Livers Of Bile Duct Ligated Rats Research Articles

MiR-22-3p and miR-29a-3p synergistically inhibit hepatic stellate cell activation by targeting AKT3.

Hepatic fibrosis (HF) is a worldwide health problem for which there is no medically effective drug treatment at present, and which is characterized by activation of hepatic stellate cells (HSCs) and excessive extracellular matrix (ECM) deposition. The HF model in cholestatic rats by ligating the common bile duct was induced and the differentially expressed miRNAs in the liver tissues were analyzed by microarray, which showed that miR-22-3p and miR-29a-3p were significantly downregulated in bile-duct ligation (BDL) rat liver compared with the sham control. The synergistic anti-HF activity and molecular mechanism of miR-22-3p and miR-29a-3p by targeting AKT serine/threonine kinase 3 (AKT3) in HSCs were explored. The expression levels of miR-22-3p and miR-29a-3p were downregulated in activated LX-2 and human primary normal hepatic fibroblasts (NFs), whereas AKT3 was found to be upregulated in BDL rat liver and activated LX-2 cells. The proliferation, colony-forming, and migration ability of LX-2 were inhibited synergistically by miR-22-3p and miR-29a-3p. In addition, cellular senescence was induced and the expressions of the LX-2 fibrosis markers COL1A1 and α-SMA were inhibited by miR-22-3p and miR-29a-3p synergistically. Subsequently, these two miRNAs binding to the 3'UTR of AKT3 mRNA was predicted and evidenced by the luciferase reporter assay. Furthermore, the proliferation, migration, colony-forming ability, and the expression levels of COL1A1 and α-SMA were promoted and cellular senescence was inhibited by AKT3 in LX-2 cells. Thus, miR-22-3p/miR-29a-3p/AKT3 regulates the activation of HSCs, providing a new avenue in the study and treatment of HF.

Changes in transporters and metabolizing enzymes in the livers of rats with bile duct ligation.

Bile duct ligation (BDL) in experimental animals is widely used as an animal model of liver cholestasis and fibrosis. The transcriptional process and plasma membrane localization of transporters are regulated by nuclear receptors and scaffold proteins, respectively. However, the detailed changes of these factors in the livers of BDL rats remain unclear. To clarify the effects of BDL on the levels of transporters and metabolizing enzymes, nuclear receptors, and scaffold proteins, we investigated changes in mRNA and protein levels of livers from BDL rats. Membrane proteins and microsomes were prepared from rats with BDL. The mRNA levels of transporters and nuclear receptors in livers of control and BDL rats were examined by real-time reverse transcription polymerase chain reaction. The protein levels of transporters, metabolizing enzymes and scaffold proteins in membrane proteins and microsomes were determined by liquid chromatography-tandem mass spectrometry-based targeted proteomics. Mdr1a mRNA was significantly decreased at 1 and 2 weeks of BDL. The mRNA levels of MRP2 were significantly decreased. The mRNA levels of nuclear receptors were significantly decreased in livers of 1-week BDL rats. The protein levels of P-gp were significantly increased by BDL. Regarding scaffold proteins, the protein levels of ezrin, moesin and EBP50 were significantly decreased at 2 weeks of BDL. The protein levels of radixin were significantly increased at 1 week of BDL. In 1-week BDL rats, the protein levels of metabolizing enzymes such as CYP and UGT were significantly decreased. This study reports the comprehensive changes of transporters, metabolizing enzymes, nuclear receptors, and ezrin/radixin/moesin proteins in the livers of BDL rats. The expression levels of nuclear receptors and radixin that regulate the transcription and localization of CYP and/or transporters were decreased by BDL.

AICAR-Induced AMPK Activation Inhibits the Noncanonical NF-κB Pathway to Attenuate Liver Injury and Fibrosis in BDL Rats.

