Combination of retinoic acid and ursodeoxycholic acid attenuates liver injury in bile duct-ligated rats and human hepatic cells.
ABSTRACT: UNLABELLED: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 ?1 (Tgf-?1), collagen 1a1 (Col1A1), matrix metalloproteinase 2 (Mmp2), cytokeratin 19, ?-smooth muscle actin (?-SMA), cytochrome P450 7A1 (Cyp7a1), tumor necrosis factor ?, and interleukin-?1. 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 ?-SMA expression and/or activity in primary human hepatic stellate cells and LX-2 cells. Furthermore, atRA reduced TGF-?1-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.
Project description:BACKGROUND & AIMS: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. METHODS:Bile duct-ligated (BDL) rats and Mdr2(Abcb4)-/- mice were treated with all-trans retinoic acid (atRA), a potent inhibitor of bile acid synthesis, 5 mg/kg/d by gavage, or Cenicriviroc (CVC), a known antagonist of CCR2 and CCR5, 50 mg/kg/d alone or in combination for 14 days and 1 month respectively. RESULTS: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-/- mice where a significant reduction in plasma liver enzymes, bilirubin, liver fibrosis, bile duct proliferation and hepatic infiltration of neutrophils and T cells and expression of cytokines were found. CONCLUSIONS: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.
Project description:The transcription factor nuclear factor-E2-related factor 2 (Nrf2) is a key regulator for induction of hepatic detoxification and antioxidant mechanisms, as well as for certain hepatobiliary transporters. To examine the role of Nrf2 in bile acid homeostasis and cholestasis, we assessed the determinants of bile secretion and bile acid synthesis and transport before and after bile duct ligation (BDL) in Nrf2(-/-) mice. Our findings indicate reduced rates of biliary bile acid and GSH excretion, higher levels of intrahepatic bile acids, and decreased expression of regulators of bile acid synthesis, Cyp7a1 and Cyp8b1, in Nrf2(-/-) compared with wild-type control mice. The mRNA expression of the bile acid transporters bile salt export pump (Bsep) and organic solute transporter (Ost?) were increased in the face of impaired expression of the multidrug resistance-associated proteins Mrp3 and Mrp4. Deletion of Nrf2 also decreased ileal apical sodium-dependent bile acid transporter (Asbt) expression, leading to reduced bile acid reabsorption and increased loss of bile acid in feces. Finally, when cholestasis is induced by BDL, liver injury was not different from that in wild-type BDL mice. These Nrf2(-/-) mice also had increased pregnane X receptor (Pxr) and Cyp3a11 mRNA expression in association with enhanced hepatic bile acid hydroxylation. In conclusion, this study finds that Nrf2 plays a major role in the regulation of bile acid homeostasis in the liver and intestine. Deletion of Nrf2 results in a cholestatic phenotype but does not augment liver injury following BDL.
Project description:Organic solute transporter alpha-beta (Ostalpha-Ostbeta) is a heteromeric bile acid and sterol transporter that facilitates the enterohepatic and renal-hepatic circulation of bile acids. Hepatic expression of this basolateral membrane protein is increased in cholestasis, presumably to facilitate removal of toxic bile acids from the liver. In this study, we show that the cholestatic phenotype induced by common bile duct ligation (BDL) is reduced in mice genetically deficient in Ostalpha. Although Ostalpha(-/-) mice have a smaller bile acid pool size, which could explain lower serum and hepatic levels of bile acids after BDL, gallbladder bilirubin and urinary bile acid concentrations were significantly greater in Ostalpha(-/-) BDL mice, suggesting additional alternative adaptive responses. Livers of Ostalpha(-/-) mice had higher messenger RNA levels of constitutive androstane receptor (Car) than wild-type BDL mice and increased expression of Phase I enzymes (Cyp7a1, Cyp2b10, Cyp3a11), Phase II enzymes (Sult2a1, Ugt1a1), and Phase III transporters (Mrp2, Mrp3). Following BDL, the bile acid pool size increased in Ostalpha(-/-) mice and protein levels for the hepatic basolateral membrane export transporters, multidrug resistance-associated protein 3 (Mrp3) and Mrp4, and for the apical bilirubin transporter, Mrp2, were all increased. In the kidney of Ostalpha(-/-) mice after BDL, the apical bile acid uptake transporter Asbt is further reduced, whereas the apical export transporters Mrp2 and Mrp4 are increased, resulting in a significant increase in urinary bile acid excretion.These findings indicate that loss of Ostalpha provides protection from liver injury in obstructive cholestasis through adaptive responses in both the kidney and liver that enhance clearance of bile acids into urine and through detoxification pathways most likely mediated by the nuclear receptor Car.
