Pathobiochemical signatures of cholestatic liver disease in bile duct ligated mice.
ABSTRACT: Disrupted bile secretion leads to liver damage characterized by inflammation, fibrosis, eventually cirrhosis, and hepatocellular cancer. As obstructive cholestasis often progresses insidiously, markers for the diagnosis and staging of the disease are urgently needed. To this end, we compiled a comprehensive data set of serum markers, histological parameters and transcript profiles at 8 time points of disease progression after bile duct ligation (BDL) in mice, aiming at identifying a set of parameters that could be used as robust biomarkers for transition of different disease progression phases.Statistical analysis of the more than 6,000 data points revealed distinct temporal phases of disease. Time course correlation analysis of biochemical, histochemical and mRNA transcript parameters (=factors) defined 6 clusters for different phases of disease progression. The number of CTGF-positive cells provided the most reliable overall measure for disease progression at histological level, bilirubin at biochemical level, and metalloproteinase inhibitor 1 (Timp1) at transcript level. Prominent molecular events exhibited by strong transcript peaks are found for the transcriptional regulator Nr0b2 (Shp) and 1,25-dihydroxyvitamin D(3) 24-hydroxylase (Cyp24a1) at 6 h. Based on these clusters, we constructed a decision tree of factor combinations potentially useful as markers for different time intervals of disease progression. Best prediction for onset of disease is achieved by fibronectin (Fn1), for early disease phase by Cytochrome P450 1A2 (Cyp1a2), passage to perpetuation phase by collagen1α-1 (Col1a1), and transition to the progression phase by interleukin 17-a (Il17a), with early and late progression separated by Col1a1. Notably, these predictions remained stable even for randomly chosen small sub-sets of factors selected from the clusters.Our detailed time-resolved explorative study of liver homogenates following BDL revealed a well-coordinated response, resulting in disease phase dependent parameter modulations at morphological, biochemical, metabolic and gene expression levels. Interestingly, a small set of selected parameters can be used as diagnostic markers to predict disease stages in mice with cholestatic liver disease.
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.
Project description:Dihydrotanshinone I (DHI), a lipophilic component of traditional Chinese medicine Salvia miltiorrhiza Bunge, has various therapeutic effects. We investigated the anti-fibrotic effect of DHI and its underlying mechanisms in vitro and in vivo.Rats subjected to bile duct ligation (BDL) were treated with DHI (25 mg·kg-1 ·day-1 , i.p.) for 14 days. Serum biochemical and liver tissue morphological analyses were performed. The human hepatic stellate cell line LX-2 served as a liver fibrosis model in vitro. Liver fibrogenic genes, yes-associated protein (YAP) downstream genes and autophagy markers were examined using western blot and real-time PCR analyses. Similar analyses were done in rat primary hepatic stellate cells (pHSCs). Autophagy flux was assessed by immunofluorescence.In BDL rats, DHI administration attenuated liver necrosis, bile duct proliferation and collagen accumulation and reduced the expression of genes associated with fibrogenesis, including Tgfb1, Mmp-2, Acta2 and Col1a1. DHI (1, 5, 10 ?mol·L-1 ) time- and dose-dependently suppressed the protein level of COL1A1, TGF?1 and ?-SMA in LX-2 cells and rat pHSCs. Furthermore, DHI blocked the nuclear translocation of YAP, which inhibited the YAP/TEAD2 interaction and its downstream fibrogenic genes, connective tissue growth factor, SOX4 and survivin. This stimulated autophagic flux and accelerated the degradation of liver collagen.DHI exerts anti-fibrotic effects in BDL rats, LX-2 cells and rat pHSCs by inhibiting the YAP and TEAD2 complex and stimulating autophagy. These findings indicate that DHI may be a potential therapeutic for the treatment of liver fibrosis.