Background To evaluate the AMP-activated protein kinase- (AMPK-) mediated signaling and NF-κB-related inflammatory pathways that contribute to cholestatic diseases in the bile duct ligation (BDL) rat model of chronic cholestasis and verify the protective role of 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR) against hepatic injury and fibrosis triggered by cholestasis-related inflammation. Methods Animals were randomly divided into three groups: sham-operated group, BDL group, and BDL+ AICAR group. Cholestatic liver injury was induced by common BDL. Two weeks later, rats in BDL+AICAR group started receiving AICAR treatment. Hepatic pathology was examined by haematoxylin and eosin (H&E) and sirius red staining and hydroxyproline assay was performed in evaluating the severity of hepatic cirrhosis. Real-time PCR and Western blot were performed for RNA gene expression of RNA and protein levels, respectively. Results The BDL group showed liver injury as evidenced by histological changes and elevation in serum biochemicals, ductular reaction, fibrosis, and inflammation. The mRNA expression of canonical NF-κB inflammatory cytokines such as TNF-α, IL-1β, TGF-β, and the protein of noncanonical NF-κB, P100, and P52 was upregulated in the livers of BDL rats. The BDL rats with the administration of AICAR could induce AMPK activation inhibiting the noncanonical NF-κB pathway to attenuate liver injury and fibrosis in BDL rats. Conclusion The BDL model of hepatic cholestatic injury resulting in activation of Kupffer cells and recruitment of immune cells might initiate an inflammatory response through activation of the NF-κB pathway. The AMPK activator AICAR significantly alleviated BDL-induced inflammation in rats by mainly inhibiting the noncanonical NF-κB pathway and thus protecting against hepatic injury and fibrosis triggered by BDL.

Endoplasmic reticulum stress induces inverse regulations of major functions in portal myofibroblasts during liver fibrosis progression

Portal myofibroblasts (PMF) form a sub-population of highly proliferative and proangiogenic liver myofibroblasts that derive from portal mesenchymal progenitors. Endoplasmic reticulum (ER) stress was previously shown to modulate fibrogenesis, notably in the liver. Our aim was to determine if ER stress occurred in PMF and affected their functions. PMF were obtained after their expansion in vivo from bile duct-ligated (BDL) rats and referred to as BDL PMF. Compared to standard PMF obtained from normal rats, BDL PMF were more myofibroblastic, as assessed by higher alpha-smooth muscle actin expression and collagen 1 production. Their proangiogenic properties were also higher, whereas their proliferative and migratory capacities were lower. CHOP expression was detected in the liver of BDL rats, at the leading edge of portal fibrosis where PMF accumulate. BDL PMF displayed ER dilatation and an overexpression of the PERK pathway downstream targets, Chop, Gadd34 and Trb3, in comparison with standard PMF. In vitro, the induction of ER stress by tunicamycin in standard PMF, caused a decrease in their proliferative and migratory activity, and an increase in their proangiogenic activity, without affecting their myofibroblastic differentiation. Conversely, the treatment of BDL PMF with the PERK inhibitor GSK2656157 reduced ER stress, which caused a decrease in their angiogenic properties, and restored their proliferative and migratory capacity. In conclusion, PMF develop ER stress as they expand with the progression of fibrosis, which further increases their proangiogenic activity, but also inhibits their proliferation and migration. This phenotypic switch may restrict PMF expansion while they support angiogenesis.

Effects of Yinchenhao decoction on self-regulation of renin-angiotensin system by targeting angiotensin converting enzyme 2 in bile duct-ligated rat liver.

