transforming-growth-factor-beta and Cholestasis--Intrahepatic

Herbal medicines harbor essential therapeutic agents for the treatment of cholestasis. In this study, we have assessed the anticholestatic potential of

Topics: Actins; Animals; Anti-Inflammatory Agents; Antifibrotic Agents; Antioxidants; Cholestasis, Intrahepatic; Common Bile Duct; Cytokines; Disease Models, Animal; Inflammation Mediators; Ligation; Liposomes; Liver; Liver Cirrhosis, Biliary; Male; Oxidative Stress; Plant Extracts; Protein Carbonylation; Rats, Wistar; Stachys; Transforming Growth Factor beta

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of β-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of β-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-β signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of β-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

Topics: Adherens Junctions; Animals; beta Catenin; Cell Line, Tumor; Cell Polarity; Cholestasis, Intrahepatic; gamma Catenin; Hepatocyte Nuclear Factor 4; Hepatocytes; Humans; Liver; Mice; Mice, Knockout; Signal Transduction; Transforming Growth Factor beta

Collagen triple helix repeat containing-1 (Cthrc1) is a documented specific inhibitor of TGF-β signaling. Based on this observation, we developed the hypothesis that knocking in/knocking out the Cthrc1 gene in murine models of cholestasis would alter the natural history of cholestatic fibrosis. To study this thesis, we studied two murine models of fibrosis, first, common bile duct ligation (CBDL) and second, feeding of 3, 5-diethoxy-carbonyl-1, 4-dihydrocollidine (DDC). In both models, we administered well-defined adenoviral vectors that expressed either Cthrc1 or, alternatively, a short hairpin RNA (shRNA)-targeting Cthrc1 either before or after establishment of fibrosis. Importantly, when Cthrc1 gene expression was enhanced, we noted a significant improvement of hepatic fibrosis, both microscopically and by analysis of fibrotic gene expression. In contrast, when Cthrc1 gene expression was deleted, there was a significant exacerbation of fibrosis. To identify the mechanism of action of these significant effects produced by knocking in/knocking out Cthrc gene expression, we thence studied the interaction of Cthrc1 gene expression using hepatic stellate cells (HSCs) and human LX-2 cells. Importantly, we demonstrate that Cthrc1 is induced by TGF-β1 via phospho-Smad3 binding to the promoter with subsequent transcription activation. In addition, we demonstrate that Cthrc1 inhibits TGF-β signaling by accelerating degradation of phospho-Smad3 through a proteosomal pathway. Importantly, the anti-fibrotic effects can be recapitulated with a truncated fragment of Cthrc1. In conclusion, our findings uncover a critical negative feedback regulatory loop in which TGF-β1 induces Cthrc1, which can attenuate fibrosis by accelerating degradation of phospho-Smad3.

Topics: Animals; Autoimmune Diseases; Cell Line; Cholestasis, Intrahepatic; Extracellular Matrix Proteins; Gene Expression Regulation; Genetic Therapy; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Liver Diseases; Mice; Mice, Knockout; Pyridines; Smad3 Protein; Transforming Growth Factor beta

Bile duct injury (BDI) is one of the most severe complications of biliary operation. This study is to investigate the correlation between the timing of bile duct repair and anastomotic bile duct stricture. Transverse BDI models were constructed in 60 dogs that were divided randomly into BDI₅, BDI₁₀, BDI₁₅, BDI₂₀, and BDI₃₀ groups according to days of injury (5, 10, 15, 20, and 30 days). The morphological and histological changes of anastomotic stoma of hepaticojejunostomy (HJ) were observed after bile duct reconstruction. TGF-β1, α-SMA, and collagen of anastomotic stoma were detected. After HJ, the concentration of direct bilirubin decreased significantly, dropping to 50% after one week, and returning to normal levels after three weeks. The anastomotic diameter shrunk from 1.5 cm to 0.6 cm without significant difference. At 3 months and 6 months after HJ, the expression of TGF-β in the anastomotic tissue in BDI₅ group was higher than that in BDI₁₀, BDI₁₅, BDI₂₀, and BDI₃₀ groups. However, no significant differences were observed (F = 1.282, P > 0.05 at 3 months; F = 1.308, P > 0.05 at 6 months). Similarly, the expression of α-SMA and collagen did not vary significantly. For obstructive BDI, repairing time is not a relevant factor for postoperative anastomotic stenosis, but surgeons and operation methods are the key factors. For patients with BDI, hospitals should focus on the experience of surgeons and the choice of operation methods in order to achieve a good long-term effect.

