Biliary Epithelial Cell Injury Research Articles

The association of immune-related genes and the potential role of IL10 with biliary atresia.

Biliary atresia (BA) is a severe immune-related disease that is characterized by biliary obstruction and cholestasis. The etiology of BA is unclear, our aim was to explore the relationship between biliary tract inflammation and immune-related genes. We selected 14 SNPs in 13 immune-related genes and investigated their associations with BA by using a large case‒control cohort with a total of 503 cases and 1473 controls from southern China. SNP rs1518111 in interleukin10 (IL10) was identified as associated with BA (P = 5.79E-03; OR: 0.80; 95% CI: 0.68-0.94). The epistatic effects of the following pairwise interactions among these SNPs were associated with BA: signal transducer and activator of transcription 4 (STAT4) and chemokine (C-X-C motif) ligand 3 (CXCL3); STAT4 and damage-regulated autophagy modulator1 (DRAM1); CXCL3 and RAD51 paralog B (RAD51B); and interferon gamma (IFNG) and interleukin26 (IL26). Furthermore, we explored the potential role of IL-10 in the pathogenesis of the neonatal mouse model of BA. IL-10 effectively prevented biliary epithelial cell injury and biliary obstruction in murine BA as well as inhibit the activation of BA-related immune cells. In conclusion, this study provided strong evidence implicating IL10 as a susceptibility gene for BA in the southern Chinese population. This study provided strong evidence implicating IL10 as a susceptibility gene for BA in the southern Chinese population. This study could infer that IL-10 may play a protective role in BA mouse model. We found that four SNPs (rs7574865, rs352038, rs4622329, and rs4902562) have genetic interactions.

Bile acid-activated macrophages promote biliary epithelial cell proliferation through integrin αvβ6 upregulation following liver injury.

Cholangiopathies caused by biliary epithelial cell (BEC) injury represent a leading cause of liver failure. No effective pharmacologic therapies exist, and the underlying mechanisms remain obscure. We aimed to explore the mechanisms of bile duct repair after targeted BEC injury. Injection of intermedilysin into BEC-specific human CD59 (hCD59) transgenic mice induced acute and specific BEC death, representing a model to study the early signals that drive bile duct repair. Acute BEC injury induced cholestasis followed by CCR2+ monocyte recruitment and BEC proliferation. Using microdissection and next-generation RNA-Seq, we identified 5 genes, including Mapk8ip2, Cdkn1a, Itgb6, Rgs4, and Ccl2, that were most upregulated in proliferating BECs after acute injury. Immunohistochemical analyses confirmed robust upregulation of integrin αvβ6 (ITGβ6) expression in this BEC injury model, after bile duct ligation, and in patients with chronic cholangiopathies. Deletion of the Itgb6 gene attenuated BEC proliferation after acute bile duct injury. Macrophage depletion or Ccr2 deficiency impaired ITGβ6 expression and BEC proliferation. In vitro experiments revealed that bile acid-activated monocytes promoted BEC proliferation through ITGβ6. Our data suggest that BEC injury induces cholestasis, monocyte recruitment, and induction of ITGβ6, which work together to promote BEC proliferation and therefore represent potential therapeutic targets for cholangiopathies.

Effects of bone marrow mesenchymal stem cells combined with normothermic mechanical perfusion on biliary epithelial cells donated after cardiac death in rats

To investigate the protective effect of bone marrow mesenchymal stem cells (BMMSC) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) in rats receiving donation after cardiac death (DCD) donor liver transplantation. The BMMSC were isolated from male Sprague-Dawley (SD) rats aged 2-3 weeks and weighing 40-60 g, and then cultured, identified and expanded to the third generation in vitro. Male SD rats aged 6-8 weeks and weighing 200-220 g were divided into sham-operated group (Sham group), static cold storage (SCS group), simple NMP group (NMP group) and BMMSC combined with NMP group (BMMSC+NMP group) by random number table method with 44 rats in each group. The DCD donor liver transplantation models in rats were reproduced with 30-minute warm ischemic time. While the rats in Sham group merely received perihepatic ligaments-separation, which did not affect their liver blood supply, and then their incisions were sutured after 30 minutes. The DCD donor grafts in SCS group were preserved in the University of Wisconsin (UW) cold storage solution for 4 hours. While the DCD donor grafts in the NMP group and the BMMSC+NMP group were perfused with the DMEM/F12-based culture solution or combined with BMMSC for 4 hours through the established ex vivo NMP system. The orthotopic liver transplantation model was reproduced, and the survival rate of the recipients was observed at 0, 1, 7 and 14 days after liver transplantation. The biochemical liver function of rats in different groups was determined at each time point after operation. The morphological changes in bile ducts of liver grafts were observed by hematoxylin-eosin (HE) staining, and the expression of cytokeratin 19 (CK19) was determined qualitatively by immunohistochemistry and quantitatively by Western Blot after protein extraction from BEC in liver samples. The morphology, differentiation function and phenotypic identification of BMMSC confirmed that the stem cells used in this experiment were standard BMMSC. The survival rates of rats in the NMP group and the BMMSC+NMP group were significantly higher than that in the SCS group at 0, 1, 7 and 14 days after operation. The increase was more significant in the BMMSC+NMP group, with 100% on postoperative day (POD) 0, and the 14-day survival rate was still significantly higher than that in the SCS group and the NMP group [80.0% (16/20) vs. 20.0% (4/20), 70.0% (14/20), both P < 0.05]. As the time after liver transplantation prolonged, the liver function parameters of rats in the SCS group were deteriorated gradually, which reached the peak at 1-7 days after operation. The damage of biliary tissue increased gradually under the microscope, and the injury was most serious on POD 7 in the SCS group, showing a lot of balloon-like changes in hepatocytes, with obvious bile duct dilatation accompanied by large area inflammatory cell infiltration. Immunohistochemistry and Western Blot showed that the expression of CK19 in BEC cytoplasm was decreased gradually in the SCS group, reached the lowest on POD 7, and then gradually increased. The BMMSC+NMP group and the NMP group were significantly better than the SCS group in terms of liver function, pathological injury of biliary tract and CK19 expression in BEC, and the improvement was more significant in the BMMSC+NMP group. These results suggested that the protective effects of BMMSC combined with NMP on BEC was significantly better than that of the SCS and NMP. Preservation of rat DCD donor liver by BMMSC combined with NMP can reduce the BEC injury after liver transplantation significantly, thus improving both the prognosis and the survival rate after transplantation.

Progress in research of mechanism of biliary epithelial cell injury in primary biliary cholangitis

Progress in research of mechanism of biliary epithelial cell injury in primary biliary cholangitis

The British Society of Gastroenterology/UK-PBC primary biliary cholangitis treatment and management guidelines

Primary biliary cholangitis (formerly known as primary biliary cirrhosis, PBC) is an autoimmune liver disease in which a cycle of immune mediated biliary epithelial cell injury, cholestasis and progressive fibrosis...

Partial characterization of proapoptotic action of biliary deteriorated lipids on biliary epithelial cells in pancreaticobiliary diseases

Lysophosphatidylcholine (LPC), a derivative of phosphatidylcholine (PC) hydrolyzed by phospholipase A2 (PLA2), is reported to be increased in bile of the patients with pancreaticobiliary maljunction or intrahepatic cholelithiasis, both of which are major risk factors for biliary tract cancers with undefined etiology. To investigate the influence of LPC on biliary epithelial cells (BECs), a human cholangiocarcinoma cell line HuCCT-1 and an immortalized human BECs line MMNK-1 were treated by LPC in vitro. The treatment of LPC exhibited cytotoxicity with significant induction of apoptosis. In addition to upregulation of Fas receptor mRNA, the activities of caspase-8 and -3 were significantly increased by LPC treatment. We also observed upregulation of Bax mRNA and significant activation of caspase-9. Interestingly, LPC significantly upregulated G2A, a member of transmembrane G protein-coupled receptor family at mRNA and protein levels, and 9-hydroxyoctadecaduenoic acid (9HODE), an oxidized free fatty acid that functions as a ligand for G2A dramatically reduced cell viability when treated together with LPC. These data suggest that PLA2, which catalyzes the hydrolysis of PC to yield LPC and free fatty acid, is supposed to be an important etiological factor in BECs injury in pancreaticobiliary maljunction or intrahepatic cholelithiasis.

Effects of warm ischemia time on biliary injury in rat liver transplantation

To investigate the effect of different secondary warm ischemia time (SWIT) on bile duct injury in liver-transplanted rats. Forty-eight male inbred Sprague-Dawley rats were randomly assigned into four groups: a sham-operation group and three groups with secondary biliary warm ischemia time of 0 min, 10 min and 20 min. A rat model of autologous liver transplantation under ether anesthesia was established, and six rats were killed in each group and blood samples and the median lobe of the liver were collected for assay at 6 h and 24 h after hepatic arterial reperfusion. With prolongation of biliary warm ischemia time, the level of vascular endothelial growth factor-A was significantly decreased, and the value at 24 h was higher than that at 6 h after hepatic arterial reperfusion, but with no significant difference. The extended biliary SWIT led to a significant increase in bile duct epithelial cell apoptosis, and a decrease in the number of blood vessels, the bile duct surrounding the blood vessels and bile duct epithelial cell proliferation in the early postoperative portal area. Pathologic examinations showed that inflammation of the rat portal area was aggravated, and biliary epithelial cell injury was significantly worsened. A prolonged biliary warm ischemia time results in aggravated injury of the bile duct and the surrounding vascular plexus in rat autologous orthotopic liver transplantation.

