Combination of retinoic acid and ursodeoxycholic acid attenuates liver injury in bile duct-ligated rats and human hepatic cells

Abstract

Cholestasis leads to liver cell death, fibrosis, cirrhosis, and eventually liver failure. Despite limited benefits, ursodeoxycholic acid (UDCA) is the only Food and Drug Administration-approved treatment for cholestatic disorders. Retinoic acid (RA) is a ligand for nuclear receptors that modulate bile salt homeostasis. RA also possesses immunomodulatory effects and is used to treat acute promyelocytic leukemia and inflammatory disorders such as psoriasis, acne, and rheumatoid arthritis. To test whether the supplementation of RA with UDCA is superior to UDCA alone for treating cholestasis, male Sprague-Dawley rats underwent common bile duct ligation (BDL) for 14 days and were treated with phosphate-buffered saline (PBS), UDCA, all-trans retinoic acid (atRA), or UDCA and atRA by gavage. Treatment with UDCA and atRA substantially improved animal growth rates, significantly reduced liver fibrosis and bile duct proliferation, and nearly eliminated liver necrosis after BDL. Reductions in the bile salt pool size and liver hydroxyproline content were also seen with treatment with atRA or atRA and UDCA versus PBS and UDCA. Furthermore, atRA and UDCA significantly reduced liver messenger RNA and/or protein expression of transforming growth factor β1 (Tgf-β1), collagen 1a1 (Col1A1), matrix metalloproteinase 2 (Mmp2), cytokeratin 19, α-smooth muscle actin (α-SMA), cytochrome P450 7A1 (Cyp7a1), tumor necrosis factor α, and interleukin-β1. The molecular mechanisms of this treatment were also assessed in human hepatocytes, hepatic stellate cells, and LX-2 cells. atRA alone or in combination with UDCA greatly repressed CYP7A1 expression in human hepatocytes and significantly inhibited COL1A1, MMP2, and α-SMA expression and/or activity in primary human hepatic stellate cells and LX-2 cells. Furthermore, atRA reduced TGF-β1-induced Smad2 phosphorylation in LX-2 cells.

Conclusion: Our findings indicate that the addition of RA to UDCA reduces the bile salt pool size and liver fibrosis and might be an effective supplemental therapy with UDCA for cholestatic diseases.

Copyright © 2010 American Association for the Study of Liver Diseases.

Figures

Figure 1

AtRA supplementation with UDCA markedly improved the liver gross appearance and histology in BDL rats. A, gross liver appearance; B, H & E stained liver histology; and C, Scores of double-blind assessment of liver histology regarding fibrosis (also confirmed by Sirius Red staining), bile duct proliferation, necrosis, and inflammation. p < 0.05, n=5–7, * to sham, † to BDL-PBS, # to BDL-UDCA, and & to BDL-atRA. Both = BDL-UDCA&atRA.

Figure 2

Biochemical and gene expression analyses. Combination of atRA and UDCA significantly reduced the expression of marker genes for liver fibrosis, bile duct proliferation and inflammation. A, liver hydroxyproline levels; B, liver mRNA expression of TGF-β1, collagen 1a1 (Col1a1), matrix metalloproteinase-2 (Mmp-2), cytokeratin 19 (Ck19); C, liver alpha-smooth muscle actin (α-Sma) protein expression detected by Western blot where data is normalized to house-keeper gene Sh-ptp1; D, liver TNF-α and IL-1β mRNA expression. p < 0.05, n=5–7, * to sham, † to BDL-PBS, # to BDL-UDCA, and & to BDL-atRA. Both = BDL-UDCA&atRA.

Figure 3

AtRA supplementation with UDCA synergistically repressed Cyp7a1 mRNA expression in vivo in BDL rat livers (A) and great repression of CYP7A1 expression in vitro in human hepatocytes by atRA +/− UDCA (B), where increased mRNA expression of SHP/Shp and FGF19/Fgf15 were also detected. For A, p < 0.05, n=5–7, * to sham, † to BDL-PBS, # to BDL-UDCA, and & to BDL-atRA. Both = BDL-UDCA&atRA.

Figure 4

AtRA alone or in combination with UDCA significantly reduced the expression of fibrotic marker genes in human hepatic stellate cell line LX-2 cells and primary human hepatic stellate cells. A, mRNA expression of collagen 1A1 (COL1A1) and matrix metalloproteinase-2 (MMP-2) in LX-2 cells; B, Western blot analysis of pro-collagen α1 and alpha-smooth muscle actin (α-SMA) expression in LX-2 cells; C, gelatin zymography assay detected reduced MMP-2 expression/activity in the culture medium of atRA treated LX-2 cells; D, atRA decreased mRNA expression of COL1A1 and MMP-2 in human hepatic stellate cells. p<0.05, n=3–4, * to medium control, † to 2 ng/ml TGF-β1 treatment control.

Figure 5

AtRA significantly reduced TGF-β1 stimulated Smad2 phosphorylation and Smad4 protein expression (A) and Smad2 nuclear translocation (B) in LX-2 cells. p<0.05, n=3–4, † to 2 ng/ml TGF-β1 treatment control.