In order to investigate whether Yinchenhao decoction (YCHD) attenuates hepatic fibrogenesis in the bile duct ligation (BDL) model via recovering and restoring the self-regulation and balance of the renin-angiotensin system (RAS), 33 specific-pathogen-free (SPF) male Sprague-Dawley rats with common BDL and scission were randomly divided into five groups as follows: G1, the sham group (n=4); G2, BDL 7-day group (n=5); G3, BDL+YCHD 430 mg/mL (n=8); G4, BDL+losartan 0.65 mg/mL (ARB group, n=8); G5, model group (BDL without any treatment, n=8). YCHD and losartan (10 mL·kg(-1)·day(-1)) were given by gastric gavage for 16 days following BDL in G3 and G4 groups, respectively. The effect of YCHD on liver fibrosis and the detailed molecular mechanisms were assessed by liver function including total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IDBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Histological changes were observed by transmission electron microscopy (TEM) and Masson trichrome staining. Western blotting was used to detect the protein expression level of the renin-angiotensin system (RAS) components including angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AT1R), ACE2, angiotensin II (AngII) as well as transforming growth factor β1 (TGFβ1). The experimental data were analyzed by principle component analytical method of pattern recognition. The results showed that biochemically, serum TBIL, DBIL, IDBIL, ALT and AST levels were markedly increased following BDL as compared with the sham group (P<0.05). Serum TBIL, IDBIL and DBIL levels in G3 group were dramatically decreased as compared with G5 and G4 groups (P<0.05). Serum AST level in G3 was significantly lowered than in G5 group (P<0.05), but there was no significant difference in ALT among G3, G4 and G5 groups (P>0.05). Histologically, livers in G3 group showed less hepatocytes necrosis, less bile duct hyperplasia and less collagen formation than in G4 and G5 groups. The protein expression levels of ACE2, ACE, AngII, AT1R and TGFβ1 in G2, G3 and G4 groups were significantly higher than in sham group (P<0.05), and lower than in G5 group (P<0.05). However, the differences among G2, G3 and G4 groups were not significant (P>0.05). ACE2 protein expression in G3 group was significantly higher than in G2 group (P<0.05) and there was no significant difference in comparison with G4 group (P>0.05). Moreover, the protein expression of TGFβ1 in G3 group was significantly lower than in G5 and G4 groups (P<0.05). Our findings suggest that the antifibrotic effects of YCHD may be associated with the decreased classical RAS pathway components and TGFβ1 downexpression so as to recover and rebuild self-regulation of the RAS by elevating the protein expression of ACE2.

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway.

(-)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro. BDL rats were treated with EGCG (25 mg·kg(-1)·d(-1), po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-β1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR. In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1β, TGF-β1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 μmol/L) dose-dependently suppressed TGF-β1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-β1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-β1-stimulated LX-2 cells. Application of LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-β1-stimulated LX-2 cells, but co-application of EGCG and LY294002 did not produce additive effects. EGCG exerts anti-fibrotic effects in BDL rats and TGF-β1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.

Antiangiogenic and antifibrogenic activity of pigment epithelium-derived factor (PEDF) in bile duct-ligated portal hypertensive rats

ObjectiveAntiangiogenic strategies have been proposed as a promising new approach for the therapy of portal hypertension and chronic liver disease. Pigment epithelium-derived factor (PEDF) is a powerful endogenous angiogenesis inhibitor...

Expression of brain derivated neurotrophic factor and of its receptors: TrKB and p75NT in normal and bile duct ligated rat liver.

Cholangiocytes are the cells lining the biliary tree from canals of Hering to larger bile ducts. By morphology, they are divided in small and large cholangiocytes, which result heterogeneous at functional and proliferative levels. Proliferating cholangiocytes acquire a neuroendocrine phe- notype, modulated by several factors including neurotrophins. Brain Derivated Neurotrophic Factor (BDNF) is a neurotrophin expressed in the nervous system and also in different types of epithelial and progenitor cells. The aim of the present study is to detect the expression of BDNF and of its two receptors (TrKB and p75NT, or p75NTR) in normal and bile duct ligated (BDL) rat livers. In normal and BDL livers, BDNF and its receptors are expressed by small and large cholan- giocytes and by hepatic progenitors cells. In cholangiocytes, the expression of BDNF and of its receptors changes after different BDL timing. After one or two weeks of BDL, both BDNF and TrKB and p75NT receptors are highly expressed, while after BDL for three weeks BDNF expression is drastically reduced and p75NT receptor prevails on TrKB. The expression of BDNF and of its receptors correlates with the proliferation rate of biliary tree during BDL. Indeed, after one or two weeks of BDL, proliferation prevails on apoptosis, whereas after BDL for three weeks, apoptosis prevails on proliferation. Our morphological results strongly suggest that BDNF plays a role in the remodeling of biliary tree during cholestasis and that it may be involved in the pathophysiology of cholestasic liver diseases.

Neutrophil-derived Matrix Metalloproteinase 8 is Required to Initiate Collagenase Activity in Bile Duct Ligated Rat Liver

Neutrophil-derived Matrix Metalloproteinase 8 is Required to Initiate Collagenase Activity in Bile Duct Ligated Rat Liver

Spironolactone Lowers Portal Hypertension by Inhibiting Liver Fibrosis, ROCK-2 Activity and Activating NO/PKG Pathway in the Bile-Duct-Ligated Rat

ObjectiveAldosterone, one of the main peptides in renin angiotensin aldosterone system (RAAS), has been suggested to mediate liver fibrosis and portal hypertension. Spironolactone, an aldosterone antagonist, has beneficial effect on hyperdynamic circulation in clinical practice. However, the mechanisms remain unclear. The present study aimed to investigate the role of spionolactone on liver cirrhosis and portal hypertension.MethodsLiver cirrhosis was induced by bile duct ligation (BDL). Spironolactone was administered orally (20 mg/kg/d) after bile duct ligation was performed. Liver fibrosis was assessed by histology, Masson's trichrome staining, and the measurement of hydroxyproline and type I collagen content. The activation of HSC was determined by analysis of alpha smooth muscle actin (α-SMA) expression. Protein expressions and protein phosphorylation were determined by immunohistochemical staining and Western blot analysis, Messenger RNA levels by quantitative real time polymerase chain reaction (Q-PCR). Portal pressure and intrahepatic resistance were examined in vivo.ResultsTreatment with spironolactone significantly lowered portal pressure. This was associated with attenuation of liver fibrosis, intrahepatic resistance and inhibition of HSC activation. In BDL rat liver, spironolactone suppressed up-regulation of proinflammatory cytokines (TNFα and IL-6). Additionally, spironolactone significantly decreased ROCK-2 activity without affecting expression of RhoA and Ras. Moreover, spironolactone markedly increased the levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS and the activity of NO effector- protein kinase G (PKG) in the liver.ConclusionSpironolactone lowers portal hypertension by improvement of liver fibrosis and inhibition of intrahepatic vasoconstriction via down-regulating ROCK-2 activity and activating NO/PKG pathway. Thus, early spironolactone therapy might be the optional therapy in cirrhosis and portal hypertension.

The effect of ursodeoxycholic acid on oxidative stress level and DNase activity in rat liver after bile duct ligation

Accumulation of hydrophobic bile acids (BAs) during cholestasis plays an important role in apoptosis initiation as well as oxidative stress increase in liver cells. Ursodeoxycholic acid (UDCA) acts as a protector in BA-induced cell injury.The aim of the study was to evaluate the effect of UDCA on oxidative stress level and DNase I and II activity caused by liver injury in bile duct ligation (BDL) rats.Wistar rats were divided in four groups: group 1, control (sham-operated); group 2, sham-operated and injected with UDCA (30 mg/kg); group 3,animals with BDL; and group 4,UDCA-treatedcholestatic rats. Animals were sacrificed after 9 days. Malondialdehyde (MDA; lipid peroxidation end-product) level and protein-molecule oxidative modification (carbonyl group content) significantly increased in BDL rat liver. Catalase (CAT) activity in liver tissue was found to be decreased in BDL rats. In addition, xanthine oxidase (XO) activity, which is thought to be one of the key enzymes producing reactive oxygen species, was found to be increased in the cholestatic group. The apoptotic effect in cholestasis was probably triggered by the increased activation of DNase I and II. The protective effect of UDCA on liver tissue damage in BDL rats, in comparison to cholestatic liver, were 1) decrease of MDA levels, 2) increased CAT activity, 3) reduced XO activity, and 4) effect on terminal apoptotic reaction, shown as a decrease in DNase I and II activity.Therefore, UDCA may be useful in the preservation of liver function in cholestasis treatment.

Assessment of liver steatosis and fibrosis in rats using integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique

We report the implementation of a unique integrated coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and two-photon excitation fluorescence (TPEF) microscopy imaging technique developed for label-free monitoring of the progression of liver steatosis and fibrosis generated in a bile duct ligation (BDL) rat model. Among the 21 adult rats used in this study, 18 rats were performed with BDL surgery and sacrificed each week from weeks 1 to 6 (n = 3 per week), respectively; whereas 3 rats as control were sacrificed at week 0. Colocalized imaging of the aggregated hepatic fats, collagen fibrils, and hepatocyte morphologies in liver tissue is realized by using the integrated CARS, SHG, and TPEF technique. The results show that there are significant accumulations of hepatic lipid droplets and collagen fibrils associated with severe hepatocyte necrosis in BDL rat liver as compared to a normal liver tissue. The volume of normal hepatocytes keeps decreasing and the fiber collagen content in BDL rat liver follows a growing trend until week 6; whereas the hepatic fat content reaches a maximum in week 4 and then appears to stop growing in week 6, indicating that liver steatosis and fibrosis induced in a BDL rat liver model may develop at different rates. This work demonstrates that the integrated CARS and multiphoton microscopy imaging technique has the potential to provide an effective means for early diagnosis and detection of liver steatosis and fibrosis without labeling.

P60 Acute lowering of portal pressure in cirrhotic rats by Anti-TNF therapy is associated with reduced NFkB-driven inflammation and improved eNOS function through the asymmetric dimethylarginine-dimethylarginine-diaminohydrolase axis

IntroductionAnti-TNF (Infliximab) monotherapy has been shown to reduce hepatic inflammation and lower portal pressure in alcoholic hepatitis patients. Although the side effect profile of Infliximab limits its clinical use in...

Oxidative stress and DNA fragmentation in hepatic injury of the bile duct-ligated rats: modulatory effect of ursodeoxycholic acid

