Farnesoid X receptor is an important target for the treatment of disorders of bile acid and fatty acid metabolism in mice with nonalcoholic fatty liver disease combined with cholestasis.

BACKGROUND AND AIM: The prevalence of nonalcoholic fatty liver disease (NAFLD) has been rising globally. NAFLD patients combined with cholestasis have more obvious liver fibrosis, impaired bile acid (BA), and fatty acid (FA) metabolism and severer liver injury; however, its therapeutic options are limited, and the underlying metabolic mechanisms are understood. Here, we aimed to investigate the effects of farnesoid X receptor (FXR) on BA and FA metabolism in NAFLD combined with cholestasis and related signaling pathways. METHODS: A mouse model of NAFLD combined with cholestasis was established by joint intervention with high-fat diet (HFD) and alpha-naphthylisothiocyanate. The effects of FXR on BA and FA metabolism were evaluated by serum biochemical analysis. Liver damage was identified by histopathology. The expression of nuclear hormone receptor, membrane receptor, FA transmembrane transporter, and BA transporter protein in mice were measured by western blot. RESULTS: NAFLD mice combined with cholestasis developed more severe cholestasis and dysregulated BA and FA metabolism. Meanwhile, the expression of FXR protein was decreased in NAFLD mice combined with cholestasis compared to the controls. Fxr-/- mice showed liver injury. HFD aggravated the liver injury with decreased BSEP expression, increased expression of NTCP, LXRα, SREBP-1c, FAS, ACC1, and CD36, and significantly increased BA and FA accumulation. CONCLUSION: All the results suggested that FXR plays a key role in both FA and BA metabolism in NAFLD combined with cholestasis and thus may be a potential target for the treatment of disorders of BA and FA metabolism in NAFLD combined with cholestasis.

Intrahepatic cholestasis induced by α-naphthylisothiocyanate can cause gut-liver axis disorders.

Clinical studies have shown that Intrahepatic cholestasis is closely related to intestinal injury. The gut-liver axis theory suggests that the intestine and liver are closely related, and that bile acids are important mediators linking the intestine and liver. We compared two cholestasis models: a single injection model that received a single subcutaneous ANIT injection (75 mg/kg), and a multiple subcutaneous injection model that received an injection of ANIT (50 mg/kg) every other day for 2 weeks. We used Transmetil (ademetionine 1,4-butanedisulfonate) to relieve intrahepatic cholestasis in the multiple injection group. In the multiple injection group, we found increased hepatic bile duct hyperplasia, increased fibrosis of the liver, increased small intestine inflammation and oxidative damage, increased harmful bile acids, decreased bile acids transporter levels. After treatment with Transmetil, the liver and gut injuries were relieved. These results suggest that intrahepatic cholestasis can cause disorders of the gut-liver axis.

Protective effect of Echinochrome against intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats / Efeito protetor de Echinochrome contra colestase intra-hepática induzida por isotiocianato de alfa-naftilo em ratos

Abstract The present study was designed to evaluate the protective effects of echinochrome (Ech) on intrahepatic cholestasis in rats induced by a single (i.p.) injection of alpha-naphthylisothiocyanate (ANIT) (75 mg/kg body weight). The rats were pre-treated orally for 48hr (one dose / 24hr) with Ech (1, 5 and 10 mg/kg body weight) or ursodeoxycholic acid (UDCA) 80 mg/kg body weight drug then, injected with ANIT. ANIT markedly increased serum activities of alanine amino transaminase (ALT), aspartate amino transaminase (AST) and alkaline phosphatase (ALP), which was accompanied by a massive inflammation of epithelial cells on bile duct at 24h after ANIT injection. ANIT also increased the levels of total protein (TP), total bilirubin (TB), direct bilirubin (DB), indirect bilirubin (IB), however decrease albumin content (ALB). In addition ANIT increased hepatic MDA and NO level and decreased GSH level and GST activity. The Ech exerted hepatoprotective and anticholestatic effects as assessed by a significant decrease in the activities of serum AST, ALT and ALP, and the levels of TP, TB, DB and IB as well as liver MDA level and NO level. In conclusion, Ech was found to possess hepatoprotective effect against intrahepatic cholestasis induced by hepatotoxin such as ANIT.

