Cytochrome P450 3A4 activity and genetic variants as predictors of liver failure in patients with obstructive jaundice.

Liver failure in patients with obstructive jaundice is a significant contributor to mortality within this patient cohort. The exact mechanism and triggers of this occurrence are yet to be fully understood. With this in mind, our study aimed to assess the correlation between the urinary 6 ß-OHC/C ratio and various biochemical parameters of liver function. Furthermore, we conducted genotyping of CYP3A4*22 (rs35599367), CYP3A5*3 (rs776746) polymorphic markers to investigate the potential effects of their variants on the probability of liver failure in obstructive jaundice. Our study included 75 patients diagnosed with severe obstructive jaundice. All test subjects underwent functional liver tests, and control blood tests were administered on the seventh day following biliary decompression. Patients were categorized into two groups: group 1 - patients without liver failure (n = 60) and group 2 - patients with liver failure (n = 15). Laboratory indexes such as 6 ß -OHC concentration and 6 ß- OHC/cortisol ratio can serve as significant predictors of liver failure in patients with moderate and severe degree obstructive jaundice after biliary decompression. Based on the study of "wild" and polymorphic variants of CYP3A4*22 (CC and CT) and polymorphism of CYP3A5*3A6986G (GG, GA, AA), it was discovered that liver failure in the CYP3A4*22 variant may be associated with the CC genotype, and in the CYP3A5*3 variant - with the GA genotype. Hence, the determination of 6ß- OHC concentration and 6ß- OHC/C ratio, as well as the analysis of polymorphic and "wild" variants of CYP3A4*22 (CC and CT) and CYP3A5*3 polymorphism A6986G (GG, GA, AA), may play a crucial role in predicting liver failure in patients with obstructive jaundice.

Limb expression 1-like (LIX1L) protein promotes cholestatic liver injury by regulating bile acid metabolism.

BACKGROUND & AIMS: Cholestatic liver diseases comprise a variety of disorders of bile formation and/or flow which generally result in progressive hepatobiliary injury. Regulation of bile acid (BA) synthesis and homeostasis is a promising strategy for the treatment of cholestatic liver disease. Limb expression 1-like protein (LIX1L) plays an important role in post-transcriptional gene regulation, yet its role in cholestatic liver injury remains unclear. METHODS: LIX1L expression was studied in patients with primary sclerosing cholangitis (PSC) or primary biliary cholangitis (PBC), and 3 murine models of cholestasis (bile duct ligation [BDL], Mdr2 knockout [Mdr2-/-], and cholic acid [CA] feeding). Lix1l knockout mice were employed to investigate the function of LIX1L in cholestatic liver diseases. Chromatin immunoprecipitation assays were performed to determine whether Egr-1 bound to the Lix1l promoter. MiRNA expression profiling was analyzed by microarray. An adeno-associated virus (AAV)-mediated hepatic delivery system was used to identify the function of miR-191-3p in vivo. RESULTS: LIX1L expression was increased in the livers of patients with PSC and PBC, and in the 3 murine models, as well as in BA-stimulated primary mouse hepatocytes. BA-induced Lix1l upregulation was dependent on Egr-1, which served as a transcriptional activator. LIX1L deficiency attenuated cholestatic liver injury in BDL and Mdr2-/- mice. MiR-191-3p was the most reduced miRNA in livers of WT-BDL mice, while it was restored in Lix1l-/--BDL mice. MiR-191-3p targets and downregulates Lrh-1, thereby inhibiting Cyp7a1 and Cyp8b1 expression. AAV-mediated hepatic delivery of miR-191-3p significantly attenuated cholestatic liver injury in Mdr2-/- mice. CONCLUSIONS: LIX1L deficiency alleviates cholestatic liver injury by inhibiting BA synthesis. LIX1L functions as a nexus linking BA/Egr-1 and miR-191-3p/LRH-1 signaling. LIX1L and miR-191-3p may be promising targets for the treatment of BA-associated hepatobiliary diseases. LAY SUMMARY: Bile acid homeostasis can be impaired in cholestatic liver diseases. Our study identified a novel mechanism of positive feedback regulation in cholestasis. LIX1L and miR-191-3p represent potential therapeutic targets for cholestatic liver diseases.

Association between the UGT1A1*28 allele and hyperbilirubinemia in HIV-positive patients receiving atazanavir: a meta-analysis.

Objectives The uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1)*28 allele in HIV-positive patients receiving atazanavir (ATV) might be associated with the risk of hyperbilirubinemia. Owing to mixed and inconclusive results, a meta-analysis was conducted to systematically summarize and clarify this association.Methods Based on a comprehensive search of PubMed, Embase and Web of Science databases, studies investigating the association between UGT1A1 alleles and hyperbilirubinemia was retrieved. We evaluated the strength of this relationship using odds ratios (ORs) with 95% confidence intervals (CIs). Sensitivity analysis was performed by removing each study one at a time and calculating the pooled ORs of the remaining studies to test the robustness of the meta-analysis results. The Q statistic and the I2 index statistic were used to assess heterogeneity. Publication bias was evaluated using Orwin's fail-safe N test.Results A total of six individual studies were included in this meta-analysis. A significantly increased risk of hyperbilirubinemia was observed in HIV-positive patients receiving ATV with the UGT1A1*1/*28 or UGT1A1*28/*28 genotype, and the risk was higher with the UGT1A1*28/*28 genotype than with the UGT1A1*1/*28 genotype. (UGT1A1*28/*28 versus UGT1A1*1/*28: OR = 3.69, 95%CI = 1.82-7.49; UGT1A1*1/*28 versus UGT1A1*1/*1: OR = 3.50, 95%CI = 1.35-9.08; UGT1A1*28/*28 versus UGT1A1*1/*1: OR = 10.07, 95%CI = 4.39-23.10). All of the pooled ORs were not significantly affected by the remaining studies and different modeling methods, indicating robust results.Conclusions This meta-analysis suggests that the UGT1A1*28 allele represents a biomarker for an increased risk of hyperbilirubinemia in HIV-positive patients receiving ATV.

Identification of novel loci for pediatric cholestatic liver disease defined by KIF12, PPM1F, USP53, LSR, and WDR83OS pathogenic variants.

PURPOSE: Genetic testing in pediatric cholestasis can be very informative but genetic causes have not been fully characterized. METHODS: Exome sequencing and positional mapping in seven families with cholestatic liver disease and negative clinical testing for known disease genes. RESULTS: KIF12, which encodes a microtubule motor protein with a tentative role in cell polarity, was found to harbor three homozygous likely deleterious variants in three families with sclerosing cholangitis. KIF12 expression is dependent on HNF-1ß, deficiency which is known to cause bile duct dysmorphogenesis associated with loss of KIF12 expression. In another extended family, we mapped an apparently novel syndrome of sclerosing cholangitis, short stature, hypothyroidism, and abnormal tongue pigmentation in two cousins to a homozygous variant in PPM1F (POPX2), a regulator of kinesin-mediated ciliary transport. In the fifth family, a syndrome of normal gamma glutamyltransferase (GGT) cholestasis and hearing loss was found to segregate with a homozygous truncating variant in USP53, which encodes an interactor with TJP2. In the sixth family, we mapped a novel syndrome of transient neonatal cholestasis, intellectual disability, and short stature to a homozygous variant in LSR, an important regulator of liver development. In the last family of three affected siblings, a novel syndrome of intractable itching, hypercholanemia, short stature, and intellectual disability was mapped to a single locus that contains a homozygous truncating variant in WDR83OS (C19orf56), known to interact with ATP13A2 and BSEP. CONCLUSION: Our results expand the genetic heterogeneity of pediatric cholestatic liver disease and highlight the vulnerability of bile homeostasis to a wide range of molecular perturbations.

Jaundice revisited: recent advances in the diagnosis and treatment of inherited cholestatic liver diseases.