Topics: Actins; Anastomosis, Surgical; Animals; Bilirubin; Biomarkers; Cholestasis, Intrahepatic; Clinical Competence; Collagen; Common Bile Duct; Constriction, Pathologic; Cystic Duct; Disease Models, Animal; Dogs; Female; Jejunostomy; Male; Reoperation; Time Factors; Time-to-Treatment; Transforming Growth Factor beta; Wound Healing

Antiinflammatory effect of statins mediated by the reduction of cytokine IL-6 in hepatocytes have been reported. Contrary to beneficial effect, statins can increase susceptibility to mitochondrial dysfunction. Extrahepatic biliary obstruction is associated with oxidative stress, pro-inflammatory response and hepatocyte mitochondrial dysfunction. The aim of our study was to verify the effect of fluvastatin on cholestatic liver injury. Cholestasis was induced in Wistar rats by bile duct ligation. Fluvastatin (1 or 5 mg/kg) was administered after surgery and then daily for 7 days. The dose of 5 mg/kg led to the deterioration of hepatocellular injury. Despite lower production of IL-6, decrease in GSH content, rise of TGFß and inhibition of respiratory complex I in mitochondria were determined. The mRNA expressions of canalicular transporter Mdr1b and basolateral transporter Mrp3 increased in cholestatic liver. Fluvastatin administration then led to the attenuation of this change. Analogously, mRNA expression of conjugative enzyme Ugt1a1 was diminished by fluvastatin administration to cholestatic rats. We can conclude that decrease in the antioxidative status and mitochondrial dysfunction could at least in part participate on the deteriorating effect of fluvastatin. Whether these processes can be a consequence of the alteration in metabolism and transport of potentially toxic substances remains to verify.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Bilirubin; Cholestasis, Intrahepatic; Fatty Acids, Monounsaturated; Fluvastatin; gamma-Glutamyltransferase; Glucuronosyltransferase; Glutathione; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Interleukin-6; Ligation; Liver; Male; Rats; Rats, Wistar; RNA, Messenger; Transforming Growth Factor beta

To investigate the influence of transforming growth factor-beta2 (TGF-beta2) on production of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) from placenta of patients with intrahepatic cholestasis of pregnancy (ICP).. Villous explants from 10 ICP patients (ICP group) and 10 normal gravidae (control group) were cultured in vitro. The concentrations of TNF-alpha and IFN-gamma in the culture medium were determined by enzyme linked immunoadsorbent assay (ELISA) after incubation with different concentrations of TGF-beta2 for 1, 24 and 48 hours.. (1) After cultured for 1, 24 and 48 hours, TNF-alpha level in ICP group was (771 +/- 187) pg/g, (2 490 +/- 575) pg/g, and (3339 +/- 1106) pg/g, respectively, while IFN-gamma level was (931 +/- 148) pg/g, (1888 +/- 545) pg/g, and (3027 +/- 667) pg/g, respectively. All were significantly higher than those in the normal group (P < 0.001). (2) TGF-beta2 could inhibit the release of above cytokines in a time- and dose-dependent manner: TNF-alpha level was (2490 +/- 575) pg/g, (1806 +/- 502) pg/g, and (1231 +/- 238) pg/g after 24 h, (3339 +/- 1106) pg/g, (2168 +/- 794) pg/g, and (1047 +/- 183) pg/g after 48 h, respectively; IFN-gamma level was (1888 +/- 545) pg/g, (1 347 +/- 405) pg/g, and (913 +/- 303) pg/g after 24 h, and (3027 +/- 667) pg/g, (2062 +/- 795) pg/g, and (1001 +/- 301) pg/g after 48 h, respectively.. (1) The excessive production of TH1 type cytokines TNF-alpha and IFN-gamma may involve in the pathogenesis of ICP; (2) TGF-beta2 may exert immunoprotection effect through inhibiting the production of TNF-alpha and IFN-gamma.

Topics: Adult; Cholestasis, Intrahepatic; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interferon-gamma; Placenta; Pregnancy; Pregnancy Complications; Time Factors; Tissue Culture Techniques; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Hydrophobic bile acids lead to generation of oxygen free radicals in mitochondria. Accordingly, this study investigated if gene delivery of superoxide dismutase (SOD) would reduce hepatic injury caused by experimental cholestasis. Rats were given adenovirus (Ad; 3 x 10(9) p.f.u., i.v.) carrying the bacterial control gene lacZ, mitochondrial Mn-SOD or cytosolic Cu/Zn-SOD genes 3 days before bile duct ligation. Both Mn- and Cu/Zn-SOD activity was increased in the liver about four-fold 3 days after viral infection. Serum alanine transaminase increased to about 710 U/l after bile duct ligation, which was blunted by about 70% in rats receiving Ad-Mn-SOD, but by only 30% in rats receiving Ad-Cu/Zn-SOD. Bile duct ligation caused focal necrosis, apoptosis and fibrosis in the liver and increased collagen alpha1 mRNA about 20-fold. These effects were reduced significantly by Ad-Mn-SOD, but not by Ad-Cu/Zn-SOD. In addition, bile duct ligation increased 4-hydroxynonenal, a product of lipid peroxidation, activated NF-kappaB and increased synthesis of TNF(alpha) and TGF-beta. These effects were also blunted significantly by Ad-Mn-SOD, but not by Ad-Cu/Zn-SOD. Taken together, it is concluded that cholestasis causes liver injury by mechanisms involving mitochondrial oxidative stress. Gene delivery of mitochondrial Mn-SOD blocks formation of oxygen radicals and production of toxic cytokines thereby minimizing liver injury caused by cholestasis.

