Antioxidant Effect of Sepia Ink Extract on Extrahepatic Cholestasis Induced by Bile Duct Ligation in Rats

Objective The aim of our study was to assess the complications of hepatic fibrosis associated with bile duct ligation and the potential curative role of sepia ink extract in hepatic damage induced by bile duct ligation. Methods Rattus norvegicus rats were divided into 3 groups: Sham-operated group, model rats that underwent common bile duct ligation (BDL), and BDL rats treated orally with sepia ink extract (200 mg/kg body weight) for 7, 14, and 28 d after BDL. Results There was a significant reduction in hepatic enzymes, ALP, GGT, bilirubin levels, and oxidative stress in the BDL group after treatment with sepia ink extract. Collagen deposition reduced after sepia ink extract treatment as compared to BDL groups, suggesting that the liver was repaired. Histopathological examination of liver treated with sepia ink extract showed moderate degeneration in the hepatic architecture and mild degeneration in hepatocytes as compared to BDL groups. Conclusion Sepia ink extract provides a curative effect and an antioxidant capacity on BDL rats and could ameliorate the complications of liver cholestasis.

Protective effects of a polymethoxy flavonoids-rich Citrus aurantium peel extract on liver fibrosis induced by bile duct ligation in mice

Objective: To evaluate the possible protective effect of Citrus aurantium peel extract(CAE) against apoptosis in cholestatic liver fibrosis induced by bile duct ligation in mice. Methods: Male ICR mice were divided to 5 groups: 1) Control group(Sham-operated mice), 2) Cholestatic liver injury group induced by bile duct ligation(BDL), 3) BDL mice treated with silymarin(200 mg/kg) for 4 weeks, 4) BDL mice treated with 50 mg/kg CAE for 4 weeks, 5) BDL mice treated with 200 mg/kg CAE for 4 weeks. Mice were sacrificed and liver fibrosis was evaluated by serum and hepatic tissue biochemistry tests and liver histopathological examination. Effects of CAE on inflammation and apoptosis gene regulation were investigated through real-time PCR. CAE effect on lipid metabolism related signaling was determined by western blot analysis. Results: In BDL mice, administration of CAE for 4 weeks markedly attenuated liver fibrosis based on histopathological alteration. Serum and hepatic tissue biochemistry results revealed that CAE(50 and 200 mg/kg) decreased the levels of alanine transaminase, aspartate transaminase, gamma glutamyl transferase, total bilirubin, nitric oxide, and thiobarbituric acid reactive substances. Real-time PCR and western blot analysis showed that CAE regulated inflammation, apoptosis, and lipid metabolism factors increased by BDL. Interleukin family, tumor necrosis factor α, and related apoptosis factors m RNA levels were increased by BDL treatment. However, these increases were suppressed by CAE administration. In addition, CAE effectively increased phosphorylation of AMPactivated protein kinase, nuclear factor E2-related factor 2, and related cytoprotective proteins. Conclusions: CAE can efficiently regulate BDL-induced liver injury with antioxidant, antiinflammatory, and antiapoptotic activities.

Multiparameter magnetic resonance imaging of liver fibrosis in a bile duct ligation mouse model

BACKGROUND Bile duct ligation(BDL)in animals is a classical method for mimicking cholestatic fibrosis.Although different surgical techniques have been described in rats and rabbits,mouse models can be more cost-effective and reproducible for investigating cholestatic fibrosis.Magnetic resonance imaging(MRI)has made great advances for noninvasive assessment of liver fibrosis.More comprehensive liver fibrotic features of BDL on MRI are important.However,the utility of multiparameter MRI to detect liver fibrosis in a BDL mouse model has not been assessed.AIM To evaluate the correlation between the pathological changes and multiparameter MRI characteristics of liver fibrosis in a BDL mouse model.METHODS Twenty-eight healthy adult male balb/c mice were randomly divided into four groups:sham,week 2 BDL,week 4 BDL,and week 6 BDL.Multiparameter MRI sequences,included magnetic resonance cholangiopancreatography,T1-weighted,T2-weighted,T2 mapping,and pre-and post-enhanced T1 mapping,were performed after sham and BDL surgery.Peripheral blood and liver tissue were collected after MRI.For statistical analysis,Student’s t-test and Pearson’s correlation coefficient were used.RESULTS Four mice died after BDL surgery;seven,six,five and six mice were included separately from the four groups.Signal intensities of liver parenchyma showed no difference on TI-and T2-weighted images.Bile duct volume,ΔT1 value,T2 value,and the rate of liver fibrosis increased steadily in week 2 BDL,week 4 BDL and week 6 BDL groups compared with those in the sham group(P<0.01).Alanine aminotransferase and aspartate transaminase levels initially surged after surgery,followed by a gradual decline over time.Strong correlations were found between bile duct volume(r=0.84),T2 value(r=0.78),ΔT1 value(r=0.62),and hepatic fibrosis rate(all P<0.01)in the BDL groups.CONCLUSION The BDL mouse model induces changes that can be observed on MRI.The MRI parameters correlate with the hepatic fibrosis rate and allow for detection of cholestatic fibrosis.