Resumo O presente estudo destinou-se a avaliar os efeitos protetores do Ech na colestase intra-hepática em ratos induzidos por uma única injeção (i.p.) de alfa-naftilisotiocianato (ANIT) (75 mg / kg de peso corporal). Os ratos foram pré-tratados oralmente durante 48 horas (uma dose / 24 horas) com cada (1, 5 e 10 mg / kg de peso corporal) e ácido ursodeoxicólico (UDCA) 80 mg / kg de peso corporal, em seguida, injetado com ANIT. ANIT atividades de soro marcadamente aumentadas de alanina amino transaminasa (ALT), aspartato de amino transaminases (AST) e fosfatase alcalina (ALP), que foi acompanhada por uma inflamação maciça de células epiteliais no ducto biliar às 24h após a injeção de ANIT. A ANIT também aumentou os níveis de proteína total (TP), bilirrubina total (TB), bilirrubina direta (DB), bilirrubina indireta (IB), no entanto, diminuem o teor de albumina (ALB). Além disso, a ANIT aumentou o nível de MDA hepático e NO e diminuiu o nível de GSH e a atividade de GST. O Ech exerceu efeitos hepatoprotectores e anticolestáticos como avaliado por uma diminuição significativa nas atividades de AST sérica, ALT e ALP e os níveis de TP, TB, DB e IB, bem como o nível de MDA no fígado e o nível de NO. Ech foi encontrado para possuir efeito protetor no fígado contra a colestase intra-hepática induzida por hepatotoxina, como a ANIT.

UPLC-MS-based metabonomic analysis of intervention effects of Da-Huang-Xiao-Shi decoction on ANIT-induced cholestasis.

ETHNOPHARMACOLOGICAL RELEVANCE: Cholestasis, caused by hepatic accumulation of bile acids, is a serious manifestation of liver diseases resulting in liver injury, fibrosis, and liver failure with limited therapies. Da-Huang-Xiao-Shi decoction (DHXSD) is a representative formula for treating jaundice and displays bright prospects in liver protective effect. AIM OF THE STUDY: This study was designed to assess the effects and possible mechanisms of DHXSD against alpha-naphthylisothiocyanate-induced liver injury based on ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) metabonomic approach. MATERIALS AND METHODS: The effects of DHXSD on serum indices (TBIL, DBIL, AST, ALT, ALP, TBA, and γ-GT) and the histopathology of the liver were analyzed. Moreover, UHPLC-Q-Orbitrap MS was performed to identify the possible effect of DHXSD on metabolites. The pathway analysis was conducted to illustrate the pathways and network by which DHXSD treats cholestasis. RESULTS: The results demonstrated that DHXSD could significantly regulate serum biochemical indices and alleviate histological damage to the liver. Twelve endogenous components, such as glycocholic acid, taurocholic acid and indoleacetaldehyde, were identified as potential biomarkers of the therapeutic effect of DHXSD. A systematic network analysis of their corresponding pathways indicates that the anti-cholestatic effect of DHXSD on alpha-naphthylisothiocyanate-induced cholestasis rats occurs mainly through regulating primary bile acid biosynthesis, arginine and proline metabolism, and arachidonic acid metabolism. CONCLUSIONS: DHXSD has exhibited favorable pharmacological effect on serum biochemical indices and pathological observation on cholestatic model by partially regulating the perturbed pathways. Moreover, these findings may help better understand the mechanisms of disease and provide a potential therapy for cholestasis.

A systemic combined nontargeted and targeted LC-MS based metabolomic strategy of plasma and liver on pathology exploration of alpha-naphthylisothiocyanate induced cholestatic liver injury in mice.