Event Abstract Back to Event Oxidative stress and DNA fragmentation in hepatic injury of the bile duct-ligated rats: modulatory effect of ursodeoxycholic acid Maja Jovic1, Marko Jovic1* and Dusan Sokolovic1 1 University of Nis, Institute of Biochemistry, Serbia Introduction. Accumulation of toxic hydrophobic bile acids (BA) during cholestasis can activate Fas death receptors directly and induce oxidative damage that causes mitochondrial dysfunction and hepatocyte apoptosis. Reactive oxygen species (ROS) induce chromatin dysfunction such as single- and double-strand DNA fragmentation. The major beneficial effects of treatment with ursodeoxycholic acid (UDCA) are protection against cytotoxicity of BA, stimulation of hepatobiliary secretion, antioxidative activity and blocking the apoptotic signal (inhibition of mitochondrial cytochrome C releasing). Aim of the present study was to evaluate the effect of UDCA on oxidative stress and DNA fragmentation in liver injury of bile duct-ligated (BDL) rats. Material and Methods. Three groups of Wistar rats were investigated: I-sham-operated, II-BDL and III-BDL+UDCA (30mg/kgBW,i.g.). The animals were killed after 7 days. Results: Lipid peroxidation (MDA) level and oxidative modification of proteins (carbonyl group content) significantly increased in BDL group (4.75±0.59 vs. sham-operated 2.84±0.31 nmol/mg prot, p<0.001 and 48.21±6.37 vs. sham-operated 39.63±5.69µmol/g prot,p<0.05). Activity of catalase, the free radicals scavenging enzymes was decreased in liver of BDL rats (5.8±2.11 vs. sham-operated 8.4±2.05 U/mg prot,p<0.001). Xanthine oxidase (XO), major enzyme of ROS production was increased in cholestatic group (2.25±0.26 vs. sham-operated 1.48±0.46 U/g prot;p<0.001). The apoptotic effect in cholestasis is probably triggered by the increase activation of alkaline-DNase I (caspase 3-activated) and acid-DNase II (3.19±0.61 vs. sham-operated 1.06±0.19 U/g prot. and 2.93±0.91 vs. sham-operated 1.53±0.24 U/g prot,p<0.001). UDCA was found to be effective in liver of BDL rats:(1) decreased MDA levels (3.26±0.67,p<0.01),(2) no significantly decreased carbonyl group (45.62±6.69), (3) increase activity of catalase (7.94±1.34, p<0.001), (4) reduces activity of XO (1.77±0.68,p<0.05). (5) effect on terminal apoptotic reaction, because of the decrease in DNase I and DNase II activity (2.37±0.34 and 1.92±0.42,p<0.05), compared with cholestatic liver. Conclusion. These results support a novel effect for UDCA in the control of endonuclease activity as a final down-stream mediator of apoptosis and therefore in the DNA stability, beside established role in reducing oxidative damage and maintained antioxidant defenses in cholestatic liver. Keywords: Cholestasis, Oxidative Stress, Ursodeoxycholic Acid, DNA, Rats 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: Jovic M, Jovic M and Sokolovic D (2010). Oxidative stress and DNA fragmentation in hepatic injury of the bile duct-ligated rats: modulatory effect of ursodeoxycholic acid. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00133 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. Marko Jovic, University of Nis, Institute of Biochemistry, Nis, Serbia, markojovic10@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 Maja Jovic Marko Jovic Dusan Sokolovic Google Maja Jovic Marko Jovic Dusan Sokolovic Google Scholar Maja Jovic Marko Jovic Dusan Sokolovic PubMed Maja Jovic Marko Jovic Dusan Sokolovic 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.

Effects of ursodeoxycholic acids on oxidative stress level in hepatic tissue of rats with extrahepatic cholestasis

Event Abstract Back to Event Effects of ursodeoxycholic acids on oxidative stress level in hepatic tissue of rats with extrahepatic cholestasis Marko Jovic1*, Maja Jovic1 and Dusan Sokolovic1 1 University of Nis, Institute of Biochemistry, Serbia Introduction. Accumulation of toxic hydrophobic bile acids (BA) during cholestasis can activate Fas death receptors and increase production of reactive oxygen species (ROS) simultaneously inducing apoptosis of hepatocytes. The major beneficial effects of treatment with ursodeoxycholic acid (UDCA) are protection against cytotoxicity of BA, stimulation of hepatobiliary secretion, antioxidative activity and blocking the apoptotic signal.The aim of the present study was to evaluate the effect of UDCA on oxidative stress in liver tissue of bile duct-ligated (BDL) rats. Methods. Four groups of Wistar rats were investigated: I-control (sham operated), II-rats treated with UDCA (25mg/kg b.w. i.g.), III-rats with common bile duct-ligation (BDL), IV-cholestatic rats treated with UDCA. The animals were killed after 9 days. Results. Increased plasma activity of GGT, AF (cholestatic markers) and concentration of BA in BDL rats was registered, while oral administration of UDCA induce decreasing of activity and concentration of all mentioned parameters (p<0.001). Lipid peroxidation-MDA and oxidative modification of proteins-carbonyl group content are significantly increased in BDL group (p<0.05). UDCA was found to be protective in liver of BDL rats. It decreases MDA levels (p<0.01); moderately decrease carbonyl group, compared with cholestatic rats. Conclusions. These results support a novel effects of UDCA in reducing oxidative damage and maintaining of antioxidant defenses in cholestatic liver. Keywords: usodeoxycholic acids, Oxidative Stress, extrahepatic cholestasis, Rats 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: Jovic M, Jovic M and Sokolovic D (2010). Effects of ursodeoxycholic acids on oxidative stress level in hepatic tissue of rats with extrahepatic cholestasis. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00132 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. Marko Jovic, University of Nis, Institute of Biochemistry, Nis, Serbia, markojovic10@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 Marko Jovic Maja Jovic Dusan Sokolovic Google Marko Jovic Maja Jovic Dusan Sokolovic Google Scholar Marko Jovic Maja Jovic Dusan Sokolovic PubMed Marko Jovic Maja Jovic Dusan Sokolovic 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.