BACKGROUND: Jaundice is a common symptom of inherited or acquired liver diseases or a manifestation of diseases involving red blood cell metabolism. Recent progress has elucidated the molecular mechanisms of bile metabolism, hepatocellular transport, bile ductular development, intestinal bile salt reabsorption, and the regulation of bile acids homeostasis. MAIN BODY: The major genetic diseases causing jaundice involve disturbances of bile flow. The insufficiency of bile salts in the intestines leads to fat malabsorption and fat-soluble vitamin deficiencies. Accumulation of excessive bile acids and aberrant metabolites results in hepatocellular injury and biliary cirrhosis. Progressive familial intrahepatic cholestasis (PFIC) is the prototype of genetic liver diseases manifesting jaundice in early childhood, progressive liver fibrosis/cirrhosis, and failure to thrive. The first three types of PFICs identified (PFIC1, PFIC2, and PFIC3) represent defects in FIC1 (ATP8B1), BSEP (ABCB11), or MDR3 (ABCB4). In the last 5 years, new genetic disorders, such as TJP2, FXR, and MYO5B defects, have been demonstrated to cause a similar PFIC phenotype. Inborn errors of bile acid metabolism also cause progressive cholestatic liver injuries. Prompt differential diagnosis is important because oral primary bile acid replacement may effectively reverse liver failure and restore liver functions. DCDC2 is a newly identified genetic disorder causing neonatal sclerosing cholangitis. Other cholestatic genetic disorders may have extra-hepatic manifestations, such as developmental disorders causing ductal plate malformation (Alagille syndrome, polycystic liver/kidney diseases), mitochondrial hepatopathy, and endocrine or chromosomal disorders. The diagnosis of genetic liver diseases has evolved from direct sequencing of a single gene to panel-based next generation sequencing. Whole exome sequencing and whole genome sequencing have been actively investigated in research and clinical studies. Current treatment modalities include medical treatment (ursodeoxycholic acid, cholic acid or chenodeoxycholic acid), surgery (partial biliary diversion and liver transplantation), symptomatic treatment for pruritus, and nutritional therapy. New drug development based on gene-specific treatments, such as apical sodium-dependent bile acid transporter (ASBT) inhibitor, for BSEP defects are underway. SHORT CONCLUSION: Understanding the complex pathways of jaundice and cholestasis not only enhance insights into liver pathophysiology but also elucidate many causes of genetic liver diseases and promote the development of novel treatments.

The usefulness of adding p53 immunocytochemistry to bile drainage cytology for the diagnosis of malignant biliary strictures.

BACKGROUND: Obstructive jaundice is frequently caused by bile duct strictures. Determination of malignant strictures is crucial for the initiation of appropriate treatment. Cytologic examination of bile drainage fluid is an easy and reproducible method of detecting malignant cells. This method, however, frequently yields indeterminate results, such as atypia or suspicious of malignancy, due to difficulties in differentiating malignancy from benign atypia. Immunocytochemical assessment of p53 expression by cells in bile drainage fluid may enhance the ability to detect malignancy. METHODS: A total of 139 samples of bile drainage fluid were obtained from 80 patients. Following cytologic examination, the samples were incubated with antibody to p53. The performance of cytology with and without p53 immunocytochemistry was evaluated, with reference to surgical or clinical findings of benign and malignant biliary strictures. RESULTS: Bile drainage cytology alone had a sensitivity of 31.6% and a specificity of 98.4% in the identification of malignant strictures, whereas the combination of p53 immunocytochemistry and bile drainage cytology had a sensitivity of 80.3% and a specificity of 92.1%. P53 immunocytochemistry alone had a sensitivity of 64.5% and a specificity of 92.7% for the identification of malignant strictures in bile drainage samples with atypical cytology, and a sensitivity of 85.0% and a specificity of 100.0% in samples with suspicious of malignancy. CONCLUSION: The addition of p53 immunocytochemistry to bile drainage cytology can be useful in identifying malignant strictures in samples showing indeterminate results on bile drainage cytology. Diagn. Cytopathol. 2017;45:592-597. © 2017 Wiley Periodicals, Inc.

Identification of Pancreatic Cancer in Biliary Obstruction Patients by FRY Site-specific Methylation.

BACKGROUND: Methylation at cg 16941656 of FRY is exclusively found in normal pancreatic tissue and has been proven to be specific for pancreatic-in-origin among several adenocarcinomas. Here, we investigated methylated DNA in the bile as a biomarker to differentiate the cause of obstruction between pancreatic cancer and benign causes. MATERIALS AND METHODS: Bile samples of 45 patients with obstructive jaundice who underwent ERCP were collected and classified into pancreatic cancer (group 1) and benign causes (group 2) in 24 and 21 patients, respectively. DNA was extracted from bile and bisul te modification was performed. After, methylation in cg 16941656 of FRY was identified by real-time PCR, with beta-actin used as a positive control. RESULTS: Methylated DNA was identified in 10/24 (41.67%) and 1/21 (4.8%) of cases in groups 1 and 2, respectively (P= 0.012). The sensitivity, specificity, positive predictive value and negative predictive value to differentiate pancreatic cancer from benign causes were 42%, 95%, 91%, and 59%, respectively. CONCLUSIONS: Detecting a methylation at cg 16941656 of FRY in bile has high specificity, with an acceptable positive likelihood rate, and may therefore be helpful in distinguishing pancreatic cancer from benign strictures.