Bile duct ligation differently regulates protein expressions of organic cation transporters in intestine,liver and kidney of rats through activation of farnesoid X receptor by cholate and bilirubin

Body is equipped with organic cation transporters(OCTs).These OCTs mediate drug transport and are also involved in some disease process.We aimed to investigate whether liver failure alters intestinal,hepatic and renal Oct expressions using bile duct ligation(BDL)rats.Pharmacokinetic analysis demonstrates that BDL decreases plasma metformin exposure,associated with decreased intestinal absorption and increased urinary excretion.Western blot shows that BDL significantly downregulates intestinal Oct2 and hepatic Oct1 but upregulates renal and hepatic Oct2.In vitro cell experiments show that chenodeoxycholic acid(CDCA),bilirubin and farnesoid X receptor(FXR)agonist GW4064 increase OCT2/Oct2 but decrease OCT1/Oct1,which are remarkably attenuated by glycine-β-muricholic acid and silencing FXR.Significantly lowered intestinal CDCA and increased plasma bilirubin levels contribute to different Octs regulation by BDL,which are confirmed using CDCA-treated and bilirubin-treated rats.A disease-based physiologically based pharmacokinetic model characterizing intestinal,hepatic and renal Octs was successfully developed to predict metformin pharmacokinetics in rats.In conclusion,BDL remarkably downregulates expressions of intestinal Oct2 and hepatic Oct1 protein while upregulates expressions of renal and hepatic Oct2 protein in rats,finally,decreasing plasma exposure and impairing hypoglycemic effects of metformin.BDL differently regulates Oct expressions via Fxr activation by CDCA and bilirubin.

Bile Acid Overload Induced by Bile Duct and Portal Vein Ligation Improves Survival after Staged Hepatectomy in Rats

Objective Compared to portal vein ligation(PVL),simultaneous bile duct and portal vein ligation(BPL)can significantly enhance hypertrophy of the intact liver.This study aimed to investigate whether BPL could improve survival after extended hepatectomy independently of an increased remnant liver.Methods We adopted rat models of 90%BPL or 90%PVL.To investigate the role of bile acids(BAs)the BA pools in the PVL and BPL groups were altered by the diet.Staged resection preserving 10%of the estimated liver weight was performed 3 days after BPL;PVL;or sham operation.Histology,canalicular network(CN)continuity;and hepatocyte polarity were evaluated.Results At 3 days after BPL;PVL;or sham operation when the volumetric difference of the intended liver remained insignificant,the survival rates after extended hepatectomy were 86.7%,47%,and 23.3%,respectively(P<0.01).BPL induced faster restoration of canalicular integrity along with an intensive but transient BA overload.Staged hepatectomy after BPL shortened the duration of the bile CN disturbance and limited BA retention.Decreasing the BA pools in the rats that underwent BPL could compromise these effects,whereas increasing the BA pools of rats that underwent PVL could induce similar effects.The changes in CN restoration were associated with activation of LKB1.Conclusion In addition to increasing the future remnant liver,BPL shortened the duration of the spatial disturbance of the CN and could significantly improve the tolerance of the hypertrophied liver to staged resection.BPL may be a safe and efficient future option for patients with an insufficient remnant liver.

Effects of Yinchenhao Decoction on Self-regulation of Renin-angiotensin System by Targeting Angiotensin Converting Enzyme 2 in Bile Duct-ligated Rat Liver

Summary： In order to investigate whether Yinchenhao decoction （YCHD） attenuates hepatic fibro- genesis in the bile duct ligation （BDL） model via recovering and restoring the self-regulation and bal- ance 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.kgl.day-1） were given by gastric gavage for 16 days following BDL in G3 and G4 groups, respec- tively. 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 ob-. served 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 in- cluding angiotensin converting enzyme （ACE）, angiotensin II type 1 receptor （AT1R）, ACE2, angio- tensin II （Ang II） as well as transforming growth factor 131 （TGF131）. The experimental data were ana- lyzed by principle component analytical method of pattern recognition. The results showed that bio- chemically, 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 sig- nificantly 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 lev- els of ACE2, ACE, Ang II, AT1R and TGF131 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 TGF131 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 TGFI31 downexpression so as to recover and rebuild self-regulation of the RAS by elevating the protein expression of ACE2.

