Cholestatic Rats Research Articles

The soluble guanylate cyclase stimulator riociguat reduces fibrogenesis and portal pressure in cirrhotic rats

In cirrhotic patients, portal hypertension (PHT) deteriorates survival, yet treatment options are limited. A major contributor to increased intrahepatic vasoconstriction in PHT is dysfunctional nitric-oxide signaling. Soluble guanylate cyclase (sGC) is the receptor of nitric-oxide and can be stimulated by riociguat. Riociguat is approved for pulmonary hypertension but has not been studied in liver cirrhosis. In this study we assessed the effects of riociguat on PHT and liver fibrosis in cholestatic (bile duct ligation, BDL) and toxic (carbon-tetrachloride, CCl4) rat models. In cirrhotic livers sGC expression was upregulated. In BDL rats, riociguat reduced liver fibrosis and decreased portal pressure without affecting systemic hemodynamics. In an early BDL disease stage, riociguat decreased bile duct proliferation, improved sinusoidal vascular dysfunction and inhibited angiogenesis. In advanced BDL riociguat exhibited anti-inflammatory effects. In CCl4 rats the beneficial effects of riociguat treatment were less pronounced and confined to an early disease stage. Similarly, in patients with cholestatic cirrhosis and PHT nitrates (that induce sGC activity) decreased portal pressure more effectively than in patients with non-cholestatic etiology. We also found an improvement of transaminases in patients with pulmonary hypertension receiving riociguat. Our findings support the clinical development of sGC stimulators in patients with cirrhotic PHT.

Yinchenhao Decoction Ameliorates Alpha-Naphthylisothiocyanate Induced Intrahepatic Cholestasis in Rats by Regulating Phase II Metabolic Enzymes and Transporters.

Yinchenhao Decoction (YCHD), a famous traditional Chinese formula, has been used for treating cholestasis for 1000s of years. The cholagogic effect of YCHD has been widely reported, but its pharmacodynamic material and underlying therapeutic mechanism remain unclear. By using ultra-high-performance liquid chromatography (UHPLC)-quadrupole time-of-flight mass spectrometry, 11 original active components and eight phase II metabolites were detected in rats after oral administration of YCHD, including three new phase II metabolites. And it indicated that phase II metabolism was one of the major metabolic pathway for most active components in YCHD, which was similar to the metabolism process of bilirubin. It arouses our curiosity that whether the metabolism process of YCHD has any relationship with its cholagogic effects. So, a new method for simultaneous quantitation of eight active components and four phase II metabolites of rhein, emodin, genipin, and capillarisin has been developed and applied for their pharmacokinetic study in both normal and alpha-naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis rats. The results indicated the pharmacokinetic behaviors of most components of YCHD were inhibited, which was hypothesized to be related to different levels of metabolic enzymes and transporters in rat liver. So dynamic changes of intrahepatic enzyme expression in cholestasis and YCHD treated rats have been monitored by an UHPLC-tandem mass spectrometry method. The results showed expression levels of UDP-glucuronosyltransferase 1-1 (UGT1A1), organic anion-transporting polypeptide 1A4 (OATP1A4), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1, sodium-dependent taurocholate cotransporter, and organic anion-transporting polypeptide 1A2 were significantly inhibited in cholestasis rats, which would account for reducing the drug absorption and the metabolic process of YCHD in cholestatic rats. A high dose (12 g/kg) of YCHD remarkably increased the expression of UGT1A1, bile salt export pump, MRP2, OATP1A4 in cholestasis rats presented it exhibited the greatest ameliorative effect on cholestasis, also particularly in histopathological examination and reducing levels of alanine transaminase, aspartate transaminase, total bilirubin, direct bilirubin, and total bile acid. Considering the metabolic process of bilirubin in vivo, the choleretic effect of YCHD is proven to be related to its regulatory action on expression of metabolic enzymes and transporters in cholestatic liver.

Ursodeoxycholate Restores Biliary Excretion of Methotrexate in Rats with Ethinyl Estradiol Induced-Cholestasis by Restoring Canalicular Mrp2 Expression.

