Liposome Extract of Stachys pilifera Benth Effectively Improved Liver Damage due to Bile Duct Ligation Rats.

Herbal medicines harbor essential therapeutic agents for the treatment of cholestasis. In this study, we have assessed the anticholestatic potential of Stachys pilifera Benth's (SPB's) hydroalcoholic extract encapsulated into liposomes using bile duct ligation- (BDL-) induced hepatic cholestasis in rats. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), malondialdehyde (MDA), total thiol (T-SH) content, protein carbonyl (PCO), total bilirubin (TBIL), albumin (ALB), and nitric oxide (NO) metabolite levels were measured in either liver tissue or plasma to assess liver damage. Moreover, expression of proinflammatory cytokines (IL-1ß and TNF-α) and liver fibrosis markers (TGF-ß and SM-α) which are driving forces of many liver disorders was also determined. The activity of AST, ALT, and ALP was significantly enhanced in the BDL group in comparison to the control group; however, treatment with liposomal (SPB) hydroalcoholic extract significantly reduced AST and ALT's activity. Increases in MDA, TBIL, and NO levels and T-SH content due to BDL were restored to control levels by liposomal (SPB) hydroalcoholic extract treatment. Similarly, hepatic and plasma oxidative marker MDA levels, significantly enhanced by BDL, were significantly decreased by liposomal (SPB) hydroalcoholic extract treatment. Moreover, histopathological findings further demonstrated a significant decrease in hepatic damage in the liposomal (SPB) hydroalcoholic extract-treated BDL group. In addition, liposomal (SPB) hydroalcoholic extract treatment decreased the liver expression of inflammatory cytokines (IL-1ß, TNF-α) and liver fibrosis markers (TGF-ß and SM-α). Since liposomal (SPB) hydroalcoholic extract treatment alleviated the BDL-induced injury of the liver and improved the hepatic structure and function more efficiently in comparison to free SPB hydroalcoholic extract, probable liposomal (SPB) hydroalcoholic extract exhibits required potential therapeutic value in protecting the liver against BDL-caused oxidative injury.

Dolomiaea souliei ethyl acetate extract protected against α-naphthylisothiocyanate-induced acute intrahepatic cholestasis through regulation of farnesoid x receptor-mediated bile acid metabolism.

BACKGROUND: Cholestasis is characterized by accumulation of bile components in liver and systemic circulation. Restoration of bile acid homeostasis via activating farnesoid x receptor (FXR) is a promising strategy for the treatment of cholestasis. FXR-SHP (small heterodimer partner) axis plays an important role in maintaining bile acid homeostasis. PURPOSE: To investigate the anti-cholestasis effect of Dolomiaea souliei (Franch.) C.Shih (D. souliei) and clarify its underlying mechanism against α-naphthylisothiocyanate (ANIT) induced acute intrahepatic cholestasis. METHODS: ANIT-induced Sprague-Dawley rats were employed to investigate the anti-cholestasis effect of D. souliei ethyl acetate extract (DSE). Ursodeoxycholic acid (UDCA) was used as positive control. Bile flow and blood biochemical parameters were measured. Liver histopathological examination was conducted via hematoxylin-eosin staining. Western blot analysis was carried out to evaluate the protein levels related to bile acids metabolism and inflammation. The interactions between FXR and costunolide or dehydrocostus lactone, were conducted by molecular docking experiments. The effect of costunolide and dehydrocostus lactone on aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels and FXR expression were also evaluated using guggulsterone-induced L02 cells. RESULTS: DSE could promote bile excretions and protect against ANIT-induced liver damage in cholestasis rats. Protein levels of FXR, SHP, Na+/taurocholate cotransporter (NTCP), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2) were increased and the expressions of cholesterol 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) were decreased by DSE. Meanwhile, the anti-inflammatory factors, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) were also significantly increased, and the pro-inflammatory factor, interleukin-10 (IL-10), was significantly decreased in rats of DSE groups. Molecular docking revealed that costunolide and dehydrocostus lactone could be well docked into the FXR protein molecule, and hydrophobic interactions played the main function. Costunolide could reverse the increased AST and ALT levels and increase the FXR expression in guggulsterone-induced L02 cells. CONCLUSION: DSE had an anti-cholestasis effect by activating FXR-SHP axis, inhibiting synthesis of bile acid, and increasing bile secretion, together with inflammatory response and improving liver injury. Costunolide may be the main active component. This study provided a potential therapeutic mechanism for D. souliei as an anti-cholestasis medicine in the treatment of cholestasis liver diseases.