Resveratrol Protects Oxidative Stress-Induced Intestinal Epithelial Barrier Dysfunction by Upregulating Heme Oxygenase-1 Expression.

BACKGROUND/AIM: Obstructive jaundice (OJ) is frequently complicated by infections and has been associated with increased bacterial translocation, intestinal epithelial hyperpermeability, and oxidative stress, but the mechanism remains unclear. The potential effect of resveratrol (Res) on modifying intestinal epithelial dysfunction was evaluated both in vitro and in vivo. METHODS: Caco-2 cells (in vitro) and male Wistar rats (n = 60; in vivo) were used to evaluate the role of Res on intestinal epithelial dysfunction. Hydrogen peroxide was used to induce oxidative stress in the Caco-2 cells. In bile duct-ligated group, OJ was successfully established on Day 7 after bile duct ligation, whereas sham-operated and vehicle-treated rats served as controls. Western blot and RT-qPCR were performed to analyze TJ proteins expression in epithelium isolated from rat intestine. RESULTS: Intestinal hyperpermeability was associated with decreased expression and phosphorylation of occludin and zonula occluden (ZO-1), but increased oxidation in Caco-2 cells and the intestinal epithelium. Res treatment increased the epithelial expression and phosphorylation of occludin and ZO-1 in a concentration-dependent manner. Moreover, Res which protected Caco-2 cells from H2O2-induced oxidative damage clearly reduced malondialdehyde level and intracellular reactive oxygen species accumulation, but increased the expression levels of superoxide dismutase and heme oxygenase-1 (HO-1). Further studies showed that Res also inhibited H2O2-induced protein kinase C activity and p38 phosphorylation. Interestingly, these effects of Res were abolished by the HO-1 inhibitor zinc protoporphyrin or knockdown of HO-1 by siRNA. CONCLUSIONS: Res protected gut barrier function possibly by initiating HO-1-dependent signaling which is essential for common expression of key tight junction proteins. It also provides a rationale to develop Res clinical applications of intestinal disorders.

Expression of renal Oat5 and NaDC1 transporters in rats with acute biliary obstruction.

AIM: To examine renal expression of organic anion transporter 5 (Oat5) and sodium-dicarboxylate cotransporter 1 (NaDC1), and excretion of citrate in rats with acute extrahepatic cholestasis. METHODS: Obstructive jaundice was induced in rats by double ligation and division of the common bile duct (BDL group). Controls underwent sham operation that consisted of exposure, but not ligation, of the common bile duct (Sham group). Studies were performed 21 h after surgery. During this period, animals were maintained in metabolic cages in order to collect urine. The urinary volume was determined by gravimetry. The day of the experiment, blood samples were withdrawn and used to measure total and direct bilirubin as indicative parameters of hepatic function. Serum and urine samples were used for biochemical determinations. Immunoblotting for Oat5 and NaDC1 were performed in renal homogenates and brush border membranes from Sham and BDL rats. Immunohistochemistry studies were performed in kidneys from both experimental groups. Total RNA was extracted from rat renal tissue in order to perform reverse transcription polymerase chain reaction. Another set of experimental animals were used to evaluate medullar renal blood flow (mRBF) using fluorescent microspheres. RESULTS: Total and direct bilirubin levels were significantly higher in BDL animals, attesting to the adequacy of biliary obstruction. An important increase in mRBF was determined in BDL group (Sham: 0.53 ± 0.12 mL/min per 100 g body weight vs BDL: 1.58 ± 0.24 mL/min per 100 g body weight, P < 0.05). An increase in the urinary volume was observed in BDL animals. An important decrease in urinary levels of citrate was seen in BDL group. Besides, a decrease in urinary citrate excretion (Sham: 0.53 ± 0.11 g/g creatinine vs BDL: 0.07 ± 0.02 g/g creatinine, P < 0.05) and an increase in urinary excretion of H(+) (Sham: 0.082 ± 0.03 µmol/g creatinine vs BDL: 0.21 ± 0.04 µmol/g creatinine, P < 0.05) were observed in BDL animals. We found upregulations of both proteins Oat5 and NaDC1 in brush border membranes where they are functional. Immunohistochemistry technique corroborated these results for both proteins. No modifications were observed in Oat5 mRNA and in NaDC1 mRNA levels in kidney from BDL group as compared with Sham ones. CONCLUSION: Citrate excretion is decreased in BDL rats, at least in part, because of the higher NaDC1 expression. Using the outward gradient of citrate generated by NaDC1, Oat5 can reabsorb/eliminate different organic anions of pathophysiological importance.