Hepatic VASP upregulation in rats with secondary biliary cirrhosis by expression in the peribiliary vascular plexus

Introduction/aims: Hepatic disorders in patients and animal models may be characterized by increased ductular proliferation and concomitant expansion of the peribiliary vascular plexus (PBP). VASP plays an essential role for the control of cell assembly during the formation of three-dimensional organ structures in the cardiovascular system. Here, we investigated hepatic VASP expression in response to bile duct ligation (BDL) of rats. Methods: BDL was performed in male rats. Sham-operated rats served as controls. After 4 weeks, hepatic VASP expression was assessed by Western-blot analysis and immunohistochemical staining. Results: Livers of BDL rats showed excessive formation of new bile ducts. VASP mRNA and protein expression was upregulated in whole liver homogenates from BDL rats. This upregulation was located to the expanding PBP in the areas of ductular proliferation. In patients with cirrhosis, hepatic VASP expression was upregulated compared to non-cirrhotic patients. Discussion: VASP may play a role for vascular morphogenesis in the expanding PBP of BDL rats.

Immunoprecipitation and MALDI-MS identification of lithocholic acid-tagged proteins in liver of bile duct-ligated rats

Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCA's known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the epsilon-amino group of lysine residues. Antibodies recognizing the 3alpha-hydroxy-5beta-steroid moiety of LCA were generated by immunizing rabbits with immunogens in which the carboxyl group of LCA was coupled to BSA via a 6-aminohexanoic acid and/or succinic acid spacer. The resulting antibodies reacted with N-alpha-(t-butoxycarbonyl)-l-lysine-epsilon-LCA, the amidated and nonamidated forms of LCA, as well as synthetically prepared LCA adducts with ovalbumin and lysozyme. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Proteins were isolated by two-dimensional electrophoresis, and their structure was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and computer-assisted programs. Proteins labeled with LCA were Rab-3, Rab-12, Rab-16, and M-Ras. Rab proteins are Ras-like small GTP binding proteins that regulate vesicle trafficking pathways. The covalent binding of the Rab proteins with LCA may influence vesicular transport or binding of vesicles to their cognate membrane and may contribute to LCA-induced liver toxicity.

Defective endothelial nitric oxide synthase signaling is mediated by rho-kinase activation in rats with secondary biliary cirrhosis

In liver cirrhosis, down-regulation of endothelial nitric oxide synthase (eNOS) has been implicated as a cause of increased intrahepatic resistance. We investigated whether Rho-kinase activation is one of the molecular mechanisms involved in defective eNOS signaling in secondary biliary cirrhosis. Liver cirrhosis was induced by bile duct ligation (BDL). We measured mean arterial pressure (MAP), portal venous pressure (PVP), and hepatic tissue blood flow (HTBF) during intravenous infusion of saline (control), 0.3, 1, or 2 mg/kg/hour fasudil for 60 minutes. In BDL rats, 1 and 2 mg/kg/hour fasudil significantly reduced PVP by 20% compared with controls but had no effect on HTBF. MAP was significantly reduced in response to 2 mg/kg/hour fasudil. In the livers of BDL rats, 1 and 2 mg/kg/hour fasudil significantly suppressed Rho-kinase activity and significantly increased eNOS phosphorylation, compared with controls. Fasudil significantly reduced the binding of serine/threonine Akt/PKB (Akt) to Rho-kinase and increased the binding of Akt to eNOS. These results show in secondary biliary cirrhosis that (1) Rho-kinase activation with resultant eNOS down-regulation is substantially involved in the pathogenesis of portal hypertension and (2) Rho-kinase might interact with Akt and subsequently inhibit the binding of Akt to eNOS.