Network pharmacology-based mechanism prediction and pharmacological validation of Xiaoyan Lidan formula on attenuating alpha-naphthylisothiocyanate induced cholestatic hepatic injury in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The well-known Chinese prescription, Xiaoyan Lidan Formula (XYLDF), possesses efficiency of heat-clearing, dampness-eliminating and jaundice-removing. It has long been used clinically for the treatment of hepatobiliary diseases due to intrahepatic cholestasis (IHC). However, the mechanism of XYLDF for its therapeutic effects remains elusive. AIM OF THE STUDY: The study aimed to explore the potential targets for liver protective mechanism of XYLDF based on network pharmacology and experimental assays in ANIT-induced cholestatic hepatic injury (CHI) in rats. MATERIALS AND METHODS: On the basis of the 29 serum migrant compounds of XYLDF elucidated by UPLC-TOF-MS/MS, a network pharmacology approach was applied for the mechanism prediction. Systematic networks were constructed to identify potential molecular targets, biological processes, and signaling pathways. And the interactions between significantly potential targets and active compounds were simulated by molecular docking. For the mechanism validation, an ANIT-induced rat model was used to evaluate the effects of XYLDF on CHI according to serum biochemistry, bile flow rates, histopathological examination, and the gene and protein expression including enzymes related to synthesis, export, and import of bile acid in liver and ileum, and those of inflammatory cytokines, analyzed by RT-qPCR and WB. RESULTS: The results of network pharmacology research indicated TNF (TNF-α), RELA (NF-κB), NR1H4 (FXR), and ICAM1 (ICAM-1) to be the important potential targets of XYLDF for cholestatic liver injury, which are related to bile metabolism and NF-κB-mediated inflammatory signaling. And the molecular docking had pre-validated the prediction of network pharmacology, as the core active compounds of XYLDF had shown strong simulation binding affinity with FXR, followed by NF-κB, TNF-α, and ICAM-1. Meanwhile, the effects of XYLDF after oral administration on ANIT-induced CHI in rats exhibited the decreased levels of transaminases (ALT and AST), TBA, and TBIL in serum, raised bile flow rates, and markedly improved hepatic histopathology. Furthermore, consistent to the above targets prediction and molecular docking, XYLDF significantly up-regulated the expression of FXR, SHP, BSEP, and MRP2, and down-regulated CYP7A1 and NTCP in liver, and promoted expression of IBABP and OSTα/ß in ileum, suggesting the activation of FXR-mediated pathway referring to bile acid synthesis, transportation, and reabsorption. Moreover, the lower levels of TNF-α in plasma and liver, as well as the reduced hepatic gene and protein expression of NF-κB, TNF-α, and ICAM-1 after XYLDF treatment revealed the suppression of NF-κB-mediated inflammatory signaling pathway, as evidenced by the inhibition of nuclear translocation of NF-κB. CONCLUSIONS: XYLDF exhibited an ameliorative liver protective effect on ANIT-induced cholestatic hepatic injury. The present study has confirmed its mechanism as activating the FXR-regulated bile acid pathway and inhibiting inflammation via the NF-κB signaling pathway.

Dehydrodiconiferyl alcohol, a lignan from Herpetospermum pedunculosum, alleviates cholestasis by activating pathways associated with the farnesoid X receptor.