The kinetic expression of lipopolysaccharide-binding protein and CD14 gene in obstructive jaundice.

BACKGROUND: Binding lipopolysaccharide (LPS) with high-affinity, lipopolysaccharide-binding protein (LBP) and CD14 lower the threshold stimulatory concentrations of LPS dramatically and enhance the rate of cytokine production markedly. This study aimed to investigate the kinetic expression of LBP/CD14 and its possible relationship with tumor necrosis factor alpha (TNF-α) to better understand the pathophysiology of obstructive jaundice. MATERIALS AND METHODS: The tissues (liver, spleen, intestine, and lung) of male Sprague-Dawley rats were harvested at pre-bile duct ligation in controls and at specific time points (24, 48, 72, 96, and 120 hr) after bile duct ligation. LBP, CD14, and TNF-α mRNA expression were measured in tissues harvested from controls and at the specific time points. RESULTS: Hepatic LBP mRNA expression increased significantly at five days after bile duct ligation. CD 14 mRNA expression increased significantly after five days of bile duct ligation in liver, lung, spleen, and ileum. TNF-α mRNA expression increased significantly in all four organs (liver, lung, spleen, and ileum) after four days of bile duct ligation. CONCLUSION: Five days of bile duct ligation upregulated CD 14 mRNA expression in liver, lung, spleen, and ileum and increased TNF-α mRNA expression simultaneously in the liver, lung, spleen, and ileum. In addition, five days of bile duct ligation also upregulated LBP mRNA expression in the liver and increased hepatic TNF-α mRNA expression simultaneously. The kinetic expressions of LBP and CD 14 in obstructive jaundice are intriguing and further evaluation is warranted.

Novel α-spectrin mutation in trans with α-spectrin causing severe neonatal jaundice from hereditary spherocytosis.

We evaluated a neonate with severe jaundice but a negative family history. Spherocytes were present and suspected hereditary spherocytosis was confirmed by osmotic fragility and eosin-5-maleimide erythrocyte staining. We found he was a compound heterozygote for two pathogenic mutations in the gene encoding α-spectrin: a previously reported α(LEPRA) inherited from his asymptomatic mother, and a novel α-spectrin mutation in intron 45 +1 disrupting the consensus splice site, from his asymptomatic father.

Nod2 deficiency protects mice from cholestatic liver disease by increasing renal excretion of bile acids.

BACKGROUND & AIMS: Chronic liver disease is characterized by fibrosis that may progress to cirrhosis. Nucleotide oligomerization domain 2 (Nod2), a member of the Nod-like receptor (NLR) family of intracellular immune receptors, plays an important role in the defense against bacterial infection through binding to the ligand muramyl dipeptide (MDP). Here, we investigated the role of Nod2 in the development of liver fibrosis. METHODS: We studied experimental cholestatic liver disease induced by bile duct ligation or toxic liver disease induced by carbon tetrachloride in wild type and Nod2(-/-) mice. RESULTS: Nod2 deficiency protected mice from cholestatic but not toxin-induced liver injury and fibrosis. Most notably, the hepatic bile acid concentration was lower in Nod2(-/-) mice than wild type mice following bile duct ligation for 3 weeks. In contrast to wild type mice, Nod2(-/-) mice had increased urinary excretion of bile acids, including sulfated bile acids, and an upregulation of the bile acid efflux transporters MRP2 and MRP4 in tubular epithelial cells of the kidney. MRP2 and MRP4 were downregulated by IL-1ß in a Nod2 dependent fashion. CONCLUSIONS: Our findings indicate that Nod2 deficiency protects mice from cholestatic liver injury and fibrosis through enhancing renal excretion of bile acids that in turn contributes to decreased concentration of bile acids in the hepatocyte.

MicroRNA-29a protects against acute liver injury in a mouse model of obstructive jaundice via inhibition of the extrinsic apoptosis pathway.

Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis, as demonstrated in human liver cirrhosis, and that its downregulation influences the activation of hepatic stellate cells. In addition, both cleaved caspase-3 production and apoptosis play a role in cholestatic liver injury. However, it is unknown if miR-29 is effective in modulating the extent of injury. We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type (WT) littermates to clarify the role of miR-29 in hepatic injury and fibrogenesis, using the bile duct-ligation (BDL) mouse model. After BDL, all three members of the miR-29 family were significantly downregulated in the livers of WT mice, and miR-29b and miR-29c were significantly downregulated in the livers of the miR-29aTg mice. Liver function, as measured by alanine transaminase and aspartate transaminase activity, was significantly improved in the miR-29aTg mice than in the WT littermates, following 1 week of obstructive jaundice. In addition, overexpression of miR-29a was associated with a significant downregulation of the expression of collagen-1α1, collagen-4α1, phospho-FADD, cleaved caspase-8, cleaved caspase-3, Bax, Bcl-2, PARP, and nuclear factor-κB, as well as an upregulation of phospho-AKT expression. In addition, there were significantly fewer TUNEL-positive liver cells in the miR-29aTg group than in the WT littermates after BDL. Our results indicate that miR-29a decreases cholestatic liver injury and fibrosis after BDL, at least partially, by modulating the extrinsic rather than intrinsic pathway of apoptosis.

Bile acid-induced expression of farnesoid X receptor as the basis for superiority of internal biliary drainage in experimental biliary obstruction.

AIMS AND OBJECTIVES: The aim is to determine the efficacy of internal and external biliary drainage (ED) with special reference to the effect of bile acid on intestinal epithelium during experimental biliary obstruction. Methods. A total of 59 male Sprague Dawley rats were randomly assigned to four groups: (I) sham operation (SH); (II) obstructive jaundice (OJ); (III) OJ and ED; and (IV) OJ and internal biliary drainage (ID). The animals underwent surgical ligation of the bile duct on day 1. They were reoperated on day 8 for biliary drainage procedure. Blood cultures were obtained from portal vein and inferior vena cava on day 15. Samples were also drawn for serum total bile acid (TBA) and white blood cell (WBC) counts. The terminal ileum was harvested to study the tight junction-associated protein ("occludin") and bile acid receptor ("farnesoid X receptor" [FXR]) using immunohistochemical method. RESULTS: Serum TBA (118.9 ± 39.0 µmol/L) and WBC (11.4 ± 2.7 × 10(9)/L) were significantly increased (p = 0.001) after bile duct ligation as compared with SH rats (38.0 ± 15.0 µmol/L and 5.5 ± 1.0 × 10(9)/L, respectively; p = 0.001). The resulting mucosal lesion was high grade and the expressions of FXR and Occludin were decreased. After ED, there was slight decrease in total WBCs, whereas TBA levels declined significantly. The expression of FXR was minimal and Occludin showed no change (ED vs. OJ: p = 0.533). However, both WBC and TBA decreased after ID. The ileal structure, grade of mucosal lesion, and expression of FXR/Occludin were comparable with SH group (p > 0.05). The rate of bacterial translocation was: 57.1% (OJ); 62.5% (ID); and 80% (ED) with identical strains in cultures from the portal vein and inferior vena cava. CONCLUSION: Downregulation of TBA/FXR expression during biliary obstruction results in damage to intestinal epithelium. Unlike ED, ID restores FXR/Occludin expression in the terminal ileum through reappearance of intestinal bile acid, which thus appears to be a key factor in maintaining integrity of the epithelial barrier.

Mitochondrial DNA damage and its effect on mitochondrial function in rats with obstructive jaundice.

This study investigated mitochondrial DNA (mtDNA) damage in rats with obstructive jaundice (OJ) and to explore its effect on mitochondrial and hepatic function. Forty-eight male Wistar rats were randomly divided into two groups: sham-operated (Sham) and bile duct ligation (BDL). Blood and tissue samples were collected from the two groups on days 1, 4, 7 and 14 following surgery. Hepatic and mitochondrial function were measured. Long and accurate PCR, restriction enzyme digestion and gene sequencing were used to analyze the locations of mtDNA deletions. In addition, quantitative fluorescent PCR was used to measure the relative amounts of total DNA in hepatocytes and mtDNA deletions. Results showed that the hepatic and mitochondrial function was compromised in the BDL group compared to the Sham group. Notably, a novel 11,194-bp mtDNA deletion (nucleotide positions 4101-15294) and fewer mtDNA copies were found compared to the Sham group. With prolonged ligation time, there was a decrease in the copy number, while the ratio of mtDNA deletions to total mtDNA levels increased in the BDL group. These changes were consistent with damage to hepatic and mitochondrial function. A novel 11,194-bp mtDNA deletion and fewer mtDNA copies were detected in hepatocytes of rats with OJ. The mtDNA deletions may therefore be an important factor leading to mitochondrial and hepatic dysfunction.