The in vivo relevance of ursodeoxycholate (UDCA) treatment (100 mg/kg/day, per oral tid for 5 days before cholestasis induction followed by the same dosing for 5 days) on hepatic function was investigated in rats with 17α-ethinylestradiol (EE, 10 mg/kg, subcutaneous for 5 days)-induced experimental cholestasis. The bile flow rate and the expression level of hepatic multidrug resistance-associated protein 2 (Mrp 2) that were decreased in cholestasis were restored after UDCA treatment. Consistent with this, the biliary excretion clearance (CLexc,bile) of a representative Mrp2 substrate—methotrexate (MTX)—was decreased in cholestatic rats but was restored after UDCA treatment. Consequently, the plasma concentrations of MTX, which were increased by cholestasis, were decreased to control levels by UDCA treatment. Thus, the restoration of CLexc,bile appears to be associated with the increase in Mrp2 expression on the canalicular membrane by UDCA treatment followed by Mrp2-mediated biliary excretion of MTX. On the other hand, the hepatic uptake clearance (CLup,liver) of MTX was unchanged by cholestasis or UDCA treatment, suggestive of the absence of any association between the uptake process and the overall biliary excretion of MTX. Since UDCA has been known to induce the expression of canalicular MRP2 in humans, UDCA treatment might be effective in humans to maintain or accelerate the hepatobiliary elimination of xenobiotics or metabolic conjugates that are MRP2 substrates.

Bacterial Translocation Is Linked to Increased Intestinal IFN-γ, IL-4, IL-17, and mucin-2 in Cholestatic Rats

Background and rationale for the study. Bacterial translocation is an important triggering factor of infection and mortality in cirrhosis. In a rat model using bile duct ligation (BDL), bacterial translocation appears within 24 h after ligation. The dynamic between TH1/TH2/TH17 cytokines and the integrity of the colonic mucosa in the context of cirrhosis is little known. This study aims to determine the link between bacterial translocation and intestinal inflammation in a cholestasis model. Additionally, alterations of the colonic mucus layer and the bacterial load were also addressed.Results. Bacterial translocation detected by microbiological cultures and MALDI-TOF showed that Escherichia coli predominates in mesenteric lymph nodes of BDL rats. Intestinal bacterial load analyzed by qPCR indicates a dramatic Escherichia/Shigella overgrowth at 8 and 30 days post-BDL. IFN-γ, IL-4, and IL-17 evaluated by Western blotting were increased at 8 and 30 days in the small intestine. In the colon, in contrast, only IFN-γ was significantly increased. The colonic mucus layer and mucin-2 expression determined by Alcian blue staining and immunohistochemistry surprisingly showed an increase in the mucus layer thickness related to increased mucin-2 expression during the entire process of liver damage. Hepatic enzymes, as well as collagen I, collagen III, TNF-α, and IL-6 liver gene expression were increased.In conclusion, bacterial overgrowth associated with bacterial translocation is linked to the over-expression of IFN-γ, IL-4, IL-17 and mucin-2. These molecules might facilitate the intestinal permeability through exacerbating the inflammatory process and disturbing tight junctions, leading to the perpetuation of the liver damage.

Dexamethasone restores transforming growth factor-β activated kinase 1 expression and phagocytosis activity of Kupffer cells in cholestatic liver injury

The role of transforming growth factor-β activated kinase 1 (TAK1) in modulating the function of Kupffer cells (KCs) within cholestatic livers remains unclear. This study examined the immunopharmacological action of dexamethasone (DEX) in modulating hepatic TAK1 expression and related signaling activity in a rat model of bile duct ligation-mimicked obstructive jaundice. The in vitro effects of DEX on porcine biliary extract (PBE)-modulated gene expression and phagocytosis of KCs were examined using a rat alveolar macrophage cell line (NR8383 cells). Although DEX therapy did not restore the downregulated TAK1 expression and phosphorylation, it significantly attenuated the upregulation of high-mobility group box 1 expression and caspase-3 activation in whole liver extracts of cholestatic rats, possibly via enhancing extracellular signal-regulated kinase-mediated signaling. Dual immunofluorescence staining of cholestatic livers and western detection on primary KCs isolated from cholestatic livers identified that DEX treatment indeed increased both the expression and phosphorylation levels of TAK1 in the KCs of cholestatic livers. In vitro studies using alveolar NR8383 macrophages with KC-characteristic gene expression further demonstrated that DEX not only repressed the pro-inflammatory cytokine production including with respect to interleukin (IL)-1β and IL-6, but also enhanced gene expression of TAK1 and a phagocytic marker, natural-resistance-associated macrophage protein 1, under PBE-mimicked cholestatic conditions. However, WST-1 assay showed that DEX did not protect NR8383 macrophages against the PBE-induced cytotoxicity. Immunofluorescence visualization of cellular F-actin by phalloidin suggested that DEX sustained the PBE-induced phagocytosis morphology of NR8383 macrophages. In conclusion, DEX treatment may pharmacologically restore the expression and activity of TAK1 in KCs, and sustain the phagocytic phenotype of KCs in cholestatic livers.