Altered Endothelin-1 Signaling in Production of Thromboxane A_(2) in Kupffer Cells from Bile Duct Ligated Rats

Kupffer cells(KCs),the liver resident macrophages accounting for 80-90%of the total population of fixed tissue macrophages in the body,not only play a key role in host defense via removing particulate materials from the portal circulation,but may also contribute to the pathogenesis of various liver diseases.We have previously demonstrated that KCs play an important role in controlling portal hypertension and hepatocellular injury via releasing thromboxane A_(2)(TXA_(2))in early fibrosis induced by one-week bile duct ligation(BDL).Production of TXA_(2) is controlled by cytosolic phospholipase A_(2)(cPLA_(2))that is activated by the interaction of entothelin-1(ET-1)with its G-protein coupled ET receptor B(ETBR).However,the signaling pathways that contribute to the ET-1-induced activation of cPLA_(2) and production of TXA_(2) in KCs in the normal healthy or injured livers are not yet clear,which are investigated in the present study using isolated KCs from one-week BDL or sham rats.The pharmacological inhibition of cPLA_(2) or chelation of intracellular calcium abrogated the ET-1 induction of TXA_(2) from KCs.Compared to those from sham rats,KCs from BDL animals displayed a significantly enhanced responsiveness of p38 MAPK to ET-1,increased ETBR and Gai subunit but decreased Gaq and Ga11 expression.Inhibition of ERK1/2 or Gq signaling abrogated significantly the ET-1 induction of TXA_(2) in sham KCs but only slightly in BDL KCs.In contrast,inhibition of p38 MAPK and Gi signaling markedly attenuated the ET-1 induction of TXA_(2) in BDL KCs but had no effect in sham KCs.Lastly,inhibition of PLC or PKC abrogated ET-1 induction of TXA_(2) in KCs from both sham and BDL groups.The hepatic stress(such as BDL)induces significant modifications in the receptor and intermediates of ET-1 signaling in KC and subsequently alters ET-1 signaling mechanisms,particularly a shift from Gq induced signaling to Gi induced signaling,in the activation of cPLA_(2) and production of TXA_(2) in response to ET-1.

Melatonin prevents oxidative stress,inflammatory activity,and DNA damage in cirrhotic rats

BACKGROUND Cirrhosis is an important health problem characterized by a significant change in liver parenchyma.In animals,this can be reproduced by an experimental model of bile duct ligation(BDL).Melatonin(MLT)is a physiological hormone synthesized from serotonin that has been studied for its beneficial properties,including its antioxidant potential.AIM To evaluate MLT’s effects on oxidative stress,the inflammatory process,and DNA damage in an experimental model of secondary biliary cirrhosis.METHODS Male Wistar rats were divided into 4 groups:Control(CO),CO+MLT,BDL,and BDL+MLT.MLT was administered(20 mg/kg)daily beginning on day 15 after biliary obstruction.On day 29 the animals were killed.Blood samples,liver tissue,and bone marrow were collected for further analysis.RESULTS BDL caused changes in biochemical and histological parameters and markers of inflammatory process.Thiobarbituric acid(0.46±0.01)reactive substance levels,superoxide dismutase activity(2.30±0.07)and nitric oxide levels(2.48±0.36)were significantly lower(P<0.001)n the groups that received MLT.DNA damage was also lower(P<0.001)in MLT-treated groups(171.6±32.9)than the BDL-only group(295.5±34.8).Tissue damage and the expression of nuclear factor kappa B,interleukin-1β,Nrf2,NQO1 and Hsp70 were significantly lower in animals treated with MLT(P<0.001).CONCLUSION When administered to rats with BDL-induced secondary biliary cirrhosis,MLT effectively restored the evaluated parameters.

TLR4 Knockout Attenuates BDL-induced Liver Cholestatic Injury through Amino Acid and Choline Metabolic Pathways

The exact mechanism by which knockout of Toll-like receptor 4(TLR4)attenuates the liver injury remains unclear.The present study aimed to examine the role of TLR4 in the pathogenesis of bile duct ligation(BDL)-induced liver cholestatic injury and the underlying mechanism.Wild type(WT)mice and TLR4 knockout(TLR4-KO)mice were used for the establishment of the BDL model.Metabolomics were applied to analyze the changes of small molecular metabolites in the serum and liver of the two groups.The serum biochemical indexes and the HE staining results of liver tissue showed that liver damage was significantly reduced in TLR4-KO mice after BDL when compared with that in WT mice.The metabolite analysis results showed that TLR4 KO could maintain the metabolisms of amino acids-and choline-related metabolites.After BDL,the amino acids-and choline-related metabolites,especially choline and 3-hydroxybutyrate,were significantly increased in WT mice(both in serum and liver),but these metabolites in the liver of TLR4-KO mice after BLD were not significant different from those before BLD.In conclusion,TLR4 KO could attenuate BDL-induced liver cholestatic injury through regulating amino acid and choline metabolic pathways.