BACKGROUND: In our previous study, we demonstrated the hepatoprotective effect of Herpetospermum pedunculosum in cholestatic rats. A bioassay-guided study also led to the identification and isolation of a lignan, dihydrodiconiferyl alcohol (DA) from the seeds of H. pedunculosum. PURPOSE: To investigate whether DA could alleviate cholestasis and determine the mechanisms underlying such action. METHODS: Male Sprague-Dawley (SD) rats were administered with DA (10, 20 or 40 mg/kg) intragastrically once daily for 7 days prior to treatment with α-naphthylisothiocyanate (ANIT) (60 mg/kg). We then evaluated the levels of a range of serum indicators, determined bile flow, and carried out histopathological analyses. Western blotting was then used to investigate the levels of inflammatory mediators and the Farnesoid X Receptor (FXR), proteins involved in the downstream biosynthesis of bile acids, and a range of transport proteins. Molecular docking was used to simulate the interaction between DA and FXR. Cell viability of human hepatocytes (L-02) cells was determined by MTT. Then, we treated guggulsterone-inhibited L-02 cells, Si-FXR L-02 cells, and FXR-overexpression cells with the FXR agonist GW4064 (6 µM) or DA (25, 50 and 100 µM) for 24 h before detecting gene and protein expression by RT-PCR and western blotting, respectively. RESULTS: DA significantly attenuated ANIT-induced cholestasis in SD rats by reducing liver function indicators in the serum, increasing bile flow, improving the recovery of histopathological injuries in the liver, and by alleviating pro-inflammatory cytokines in the liver. DA also increased the expression levels of FXR and altered the levels of downstream proteins in the liver tissues, thus indicating that DA might alleviate cholestasis by regulating the FXR. Molecular docking simulations predicted that DA was as an agonist of FXR. In vitro mechanical studies further showed that DA increased the mRNA and protein expression levels of FXR, Small Heterodimer Partner 1/2, Bile Salt Export Pump, Multidrug Resistance-associated Protein 2, and Na+/taurocholate Co-transporting Polypeptide, in both guggulsterone-inhibited and Si-FXR L-02 cells. Moreover, DA enhanced the mRNA and protein expression of FXR, and its downstream genes and proteins, in L-02 cells containing an FXR-overexpression plasmid. CONCLUSION: DA may represent an effective agonist for FXR has significant therapeutic potential for the treatment of cholestatic liver injury.

Ginsenoside Rg1 alleviates ANIT-induced intrahepatic cholestasis in rats via activating farnesoid X receptor and regulating transporters and metabolic enzymes.

Ginsenoside Rg1 is an active ingredient extracted from the roots of ginsenoside, and an α-naphthylisothiocyanate (ANIT)-induced rat model of intrahepatic cholestasis was used to investigate the protective effect of Rg1 on cholestasis. 48 SD male rats were randomly divided into 6 groups: control group, model group, UDCA group (ursodeoxycholic acid), low-dose Rg1 group (10 mg/kg), medium-dose Rg1 group (20 mg/kg) and high-dose Rg1 group (40 mg/kg). The model group, the UDCA group and all the Rg1 group were then intragastrically administered with 80 mg/kg ANIT, and the control group were given equal volume of olive oil. Then the pathological changes in liver tissue were observed, the secretion of bile in the bile duct was measured, and the biochemical markers in serum were quantified, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), glutamyl transfer peptidase (GTP) and the content of total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBA). The contents of inflammatory mediators in serum were quantified, including tumor necrosis factor (TNF-α), γ-interferon (IFN-γ) and interleukin-1ß (IL-1ß). The contents of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in liver homogenate were quantified. Expression of farnesoid X receptor (FXR), transporters and metabolic enzymes in liver tissue was monitored. Rg1 treatment improved liver tissue pathological damage, promoted bile secretion and significantly reduced serum levels of the intrahepatic cholestasis markers ALT, AST, ALP, GTP, TBIL, DBIL and TBA. Rg1 increased the activity of SOD and GSH-Px in liver homogenate, while, reducing the serum levels of MDA and inflammatory mediators. Rg1 also regulated the expression of FXR, bile acid transporters and metabolic enzymes. Overall, Rg1 alleviated liver injury by improving secretion of bile and normalizing the activity of enzymes in the serum. The protective mechanism appeared to be related to the activation of FXR and regulation of liver transporters and metabolic enzymes.