Gene expression profiling in whole liver of bile duct ligated rats: VEGF‐A expression is up‐regulated in hepatocytes adjacent to the portal tracts

It would be of clinical importance to clarify molecular mechanisms of cholangiocytes proliferation for the treatment of intractable cholestatic diseases. The aim of this study was to elucidate gene expression profiling in the whole liver of bile duct ligated (BDL) rats using microarray analysis. In addition, the localization and time course of up-regulated expression of vascular endothelial growth factor (VEGF) was investigated. Male Sprague-Dawley rats were used. The whole liver was removed from BDL and sham-operated rats at day 2 after the procedure, and microarray analysis was performed using an array on which 3757 rat cDNA clones spotted. The up-regulation of VEGF expression was investigated by RT-PCR using livers at day 1, 2, 4 and 7, and immunoblotting and immunohistochemistry at day 2. Marked proliferation of bile ducts was observed in livers of BDL rats. By microarray analysis, 38 up-regulated and 17 down-regulated transcripts were detected in whole liver of the BDL rat. The expression of VEGF-A was significantly elevated in the BDL rats at day 2; the VEGF-A/GAPDH ratio was 4.030 +/- 2.493 in BDL rats and 1.159 +/- 0.125 in sham-operated rats (P = 0.0330). The up-regulation of VEGF-A expression was maximal at day 2. Immunoblotting also demonstrated up-regulated expression of VEGF-A at the protein level. Immunostaining of VEGF revealed that the expression was evident in hepatocytes adjacent to the portal tracts, and scarcely observed in hepatocytes at the centrilobular area or cholangiocytes. Gene expression profiling in the whole liver of the BDL rats revealed 38 up-regulated and 17 down-regulated transcripts. In addition, the up-regulated expression of VEGF was mainly observed in hepatocytes surrounding to the portal tracts.

Hepatic stellate cell‐targeted delivery of M6P‐HSA‐glycyrrhetinic acid attenuates hepatic fibrogenesis in a bile duct ligation rat model

Hepatic stellate cells (HSCs) play a key role in fibrogenesis. Here, we used mannose-6-phosphate-modified human serum albumin (M6P(26)-HSA) as a selective carrier to deliver antifibrotic drug 18beta-glycyrrhetinic acid (18beta-GA) in experimental fibrosis animals, and tested its effect in injured liver tissues. Bile duct ligation (BDL) was performed to induce liver damage in rats. Masson's stain and immunocytochemistry were used to assess hepatic collagen deposits and uptakes of M6P(26)-HSA-GA in HSCs in rat livers. Gene expression profiles of procollagen type I alpha2, smooth muscle actin (SMA), and transforming growth factor-beta1 (TGF-beta1) were analysed by TaqMan and quantitative polymerase chain reaction assays. The depositions of M6P(26)-HSA-GA in the HSC-T6 cell line and primary HSCs were assessed by immunofluorescent staining. Treatment with M6P(26)-HSA-GA at 10 mg/kg (three times/week for 2 weeks), but not the equivalent doses of free 18beta-GA and M6P(26)-HSA carrier alone, could significantly attenuate collagen deposits in BDL rat liver. Masson's stain and TaqMan assay revealed significant modulation of procollagen type I alpha2 in the BDL-injured liver. The depositions of M6P(26)-HSA-GA in HSCs were revealed by immunostaining with HSA and SMA markers. M6P(26)-HSA bound activated HSCs in vitro and the immunoreactivity of M6P(26)-HSA-GA was detected in the cytoplasm and cell surface of HSCs and HSC-T6 cells. The gene transcript levels of SMA and TGF-beta1 were modulated in HSC-T6 cells treated with M6P(26)-HSA-GA. The M6P(26)-HSA holds promise as a targeting carrier for the liver or HSCs, which may be used to deliver 18beta-GA as a therapeutic agent to treat liver fibrosis.