Project description:Organic anion transporting polypeptide 1a1 (Oatp1a1) is predominantly expressed in livers of mice and is thought to transport bile acids (BAs) from blood into liver. Because Oatp1a1 expression is markedly decreased in mice after bile duct ligation (BDL). We hypothesized that Oatp1a1-null mice would be protected against liver injury during BDL-induced cholestasis due largely to reduced hepatic uptake of BAs. To evaluate this hypothesis, BDL surgeries were performed in both male wild-type (WT) and Oatp1a1-null mice. At 24 h after BDL, Oatp1a1-null mice showed higher serum alanine aminotransferase levels and more severe liver injury than WT mice, and all Oatp1a1-null mice died within 4 days after BDL, whereas all WT mice survived. At 24 h after BDL, surprisingly Oatp1a1-null mice had higher total BA concentrations in livers than WT mice, suggesting that loss of Oatp1a1 did not prevent BA accumulation in the liver. In addition, secondary BAs dramatically increased in serum of Oatp1a1-null BDL mice but not in WT BDL mice. Oatp1a1-null BDL mice had similar basolateral BA uptake (Na(+)-taurocholate cotransporting polypeptide and Oatp1b2) and BA-efflux (multidrug resistance-associated protein [Mrp]-3, Mrp4, and organic solute transporter ?/?) transporters, as well as BA-synthetic enzyme (Cyp7a1) in livers as WT BDL mice. Hepatic expression of small heterodimer partner Cyp3a11, Cyp4a14, and Nqo1, which are target genes of farnesoid X receptor, pregnane X receptor, peroxisome proliferator-activated receptor alpha, and NF-E2-related factor 2, respectively, were increased in WT BDL mice but not in Oatp1a1-null BDL mice. These results demonstrate that loss of Oatp1a1 function exacerbates cholestatic liver injury in mice and suggest that Oatp1a1 plays a unique role in liver adaptive responses to obstructive cholestasis.
Project description:This study clarified the role of Cygb, the fourth globin in mammals originally discovered in rat hepatic stellate cells (HSCs), in cholestatic liver disease. Bile duct ligation (BDL) augmented inflammatory reactions as revealed by increased infiltrating neutrophils, CD68+-macrophages, and chemokine expression in Cygb-/- mice. In these mice, impairment of bile canalicular indicated by the loss of CD10 expression, down-regulation of bile salt transporters, increased total bile acid, and massive apoptotic and necrotic hepatocytes occurred with the release of cytochrome c, activation of caspase 3, resulting in reduced animal survival compared to wild-type mice. In Cygb-/- mouse liver, all of NO metabolites and oxidative stress were increased. Treatment with NO inhibitor restrained all above phenotypes and restored CD10 expression in BDL Cygb-/- mice, while administration of NO donor aggravated liver damage in BDL-wild type mice to the same extent of BDL-Cygb-/- mice. N-acetylcysteine administration had a negligible effect in all groups. In mice of BDL for 1-3 weeks, expression of all fibrosis-related markers was significantly increased in Cygb-/- mice compared with wild-type mice. Thus, Cygb deficiency in HSCs enhances hepatocyte damage and inflammation in early phase and fibrosis development in late phase in mice subjected to BDL, presumably via altered NO metabolism.
Project description:Accumulating evidence indicates that oxidative stress plays a critical role in initiating the progression of inflammatory and fibrotic liver diseases, including cholestatic hepatitis. Peroxiredoxin 4 (PRDX4) is a secretory antioxidase that protects against oxidative damage by scavenging reactive oxygen species (ROS) in both the intracellular compartments and extracellular space. In this study, we examined the in vivo net effects of PRDX4 overexpression in a murine model of cholestasis. To induce cholestatic liver injury, we subjected C57BL/6J wild-type (WT) or human PRDX4 (hPRDX4) transgenic (Tg) mice to sham or bile duct ligation (BDL) surgery for seven days. Our results showed that the liver necrosis area was significantly suppressed in Tg BDL mice with a reduction in the severity of liver injuries. Furthermore, PRDX4 overexpression markedly reduced local and systemic oxidative stress generated by BDL. In addition, suppression of inflammatory cell infiltration, reduced proliferation of hepatocytes and intrahepatic bile ducts, and less fibrosis were also found in the liver of Tg BDL mice, along with a reduced mortality rate after BDL surgery. Interestingly, the composition of the hepatic bile acids (BAs) was more beneficial for Tg BDL mice than for WT BDL mice, suggesting that PRDX4 overexpression may affect BA metabolism during cholestasis. These features indicate that PRDX4 plays an important role in protecting against liver injury following BDL and might be a promising therapeutic modality for cholestatic diseases.