TianJiu therapy for α-naphthyl isothiocyanate-induced intrahepatic cholestasis in rats treated with fresh Ranunculus sceleratus L.

ETHNOPHARMACOLOGICAL RELEVANCE: TianJiu (TJ) therapy, one type of cold moxibustion, applies to specific acupuncture points with herbal patches of hot nature, providing a constant irritant to the skin until the presence of hyperemia and blistering. Traditional and clinical reports suggest that TJ is an effective therapy for the treatment of jaundice with fresh Ranunculus sceleratus L. (RS), in which protoanemonin is one of the main irritant constituents. However, the therapeutic effect of TJ treatment with fresh RS against intrahepatic cholestasis has not been studied in animal experiments. AIM OF THE STUDY: Present study was undertaken to investigate the effect of TJ treatment with fresh RS against intrahepatic cholestasis in rats and provide an experimental basis for the underlying mechanism of TJ therapy. MATERIALS AND METHODS: Male intrahepatic cholestatic Sprague-Dawley rats induced by 2% α-naphthylisothiocyanate (ANIT, 80 mg/kg B.W.) were treated by TJ therapy with fresh RS. The levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), direct bilirubin (DBIL), total bilirubin (TBIL), total bile acid (TBA), hepatic malondialdehyde (MDA) and nitric monoxide (NO), as well as hepatic body ratio, bile flow and hepatic histopathological assay were measured and evaluated to investigate the therapeutic effect of TJ treatment with fresh RS. Phytochemical analysis of fresh and dried RS was performed by gas chromatography-mass spectrometer (GC-MS). RESULTS: After TJ treatment with fresh RS, the abnormally elevated levels of serum AST, ALT, ALP, DBIL, TBIL and TBA, as well as hepatic MDA and NO at 108 h were reduced significantly (versus model group, P < 0.01). The hepatic body ratio, bile flow and hepatic pathological change of cholestatic rats at 108 h in TJ group were restored when compared with those of model group. Thirty-one compounds including lactones, flavonoids and phenolic acids were identified and determined by GC-MS analysis. The content of protoanemonin in fresh RS (9.49%) was about 25-fold higher than that in dried RS (0.38%). CONCLUSIONS: TJ treatment with fresh RS exhibited good therapeutic effect on ANIT-induced intrahepatic cholestasis in rats, which may be due to the attenuated oxidative stress in the liver tissue. It is rational for the ancients to choose fresh RS as TJ herbal patches because of its abundant protoanemonin with the character of irritant. The qualitative and quantitative results of GC-MS analysis provided the chemical basis of TJ therapy with fresh RS, which can be regarded as a simple and efficient method for the treatment of cholestasis hepatitis.

Xiaoyan lidan formula ameliorates α-naphthylisothiocyanate-induced intrahepatic cholestatic liver injury in rats as revealed by non-targeted and targeted metabolomics.