[Obstructive jaundice by genodermatosis--case report]. / Icter obstructiv prin genodermatoza--caz clinic.

BACKGROUND: Neurofibromatosis type I, or Recklinnghausen disease, is the most frequently occurring neurofibromatosis, in 1/3000-11,5000 of children born. This disease is a genodermatosis with 1/3000-1/5000 autosomal dominant transmission. Incriminated in the pathological appearance of the disease gene is located on chromosome 17, gene product, neurofibromina, is a protein involved in controlling cell differentiation and proliferation. Skin manifestations can be associated with the same papillary tumors and the internal organ. Treatment is surgery for larger tumors. Worse prognosis in malignant developpment, with the lower quality of life in the presence of complications, as in this case: mechanical obstructive jaundice. MATERIAL AND METHOD: Patients aged 75 years, admitted for obstructive jaundice (progressive, pruritic), cutaneous papillomas (0.5-3 cm) on the trunk and several hyperpigmented brown spots (5-6 cm diameter). Cutaneous lesions (45 years old) have been previously diagnosed by histological examination. RESULTS: We did surgery under general anesthesia: cholecystectomy, intraoperative choledocoscopy of bile duct. In the last portion of bile duct we found pedicled tumors. We did partial excision of tumors and coledoco-duodenoanastomosis in healthy tissue. Histological examination showed neurofibrodermatoza type I. Discharge 12 days postoperatively. CONCLUSIONS: Preoperative diagnosis suggested the possibility of mechanical jaundice by malignancy. Etiologic diagnosis of this rare form of obstructive jaundice could not be established before surgery, only by histological examination of the excised tumors.

Selective activation of Toll-like receptor 7 in activated hepatic stellate cells may modulate their profibrogenic phenotype.

Cholestatic liver injury may activate HSCs (hepatic stellate cells) to a profibrogenic phenotype, contributing to liver fibrogenesis. We have previously demonstrated the involvement of TLR (Toll-like receptor) 7 in the pathogenesis of biliary atresia. In the present study we investigated the ability of TLR7 to modulate the profibrogenic phenotype in HSCs. Obstructive jaundice was associated with significant down-regulation of TLR7. Primary HSCs isolated from BDL (bile duct ligation) rats with obstructive jaundice exhibited reduced expression of TLR7 and increased expression of α-SMA (α-smooth muscle actin) and collagen-α1 compared with sham rats, reflecting HSC-mediated changes. Treatment of primary activated rat HSCs and rat T6 cells with CL075, a TLR7 and TLR8 ligand, significantly decreased expression of MCP-1 (monocyte chemotactic protein-1), TGF-ß1 (transforming growth factor-ß1), collagen-α1 and MMP-2 (matrix metalloproteinase-2), and inhibited cell proliferation and migration. In contrast, silencing TLR7 expression with shRNA (short hairpin RNA) in T6 cells effectively blocked the effects of CL075 stimulation, reversing the changes in MCP-1, TGF-ß1 and collagen-α1 expression and accelerating cell migration. Our results indicate that obstructive jaundice is associated with down-regulation of TLR7 and up-regulation of profibrogenic gene expression in HSCs. Selective activation of TLR7 may modulate the profibrogenic phenotype in activated HSCs associated with cholestatic liver injury.

Effects of Kupffer cell blockade on the hepatic expression of metallothionein and heme oxygenase genes in endotoxemic rats with obstructive jaundice.

