tretinoin and Cholestasis

Cholestatic liver injury is mediated by bile acid-induced inflammatory responses. We hypothesized that superior therapeutic effects might be achieved by combining treatments that reduce the bile acid pool size with one that blocks inflammation.. All-trans retinoic acid alone reduced bile acid pool size and liver necrosis in BDL rats. However, the combination with CVC further reduced liver to body weight ratio, bile acid pool size, plasma liver enzyme, bilirubin, liver necrosis and fibrosis when compared to the atRA treatment. The assessment of hepatic hydroxyproline content further confirmed the reduced liver injury concurrent with reduction of pro-inflammatory cytokines emphasizing the synergistic effects of these two agents. Profiling of hepatic inflammatory cells revealed that combination therapy reduced neutrophils and T cells but not macrophages. The superior therapeutic effects of combination treatment were also confirmed in Mdr2. Multitargeted therapy is an important paradigm for treating cholestatic liver injury. The combination of CVC with atRA or other FXR activators may warrant a clinical trial in patients with cholestatic liver disease.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Bile Acids and Salts; Cholestasis; Disease Models, Animal; Drug Therapy, Combination; Imidazoles; Ligation; Liver; Liver Diseases; Male; Mice; Mice, Knockout; Rats; Rats, Sprague-Dawley; Receptors, Cytokine; Sulfoxides; Tretinoin

The organic solute transporter-α/β (OSTα/β) is a heteromeric transporter that is essential for bile acid and sterol disposition and for the enterohepatic circulation. To better understand the mechanism underlying OST gene regulation, the effects of retinoic acid (RA) on OSTα/β gene expression were investigated. The results show a dose-dependent induction of OSTβ but not OSTα expression in both Huh7 and HepG2 cells by RA treatment. A novel functional RA receptor response element (RARE; so-called DR5) in the promoter of OSTβ gene was identified. The interaction of RARα/RXRα with the RARE was verified by electrophoretic mobility shift and chromatin immunoprecipitation assays and its functional importance by hOSTβ promoter activation in luciferase reporter assays. The studies demonstrated that the RARE is also a constitutive androstane receptor (CAR) binding site for OSTβ gene regulation. These results suggest that OSTβ is a target of both FXR-mediated (by binding to IR-1 element) and RARα- and CAR-mediated (by binding to DR5 element) gene regulation pathways. In summary, this study has uncovered a novel RARE (DR5) element in the promoter of OSTβ that binds RARα or CAR heterodimerized with RXRα and appears to function synergistically with the IR-1 element to provide maximal induction of OSTβ in response to RA. These findings demonstrate a role for RARα and CAR in controlling OSTβ expression levels.

Topics: Base Sequence; Blotting, Western; Cell Line, Tumor; Cholestasis; Cholesterol; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Genes, Reporter; Humans; Membrane Transport Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Real-Time Polymerase Chain Reaction; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Response Elements; Retinoic Acid Receptor alpha; Retinoid X Receptor alpha; RNA; RNA, Small Interfering; Transfection; Tretinoin; Xenobiotics

Chronic cholestasis results in liver injury and eventually liver failure. Although ursodeoxycholic acid (UDCA) showed limited benefits in primary biliary cirrhosis, there is an urgent need to develop alternative therapy for chronic cholestatic disorders. Previous studies from our laboratory demonstrated that all-trans-retinoic acid (atRA) is a potent suppressor of CYP7A1, the rate-limiting enzyme in bile acid synthesis. atRA also repressed the expression of tumor growth factor-β and collagen 1A1 in activated primary human stellate cells and LX2 cells. When administered together with UDCA to bile duct-ligated rats, this combined therapy significantly reduced the bile acid pool size and improved liver conditions. To further examine whether atRA alone or in combination with UDCA has greater beneficial effects than UDCA treatment alone, we assessed this treatment in two additional chronic cholestatic rodent models: α-naphthylisothiocyanate (ANIT)-treated rats and the Mdr2(-/-) (Abcb4(-/-)) knockout mouse. atRA alone significantly reduced bile duct proliferation, inflammation, and hydroxyproline levels in ANIT-treated rats, whereas the combination of atRA and UDCA significantly reduced plasma bile salt level compared with UDCA treatment. atRA alone or in combination with UDCA significantly reduced plasma levels of alkaline phosphatase and bile salts in 12-week-old Mdr2(-/-) mice. Reduced bile duct proliferation and inflammation were also observed in the livers of these mice. Together, atRA alone or in combination with UDCA significantly reduced the severity of liver injury in these two animal models, further supporting the combination treatment of atRA and UDCA as a potential new therapy for patients with chronic cholestatic liver disease who have not responded fully to UDCA.

Topics: 1-Naphthylisothiocyanate; Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Bile Acids and Salts; Cell Proliferation; Cholagogues and Choleretics; Cholestasis; Chronic Disease; Disease Models, Animal; Drug Therapy, Combination; Liver; Male; Mice; Mice, Knockout; Rats; Rats, Sprague-Dawley; Tretinoin; Ursodeoxycholic Acid

Hepatic stellate cells (HSC) participate in liver fibrogenesis via myofibroblastic activation, the extent of which appears to correlate with the loss of cellular vitamin A. The present study has tested a hypothesis that HSC activation is associated with diminished retinoic acid (RA) signaling. Pure HSC were isolated from rats with cholestatic liver fibrosis induced by bile duct ligation (BDL) and sham-operated animals (Sham). Northern blot analysis of HSC RNA from BDL confirmed fibrogenic activation of the cells with enhanced mRNA levels for procollagen-alpha1(I) and transforming growth factor-beta1 (TGF-beta1). Competitive polymerase chain reaction analysis showed selective reductions in the mRNA levels of RA receptor (RAR)-beta and retinoid X receptor (RXR)-alpha to 20 and 17% of the Sham HSC. The mRNA level for cellular retinol binding protein I, a gene with RA responsive element (RARE), was also suppressed by 78% in BDL. The concentrations of all-trans-RA and 9-cis-RA were decreased in HSC from BDL. Nuclear extracts of these cells showed diminished binding activity to the RARE, whereas activity of AP-1, a transcription factor known to be antagonized by RAR and RXR, was enhanced. These results demonstrate diminished RA signaling in HSC from cholestatic liver fibrosis, which appeared to have resulted from RA deficiency and suppressed expression of RAR-beta and RXR-alpha. Furthermore, the reciprocal enhancement of AP-1 activity and coordinately increased expression of an AP-1 responsive gene, TGF-beta1, suggest a permissive role of the diminished RA signaling in promoting AP-1 activity and TGF-beta1 expression.

Topics: Animals; Base Sequence; Cholestasis; DNA-Binding Proteins; Liver; Liver Cirrhosis, Biliary; Male; Molecular Sequence Data; Procollagen; Rats; Rats, Wistar; Receptors, Retinoic Acid; Retinoid X Receptors; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; RNA, Messenger; Signal Transduction; Transcription Factor AP-1; Transcription Factors; Transforming Growth Factor beta; Tretinoin; Up-Regulation