Mechanisms of canalicular transporter endocytosis in the cholestatic rat liver

Impaired canalicular secretion due to increased endocytosis and intracellular retention of canalicular transporters such as BSEP and MRP2 is a main, common pathomechanism of cholestasis. Nevertheless, the mechanisms governing this process are unknown. We characterized this process in estradiol 17 β-d-glucuronide (E17G)-induced cholestasis, an experimental model which partially mimics pregnancy-induced cholestasis. Inhibitors of clathrin-mediated endocytosis (CME) such as monodansylcadaverine (MDC) or K+ depletion, but not the caveolin-mediated endocytosis inhibitors filipin and genistein, prevented E17G-induced endocytosis of BSEP and MRP2, and the associated impairment of activity of these transporters in isolated rat hepatocyte couplets (IRHC). Immunofluorescence and confocal microscopy studies showed that, in E17G-treated IRHC, there was a significant increase in the colocalization of MRP2 with clathrin, AP2, and Rab5, three essential members of the CME machinery. Knockdown of AP2 by siRNA in sandwich-cultured rat hepatocytes completely prevented E17G-induced endocytosis of BSEP and MRP2. MDC significantly prevented this endocytosis, and the impairment of bile flow and biliary secretion of BSEP and MRP2 substrates, in isolated and perfused livers. BSEP and MRP2, which were mostly present in raft (caveolin-enriched) microdomains in control rats, were largely found in non-raft (clathrin-enriched) microdomains in livers from E17G-treated animals, from where they can be readily recruited for CME. In conclusion, our findings show that CME is the mechanism responsible for the internalization of the canalicular transporters BSEP and MRP2 in E17G-induced cholestasis. The shift of these transporters from raft to non-raft microdomains could be a prerequisite for the transporters to be endocytosed under cholestatic conditions.

Galactosylated Pro-Drug of Ursodeoxycholic Acid: Design, Synthesis, Characterization, and Pharmacological Effects in a Rat Model of Estrogen-Induced Cholestasis.

Ursodeoxycholic acid (UDCA) is considered the first-choice therapy for cholestatic disorders. To enhance solubility and exploit specific transporters in liver, we synthesized a new galactosyl pro-drug of UDCA (UDCAgal). Ethinylestradiol (EE)-induced cholestasis was used to study and compare the effects of UDCAgal with UDCA on bile flow, hepatic canalicular efflux transporter expression, and inflammation. UDCAgal resulted quite stable both at pH 7.4 and 1.2 and regenerated the parent drug after incubation in human plasma. Its solubility, higher than UDCA, was pH- and temperature-independent. UDCAgal displayed a higher cell permeation compared to UDCA in liver HepG2 cells. Moreover, in cholestatic rats, UDCAgal showed a higher potency compared to UDCA in reducing serum biomarkers (AST, ALT, and ALP) and cytokines (TNF-α and IL-1β). The higher effect of UDCAgal on the increase in bile salt export pump and multidrug resistance-associated protein 2 transcription indicated an improved spillover of bile acids from the liver. UDCAgal showed a reduction in CCL2, as well as TNF-α, IL-1β, and cyclooxygeanse-2 mRNAs, indicating a reduction in hepatic neutrophil accumulation and inflammation. Moreover, UDCAgal, similarly to UDCA, heightens bile flow and modulates biliary acids secretion. These results indicate that UDCAgal has a potential in the treatment of cholestatic disease.

