Glycochenodeoxycholate Promotes Liver Fibrosis in Mice with Hepatocellular Cholestasis.

Hydrophobic bile salts are considered to promote liver fibrosis in cholestasis. However, evidence for this widely accepted hypothesis remains scarce. In established animal models of cholestasis, e.g., by Mdr2 knockout, cholestasis and fibrosis are both secondary to biliary damage. Therefore, to test the specific contribution of accumulating bile salts to liver fibrosis in cholestatic disease, we applied the unique model of inducible hepatocellular cholestasis in cholate-fed Atp8b1G308V/G308V mice. Glycochenodeoxycholate (GCDCA) was supplemented to humanize the murine bile salt pool, as confirmed by HPLC. Biomarkers of cholestasis and liver fibrosis were quantified. Hepatic stellate cells (HSC) isolated from wild-type mice were stimulated with bile salts. Proliferation, cell accumulation, and collagen deposition of HSC were determined. In cholestatic Atp8b1G308V/G308V mice, increased hepatic expression of αSMA and collagen1a mRNA and excess hepatic collagen deposition indicated development of liver fibrosis only upon GCDCA supplementation. In vitro, numbers of myofibroblasts and deposition of collagen were increased after incubation with hydrophobic but not hydrophilic bile salts, and associated with EGFR and MEK1/2 activation. We concluded that chronic hepatocellular cholestasis alone, independently of biliary damage, induces liver fibrosis in mice in presence of the human bile salt GCDCA. Bile salts may have direct pro-fibrotic effects on HSC, putatively involving EGFR and MEK1/2 signaling.

Biliverdin Reductase inhibitors did not improve severe unconjugated hyperbilirubinemia in vivo.

We aimed to identify potent biliverdin reductase (BVRA) inhibitors as a novel concept for the treatment of severe unconjugated hyperbilirubinemia. 1280 FDA-approved compounds were screened in vitro for their ability to inhibit human and rat BVRA activity and 26 compounds were identified as BVRA inhibitors. Montelukast and Disulfiram were selected as potentially clinically applicable drugs and tested to reduce serum unconjugated bilirubin (UCB) levels in the Ugt1a1-deficient rat, a model for chronic unconjugated hyperbilirubinemia. Oral administration of Disulfiram was toxic in the Ugt1a1-deficient rat (weight loss, transaminase elevation). Oral Montelukast administration led to low serum concentrations and did not alter serum UCB levels. Intraperitoneal injections of Montelukast resulted in concentrations up to 110 µmol/L in serum and 400 µmol/L in the liver. Still, serum UCB levels remained unaltered. This first study on biliverdin reductase inhibition as a novel concept for treatment of unconjugated hyperbilirubinemia identified putative in vitro BVRA inhibitors. Montelukast, the clinically most suitable inhibitor, did not result in reduction of serum UCB in the Ugt1a1-deficient rat. The proposed treatment strategy will not result in amelioration of severe unconjugated hyperbilirubinemia in humans without the identification or development of more potent BVRA inhibitors.

Yin Zhi Huang and other plant-derived preparations: where herbal and molecular medicine meet.

A traditional herbal medicine enhances bilirubin clearance by activating the nuclear receptor CAR. Yin Zhi Huang, a decoction of Yin Chin (Artemisia capillaris) and three other herbs, is widely used in Asia to prevent and treat neonatal jaundice. We recently identified the constitutive androstane receptor (CAR, NR1I3) as a key regulator of bilirubin clearance in the liver. Here we show that treatment of WT and humanized CAR transgenic mice with Yin Zhi Huang for 3 days accelerates the clearance of intravenously infused bilirubin. This effect is absent in CAR knockout animals. Expression of bilirubin glucuronyl transferase and other components of the bilirubin metabolism pathway is induced by Yin Zhi Huang treatment of WT mice or mice expressing only human CAR, but not CAR knockout animals. 6,7-Dimethylesculetin, a compound present in Yin Chin, activates CAR in primary hepatocytes from both WT and humanized CAR mice and accelerates bilirubin clearance in vivo. We conclude that CAR mediates the effects of Yin Zhi Huang on bilirubin clearance and that 6,7-dimethylesculetin is an active component of this herbal medicine. CAR is a potential target for the development of new drugs to treat neonatal, genetic, or acquired forms of jaundice. [Abstract reproduced by permission of J Clin Invest 2004;113:137-143].