The pathology of cholestatic liver injury (CLI) was complicated, which has limited the development of anti-cholestatic drugs for a long period. Metabolomic researches focused on global and dynamic changes of the organism could shed some light on mechanism investigation. In order to characterize and validate metabolite alterations of alpha-naphthylisothiocyanate (ANIT) induced CLI in C57BL/6 mice, a systemic metabolomic approach combining nontargeted HPLC-ESI-QTOF-MS and targeted UFLC-ESI-MS/MS technologies were developed innovatively. Multivariate data analysis was applied to determine the changes of metabolites in processed plasma and liver samples between control and model groups. Afterwards, 38 potential plasma biomarkers and 17 potential liver biomarkers involved in bile acid (BA) biosynthesis, phospholipid biosynthesis, sphingolipid metabolism, alpha linolenic acid and linoleic acid metabolism, as well as arachidonic acid metabolism were found and attributed as potential biomarkers and influential pathways of cholestasis. Based on correlation analysis, BA biosynthesis played the most important role in ANIT induced CLI, thereinto, major BAs were carried out with quantitative analysis. Targeted metabolomic results showed that the increase of BAs might have an impact on intestinal microbial ecology which could aggravate liver injury probably, among which cholic acid (CA) and taurocholic acid (TCA) were the most sensitive indicators of ANIT induced CLI in both plasma and liver. In conclusion, CLI might correlate significantly with hepatocyte necrosis, metabolic disorders and imbalance of intestinal microbiome ecology triggered by BA accumulation.

Paracrine Fibroblast Growth Factor 1 Functions as Potent Therapeutic Agent for Intrahepatic Cholestasis by Downregulating Synthesis of Bile Acid.

Endocrine fibroblast growth factor (FGF) 19 has been shown to be capable of maintaining bile acid (BA) homeostasis and thus hold promise to be a potential therapeutic agent for cholestasis liver disease. However, whether paracrine FGFs possess this BA regulatory activity remains to be determined. In our study, we identified that paracrine fibroblast growth factor 1 (FGF1) was selectively downregulated in the liver of alpha naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis mice, suggesting a pathological relevance of this paracrine FGF with abnormal BA metabolism. Therefore, we evaluated the effects of engineered FGF1 mutant - FGF1ΔHBS on the metabolism of hepatic BA and found that this protein showed a more potent inhibitory effect of BA biosynthesis than FGF19 without any hepatic mitogenic activity. Moreover, the chronic administration of FGF1ΔHBS protected liver against ANIT-induced injury by reducing hepatic BA accumulation. Taken together, these data suggest that FGF1ΔHBS may function as a potent therapeutic agent for intrahepatic cholestasis liver disease.

Basal PPARα inhibits bile acid metabolism adaptation in chronic cholestatic model induced by α-naphthylisothiocyanate.

Cholestasis is one of the most challenging diseases to be treated in current hepatology. However little is known about the adaptation difference and the underlying mechanism between acute and chronic cholestasis. In this study, wild-type and Pparα-null mice were orally administered diet containing 0.05% ANIT to induce chronic cholestasis. Biochemistry, histopathology and serum metabolome analysis exhibited the similar toxic phenotype between wild-type and Pparα-null mice. Bile acid metabolism was strongly adapted in Pparα-null mice but not in wild-type mice. The Shp and Fxr mRNA was found to be doubled in cholestatic Pparα-null mice compared with the control group. Western blot confirmed the up-regulated expression of FXR in Pparα-null mice treated with ANIT. Inflammation was found to be stronger in Pparα-null mice than those in wild-type mice in chronic cholestasis. These data chain indicated that bile acid metabolism and inflammation signaling were different between wild-type and Pparα-null mice developing chronic cholestasis, although their toxic phenotypes could not be discriminated. So basal PPARα cross-talked with FXR and inhibited bile acid metabolism adaptation in chronic cholestasis.

Effects of human amnion-derived mesenchymal stem cells and conditioned medium in rats with sclerosing cholangitis.

Mesenchymal stem cells (MSCs) represent a valuable cell source in regenerative medicine, and large numbers of MSCs can be isolated from the amnion noninvasively. Sclerosing cholangitis is a chronic cholestatic disease and characterized by progressive biliary destruction leading to cirrhosis. Many factors are involved in the development of sclerosing cholangitis; however, effective medical therapy is not established. We investigated the effects of human amnion-derived MSCs (hAMSCs) and conditioned medium (CM) obtained from hAMSC cultures in rats with sclerosing cholangitis. Sclerosing cholangitis was induced via the intragastric administration of 100 mg/kg alpha-naphthylisothiocyanate (ANIT) twice weekly for 4 weeks. One million hAMSCs or 200 µL of CM were intravenously administered on days 15 and 22. Rats were sacrificed on day 29 and evaluated via histological, immunohistochemical, and mRNA expression analyses. hAMSC transplantation and CM administration significantly improved the histological score. In addition, these two interventions significantly improved biliary hyperplasia, peribiliary fibrosis, and inflammation in Glisson's sheath. Accordingly, CK19, MMP-9, and TNF-α, and MCP-1 expression in the liver was also decreased by hAMSC and CM administration. In conclusion, hAMSC and CM administration ameliorated biliary hyperplasia, peribiliary fibrosis, and inflammation in a rat model of sclerosing cholangitis. hAMSCs and CM may represent new modalities for treating sclerosing cholangitis.