Resveratrol modifies biliary secretion of cholephilic compounds in sham-operated and cholestatic rats.

AIMTo investigate the effect of resveratrol on biliary secretion of cholephilic compounds in healthy and bile duct-obstructed rats.METHODSResveratrol (RSV) or saline were administered to rats by daily oral gavage for 28 d after sham operation or reversible bile duct obstruction (BDO). Bile was collected 24 h after the last gavage during an intravenous bolus dose of the Mdr1/Mrp2 substrate azithromycin. Bile acids, glutathione and azithromycin were measured in bile to quantify their level of biliary secretion. Liver expression of enzymes and transporters relevant for bile production and biliary secretion of major bile constituents and drugs were analyzed at the mRNA and protein levels using qRT-PCR and Western blot analysis, respectively. The TR-FRET PXR Competitive Binding Assay kit was used to determine the agonism of RSV at the pregnane X receptor.RESULTSRSV increased bile flow in sham-operated rats due to increased biliary secretion of bile acids (BA) and glutathione. This effect was accompanied by the induction of the hepatic rate-limiting transporters for bile acids and glutathione, Bsep and Mrp2, respectively. RSV also induced Cyp7a1, an enzyme that is crucial for bile acid synthesis; Mrp4, a transporter important for BA secretion from hepatocytes to blood; and Mdr1, the major apical transporter for xenobiotics. The findings were supported by increased biliary secretion of azithromycin. The TR-FRET PXR competitive binding assay confirmed RSV as a weak agonist of the human nuclear receptor PXR, which is a transcriptional regulator of Mdr1/Mrp2. RSV demonstrated significant hepatoprotective properties against BDO-induced cirrhosis. RSV also reduced bile flow in BDO rats without any corresponding change in the levels of the transporters and enzymes involved in RSV-mediated hepatoprotection.CONCLUSIONResveratrol administration for 28 d has a distinct effect on bile flow and biliary secretion of cholephilic compounds in healthy and bile duct-obstructed rats.

Reduced triglyceride accumulation due to overactivation of farnesoid X receptor signaling contributes to impaired liver regeneration following 50% hepatectomy in extra‑cholestatic liver tissue.

The aim of the present study was to investigate the role of triglyceride metabolism in the effect of obstructive cholestasis on liver regeneration following 50% partial hepatectomy (PH). Obstructive cholestatic rat models were achieved via ligation of the common bile duct (BDL). Following comparisons between hepatic pathological alterations with patients with perihilar cholangiocarcinoma, rats in the 7day post‑BDL group were selected as the BDL model for subsequent experiments. Liver weight restoration, proliferating cell nuclear antigen labeling index, cytokine and growth factor expression levels, and hepatic triglyceride content were evaluated to analyze liver regeneration post‑PH within BDL and control group rats. The results of the present study revealed that obstructive cholestasis impaired liver mass restoration, which occurred via inhibition of early stage hepatocyte proliferation. In addition, reduced triglyceride content and inhibited expression of fatty acid β‑oxidation‑associated genes, peroxisome proliferator activated receptor α and carnitine palmitoyltransferase, were associated with an insufficient energy supply within the BDL group post‑PH. Notably, the expression levels of fatty acid synthesis‑associated genes, including sterol‑regulatory element‑binding protein‑1c, acetyl‑coA carboxylase 1 and fatty acid synthase were also reduced within the BDL group, which accounted for the reduced triglyceride content and fatty acid utilization. Further investigation revealed that overactivated farnesoid X receptor (FXR) signaling may inhibit fatty acid synthesis within BDL group rats. Collectively, the role of triglycerides in liver regeneration following PH in extra‑cholestatic livers was identified in the present study. Additionally, the results indicated that overactivated FXR signaling‑induced triglyceride reduction is associated with insufficient energy supply and therefore contributes to the extent of impairment of liver regeneration following PH within extra‑cholestatic livers.

Adenovirus-mediated human aquaporin-1 expression in hepatocytes improves lipopolysaccharide-induced cholestasis.