Topics: Animals; Cholestasis, Intrahepatic; Collagen Type I; Fibrosis; Genetic Therapy; Genetic Vectors; Lipid Peroxidation; Liver; Male; NF-kappa B; Rats; Rats, Sprague-Dawley; RNA, Messenger; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Postoperative cholangitis is one of the most common complications after bile duct reconstruction. The pathogenesis and early consequences of ascending cholangitis still are unidentified.. Male Sprague-Dawley rats were divided into 5 treatment groups: control (n = 4), blood sampling and liver biopsy only; group I, [BDL/Eschericha coli; n = 6], ligation of common bile duct (BDL) for a week, followed by Roux-en-Y choledochojejunostomy (RYCJ) and injection of E coli (ATCC 25922) into Roux limb after 24 hours; group II, [BDL/NS; n = 5], same procedures as in group I, with injection of normal saline (NS) into Roux limb; group III, [SBDL/E coli; n = 6], primary RYCJ was constructed 1 week after sham ligation of common bile duct (SBDL) followed by the same treatment as group I; Group IV, [SBDL/N.S; n = 6], same procedures as in group III, but injecting NS into Roux limb. All animals were killed after 24 hours of treatment. Blood was sampled for culture and serum cytokine levels. The liver was harvested for quantitative bacterial culture, as well as for MCP-1, interleukin (IL)-8 (CINC in the rat) and transforming growth factor beta1 mRNA expression by reverse transcriptase polymerase chain reaction (RT-PCR) and for immunohistochemistry. The choledochojejunostomy was resected for culture. Serum cytokine levels were detected by ELISA kits.. A significant increase of E coli ATCC 25922, occurred in the livers of group I rats, compared with group IV (P =.037). MCP-1 expression increased in all groups, compared with control (P =.000). The IL-8 mRNA expression was significantly higher in group I than in control (P =.021). The expression of TGF-beta1 mRNA was similar among the groups (P =.361), consistent with the immunohistochemistry results. The serum MCP-1 and IL-8 levels were higher in the 4 groups than in the control (P =.000) and were significantly higher in group I than in group IV (P =.001).. This study found that a significant colonization of E coli of the same strain was present in the cholestatic rat liver injected into the Roux limb, which was associated with a higher expression of liver MCP-1 and IL-8 mRNA, a significant increase of serum MCP-1 and IL-8, and a more evident inflammatory cell infiltration into the porta hepatis.

Topics: Anastomosis, Roux-en-Y; Animals; Chemokine CCL2; Cholangitis; Choledochostomy; Cholestasis, Intrahepatic; Common Bile Duct; Escherichia coli; Escherichia coli Infections; Interleukin-8; Ligation; Liver Cirrhosis; Male; Postoperative Complications; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

1. Cytokines are soluble factors whose action has been documented in physiological and pathological conditions. Some may be involved in the pathogenesis of cholestasis, whether of acute or chronic origin. 2. The aim of the present study was to evaluate the influence of epidermal growth factor (EGF), transforming growth factor (TGF)-beta 1, interleukin (IL)-6 and tumour necrosis factor (TNF) on cholestasis. Findings from Sprague-Dawley rats submitted to bile duct ligation for 1-28 days were compared with those from controls, which underwent laparotomy but not bile duct ligation. 3. Biochemical and morphological findings confirmed that the experimental procedure was successful. At the end of each follow-up period, the hepatic levels of the cytokines were determined and compared with liver histology findings. 4. The four cytokines studied showed different patterns of activation: hepatic levels of EGF, higher in the experimental than the control group, were comparable with the proliferative picture. The TGF-beta 1 pattern was correlated with data of periportal, perivenular and perineoductular fibrosis, confirming that this cytokine has a role in mediating the synthesis of matrix proteins. A fluctuating, phasic pattern was found for TNF in the experimental group, with high values on day 0, a decrease on the first and second postoperative days and then two peaks on days 8 and 14. Finally, immediately after surgical manipulation, high levels of IL-6 were found in the experimental group, followed by a decrease in levels until zero values were obtained. 5. This suggests that the obstructive condition produces several cytokine responses, each of which contributes to determine the cholestatic condition.

Topics: Animals; Bile Ducts, Intrahepatic; Cholestasis, Intrahepatic; Cytokines; Disease Progression; Epidermal Growth Factor; Interleukin-6; Ligation; Liver; Male; Necrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha