Volume 39, Issue 5, Pages 756-764 (November 2003) Lipid peroxidation, stellate cell activation and hepatic fibrogenesis in a rat model of chronic steatohepatitis  Jacob George, Natasha Pera, Nghi Phung, Isabelle Leclercq, Jing Yun Hou, Geoffrey Farrell  Journal of Hepatology  Volume 39, Issue 5, Pages 756-764 (November 2003) DOI: 10.1016/S0168-8278(03)00376-3

Fig. 1 Serum ALT in rats fed the MCD diet (■) and their pair-fed controls (□). Values are expressed as mean±S.E.M. for between three and six separate experiments. *P<0.01. By post hoc analysis, ALT levels in rats fed the MCD diet for 2 weeks were lower (P<0.05) than at other time points. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 2 Representative liver sections and collagen quantitation in rats fed the control or MCD diet. (A) Rat fed the control diet for 5 weeks. H&E (original magnification, 60×). (B) Rat fed the MCD diet for 5 weeks. H&E (60×). Steatosis is present predominantly in zones 2 and 3 and there is a mixed inflammatory cell infiltrate in the hepatic lobule (see inset, 400×, arrows).(C) Rat fed the control diet for 17 weeks. Sirius red staining (60×). There is no fibrosis. (D) Rat fed the MCD-diet for 12 weeks. Sirius red staining (400×). Peri-cellular ‘chicken-wire’ fibrosis is evident around zone 3 hepatocytes (arrow). Central vein is indicated by CV. (E) Rat fed the MCD-diet for 12 weeks. Sirius red staining (60×). Portal-portal and central portal bridging fibrosis is evident (arrows). (F) Rat fed the MCD-diet for 17 weeks, Sirius red staining (60×). In this liver, cirrhosis is present. (G) Quantitation of collagen deposition in Sirius red-stained sections of rats fed the MCD diet (■) and their pair-fed controls (□). Values are expressed as mean±S.E.M. for between three and four separate experiments. *P<0.05. †The difference between MCD and control rats at week 17 was significant (P=0.03) after exclusion of the cirrhotic rat liver sample. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 3 α-Smooth muscle actin (α-SMA) immunostaining in liver sections. (A) Rat fed the control diet for 17 weeks (original magnification, 200×). α-SMA expression, indicated by the brown staining, is confined to the portal tracts (PT) and central vein (CV). (B) Rat fed the MCD-diet for 12 weeks (200×). α-SMA is also expressed in the hepatic lobule (arrows), distributed around steatotic hepatocytes, in the same distribution as collagen fibrils (see Fig. 2D). (C) Rat fed the MCD-diet for 17 weeks (200×). In this liver, cirrhosis is present and smooth muscle actin staining is more extensive in the lobule. (D) Rat fed the MCD-diet for 17 weeks (400×). Higher power view of the cirrhotic liver lobule. (E) Quantitation of α-smooth muscle actin in liver lobules of rats fed the MCD diet (■) and their pair-fed controls (□). Values are expressed as mean±S.E.M. for three separate experiments. *P<0.05. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 4 Expression of collagen α1(I) in whole liver of rats fed the MCD diet (■) and their pair-fed controls (□). (A) A representative RNase protection assay showing samples at 12 and 17 weeks, using 20 μg of total RNA for the collagen α1(I) probe and 5 μg for S14. Yeast tRNA is a negative control. (B) Collagen α1 (I) mRNA expression normalised against S14 mRNA expression. Values are expressed as mean±S.E.M. for between three and five separate experiments. *P<0.05; †the difference between MCD and control rats at week 17 was significant (P=0.04) after exclusion of the cirrhotic rat liver sample. By post-hoc analysis, collagen α1(I) mRNA expression in MCD diet-fed rats was different (P<0.05) from each other at the various time points. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 5 Expression of TIMP-1 in whole liver of rats fed the MCD diet (■) for 12 and 17 weeks and their pair-fed controls (□). (A) A representative RNase protection assay using 30 μg of total RNA for the TIMP-1 probe and 5 μg for S14. Yeast tRNA is a negative control. (B) TIMP-1 mRNA expression normalised against S14 mRNA expression. Values are expressed as mean±S.E.M. for four separate experiments. *P<0.05. By post hoc analysis TIMP-1 mRNA expression was not different between week 12 and 17 in MCD diet-fed rats. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 6 Expression of TIMP-2 in whole liver of rats fed the MCD diet (■) for 5, 12 and 17 weeks and their pair-fed controls (□). (A) A representative RNase protection assay using 30 μg of total RNA for the TIMP-2 probe and 5 μg for S14. Yeast tRNA is a negative control. (B) TIMP-2 mRNA expression normalised against S14 mRNA expression. Values are expressed as mean±S.E.M. for six separate experiments. *P<0.05. By post hoc analysis, TIMP-2 mRNA expression in MCD diet-fed rats was greater at weeks 12 and 17 compared to week 5 (P<0.05), but not different from each other. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 7 Expression of collagen α1(I) in hepatic non-parenchymal cells from rats fed the MCD diet (■) for 12 and 17 weeks, and their pair-fed controls (□). (A) A representative RNase protection assay showing samples at 12 weeks using 20 μg of total RNA for the collagen α1(I) probe and 5 μg for S14. Yeast tRNA is a negative control. (B) Collagen α1 (I) mRNA expression normalised against S14 mRNA expression. Values are expressed as mean±S.E.M. for between four and five separate experiments. *P<0.05. By post hoc analysis, collagen α1(I) mRNA expression in stellate cells was greater (P<0.005) than that in Kupffer and endothelial cells at the corresponding time point. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 8 Expression of TIMP-1 in stellate cells from rats fed the MCD diet (■) for 12 and 17 weeks, and their pair-fed controls (□). (A) A representative RNase protection assay using 15 μg of total RNA for the TIMP-1 probe and 5 μg for S14. Yeast tRNA is a negative control. (B) TIMP-1 mRNA expression normalised against S14 mRNA expression. Values are expressed as mean±S.E.M. for between three and seven separate experiments; *P<0.05. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 9 Expression of TGFβ1 mRNA relative to GAPDH in whole liver of rats fed the MCD or control diets over 17 weeks. Amplification was carried out using reverse transcription PCR over 32 cycles with 56 °C as the annealing temperature. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 10 Expression of TGFβ1 mRNA normalised against GAPDH in whole liver and cell isolates of rats pair-fed the MCD- (■) and control- (□) diet for 12 weeks. Amplification of cDNA prepared from total RNA was carried out using real time PCR as outlined in Materials and methods. Values are expressed as mean±S.E.M. for between three and six separate experiments; *P<0.05. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 11 TBARS levels in whole liver during pair-feeding of the MCD- (■) and control- (□) diet. Values are expressed as mean±S.E.M. for at least six separate experiments; *P<0.05. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)

Fig. 12 TBARS levels in cell isolates from rats pair-fed the MCD- (■) and control- (□) diet for 12 weeks. Values are expressed as mean±S.E.M. for at least three separate experiments; *P<0.05. Journal of Hepatology 2003 39, 756-764DOI: (10.1016/S0168-8278(03)00376-3)