Different effects of ursodeoxycholic acid on intrahepatic cholestasis in acute and recovery stages induced by alpha-naphthylisothiocyanate in mice.

The aim of this study was to determine the effect of ursodeoxycholic acid (UDCA) on the alpha-naphthylisothiocyanate (ANIT)-induced acute and recovery stage of cholestasis model mice. In the acute stage of model mice, pretreatment with UDCA (25, 50, and 100 mg·kg-1, ig) for 12 days prior to ANIT administration (50 mg·kg-1, ig) resulted in the dramatic increase in serum biochemistry, with aggrevation of bile infarcts and hepatocyte necrosis. The elevation of beta-muricholic acid (ß-MCA), cholic acid (CA), and taurocholic acid (TCA) in serum and liver, and reduction of these bile acids (BAs) in bile was observed. In contrast, in the recovery stage of model mice, treatment with UDCA (25, 50, and 100 mg·kg-1, ig) for 7 days after ANIT administration (50 mg·kg-1, ig) resulted in the significant decrease in levels of serum alanine aminotransferase (ALT) and total bile acid (TBA). Liver injury was attenuated, and the levels of TBA, CA, TCA, and ß-MCA in the liver were significantly decreased. Additionally, UDCA can upregulate expression of BSEP, but it cannot upregulate expression of AE2. UDCA, which induced BSEP to increase bile acid-dependent bile flow, aggravated cholestasis and liver injury when the bile duct was obstructed in the acute stage of injury in model mice. In contrast, UDCA alleviated cholestasis and liver injury induced by ANIT when the obstruction was improved in the recovery stage.

Characterization of a rat model of moderate liver dysfunction based on alpha-naphthylisothiocyanate-induced cholestasis.

Plasma amino acid level changes occur in mild, moderate and severe stages of liver injury in human patients. In animal models, however, data are mainly restricted to severe liver injury models in rats. Here we present the characterization of a rat model of moderate liver dysfunction secondary to alpha-napthylisothiocyanate (ANIT)-induced cholestasis. Rats treated with 30 mg/kg/day ANIT for 3 weeks exhibited a time-dependent increase in plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bilirubin levels and a decrease in albumin concentration. According to a liver dysfunction evaluation based on the human Child-Pugh-Score, animals developed a moderate liver dysfunction in the first two weeks of ANIT treatment, while only a mild dysfunction was observed at the end of week 3 despite ongoing ANIT administration. Univariate analysis of branched-chain amino acid plasma levels indicated that reduced levels of branched chain amino acids were associated with the ANIT treatment. These data may set the stage for further research of amino acid disturbances and requirements in non-severe cholestasis.

Early indications of ANIT-induced cholestatic liver injury: Alteration of hepatocyte polarization and bile acid homeostasis.

Hepatocyte polarization is essential for biliary secretion, and loss of polarity causes bile secretory failure and hepatotoxicity. Here, we showed that alpha-naphthyl isothiocyanate (ANIT)-induced liver injury was accompanied by the dynamic interruption of bile acid homeostasis in rat plasma, liver and bile, which was characterized by the redistribution of bile acids in plasma and bile and a small range of fluctuations in the liver. Molecular mechanism studies indicated that these factors are dynamically mediated by the disruption of bile acid transporters and hepatic tight junctions. Dynamic changes in tight junction (TJ) permeability were observed by hepatobiliary barrier function assessment. Hepatocyte polarization was disrupted by ANIT before the development of cholestatic hepatotoxicity and alteration of bile acid metabolic profiles, which were assayed by high-performance liquid chromatography-tandem mass spectrometry, further verifying TJ deficiency. S1PR1 activation with SEW2871 reduced ANIT-induced liver injury by reducing the total serum bile acid concentration, liver functional enzyme activity and inflammation. Our data suggest that hepatocyte polarization plays an important role in maintaining bile acid homeostasis before the development of cholestatic hepatotoxicity and that TJs were more prominent in the early stage of cholestasis. S1PR1 may be a potential target for the prevention of drug-induced cholestatic liver injury.

