Pentoxifylline downregulates profibrogenic cytokines and procollagen I expression in rat secondary biliary fibrosis.

BACKGROUND: The trisubstituted methylxanthine derivative pentoxifylline inhibits hepatic stellate cell proliferation and collagen synthesis in vitro. The antifibrotic effect of pentoxifylline in a suitable in vivo model of chronic liver fibrogenesis remains to be tested. METHODS: Groups of adult rats (n=20-23) received oral pentoxifylline at a dose of 8 mg/kg/day from week 1 to week 6, and 16 mg/kg/day from week 1 to week 6 or week 4 to week 6 after complete bile duct occlusion. Animals who underwent sham operation that received 16 mg/kg/day pentoxifylline and untreated rats with bile duct occlusion alone served as controls. After six weeks, animals were sacrificed and parameters of fibrogenesis determined. RESULTS: Bile duct occlusion caused portal cirrhosis with a 10-fold increased hepatic collagen content in the absence of inflammation or necrosis. This was accompanied by an 11-fold elevated serum aminoterminal procollagen III peptide (PIIINP). The drug induced a dramatic eightfold downregulation of procollagen I mRNA, and suppression of the fibrogenic factors transforming growth factor beta1 and connective tissue growth factor by 60-70%. However, profibrogenic tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA was increased twofold, resulting in only a moderate decrease in liver collagen, fibrosis score, and PIIINP. CONCLUSIONS: We conclude that targeting pentoxifylline to the fibrogenic cells, thereby avoiding upregulation of TIMP-1, could become a potent antifibrogenic tool in chronic liver disease.

Antifibrotic effect of silymarin in rat secondary biliary fibrosis is mediated by downregulation of procollagen alpha1(I) and TIMP-1.

BACKGROUND/AIMS: Silymarin reduces hepatic collagen accumulation by 35% in rats with secondary biliary cirrhosis. The aim of the present study was to explore its antifibrotic mechanism. METHODS: Thirty female adult Wistar rats were allocated to (1) bile duct occlusion, (2) bile duct occlusion and oral silymarin at 50 mg/kg per day, and (3) sham operation and oral silymarin at 50 mg/kg per day. Steady-state mRNA levels for procollagen alpha1(I), tissue inhibitor of metalloproteinases-1 (TIMP-1), and transforming growth factor (TGF) beta1 were determined by multi-probe ribonuclease protection assay. RESULTS: After 6 weeks of bile duct occlusion, liver collagen content was increased 12-fold, when compared with the sham-operated controls. These animals displayed 17-, 6.5- and 16-fold higher transcript levels for procollagen alpha1(I), TIMP-1 and TGFbeta1 (P < 0.01). Silymarin downregulated elevated procollagen alpha1(I), TIMP-1 and TGFbeta1 mRNA levels by 40-60% (P < 0.01). These lowered hepatic profibrogenic transcript levels correlated with decreased serum levels of the aminoterminal propeptide of procollagen type III. CONCLUSIONS: Silymarin suppresses expression of profibrogenic procollagen alpha1(I) and TIMP-1 most likely via downregulation of TGFbeta1 mRNA in rats with biliary fibrosis. The serum procollagen type III propeptide level mirrors profibrogenic mRNA expression in the liver.

[Expression of collagen XVIII mRNA in rat liver fibrosis].

OBJECTIVE: To measure and study quantitatively the collagen XVIII mRNA in normal and fibrotic rat livers. METHODS: We used ribonuclease protection assay to investigate the collagen XVIII mRNA expression in rat liver fibrosis induced by complete bile duct occlusion (BDO). The expression level of procollagen 1 (XVIII) mRNA was compared with that of procollagen 1 (I) and tissue inhibitor of metalloproteinase 1 (TIMP1). RESULTS: mRNA levels of procollagen and TIMP 1 increased 20- and 4-fold in BDO rat livers, respectively. In contrast, hepatic procollagen 1 mRNA level increased only 1.8-fold in fibrotic rat livers. CONCLUSION: C XVIII mRNA is upregulated slightly in liver fibrosis, which is probably correlated with the fact that CXVIII is mainly expressed by hepatocytes.

[Cloning of partial cDNA of rat connective tissue growth factor and expression of its mRNA in experimental liver fibrosis].

OBJECTIVE: To clone the partial cDNA sequence of rat connective tissue growth factor (CTGF) and investigate the mRNA expression of CTGF and transforming growth factor-beta 1 (TGF-beta 1) in rat experimental liver fibrosis. METHODS: A 430-base pair sequence of rat CTGF Cdna was cloned by reverse transcription-polymerase chain reaction. Sixteen female Wistar rats were allocated into bile duct occlusion group (n=8) and sham-operated group (n=8). The liver tissue mRNA levels of CTGF and TGF-beta 1 were measured by multiprobe ribonuclease protection assay (RPA). RESULTS: The cloned partial cDNA sequence of rat CTGF was 95% homology (at nucleic acid level) with the mouse counterpart. In the liver of rats with biliary fibrosis mRNA levels of TGF-beta 1 and CTGF were 4 and 7 times as high as in those of sham-operated rats, respectively. CONCLUSION: The mRNA expression of CTGF is remarkably upregulated in rat liver fibrogenesis.

Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats.

Silymarin (SIL), a standardized plant extract containing about 60% polyphenole silibinin, is used as a hepatoprotective agent. Its antifibrotic potential in chronic liver diseases has not been explored. Therefore, we applied SIL to adult Wistar rats that were subjected to complete bile duct occlusion (BDO) by injection of sodium amidotrizoate (Ethibloc). This treatment induces progressive portal fibrosis without significant inflammation. Rats with sham-operation that received SIL at 50 mg/kg/d (n = 10) and rats with BDO alone (n = 20) served as controls, whereas groups of 20 animals were fed SIL at a dose of 25 and 50 mg/kg/d during weeks 1 through 6 or doses of 50 mg/kg/d during weeks 4 through 6 of BDO. Animals were sacrificed after 6 weeks for determination of blood chemistries, total and relative liver collagen (as hydroxyproline [HYP]), and the serum aminoterminal propeptide of procollagen type III (PIIINP). BDO in untreated rats caused an almost ninefold increase in total liver collagen (16.1 +/- 3.1 vs. 1.8 +/- 0.4 mg HYP, P < .001). SIL at 50 mg/kg/d reduced total HYP by 30% to 35%, either when given from week 1 through 6 or from week 4 through 6 after BDO (10.6 +/- 2.7 and 10.2 +/- 3.9 mg HYP, both P < .01 vs. BDO alone), whereas 25 mg/kg/d were ineffective. Because SIL at 50 mg/kg/d also reduced the collagen content per gram of liver tissue, it acted as a true antifibrotic agent. The single value of PIIINP at killing paralleled the antifibrotic activity of SIL with 11.6 +/- 3.8 and 9.9 +/- 3.7 vs. 15.3 +/- 5.2 microg/L in both high-dose groups (P < .05 and P < .01, respectively, vs. rats with BDO alone). Except for a decreased alkaline phosphatase and a lower histological fibrosis score in the groups that received SIL, clinical-chemical parameters were not different among all groups with BDO. We therefore conclude that 1) BDO with Ethibloc is a suitable model to test for pure antifibrotic drugs because it induces progressive rat secondary biliary fibrosis without major inflammation; 2) oral SIL can ameliorate hepatic collagen accumulation even in advanced (biliary) fibrosis; and 3) PIIINP appears to be a suitable serum marker to monitor the inhibition of hepatic fibrogenesis in this model of biliary fibrosis.