides were washed slowly under running distilled water for 6 minutes, dehydrated (3 minutes for each step), mounted, and examined by light microscopy. Red-stained collagen fibers were quantitated by digital image analysis as described below.

The Bacus Laboratories Inc. Slide Scanner (BLISS) system (Lombard, Illinois, USA) allows for the capture and quantitation of histologic images as tiles. The tissue section on the slide was automatically scanned using a preselected magnification lens (×40), and the captured bright-field images were digitized into a series of picture elements (pixels) each of which has a specific optical density. Through mathematical manipulation of the pixels using the instrument’s software, the captured images were quantified. For sirius red, quantitation was based on determining the optical densities of the chromogen. Quantitative histomorphometry was performed on the 6-μm–stained sections. The methodology was similar to that previously described (21, 22). Two cross sections, from two lobes from each animal were scanned. The scanned areas were captured as ×40 tiles, and the evaluation by BLISS was performed without knowledge of the specimens. All images were captured with a ×40 lens, the highest magnification available, as tiles with 480 × 752 pixel resolution. The computer automatically measured histologic features from each tile. Each captured tile was analyzed by the Histology Grade software on the instrument with separation of green and red-green-blue (RGB) filters. The red area was mathematically divided by the RGB area and multiplied by 100%. This determined the percentage area staining positively for collagen fibers, providing a quantitative value on a continuous scale. The areas scanned were captured using the BLISS system. Quantitative histomorphometry was evaluated on the individual images (pixel resolution 480 × 752) for the sirius red chromogen as previously described (18).

Immunohistochemistry. The sections (5 μm thick) were stained for myeloperoxidase (MPO) using a rabbit polyclonal antibody (NeoMarkers, Fremont, California, USA), which is prediluted by the manufacturer for staining formalin-fixed, paraffin-embedded tissues. The sections were incubated with the antibody overnight at 4°C. Negative control slides were incubated with nonimmune immunoglobulin under the same conditions. Secondary reagents were obtained from the DAKO LSAB2 kit (DAKO Corp., Carpinteria, California, USA), and 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) was used for visualization. Finally, the tissue was counterstained with hematoxylin.

Statistics. All data represent at least four independent experiments and are expressed as the mean ± SD unless otherwise indicated. Differences between groups were compared using ANOVA for repeated measures and a post hoc Bonferroni test to correct for multiple comparisons.