Copper impairs biliary epithelial cells and induces lipid peroxidation, protein oxidation and oxidative DNA damage in the isolated perfused rat liver

Event Abstract Back to Event Copper impairs biliary epithelial cells and induces lipid peroxidation, protein oxidation and oxidative DNA damage in the isolated perfused rat liver Albena Alexandrova1*, Lubomir Petrov1, Margarita Kirkova1, Elina Tzvetanova1, Almira Georgieva1, Galina Nenkova1 and Stefani Vircheva1 1 Bulgarian Academy of Sciences, Institute of Physiology, Bulgaria The liver can be damaged by a wide variety of toxicants, including heavy metals. Although copper is an essential micronutrient, it can be harmful in excess. The isolated perfused rat liver model is considered to be a sensitive system for studying chemically-induced hepatotoxicity. In literature, however, there are rather scarce data about copper effects on isolated perfused rat liver. The hepatic injury, caused by copper overload is hypothesized to result from its role in the generation of reactive oxygen species (ROS). Being highly reactive ROS are able to impair all cellular constituents – lipids, proteins and DNA. The aim of the present study was to investigate whether CuSO4 (0.01, 0.03, and 0.1 mM) in perfused rat liver (for 60 min) was associated with lipid peroxidation, protein oxidation, and oxidative DNA damage, as well as with biliary epithelial cell injury. The results regarding liver viability and function (copper decreased the portal flow and bile secretion and led to liver swelling) showed a concentration-response effect. The highest tested concentration of copper caused complete blockage of portal flow and bile production within 30 min of perfusion. In confirmation of copper-induced injury, increased leakage of lactate dehydrogenase, aspartate transaminase, and alanine transaminase (indexes of hepatocellular membrane integrity) into perfusate were registered. Compared to controls, 0.01 and 0.03mM concentrations of copper increased the amount of thiobarbituric acid reacting substances, a marker of lipid peroxidation, tissue protein carbonyl groups, an index of protein oxidation, and 8-oxo-7,8-dihydro-2-deoxyguanosine, a marker of oxidative DNA damage; the biliary levels of gamma-glutamyltransferase, an index of biliary epithelial cell integrity, were also increased. The results suggest that copper toxicity in the isolated perfused rat liver could be a consequence of its ability to impair the integrity of biliary epithelial cells and is associated with the enhanced lipid peroxidation, protein oxidation, and oxidative DNA damage. Keywords: Copper, Bile, Oxidative Stress Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Poster Topic: Xenobiotic toxicity Citation: Alexandrova A, Petrov L, Kirkova M, Tzvetanova E, Georgieva A, Nenkova G and Vircheva S (2010). Copper impairs biliary epithelial cells and induces lipid peroxidation, protein oxidation and oxidative DNA damage in the isolated perfused rat liver. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00099 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Albena Alexandrova, Bulgarian Academy of Sciences, Institute of Physiology, Sofia, Bulgaria, a_alexandrova_bas@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva Google Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva Google Scholar Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva PubMed Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Copper impairs biliary epithelial cells and induces protein oxidation and oxidative DNA damage in the isolated perfused rat liver

Copper is one of the major metals causing environmental contamination. Previous studies showed that copper induced toxic effects in isolated perfused rat liver models and these effects were associated with lipid peroxidation. Here we investigated whether effects of copper (at concentrations of 0.01, 0.03, and 0.1 mM of Cu 2+ in Krebs–Henseleit buffer perfusing the isolated rat liver for 60 min), were associated with biliary epithelial cell injury, as well as protein oxidation and oxidative DNA damage. The highest concentration of copper in perfusate (0.1 mM) did not allow complete evaluation of all parameters because it blocked portal flow within 30 min of perfusion, indicating severe microcirculatory disturbances. Further, copper decreased secretion of bile and it increased lactate dehydrogenase, aspartate transaminase, and alanine transaminase leakage into perfusate as well as liver weight in a dose-dependent manner. Biliary γ-glutamyltransferase, an index of biliary epithelial cell integrity increased similarly at 0.01 and 0.03 mM copper concentrations in perfusate. Compared to controls, 0.01 and 0.03 mM concentrations of copper increased the amount of thiobarbituric acid reacting substances, a marker of lipid peroxidation, tissue protein carbonyl groups, an index of protein oxidation, and 8-oxo-7,8-dihydro-2′-deoxyguanosine, a marker of oxidative DNA damage. The results suggest that toxic effects of copper in the isolated perfused rat liver may involve biliary epithelial cells and they are associated with lipid peroxidation, protein oxidation, and oxidative DNA damage.