Intrahepatic cholestasis is a clinical syndrome of liver damage with systemic circulation and intrahepatic accumulation of excessive toxic bile acids without effective therapeutic methods so far. Xiaoyan Lidan Formula (XYLDF), a traditional Chinese prescription, has long been clinically applied for hepatobiliary disorders due to cholestasis. But its mechanism remains unknown. In this study, a non-targeted metabolomics approach based on UHPLC-Q-TOF-MS/MS combined with a bile acids (BAs) - targeted metabolomics approach based on UHPLC-MS/MS were performed to elucidate the functional mechanisms of XYLDF on α-naphthylisothiocyanate(ANIT)-induced intrahepatic cholestasis rats. The results showed that a total of 39 endogenous metabolites with significant difference (VIP > 1.00, P < 0.05) were identified as biomarkers of ANIT-induced intrahepatic cholestasis in rats. After treatment by XYLDF, 22 biomarkers were reversed to the control-like levels, which involved in primary BA biosynthesis, bile acid metabolism and excretion, steroids metabolism, retinol metabolism, starch and sucrose metabolism, inter conversions between pentose and glucoronate as well as arachidonic acid metabolism. Meanwhile, the results of contents variation of BAs in liver and serum showed that both hydrophilic and hydrophobic BAs were markedly increased in the model rats, while XYLDF treatment could restore the increase induced by ANIT, which suggested that one of the mechanisms of XYLDF on cholestasis referred to regulation of metabolic homeostasis of cholic acid.

Protective effect of herbal medicine Huangqi decoction against chronic cholestatic liver injury by inhibiting bile acid-stimulated inflammation in DDC-induced mice.

BACKGROUND: Huangqi decoction (HQD), a classic traditional herbal medicine, has been used for liver fibrosis, but its effect on intrahepatic chronic cholestatic liver injury remains unknown. PURPOSE: In the present study, we investigated the hepatoprotective effect of HQD and the underlying molecular mechanisms in 3, 5-diethoxycarbonyl-1, 4-dihydroxychollidine (DDC)-induced chronic cholestatic mice. METHODS: The DDC-induced cholestatic mice were administrated HQD for 4 or 8 weeks. Serum biochemistry and morphology were investigated. The serum and liver bile acid (BA) levels were detected by ultra performance liquid chromatography-tandem mass spectrometry. The liver expression of BA metabolizing enzymes and transporters, and inflammatory and fibrotic markers was measured by real-time polymerase chain reaction, western blotting, and immunohistochemistry. RESULTS: HQD treatment for 4 or 8 weeks ameliorated DDC-induced liver injury by improving impaired hepatic function and tissue damage. HQD treatment for 8 weeks further decreased the liver expression of cytokeratin 19, tumor growth factor (TGF)-ß, collagen I, and α-smooth muscle actin, and ameliorated ductular reaction and liver fibrosis. HQD markedly decreased the accumulation of serum and liver BA. The expression of BA-metabolizing enzymes, cytochrome P450 2b10 and UDP glucuronosyltransferase 1 A1, and multidrug resistance-associated protein 2, Mrp3, and Mrp4 involved in BA homeostasis was increased by 4 weeks of HQD treatment. The expression of BA uptake transporter Na+-taurocholate cotransporting polypeptide was decreased and that of Mrp4 was increased after 8 weeks of HQD treatment. Nuclear factor-E2-related factor-2 (Nrf2) was remarkably induced by HQD treatment. Additionally, HQD treatment for 8 weeks decreased the liver expression of inflammatory factors, interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, monocyte chemoattractant protein-1, and intracellular adhesion molecule-1. HQD suppressed the nuclear factor (NF)-κB pathway. CONCLUSION: HQD protected mice against chronic cholestatic liver injury and biliary fibrosis, which may be associated with the induction of the Nrf2 pathway and inhibition of the NF-κB pathway, ameliorating BA-stimulated inflammation.

Alisma orientale extract exerts the reversing cholestasis effect by activation of farnesoid X receptor.