AIMS: Heme oxygenase (HO) and metallothionein (MT) genes are rapidly upregulated in the liver by pro-inflammatory cytokines and/or endotoxin as protection against cellular stress and inflammation. Gadolinium chloride (GdCl3)-induced Kupffer cell blockade has beneficial consequences in endotoxemia following bile duct ligation. Herein we further characterized the effects of Kupffer cell inhibition on the activation of the antioxidant defense system (HO and MT gene expressions, and antioxidant enzyme activities) in response to endotoxemia and obstructive jaundice. MAIN METHODS: The isoform-specific expression of MT and HO genes was assessed (RT-PCR) in rat livers following 3-day bile duct ligation, 2-h lipopolysaccharide treatment (1mg/kg) or their combination, with or without GdCl3 pretreatment (10 mg/kg, 24h before endotoxin). Lipid peroxidation, DNA damage and hepatic antioxidant enzyme activities were also assessed. KEY FINDINGS: All these challenges induced similar extents of DNA damage, whereas the lipid peroxidation increased only when endotoxemia was combined with biliary obstruction. The MT and HO mRNA levels displayed isoform-specific changes: those of MT-1 and HO-2 did not change appreciably, whereas those of MT-2 and HO-1 increased significantly in 2-h endotoxemia, with or without obstructive jaundice. Among the enzymes reflecting the endogenous protective mechanisms, the catalase and copper/zinc-superoxide dismutase levels decreased, while that of Mn-SOD slightly increased. Interestingly, GdCl3 alone induced lipid peroxidation, DNA damage and MT-2 expression. In response to GdCl3, HO-1 induction was significantly lower in each model. SIGNIFICANCE: Despite its moderate hepatocellular toxicity, the ameliorated stress-induced hepatic reactions provided by GdCl3 may contribute to its protective effects.

Identification of obstructive jaundice-related microRNAs in mouse liver.

BACKGROUND/AIMS: Although microRNAs are known to be post-transcriptional regulators in physiological and pathological events in the liver, their role in the obstructive jaundice liver remains unclear. METHODOLOGY: We sequenced the small RNA libraries of the bile duct ligation (BDL) mouse liver to detect the in vivo microRNA expression profiles of obstructive jaundice. We also validated the differential expression of microRNAs in the BDL liver using real-time PCR. Laser microdissection was performed to identify the origin of BDL-related microRNAs. An IL6-treated normal intrahepatic biliary epithelial cell line was used as an in vitro model of obstructive jaundice. RESULTS: We found microRNAs that were upregulated in the BDL liver (let-7a, let-7d, let-7f, let-7g, miR-21, miR-125a-5p, miR-125b-5p, miR-194, miR-199a-3p, miR-199a-5p, miR-214, miR-221, and miR-486). Furthermore, laser-microdissection analysis showed that miR-199a-5p was significantly upregulated in the intrahepatic bile duct of the BDL liver. The in vitro expression of miR-199a-5p was appreciably elevated in accordance with increased proliferation of IL6-treated cells. CONCLUSIONS: We revealed dynamic changes in microRNA expression during obstructive jaundice using the BDL model. MiR-199a-5p was likely associated with the proliferation of intrahepatic bile ducts. Our data will facilitate further study of the pathophysiological role(s) of microRNAs in the obstructive jaundice liver.

Decreased expression of intestinal chemokine TECK/CCL25 in experimental obstructive jaundice and its reversal following internal biliary drainage.

BACKGROUND: Although bacterial translocation is a significant problem in patients with obstructive jaundice, how translocation is promoted in this situation is not clearly understood. We previously reported the recovery of gut mucosal T-lymphocyte numbers in jaundiced rats following internal biliary drainage. This suggests that bile in the intestinal lumen promotes T-lymphocyte redistribution into the gut mucosa. To test this hypothesis, we have examined the expression patterns of chemokines that play an important role in lymphocyte recruitment into the small intestine. METHODS: Four groups of rats receiving one of the following surgical procedures were studied: a sham operation (SHAM), common bile duct ligation (CBDL), CBDL followed by external drainage, or CBDL followed by internal drainage. Expression levels of intestinal mRNAs encoding TECK, MECK, and LARC chemokines were assessed using real-time polymerase chain reaction. Distribution of chemokine mRNA in the rat ileum was examined using in situ hybridization (ISH). RESULTS: Following surgery, the expression levels of TECK mRNA decreased significantly in the CBDL group compared with in the SHAM group. While TECK expression did not recover after external drainage, it recovered to a near-normal level after internal drainage. Expression levels of MECK and LARC mRNAs were similar among all groups. ISH confirmed strong expression of TECK mRNA in the epithelial cells of the small intestine. CONCLUSIONS: These results indicate that bile may contribute to high expression levels of TECK/CCL25 mRNA in the small intestine. Bile may also have a role in regulating the distribution of gut mucosal T lymphocytes by promoting TECK production from epithelial cells.