Lipopolysaccharides (LPS) are known to cause cholestasis in sepsis. There is evidence that a defective expression of canalicular aquaporin water channels contributes to bile secretory failure in LPS-induced cholestasis. Thus, we studied whether the hepatic adenovirus-mediated transfer of human aquaporin-1 gene (haqp1) can improve the cholestasis induced by LPS. Adenoviral vector encoding hAQP1 (AdhAQP1) or control vector was administered to rats by retrograde intrabiliary infusion. Hepatocyte canalicular hAQP1 expression was assessed by liver immunostaining and immunoblotting in purified plasma membranes. LPS reduced bile flow and biliary bile acid excretion by 30% and 45%, respectively. AdhAQP1-treatment normalized both bile flow and biliary bile acid excretion in LPS-induced cholestasis. Moreover, markedly elevated serum bile acid levels in cholestatic rats, were also normalized with the AdhAQP1 hepatic transduction. Bile flow and serum or biliary bile acids in normal rats were not significantly altered by AdhAQP1. AdhAQP1 delivery unaffected the downregulated protein expression of canalicular bile salt export pump (BSEP/ABCB11) in cholestasis, but improved its transport activity restoring reduced canalicular cholesterol content. Our data suggest that the adenovirus-mediated hepatocyte hAQP1 expression improves LPS-induced cholestasis in rats by stimulating the BSEP/ABCB11-mediated biliary bile acid excretion; a finding that might contribute to the understanding and treatment of sepsis-associated cholestatic diseases. © 2017 IUBMB Life, 69(12):978-984, 2017.

Melatonin inhibits hypothalamic gonadotropin-releasing hormone release and reduces biliary hyperplasia and fibrosis in cholestatic rats.

Melatonin is a hormone produced by the pineal gland with increased circulating levels shown to inhibit biliary hyperplasia and fibrosis during cholestatic liver injury. Melatonin also has the capability to suppress the release of hypothalamic gonadotropin-releasing hormone (GnRH), a hormone that promotes cholangiocyte proliferation when serum levels are elevated. However, the interplay and contribution of neural melatonin and GnRH to cholangiocyte proliferation and fibrosis in bile duct-ligated (BDL) rats have not been investigated. To test this, cranial levels of melatonin were increased by implanting osmotic minipumps that performed an intracerebroventricular (ICV) infusion of melatonin or saline for 7 days starting at the time of BDL. Hypothalamic GnRH mRNA and cholangiocyte secretion of GnRH and melatonin were assessed. Cholangiocyte proliferation and fibrosis were measured. Primary human hepatic stellate cells (HSCs) were treated with cholangiocyte supernatants, GnRH, or the GnRH receptor antagonist cetrorelix acetate, and cell proliferation and fibrosis gene expression were assessed. Melatonin infusion reduced hypothalamic GnRH mRNA expression and led to decreased GnRH and increased melatonin secretion from cholangiocytes. Infusion of melatonin was found to reduce hepatic injury, cholangiocyte proliferation, and fibrosis during BDL-induced liver injury. HSCs supplemented with BDL cholangiocyte supernatant had increased proliferation, and this increase was reversed when HSCs were supplemented with supernatants from melatonin-infused rats. GnRH stimulated fibrosis gene expression in HSCs, and this was reversed by cetrorelix acetate cotreatment. Increasing bioavailability of melatonin in the brain may improve outcomes during cholestatic liver disease.NEW & NOTEWORTHY We have previously demonstrated that GnRH is expressed in cholangiocytes and promotes their proliferation during cholestasis. In addition, dark therapy, which increases melatonin, reduced cholangiocyte proliferation and fibrosis during cholestasis. This study expands these findings by investigating neural GnRH regulation by melatonin during BDL-induced cholestasis by infusing melatonin into the brain. Melatonin infusion reduced cholangiocyte proliferation and fibrosis, and these effects are due to GNRH receptor 1-dependent paracrine signaling between cholangiocytes and hepatic stellate cells.

D-chiro-inositol effectively attenuates cholestasis in bile duct ligated rats by improving bile acid secretion and attenuating oxidative stress.