Lymphocytes contribute to biliary injury and fibrosis in experimental xenobiotic-induced cholestasis.

The etiology of chronic bile duct injury and fibrosis in patients with autoimmune cholestatic liver diseases is complex, and likely involves immune cells such as lymphocytes. However, most models of biliary fibrosis are not autoimmune in nature. Biliary fibrosis can be induced experimentally by prolonged exposure of mice to the bile duct toxicant alpha-naphthylisothiocyanate (ANIT). We determined whether lymphocytes contributed to ANIT-mediated biliary hyperplasia and fibrosis in mice. Hepatic accumulation of T-lymphocytes and increased serum levels of anti-nuclear-autoantibodies were evident in wild-type mice exposed to ANIT (0.05% ANIT in chow). This occurred alongside bile duct hyperplasia and biliary fibrosis. To assess the role of lymphocytes in ANIT-induced biliary fibrosis, we utilized RAG1-/- mice, which lack T- and B-lymphocytes. ANIT-induced bile duct injury, indicated by increased serum alkaline phosphatase activity, was reduced in ANIT-exposed RAG1-/- mice compared to ANIT-exposed wild-type mice. Despite this reduction in biliary injury, ANIT-induced bile duct hyperplasia was similar in wild-type and RAG1-/- mice. However, hepatic induction of profibrogenic genes including COL1A1, ITGß6 and TGFß2 was markedly attenuated in ANIT-exposed RAG1-/- mice compared to ANIT-exposed wild-type mice. Peribiliary collagen deposition was also reduced in ANIT-exposed RAG1-/- mice. The results indicate that lymphocytes exacerbate bile duct injury and fibrosis in ANIT-exposed mice without impacting bile duct hyperplasia.

Dose-dependent effects of alpha-naphthylisothiocyanate disconnect biliary fibrosis from hepatocellular necrosis.

Exposure of rodents to the xenobiotic α-naphthylisothiocyanate (ANIT) is an established model of experimental intrahepatic bile duct injury. Administration of ANIT to mice causes neutrophil-mediated hepatocellular necrosis. Prolonged exposure of mice to ANIT also produces bile duct hyperplasia and liver fibrosis. However, the mechanistic connection between ANIT-induced hepatocellular necrosis and bile duct hyperplasia and fibrosis is not well characterized. We examined impact of two different doses of ANIT, by feeding chow containing ANIT (0.05%, 0.1%), on the severity of various liver pathologies in a model of chronic ANIT exposure. ANIT-elicited increases in liver inflammation and hepatocellular necrosis increased with dose. Remarkably, there was no connection between increased hepatocellular necrosis and bile duct hyperplasia and peribiliary fibrosis, as these pathologies increased similarly in mice exposed to either dose of ANIT. The results indicate that the severity of hepatocellular necrosis does not dictate the extent of bile duct hyperplasia/fibrosis in ANIT-exposed mice.

Evaluation of hepatoprotective effects of different extracts in root of Gardenia jasminoides based on partial least squares method and multi-attribute comprehensive index method / 中草药