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:Contribution of hepatic stellate cells (HSCs), portal fibroblasts (PFs), and mesothelial cells (MCs) to myofibroblasts is not fully understood due to insufficient availability of markers and isolation methods. The present study aimed to isolate these cells, characterize their phenotypes, and examine their contribution to myofibroblasts in liver fibrosis.Liver fibrosis was induced in Collagen1a1-green fluorescent protein (Col1a1(GFP)) mice by bile duct ligation (BDL), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, or CCl4 injections. Combining vitamin A (VitA) lipid autofluorescence and expression of GFP and glycoprotein M6a (GPM6A), we separated HSCs, PFs, and MCs from normal and fibrotic livers by fluorescence-activated cell sorting (FACS).Normal Col1a1(GFP) livers broadly expressed GFP in HSCs, PFs, and MCs. Isolated VitA+ HSCs expressed reelin, whereas VitA-GFP+GPM6A- PFs expressed ectonucleoside triphosphate diphosphohydrolase-2 and elastin. VitA-GFP+GPM6A+ MCs expressed keratin 19, mesothelin, and uroplakin 1b. Transforming growth factor (TGF)-?1 treatment induced the transformation of HSCs, PFs, and MCs into myofibroblasts in culture. TGF-?1 suppressed cyclin D1 mRNA expression in PFs but not in HSCs and MCs. In biliary fibrosis, PFs adjacent to the bile duct expressed ?-smooth muscle actin. FACS analysis revealed that HSCs are the major source of GFP+ myofibroblasts in the injured Col1a1(GFP) mice after DDC or CCl4 treatment. Although PFs partly contributed to GFP+ myofibroblasts in the BDL model, HSCs were still dominant source of myofibroblasts.HSCs, PFs, and MCs have distinct phenotypes, and PFs partly contribute to myofibroblasts in the portal triad in biliary fibrosis.
Project description:Bile duct ligation (BDL) is an experimental procedure that mimics obstructive cholestatic disease. One of the early consequences of BDL in rodents is the appearance of so-called bile infarcts that correspond to Charcot-Gombault necrosis in human cholestasis. The mechanisms causing bile infarcts and their pathophysiological relevance are unclear. Therefore, intravital two photon-based imaging of BDL mice was performed with fluorescent bile salts (BS) and non-BS organic anion analogues. Key findings were followed up by matrix-assisted laser desorption ionization imaging, clinical chemistry, immunostaining, and gene expression analyses. In the acute phase, 1-3 days after BDL, BS concentrations in bile increased and single-cell bile microinfarcts occurred in dispersed hepatocytes throughout the liver caused by the rupture of the apical hepatocyte membrane. This rupture occurred after loss of mitochondrial membrane potential, followed by entry of bile, cell death, and a "domino effect" of further death events of neighboring hepatocytes. Bile infarcts provided a trans-epithelial shunt between bile canaliculi and sinusoids by which bile constituents leaked into blood. In the chronic phase, ≥21 days after BDL, uptake of BS tracers at the sinusoidal hepatocyte membrane was reduced. This contributes to elevated concentrations of BS in blood and decreased concentrations in the biliary tract. Conclusion: Bile microinfarcts occur in the acute phase after BDL in a limited number of dispersed hepatocytes followed by larger infarcts involving neighboring hepatocytes, and they allow leakage of bile from the BS-overloaded biliary tract into blood, thereby protecting the liver from BS toxicity; in the chronic phase after BDL, reduced sinusoidal BS uptake is a dominant protective factor, and the kidney contributes to the elimination of BS until cholemic nephropathy sets in.