Cholestatic liver diseases are important causes of liver cirrhosis and liver transplantation, but few drugs are available for treatment. D-chiro-inositol (DCI), an isomer of inositol found in many Leguminosae plants and in animal viscera, is used clinically for the treatment of polycystic ovary syndrome (PCOS) and diabetes mellitus. In this study, we investigated whether DCI exerted an anti-cholestatic effect and its underlying mechanisms. A cholestatic rat model was established via bile duct ligation (BDL). After the surgery, the rats were given DCI (150 mg·kg-1·d-1) in drinking water for 2 weeks. Oral administration of DCI significantly decreased the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and attenuated bile duct proliferation, parenchymal necrosis and fibrosis in BDL rats. Furthermore, DCI treatment significantly increased the serum and bile levels of total bile acid (TBA), and decreased TBA levels in the liver. Moreover, DCI treatment significantly increased expression of the genes encoding bile acid transporters BSEP (Abcb11) and MRP2 (Abcc2) in liver tissues. DCI treatment also markedly decreased hepatic CD68 and NF-kappaB (NF-κB) levels, significantly decreased the serum and hepatic MDA levels, markedly increased superoxide dismutase activity in both serum and liver tissues. Using whole-genome oligonucleotide microarray, we revealed that DCI treatment altered the expression profiles of oxidation reduction-related genes in liver tissues. Collectively, DCI effectively attenuates BDL-induced hepatic bile acid accumulation and decreases the severity of injury and fibrosis by improving bile acid secretion, repressing inflammation and decreasing oxidative stress. The results suggest that DCI might be beneficial for patients with cholestatic disorders.

Hepatoprotective effect of boldine in a bile duct ligated rat model of cholestasis/cirrhosis

Liver fibrosis is a debilitating disease associated with chronic liver injury. Oxidative stress is known as a pivotal mechanism in the initiation and propagation of liver fibrosis. Boldine is a potent antioxidant molecule with several pharmacological effects. The current investigation aimed to evaluate the effect of boldine in bile duct ligated (BDL) rats as a model of cholestasis and cirrhosis. BDL animals received boldine (5, 10, and 20mg/kg/day, oral) for 14days (Cholestatic rats) and 28days (Cirrhotic rats). The serum biomarkers of liver injury were drastically increased in the BDL group. Moreover, the level of oxidative stress markers was significantly increased in BDL animals. Severe bridging fibrosis, tissue necrosis, and inflammation were also detected in BDL rats. It was found that boldine (5, 10, and 20mg/kg/day, oral) restored the BDL-induced depletion of glutathione content and tissue antioxidant capacity. Moreover, histopathological changes and collagen deposition were markedly attenuated by the boldine treatment. The beneficial effects of boldine administration in cholestasis/cirrhosis might be associated with anti-fibrotic properties via antioxidant activities.

Increased hepatic ABCA1 transporter is associated with hypercholesterolemia in a cholestatic rat model and primary biliary cholangitis patients.

Hepatic ATP-binding cassette A1 (ABCA1) transporter is the modulator of intrahepatic cholesterol levels via the efflux of cholesterol into plasma. This study aimed to determine the expression of hepatic ABCA1 levels in a cholestatic rat model and patients with primary biliary cholangitis (PBC). A cholesterol efflux study was conducted with Abca1 knock down using siRNA in WIF9 cells. Cholesterol levels in the ABCA1 siRNA cells in the medium were significantly decreased compared with those in controls (P<0.05). Hepatic ABCA1 mRNA levels were significantly higher in BDL rats than in control rats (P<0.05). Furthermore, the protein expression level of hepatic ABCA1 was also significantly increased by 200% in BDL rats (P<0.05). In PBC patients, expression of hepatic ABCA1 mRNA was 2.2-fold higher than that in controls (P<0.05). The level of hepatic liver X receptor (LXR)β mRNA was correlated with ABCA1 mRNA levels in PBC patients. The expression of hepatic ABCA1 transporter was upregulated in both the cholestatic rat model and PBC patients. Upregulated hepatic ABCA1 may lead to efflux of cholesterol into plasma, thus explaining the mechanism of cholestasis leading to hypercholesterolemia.