Correction to“Inhibiting heme oxygenase-1 attenuates rat liver fibrosis by removing iron accumulation”

We found a mistake in Figure 6. Panels A (Sham group) and F (DFX group) (180degrees rotated) is same images. We have replaced the incorrect images (Panels F)with the correct Figure. This error does not change the meaning of the picture orthe conclusion of the manuscript. We apologize for our unintentional mistakes,which caused great inconvenience.

Evaluating the regulation of transporter proteins and P-glycoprotein in rats with cholestasis and its implication for digoxin clearance

BACKGROUND Cardiac and hepatic functionality are intertwined in a multifaceted relationship.Pathologic processes involving one may affect the other through a variety of mechanisms,including hemodynamic and membrane transport effects.AIM To better understand the effect of extrahepatic cholestasis on regulations of membrane transporters involving digoxin and its implication for digoxin clearance.METHODS Twelve adult rats were included in this study;baseline hepatic and renal laboratory values and digoxin pharmacokinetic(PK)studies were established before evenly dividing them into two groups to undergo bile duct ligation(BDL)or a sham procedure.After 7 d repeat digoxin PK studies were completed and tissue samples were taken to determine the expressions of cell membrane transport proteins by quantitative western blot and real-time polymerase chain reaction.Data were analyzed using SigmaStat 3.5.Means between pre-surgery and post-surgery in the same experimental group were compared by paired t-test,while independent t-test was employed to compare the means between sham and BDL groups.RESULTS Digoxin clearance was decreased and liver function,but not renal function,was impaired in BDL rats.BDL resulted in significant up-regulation of multidrug resistance 1 expression in the liver and kidney and its down-regulation in the small intestine.Organic anion transporting polypeptides(OATP)1A4 was up-regulated in the liver but down-regulated in intestine after BDL.OATP4C1 expression was markedly increased in the kidney following BDL.CONCLUSION The results suggest that cell membrane transporters of digoxin are regulated during extrahepatic cholestasis.These regulations are favorable for increasing digoxin excretion in the kidney and decreasing its absorption from the intestine to compensate for reduced digoxin clearance due to cholestasis.

Granulocyte colony-stimulating factor reduces biliary fibrosis and ductular reaction in a mouse model of chronic cholestasis

Background:Biliary atresia is a rare congenital bile duct disease that is the leading cause of liver fibrosis in neonates.Granulocyte colony-stimulating factor(GCSF)is a potential therapy for hepatocellular diseases,but data on GCSF for cholestatic conditions remain limited.Materials and methods:The current study examines the role of GCSF in improving bile duct obstruction in mice.Two doses were administered:10.0 mg/kg/day and 61.5 mg/kg/day,which is the animal equivalent dose of 5.0 mg/kg in humans.Seven days(D7)after bile duct ligation(BDL),Swiss mice were treated with phosphate buffered saline or GCSF for 5 days.The intrahepatic adaptive response of BDL mice was evaluated on postsurgical days D12,D19,and D26.Results:Treatment with 61.5 mg/kg of GCSF resulted in a significant increase in circulating leukocytes and neutrophils on D12.Amelioration of liver injury,as shown by reduced aspartate aminotransferase levels,increased albumin levels and survival rate,as well as reduced intrahepatic inflammation and hepatic myeloperoxidase expression,downregulated ductular proliferation,periportal fibroblast activation,and fibrosis,enhanced expressions of hepatocyte growth factor,peroxisome proliferator-activated receptoralpha,and ki67,and suppressed expression of cleaved caspase-3 protein,was noted after treatment with 61.5 mg/kg of GCSF.Additionally,GCSF treatment was associated with an increased number of intrahepatic cd3-Sca1tc-Kitt bone marrow cells.

Limited potential of resolvin D1 in treatment of cholestatic liver fibrosis

Background:Several studies suggest a role for EPA-and DHA-derived pro-resolving mediators like resolvins in reversing metabolic and inflammatory disturbances seen in various chronic diseases.Here,we investigated the effects of resolvin D1(RvD1)on bile duct ligation(BDL)-induced cholestatic liver injury.Methods:Mice were treated daily with RvD1 or 0.1%ethanol(control)from the day of BDL until the final observation time points.Blood and liver tissue were collected 2,5 and 14 days after BDL for different analyses.Results:RvD1 treatment of mice had no impact on the extent of cholestatic liver injury upon BDL,neither in the acute phase nor in the progressive state of liver fibrosis.Although RvD1 treatment resulted in a significantly reduced activity of hepatic stellate cells as well as reduced deposition of extracellular matrix 2 days after BDL,mice were not protected from inflammation and further fibrosis progression.Conclusions:These data indicate that RvD1 has a limited therapeutic potential to treat cholestatic liver diseases,as it has no significant impact on regression of hepatic necroinflammation and fibrotic changes in bile duct-ligated mice.