Objective To compare the hepatoprotective effects of different extracts in root of Gardenia jasminoides including ethanol extract, petroleum ether fraction, ethyl acetate, chloroform extract, n-butanol extract, and surplus water extract on the jaundice hepatitis mice by the partial least squares (PLS) method and multi index comprehensive evaluation method. Methods Setting up two groups of high and low doses (9 and 3 g/kg) in different extraction sites in root of G. jasminoides respectively, and administered to the stomach for 7 d. Copying a icteric hepatitis model of mice by alpha naphthylisothiocyanate (ANIT) in 5th d. Then, the alanine aminotransferase (ALT), aspartic transaminase (AST), alkaline phosphatase (AKP), γ-glutamyl transpeptidase (γ-GT), total bile acid (TBA), total bilirubin (TBIL), superoxide dismutase (SOD), malonic dialdehyde (MDA), glutathione (GSH) activity were determinated in serum and liver, and pathological changes were observed in mouse liver by HE stain, total hepatoprotective effect of the different extracts in root of G. jasminoides was compared by PLS and multi index comprehensive evaluation method. Results In addition to the high dose group of petroleum ether and low dose group of chloroform, the different extracts in root of G. jasminoides were improved on some indexes or multiple indicators to an extent and compared with the model group, the pathological damage of liver tissue was alleviated obviously, multi index comprehensive evaluation showed that the high dose group of ethyl acetatein root of G. jasminoides had the best hepatoprotective effect. Conclusion The ethyl acetate extract and n-butanol extract in root of G. jasminoides have better inhibitory effect on icteric hepatitis, and the high dose group of ethyl acetate has the best effect, which may be the active site of liver protection, and mechanism may be related to improving the ability of eliminating oxygen free radical, inhibiting lipid peroxidation and enhancing bile bilirubin metabolism and secretion.

Protective effect of Echinochrome against intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats

Abstract The present study was designed to evaluate the protective effects of echinochrome (Ech) on intrahepatic cholestasis in rats induced by a single (i.p.) injection of alpha-naphthylisothiocyanate (ANIT) (75 mg/kg body weight). The rats were pre-treated orally for 48hr (one dose / 24hr) with Ech (1, 5 and 10 mg/kg body weight) or ursodeoxycholic acid (UDCA) 80 mg/kg body weight drug then, injected with ANIT. ANIT markedly increased serum activities of alanine amino transaminase (ALT), aspartate amino transaminase (AST) and alkaline phosphatase (ALP), which was accompanied by a massive inflammation of epithelial cells on bile duct at 24h after ANIT injection. ANIT also increased the levels of total protein (TP), total bilirubin (TB), direct bilirubin (DB), indirect bilirubin (IB), however decrease albumin content (ALB). In addition ANIT increased hepatic MDA and NO level and decreased GSH level and GST activity. The Ech exerted hepatoprotective and anticholestatic effects as assessed by a significant decrease in the activities of serum AST, ALT and ALP, and the levels of TP, TB, DB and IB as well as liver MDA level and NO level. In conclusion, Ech was found to possess hepatoprotective effect against intrahepatic cholestasis induced by hepatotoxin such as ANIT.

Resumo O presente estudo destinou-se a avaliar os efeitos protetores do Ech na colestase intra-hepática em ratos induzidos por uma única injeção (i.p.) de alfa-naftilisotiocianato (ANIT) (75 mg / kg de peso corporal). Os ratos foram pré-tratados oralmente durante 48 horas (uma dose / 24 horas) com cada (1, 5 e 10 mg / kg de peso corporal) e ácido ursodeoxicólico (UDCA) 80 mg / kg de peso corporal, em seguida, injetado com ANIT. ANIT atividades de soro marcadamente aumentadas de alanina amino transaminasa (ALT), aspartato de amino transaminases (AST) e fosfatase alcalina (ALP), que foi acompanhada por uma inflamação maciça de células epiteliais no ducto biliar às 24h após a injeção de ANIT. A ANIT também aumentou os níveis de proteína total (TP), bilirrubina total (TB), bilirrubina direta (DB), bilirrubina indireta (IB), no entanto, diminuem o teor de albumina (ALB). Além disso, a ANIT aumentou o nível de MDA hepático e NO e diminuiu o nível de GSH e a atividade de GST. O Ech exerceu efeitos hepatoprotectores e anticolestáticos como avaliado por uma diminuição significativa nas atividades de AST sérica, ALT e ALP e os níveis de TP, TB, DB e IB, bem como o nível de MDA no fígado e o nível de NO. Ech foi encontrado para possuir efeito protetor no fígado contra a colestase intra-hepática induzida por hepatotoxina, como a ANIT.