Notch signaling pathway participates in the differentiation of hepatic progenitor cells into bile duct epithelial cells and progression of hepatic fibrosis in cholestatic liver fibrosis rat

Objective: To investigate differentiation direction of hepatic progenitor cells (HPCs) in cholestatic liver fibrosis (CLF), and the role of Notch signaling pathway in the differentiation of HPCs. Methods: A CLF rat model was established by bile duct ligation (BDL) followed by monitoring changes of Notch signal pathway and the cellular origin of proliferating cholangiocytes. After intraperitoneal injection of DAPT (a Notch signaling inhibitor) after bile duct ligation, the progress of liver fibrosis and the proliferation of cholangiocytes after inhibition of the Notch pathway were analyzed. Results: Data showed that bile duct proliferation gradually increased along with inflammatory cell infiltration and proliferating bile duct cells surrounded by abundant collagen in the BDL group. Immunostaining confirmed markedly increased expression of CK19, OV6, Sox9 and EpCAM. In addition, RT-PCR results showed that Notch signaling pathway was activated significantly. Once the Notch signaling pathway was inhibited by DAPT, bile duct proliferation markedly suppressed along with significantly decreased the mRNA expression of CK19, OV6, Sox9 and EpCAM, compared with BDL group [(10.2±0.7) vs. (22.3±0.8), (7.6±1.5) vs. (18.1±3.7), (1.4±0.4) vs. (4.1±1.1), (1.3±0.3) vs. (5.0±1.4), respectively, P<0.01]. Moreover, liver fibrosis was also reduced significantly. Conclusion: Notch signaling activation is required for HPCs differentiation into cholangiocytes in CLF and inhibition of the Notch signaling pathway may offer a therapeutic option for treating CLF.

Adipose-derived mesenchymal stem cells slow disease progression of acute-on-chronic liver failure.

A serious complication of chronic hepatic insufficiency is acute-on-chronic liver failure, a recognized syndrome characterized by acute decompensation of cirrhosis and organ/system failure. We investigated the use of adipose-derived mesenchymal stem cells (AD-MSCs) in an experimental model of acute-on-chronic liver failure, developed by microsurgical extrahepatic cholestasis in rats. Rats undergoing microsurgical extrahepatic cholestasis were treated by intraparenchymal liver injection of human or rat AD-MSCs, undifferentiated or previously differentiated in vitro toward the hepatocyte lineage. The groups treated with rat AD-MSCs showed less ascites, lower hepato- and splenomegaly, less testicular atrophy, and an improvement in serum biochemical hepatic parameters. There was also an improvement in histological liver changes, in which the area of fibrosis and bile duct proliferation were significantly decreased in the group treated with predifferentiated rat AD-MSCs. In conclusion, an isograft of hepatocyte-predifferentiated AD-MSCs injected intraparenchymally 2 weeks after microsurgery in extrahepatic cholestatic rats prevents secondary complications of acute-on-chronic hepatic failure. These data support the potential use of autologous AD-MSCs in the treatment of human cholestasis, and specifically of newborn biliary atresia, which could be beneficial for patients awaiting transplant.

Atorvastatin does not protect against ischemia-reperfusion damage in cholestatic rat livers

BackgroundExtrahepatic cholestasis sensitizes the liver to ischemia/reperfusion (I/R) injury during surgery for perihilar cholangiocarcinoma. It is associated with pre-existent sterile inflammation, microvascular perfusion defects, and impaired energy status. Statins have been shown to protect against I/R injury in normal and steatotic mouse livers. Therefore, the hepatoprotective properties of atorvastatin were evaluated in a rat model of cholestatic I/R injury.MethodsMale Wistar rats were subjected to 70% hepatic ischemia (during 30 min) at 7 days after bile duct ligation. Rats were randomized to atorvastatin treatment or vehicle-control in three test arms: (1) oral treatment with 5 mg/kg during 7 days after bile duct ligation; (2) intravenous treatment with 2.5, 5, or 7.5 mg/kg at 24 h before ischemia; and (3) intravenous treatment with 5 mg/kg at 30 min before ischemia. Hepatocellular damage was assessed by plasma alanine aminotransferase (ALT) and histological necrosis.ResultsI/R induced severe hepatocellular injury in the cholestatic rat livers (~10-fold increase in ALT at 6 h after I/R and ~30% necrotic areas at 24 h after I/R). Both oral and intravenous atorvastatin treatment decreased ALT levels before ischemia. Intravenous atorvastatin treatment at 5 mg/kg at 24 h before ischemia was the only regimen that reduced ALT levels at 6 h after reperfusion, but not at 24 h after reperfusion. None of the tested regimens were able to reduce histological necrosis at 24 h after reperfusion.ConclusionPre-treatment with atorvastatin did not protect cholestatic livers from hepatocellular damage after I/R. Clinical studies investigating the role of statins in the protection against hepatic I/R injury should not include cholestatic patients with perihilar cholangiocarcinoma. These patients require (pharmacological) interventions that specifically target the cholestasis-associated hepatopathology.