Mitochondrial Dysfunction Occurs before Transport or Tight Junction Deficits in Biliary Epithelial Cells Exposed to Bile from Methylenedianiline-Treated Rats

Methylenedianiline (DAPM) rapidly injures biliary epithelial cells (BEC) in vivo. Prior to evident BEC injury, biliary glucose and inorganic phosphate appreciably rise, which could stem from loosened tight junctions (TJ). Concurrently, ultrastructural abnormalities in BEC mitochondria of DAPM-treated animals are observed, suggesting other impairments. Our objective was to develop an in vitro BEC model to assess the time course of impairments in TJ integrity, glucose uptake, and mitochondrial function following DAPM exposure. We exposed monolayers of primary, polarized rat BEC to bile collected from rats prior to (Basal Bile) or after oral treatment (DAPM-Bile) with 50 mg DAPM/kg. DAPM-Bile collected during 0-60 min (1st Hr) and during 61-120 min (2nd Hr) after treatment was pooled from four to six rats. When monolayers were exposed to 1st Hr DAPM-Bile for 120 min, metabolic activity (XTT assay) decreased approximately 75%, and transepithelial resistance decreased approximately 16% in agreement with an approximately 65% increase in leakage of a glucose analog, methyl-alpha-D-glucopyranoside (AMG), from apical to basolateral media. By 60 min, AMG uptake was decreased approximately 40%. Mitochondrial function was very rapidly compromised, with approximately 120% increases in the green-to-red fluorescence ratio of JC-1 (mitochondrial membrane potential dye) at 15 min and approximately 55% decreases in ATP levels at 30 min. This sequence of events indicates that DAPM impairs BEC mitochondria prior to impairments in glucose uptake or TJ integrity. Thus, our in vitro primary rat BEC/bile exposure model mimics in vivo observations and yields basic information about the time course of events that occur during DAPM-induced injury.

Is extrahepatic biliary atresia related to viral infections?

Extrahepatic biliary atresia is characterized by obliteration or absence of the extrahepatic bile ducts in very young infants and ultimately lead to liver cirrhosis and failure. The cause of this disease is currently unknown. There are several hypotheses for the development of extrahepatic biliary atresia. Failure of development or canalization of extrahepatic biliary tree is the unconfirmed old theory. Perinatal insults like viral infections have been suspected as possible etiologies including reovirus type 3, cytomegalovirus and rotavirus, although none of them has been concluded as playing a significant role in the pathogenesis of this disease. By using cultured murine extrahepatic biliary epithelial cell, a virally-induced, immune mediated extrahepatic biliary epithelial cell injury has been demonstrated in our recent work, which may be implicated in the pathogenesis of extrahepatic biliary atresia. Further studies are needed to develop a curative treatment for this serious disorder in the future.

The utility of noninvasive serologic markers in the management of early allograft rejection in liver transplantation recipients.

Early allograft rejection after orthotopic liver transplantation (OLT) currently requires a biopsy for diagnosis. Alpha-glutathione S-transferase (alpha-GST) and Pi-glutathione S-transferase (Pi-GST) are potential noninvasive markers of hepatocyte and biliary epithelial cell injury. Our aim was to determine the utility of noninvasive serologic markers in the management of early hepatic allograft rejection. Forty-four of 52 consecutive adult patients undergoing primary OLT at the University of Florida were included in the study. All had protocol liver biopsies between days 6 and 8 after OLT. Serum alpha-GST and plasma Pi-GST were determined using a sandwich enzyme immunoassay (Biotrin International, Dublin, Ireland). All biopsy specimens were retrospectively reviewed and scored for rejection and cholestasis. The biopsy specimens were scored for rejection as moderate to severe in 14 patients (group 1) or none to mild in 30 patients (group 2). Group 1 had statistically higher mean levels than group 2 for alpha-GST on days 6, 7, and 9; alanine aminotransferase on days 6 and 9; aspartate aminotransferase (AST) on days 6 and 7; alkaline phosphate (AP) on days 3 through 7, 9, and 10; and gamma-glutamyl transferase on day 3. No differences between groups were seen with Pi-GST or total bilirubin. Between days 6 and 8, the following values were found more frequently in group 1 than group 2: alpha-GST level >15 ng/ml (11/14 vs. 14/30; P<0.01); AST >100 U/L (8/14 vs. 2/30; P=0.002); and AP >120 U/L (14/14 vs. 17/30). Combining AP with either alpha-GST or AST led to improved detection of rejection over any single marker alone. In the first week after the initiation of rejection treatment, alpha-GST was the only marker that accurately predicted response. Serum alpha-GST may be useful in the management of early hepatic allograft rejection. A combination of noninvasive markers may be beneficial to diagnose early hepatic allograft rejection.