Target profiling analyses of bile acids in the evaluation of hepatoprotective effect of gentiopicroside on ANIT-induced cholestatic liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiopicroside (GPS), one of iridoid glucoside representatives, is the most potential active component in Gentiana rigescens Franch. ex Hemsl and Gentiana macrophylla Pall. These two herbs have been used to treat jaundice and other hepatic and billiary diseases in traditional Chinese medicine for thousands of years. AIM OF THE STUDY: This study aimed to investigate the protective effects and mechanisms of GPS on α-naphthylisothiocyanate (ANIT) induced cholestatic liver injury in mice. MATERIALS AND METHODS: Mice were treated with GPS (130mg/kg, ig) for 5 consecutive days. On the third day, mice were given a single dose of Alpha-naphthylisothiocyanate (75mg/kg, ig). Serum biochemical markers and individual bile acids in serum, liver, urine and feces were measured at different time points after ANIT administration. The expression of hepatic bile acid synthesis, uptake and transporter genes as well as ileum bile acid transporter genes were assayed. RESULTS: In this study, ANIT exposure resulted in serious cholestasis with liver injury, which was demonstrated by dramatically increased serum levels of ALT, ALP, TBA and TBIL along with TCA CA, MCAs and TMCAs accumulation in both liver and serum. Furthermore, ANIT significantly decreased bile acid synthesis related gene expressions, and increased expression of bile acid transporters in liver. Continuous treatment with GPS attenuated ANIT-induced acute cholestasis as well as liver injury and correct the dyshomeostasis of bile acids induced by ANIT. Our data showed that GPS significantly upregulated the hepatic mRNA levels of synthesis enzymes (Cyp8b1 and Cyp27a1) and transporters (Mrp4 Mdr1 and Ost-ß) as well as ileal bile acid circulation mediators (Asbt and Fgf15), accompanied by serum and hepatic bile acid levels decrease and further urinary and fecal bile acid levels increase. CONCLUSION: GPS can change bile acids metabolism which highlights its importance in mitigating cholestasis, resulting in the marked decrease of intracellular bile acid pool back toward basal levels. And the protective mechanism was associated with regulation of bile acids-related transporters, but the potential mechanism warrants further investigation.

Effects of andrographolide on intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats.

Cholestasis is a cardinal manifestation of liver diseases but effective therapeutic approaches are limited. Therefore, alternative therapy for treating and preventing cholestatic liver diseases is necessary. Andrographolide, a promising anticancer drug derived from the medicinal plant Andrographis paniculata, has diverse pharmacological properties and multi-spectrum therapeutic applications. However, it is unknown whether andrographolide has a hepatoprotective effect on intrahepatic cholestasis. The aims of this study were to investigate the protective effect and possible mechanisms of andrographolide in a rat model of acute intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT). Andrographolide was administered intragastrically for four consecutive days, with a single intraperitoneal injection of ANIT on the second day. Liver injury was evaluated biochemically and histologically together with hepatic gene and protein expression analysis. Rats pretreated with andrographolide prior to ANIT injection demonstrated lower levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, as well as bilirubin and bile acids as compared to rats treated with ANIT alone. Andrographolide also decreased the incidence and extent of periductular fibrosis and bile duct proliferation. Analysis of protein expression in livers from andrographolide-treated cholestatic rats revealed markedly decreased expression of alpha-smooth muscle actin and nuclear factor kappa-B (NF-κB). In conclusion, andrographolide has a potent protective property against ANIT-induced cholestatic liver injury. The mechanisms that underlie this protective effect are mediated through down-regulation of NF-κB expression and inhibition of hepatic stellate cell activation. These findings suggest that andrographolide could be a promising therapeutic option in prevention and slowing down the progression of cholestatic liver diseases.

Acute Alpha-Naphthylisothiocyanate-induced Liver Toxicity in Germfree and Conventional Male Rats.