Colestasis microquirúrgica: un modelo de inflamación esplácnica

Introduction: The ligation of the common bile duct in rats is a reliable experimental model of the cirrhosis associated with portal hypertension. This condition induces a splanchnic inflammation, in which mast cells might be involved. Methods: In order to verify this hypothesis, were measured the density of connective tissue mast cells (RMCP-I) (RMCP: rat mast cell protease) and mucosal mast cells (RMCP-II) in the splanchnic organs of pseudo-operated rats and in rats with microsurgical extrahepatic cholestasis. Results: A statistically significant increase (p<0.05) in the density of both types cells in the liver and ileum of cholestatic rats with reference to the pseudo-operated rats was observed. Conclusions: These results suggest the possible key role that mast cells could play in the visceral organs. Being splanchnic modulators of the inflammatory response, angiogenesis and fibrotic remodelling, which occur in cirrhosis.

Protective effects of petroleum ether extracts of Herpetospermum caudigerum against α-naphthylisothiocyanate-induced acute cholestasis of rats

Ethnopharmacological relevanceThe ripe seeds of Herpetospermum caudigerum have been used in Tibetan folk medicine for treatment of bile or liver diseases including jaundice, hepatitis, intumescences or inflammation. Previously reports suggested that the seed oil and some lignans from H. caudigerum exhibited protective effects against carbon tetrachloride (CCl4)-induced hepatic damage in rats, which may be related to their free radical scavenging effect. However, the protective effect of H. caudigerum against cholestasis is still not revealed. The aim of the present study was to investigate the pharmacological effect and the chemical constituents of the petroleum ether extract (PEE) derived from H. caudigerum against α-naphthylisothiocyanate (ANIT)-induced acute cholestasis in rats. Materials and methodsMale cholestatic Sprague-Dawley (SD) rats induced by ANIT (60mg/kg) were orally administered with PEE (350, 700 and 1400mg/kg). Levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-Glutamyl transpeptidase (γ-GTP), total bilirubin (TBIL), direct bilirubin (DBIL) and total bile acid (TBA), as well as bile flow, and histopathological assay were evaluated. Hepatic malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione S-transferase (GST), and nitric monoxide (NO) in liver were measured to explore the possible protective mechanisms. Phytochemical analysis of PEE was performed by gas chromatography-mass spectrometer (GC-MS). ResultsPEE have exhibited significant and dose-dependent protective effect on ANIT-induced liver injury by reduce the increases in serum levels of ALT, AST, ALP, γ-GTP, TBIL, DBIL and TBA, restore the bile flow in cholestatic rats, and reduce the severity of the pathological tissue damage induced by ANIT. Hepatic MDA, MPO and NO contents in liver tissue were reduced, while SOD and GST activities were elevated in liver tissue. 49 compounds were detected and 39 of them were identified by GC-MS analysis, in which long-chain fatty acids were the main constituents. ConclusionsPEE exhibited a dose-dependently protective effect on ANIT-induced liver injury in cholestatic rats with the potential mechanism of attenuated oxidative stress in the liver tissue, and the possible active compounds were long-chain fatty acids.

Comparison of bioavailability and efficacy of silymarin and silymarin-loaded solid dispersion formulation in cholestatic rats

Comparison of bioavailability and efficacy of silymarin and silymarin-loaded solid dispersion formulation in cholestatic rats