Chondroitin Sulfate Protects the Liver in an Experimental Model of Extra-Hepatic Cholestasis Induced by Common Bile Duct Ligation

Abstract

During liver fibrogenesis, there is an imbalance between regeneration and wound healing. The current treatment is the withdrawal of the causing agent; thus, investigation of new and effective treatments is important. Studies have highlighted the action of chondroitin sulfate (CS) in different cells; thus, our aim was to analyze its effect on an experimental model of bile duct ligation (BDL). Adult Wistar rats were subjected to BDL and treated with CS for 7, 14, 21, or 28 days intraperitoneally. We performed histomorphometric analyses on Picrosirius-stained liver sections. Cell death was analyzed according to caspase-3 and cathepsin B activity and using a TUNEL assay. Regeneration was evaluated using PCNA immunohistochemistry. BDL led to increased collagen content with corresponding decreased liver parenchyma. CS treatment reduced total collagen and increased parenchyma content after 21 and 28 days. The treatment also promoted changes in the hepatic collagen type III/I ratio. Furthermore, it was observed that CS treatment reduced caspase-3 activity and the percentage of TUNEL-positive cells after 14 days and cathepsin B activity only after 28 days. The regeneration increased after 14, 21, and 28 days of CS treatment. In conclusion, our study showed a promising hepatoprotective action of CS in fibrogenesis induced by BDL.

Keywords: apoptosis; chondroitin; fibrosis; inflammation; liver; regeneration.

Figure 1

Liver histology and quantification of fibrogenesis of BDL animals treated with vehicle (BDL group) or chondroitin sulfate (BDL-CS group). (A–H) Representative images of the Picrosirius-stained liver sections from BDL and BDL-CS groups (100× magnification). (I) Histomorphometric analysis of the liver parenchyma and collagen contents (% of field area) from BDL and BDL-CS animals. Values are expressed as mean ± SEM, n minimal of 4 animals/group. * p < 0.001, multiple t-test. Scale bar: 250 µm.

Figure 2

Collagen fibers organization in the portal triads of picrous–sirius (PS)-stained liver sections from BDL rats treated with vehicle (BDL group) or chondroitin sulfate (BDL-CS group) analyzed by polarized light microscopy. (A–H) Representative images of portal triads in polarized light-analyzed liver sections from BDL and BDL-CS groups (200× magnification), with type I (yellow and red) and type III (green) collagens. (I) Collagen type III/type I relation in the portal triads from liver sections from BDL and BDL-CS groups. Values are expressed as mean ± SEM, (n = 4 animals/group). ANOVA with Tukey post hoc test: Scale bar: 400 µm.

Figure 3

Cell death pathway analysis in the liver homogenates from BDL animals treated with vehicle (BDL group) or chondroitin sulfate (BDL-CS group). Enzymatic activity of cathepsin B (A) and caspase-3 (B) in the liver homogenates (fold-increase compared to the sham group). Panel (C) shows the quantification of TUNEL-positive hepatocytes in the liver from BDL or BDL-CS groups at 7, 14, 21, and 28 days after induction. Values are expressed as mean ± SEM. * ANOVA with Tukey post hoc test.

Figure 4

PCNA-positive hepatocytes in the BDL-liver of rats subjected to different periods of treatment with vehicle (BDL group) or chondroitin sulfate (BDL-CS group). Representative images of PCNA immunohistochemistry in the liver sections from BDL rats treated with vehicle (upper line) or CS (bottom line) for 7 (A,B), 14 (C,D), 21 (E,F), and 28 days (G,H). (I) Percentage of PCNA-positive hepatocytes quantified in relation to the total number of hepatocytes of field. Values are expressed as mean ± SEM. *ANOVA with Tukey post hoc test. Scale bar: 50 µm.

Figure 5

Experimental design.