Differences in the responses of conventional and germfree male Sprague-Dawley rats to acute injury induced by alpha-naphthylisothiocyanate (ANIT), a well-characterized biliary epithelial toxicant, were evaluated. Conventional and germfree rats were dosed once orally with 50 mg/kg of ANIT or corn oil alone and serially sacrificed daily for the next 3 days. Germfree rats treated with ANIT tended to have greater increases in virtually all liver and biliary-related analytes compared with conventional rats treated with ANIT; however, significant differences were found only in a few of these analytes including increased bile acids on day 3, total bilirubin on day 4, glutamate dehydrogenase (GLDH) on day 3, and reduced paraoxonase 1 (PON1) on days 2 and 3. Histologic differences between the conventional and germfree rats were modest, but most pronounced on day 2 (24-hr post dosing). Based on subjective scoring, biliary necrosis, neutrophilic cholangitis, and portal tract edema were more severe in germfree rats at 24 hr post dosing compared with conventional rats. Biliary epithelial replication did not differ between treated groups, however. Overall, germfree rats had a modestly greater level of biliary tract injury based on subjective histologic scoring and clinical chemistry measurements following an acute exposure to the well-characterized biliary toxin, ANIT; however, the difference between the ANIT-treated germfree and conventional groups was modest and most evident only within the first day following exposure. These findings suggest that the microbiome did not significantly affect ANIT-induced acute biliary tract injury in the conditions of this study.

Alpha-naphthylisothiocyanate modulates hepatobiliary transporters in sandwich-cultured rat hepatocytes.

Alpha-naphthylisothiocyanate (ANIT) induces intra-hepatic cholestasis mixed with hepatocellular injury mainly by bile ductular damage. However, its direct effect on hepatic parenchymal cells (hepatocytes) is unclear. Sandwich-cultured rat hepatocytes (SCRH) were applied to clarify this question. Though cytotoxicity was not observed (0-180 µM) in ANIT-treated SCRH, metabonomics analysis of the hepatocytes revealed a shift in the metabolic pattern and a decrease in cellular cholesterol level, accompanied by an increase in total bile acids after 48 h ANIT (5-45 µM) treatment. To assess the function of major hepatic bile acid transporters, the accumulation and efflux of [D-Pen(2,5)]-enkephalin (DPDPE), 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) diacetate promoiety and deuterium-labeled sodium taurocholate (d8-TCA) were measured. ANIT incubation for either 30 min or 48 h led to dose-dependent decreases in the biliary excretion index (BEI) of DPDPE and CDF, as well as the intracellular accumulation of d8-TCA, CDF and DPDPE. The basolateral efflux of d8-TCA was also decreased with its BEI barely changed. mRNA expression of multiple uptake transporters and bile acid synthesizing enzymes was down-regulated after 48 h incubation. In conclusion, ANIT could directly induce retention of bile acids in hepatocytes by inhibiting the function of bile acid transporters, which might contribute to its cholestatic effect.

Deleterious effect of oltipraz on extrahepatic cholestasis in bile duct-ligated mice.

BACKGROUND & AIMS: Oltipraz (4-methyl-5(pyrazinyl-2)-1-2-dithiole-3-thione), a promising cancer preventive agent, has an antioxidative activity and ability to enhance glutathione biosynthesis, phase II detoxification enzymes and multidrug resistance-associated protein-mediated efflux transporters. Oltipraz can protect against hepatotoxicity caused by carbon tetrachloride, acetaminophen and alpha-naphthylisothiocyanate. Whether oltipraz has hepato-protective effects on obstructive cholestasis is unknown. METHODS: We administered oltipraz to mice for 5 days prior to bile duct ligation (BDL) for 3 days. Liver histology, liver function markers, bile flow rates and hepatic expression of profibrogenic genes were evaluated. RESULTS: Mice pretreated with oltipraz prior to BDL demonstrated higher levels of serum aminotransferases and more severe liver damage than in control mice. Higher bile flow and glutathione secretion rates were observed in unoperated mice treated with oltipraz than in control mice, suggesting that liver necrosis in oltipraz-treated BDL mice may be related partially to increased bile-acid independent flow and biliary pressure. Oltipraz treatment in BDL mice enhanced α-smooth muscle actin expression, consistent with activation of hepatic stellate cells and portal fibroblasts. Matrix metalloproteinases (Mmp) 9 and 13 and tissue inhibitors of metalloproteinases (Timp) 1 and 2 levels were increased in the oltipraz-treated BDL group, suggesting that the secondary phase of liver injury induced by oltipraz might be due to excessive Mmp and Timp secretions, which induce remodeling of the extracellular matrix. CONCLUSIONS: Oltipraz treatment exacerbates the severity of liver injury following BDL and should be avoided as therapy for extrahepatic cholestatic disorders due to bile duct obstruction.