Infusion of bile from methylene dianiline-treated rats into the common bile duct injures biliary epithelial cells of recipient rats

Methylene dianiline (4,4′-diaminodiphenylmethane, DAPM) rapidly causes cholestasis and injury to biliary epithelial cells (BEC) in the liver and common bile duct of rats. Our objective was to determine if the proximate toxicants) was present in bile. Bile from DAPM-treated or control rats was infused through the common bile duct of untreated rats via inflow and outflow cannulas for 4 h. Cholestasis, increases in serum constituents, and intrahepatic BEC injury in the livers of DAPM-treated donor rats at 4 h were comparable to previous studies (Kanz et al., Toxicol. Appl. Pharmacol. 117 (1992) 88–97) [1]. BEC injury in the common bile duct of rats receiving DAPM bile or control bile was assessed by point counting. Percent necrosis was > 28% in the common bile duct of rats receiving DAPM bile compared to < 5% in rats receiving control bile. These results indicate that bile is a major route of BEC exposure to DAPM proximate toxicant(s) and demonstrate the utility of a new method for investigating mechanisms of biliary toxicants.

Platelets and α-Naphthylisothiocyanate-Induced Liver Injury

Administration of alpha-naphthylisothiocyanate (ANIT) to rats results in periportal cholangiolitic hepatopathy. Inflammation is a hallmark of the liver injury, and expression of toxicity is dependent on blood neutrophils. The role of other cellular mediators of inflammation in ANIT-induced hepatic insult is unknown. We hypothesized that platelets participate in the expression of ANIT hepatotoxicity. To test this, circulating platelets were decreased by administration of anti-rat platelet serum (PAb) prior to treatment of rats with ANIT. The PAb treatment regimen effectively reduced circulating thrombocytes over the course of the experiment. Twenty-four hours after oral ANIT administration, rats were euthanized and liver injury was estimated by increases in serum alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) activities. Cholestasis was assessed by measurement of serum total bilirubin concentration and bile flow. Reduction in platelet numbers was associated with attenuation of the increases in plasma ALT activity and bilirubin concentration seen after ANIT administration. However, PAb treatment did not attenuate the increase in plasma GGT, a marker of biliary epithelial cell injury. ANIT-induced changes in platelet function were assessed by evaluating platelet aggregation responses in platelet-rich plasma from rats treated with ANIT in vivo. ANIT treatment modestly decreased ex vivo platelet aggregation in response to ADP and collagen stimuli. To address further the role of platelet-derived cyclooxygenase products in ANIT hepatotoxicity, rats were treated with aspirin or ibuprofen. Neither pretreatment ameliorated ANIT-induced hepatic insult. These results suggest that platelets contribute to the expression of ANIT-induced liver injury, but they do not appear to act through the production of cyclooxygenase metabolites.

Menadione and cumene hydroperoxide induced cytotoxicity in biliary epithelial cells isolated from rat liver

Biliary epithelial cells (BEC) and parenchymal cells isolated from normal rat liver were exposed in vitro to a number of toxic compounds. BEC were found to be highly sensitive to concentrations of menadione (100 μM) and cumene hydroperoxide (10 μM) that are usually not effective as toxic agents in hepatocytes under nonnoxic conditions. On the other hand, BEC were not affected by concentrations of carbon tetrachloride or 7-ethoxycoumarin that are known to exert toxic effects on hepatocytes. The development of both menadione- and cumene hydroperoxide-induced toxic injury in BEC followed a common and time-correlated pattern, and included a strong depletion of GSH, depletion of protein thiols and an increase in the extent of cell death. The damage induced by cumene hydroperoxide was found to be independent of lipid peroxidative processes and was prevented by a pre-incubation with desferrioxamine. The cytotoxicity of menadione was further exacerbated by dicoumarol but was not prevented by desferrioxamine or promethazine. The mechanisms underlying BEC injury and death induced by the quinone and by the organic hydroperoxide are discussed in relation to the known biochemical characteristics of BEC.