Project description:Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder. It is uncertain if simple steatosis, the initial and prevailing form of NAFLD, sensitizes the liver to cholestasis. Here, we compared the effects of obstructive cholestasis in rats with a normal liver versus rats with simple steatosis induced by a methionine/choline-deficient diet. We found that plasma liver enzymes were higher and hepatic neutrophil influx, inflammation, and fibrosis were more pronounced in animals with combined steatosis and cholestasis compared to cholestasis alone. Circulating bile salt levels were markedly increased and hepatic bile salt composition shifted from hydrophilic tauro-?-muricholate to hydrophobic taurocholate. This shift was cytotoxic for HepG2 hepatoma cells. Gene expression analysis revealed induction of the rate-limiting enzyme in bile salt synthesis, cytochrome P450 7a1 (CYP7A1), and modulation of the hepatic bile salt transport system. In conclusion, simple steatosis sensitizes the liver to cholestatic injury, inflammation, and fibrosis in part due to a cytotoxic shift in bile salt composition. Plasma bile salt levels were elevated, linked to dysregulation of bile salt synthesis and enhanced trafficking of bile salts from the liver to the systemic circulation.
Project description:There are limited murine models of cholestatic liver diseases characterized by chronic biliary obstruction and resumption of bile flow. While murine bile duct ligation (BDL) is a well-established model of obstructive cholestasis, current models of BDL reversal (BDLR) alter biliary anatomy. We aimed to develop a more physiologic model of BDLR to evaluate the time course and mechanism for resolution of hepatic injury after biliary obstruction. In the present study, we restored bile flow into the duodenum without disruption of the gall bladder after murine BDL using biocompatible PE-50 tubing. After establishing the technique, overall survival for BDLR at 7 or 14 days after BDL was 88%. Sham laparotomy was performed in control mice. Laboratory data, liver histology, and hepatic gene expression were compared among BDL, BDLR, and controls. Laboratory evidence of cholestatic liver injury was observed at day 7 after BDL and rapid improvement occurred within 48 hr of BDLR. After BDLR there was also enhanced gene expression for the bile acid transporter Abcb11, however, bile duct proliferation persisted. Assessment of the immune response showed increased gene and protein expression for the general immune cell marker Cd45 in BDLR versus BDL mice suggesting a reparative immune response after BDLR. In summary, we have established a novel murine model of BDLR that allows for the investigation into bile acid and immune pathways responsible for hepatic repair following obstructive cholestasis. Future studies with our model may identify targets for new therapies to improve outcome in pediatric and adult cholestatic liver disease.
Project description:Niemann-Pick disease type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in the NPC1 or NPC2 genes. Liver disease is also a common feature of NPC that can present as cholestatic jaundice in the neonatal period. Liver enzymes can remain elevated above the normal range in some patients as they age. We recently reported suppression of the P450 detoxification system in a mouse model of NPC disease and in post-mortem liver from NPC patients. As bile acids regulate the P450 system, we tested bile acid treatment using ursodeoxycholic acid (UDCA; 3?, 7?-dihydroxy-5?-cholanic acid), a hydrophilic bile acid, which is used to treat several cholestatic disorders. In this study, we compared UDCA treatment with the bile acid cholic acid (CA), and found unexpected hepatotoxicity in response to CA in Npc1 mice, but not to UDCA, suggesting that only UDCA should be used as an adjunctive therapy in NPC patients.
Project description:Dehydroandrographolide (DA) is the main contributor to the therapeutic properties of the medicinal plant Andrographis paniculata (AP). However, it is unknown whether DA has a hepatoprotective effect on obstructive cholestasis in mice and humans.We administered DA to mice for 5 days prior to bile duct ligation (BDL) and for the 7 days. Liver function markers, liver histology and necrosis, compensatory responses of hepatocytes, liver fibrosis and the expression of hepatic fibrogenesis markers were evaluated in BDL mice and/or human LX-2 cells.Mice treated with DA demonstrated lower levels of serum alanine transarninase (ALT), milder liver damage, liver necrosis and fibrosis formation than in vehicle control with carboxymethylcellulose (CMC) mice after BDL. DA treatment also enhanced the Mrp3 expression of hepatocytes but not Mrp4 following BDL. Further, DA treatment in BDL mice significantly reduced liver mRNA and/or protein expression of Tgf-?, Col1a1, ?-Sma and Mmp2. This result was also supported by hydroxyproline analysis. The molecular mechanisms of DA treatment were also assessed in human hepatic stellate cell line (LX-2 cell). DA treatment significantly inhibited Tgf-?-induced Col1a1, Mmp2 and ?-Sma expression in human LX-2 cells. These data suggested that DA treatment reduced liver damage through development of a hepatic adaptive response and inhibition of the activation of HSCs, which led to a reduction in liver fibrosis formation in BDL mice.DA treatment protected against liver damage and fibrosis following BDL and might be an effective therapy for extrahepatic cholestasis due to bile duct obstruction.