Project description:Oltipraz (4-methyl-5(pyrazinyl-2)-1-2-dithiole-3-thione), a promising cancer preventive agent, has an antioxidative activity and ability to enhance glutathione biosynthesis, phase II detoxification enzymes and multidrug resistance-associated protein-mediated efflux transporters. Oltipraz can protect against hepatotoxicity caused by carbon tetrachloride, acetaminophen and alpha-naphthylisothiocyanate. Whether oltipraz has hepato-protective effects on obstructive cholestasis is unknown.We administered oltipraz to mice for 5 days prior to bile duct ligation (BDL) for 3 days. Liver histology, liver function markers, bile flow rates and hepatic expression of profibrogenic genes were evaluated.Mice pretreated with oltipraz prior to BDL demonstrated higher levels of serum aminotransferases and more severe liver damage than in control mice. Higher bile flow and glutathione secretion rates were observed in unoperated mice treated with oltipraz than in control mice, suggesting that liver necrosis in oltipraz-treated BDL mice may be related partially to increased bile-acid independent flow and biliary pressure. Oltipraz treatment in BDL mice enhanced ?-smooth muscle actin expression, consistent with activation of hepatic stellate cells and portal fibroblasts. Matrix metalloproteinases (Mmp) 9 and 13 and tissue inhibitors of metalloproteinases (Timp) 1 and 2 levels were increased in the oltipraz-treated BDL group, suggesting that the secondary phase of liver injury induced by oltipraz might be due to excessive Mmp and Timp secretions, which induce remodeling of the extracellular matrix.Oltipraz treatment exacerbates the severity of liver injury following BDL and should be avoided as therapy for extrahepatic cholestatic disorders due to bile duct obstruction.
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: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:Biliary atresia is a rare, devastating disease of infants where a fibroinflammatory process destroys the bile ducts, leading to fibrosis and biliary cirrhosis, and death if untreated. The cause and pathogenesis remain largely unknown. We tried to investigate factors involved in biliary atresia, especially forkhead box A3 (Foxa3), which might exert a role in the treatment of liver disease. We used RNA sequencing to sequence the whole transcriptomes of livers from six biliary atresia and six choledochal cysts patients. Then, we employed a rat disease model by bile duct ligation (BDL) and adenovirus transduction to address the function of Foxa3 in biliary atresia. We found that tight junction, adherence junction, cell cycle, apoptosis, chemokine singling, VEGF and MAPK signaling pathways were enriched in biliary atresia livers. We showed that Foxa3 expression was notably decreased in liver samples from biliary atresia patients. More importantly, we found that its lower expression predicted a poorer overall survival of biliary atresia patients. Rats that received BDL surgery and Foxa3 expression adenovirus resulted in a significant decrease in the deposition of collagen, and expression of profibrotic cytokines (transforming growth factor-? and connective tissue growth factor) and fibrosis markers (?-smooth muscle actin, collagen I and collagen III), as compared with rats that received BDL surgery and control adenovirus. Our data suggested a protection role for Foxa3 during the progression of liver fibrosis in biliary atresia, and thereby supported increasing Foxa3 as a targeted treatment strategy.
Project description:Insulin receptor substrate 2 (IRS2) is a key downstream mediator of insulin and insulin-like growth factor 1 (IGF1) signalling pathways and plays a major role in liver metabolism. The aim of this study was to investigate whether IRS2 had an impact on the hepatic fibrotic process associated with cholestatic injury. Bile duct ligation (BDL) was performed in wild-type (WT) and Irs2-deficient (IRS2KO) female mice. Histological and biochemical analyses, together with fibrogenic and inflammatory responses were evaluated in livers from mice at 3, 7 and 28 days following BDL. We also explored whether activation of human hepatic stellate cells (HSCs) induced by IGF1 was modulated by IRS2. IRS2KO mice displayed reduced disruption of liver histology, such hepatocyte damage and excess deposition of extracellular matrix components, compared with WT mice at 3 and 7 days post-BDL. However, no histological differences between genotypes were found at 28 days post-BDL. The less pro-inflammatory profile of bile acids accumulated in the gallbladder of IRS2KO mice after BDL corresponded with the reduced expression of pro-inflammatory markers in these mice. Stable silencing of IRS2 or inhibition of ERK1/2 reduced the activation of human LX2 cells and also reduced induction of MMP9 upon IGF1 stimulation. Furthermore, hepatic MMP9 expression was strongly induced after BDL in WT mice, but only a slight increase was found in mice lacking IRS2. Our results have unravelled the signalling pathway mediated by IGF1R-IRS2-ERK1/2-MMP9 as a key axis in regulating HSC activation, which might be therapeutically relevant for targeting liver fibrosis.