BACKGROUND: Cholestasis is a clinical syndrome of liver damage that is caused by accumulation of bile acids in the liver and systemic circulation. Farnesoid X receptor (FXR) can regulate synthesis, metabolism, and excretion of bile acids. The rhizomes of Alisma orientale is a well-known traditional Chinese medicine to treat edema, obesity, gonorrhea, leukorrhea, diarrhea, hyperlipidemia, and diabetes in China. HYPOTHESIS/PURPOSE: We hypothesized Alisma orientale extract (AOE) to exert hepatoprotective effect against α-naphthylisothiocyanate (ANIT) induced cholestasis in rat. We aimed to investigate the mechanism of AOE. STUDY DESIGN: Male Sprague Dawley rats with intrahepatic cholestasis induced by ANIT were treated with AOE (150, 300, or 600 mg/kg). Rats receiving vehicle (0.5% CMC-Na) served as control. METHODS: 48 h after ANIT administration, rats were sacrificed. Blood was collected to obtain serum and livers were removed for histopathology and protein preparation. Biochemical indicators in serum were determined using commercial kits and triterpenoids were determined by liquid chromatography tandem Qtrap mass spectrometry. Proteomics was analyzed by liquid chromatography tandem ion-trap mass spectrometry. The differently expressed proteins were analyzed via the network database and verified by western blotting. The interaction between triterpenoids and FXR were evaluated by luciferase assay and molecular docking. RESULTS: AOE treatment significantly decreased the serum AST, ALT, TBIL, and intrahepatic TBA and improved the liver pathologic change induced by ANIT. Proteomics analysis indicated that AOE regulated proteins related to bile acid homeostasis via activating farnesoid X receptor (FXR) signaling pathway. Luciferase assay and molecular docking results indicated that triterpenoids could activate FXR, which resulting in ameliorative accumulation of bile acids in the liver by increase of metabolism and transportation for bile acids, and decrease of synthesis for bile acids. CONCLUSION: AOE protected against rat liver injury and cholestasis induced by ANIT by activation of farnesoid X receptor, suggesting that A. orientale could be regarded as a potential hepatoprotective drug.

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.

[Protective effect of emodin pretreatment in young rats with intrahepatic cholestasis].

OBJECTIVE: To investigate the protective effect of emodin in young rats with intrahepatic cholestasis. METHODS: A total of 120 young Sprague-Dawley rats were randomly divided into control, model, and high-, medium-, and low-dose emodin groups, with 24 rats in each group. The rats in the control and model groups were given sodium carboxymethyl cellulose solution by gavage, while the other groups were given different doses of emodin solution by gavage. On the 5th day of experiment, alpha-naphthylisothiocyanate (ANIT, 50 mg/kg) was applied by gavage to establish the model of intrahepatic cholestasis in all groups except the control group. At 24, 48, and 72 hours after gavage, 8 rats in each group were sacrificed. Colorimetry was used to measure the serum levels of total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBA), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in each group, and hematoxylin-eosin staining was applied to observe the morphological changes of the liver under a light microscope at different time points. RESULTS: Compared with the control group, the model group had significantly increased serum levels of TBIL, DBIL, TBA, ALP, GGT, ALT, and AST at the 24-hour, 48-hour, and 72-hour time points (P<0.01). In the model group, the serum levels of TBIL, DBIL, TBA, ALT, and AST showed varying degrees of increase at 48 hours after establishment of model, compared with the values at 24 and 72 hours (P<0.05). At 24, 48, and 72 hours, the high-, medium-, and low-dose emodin groups had varying degrees of reductions in the serum levels of TBIL and TBA compared with the model group (P<0.05); the high- and low-dose emodin groups had significantly increased serum levels of TBA compared with the medium-dose emodin group (P<0.05). The model group had the most severe pathological changes at 48 hours. Compared with the model group, the high-, medium-, and low-dose emodin groups showed certain improvement in pathological changes of the liver at each time point, and the medium-dose emodin group had better improvement compared with the high- and low-dose emodin groups. CONCLUSIONS: Emodin can effectively improve ANIT-induced intrahepatic cholestasis in young rats, and medium-dose emodin shows the best effect.

Byler disease: early natural history.

OBJECTIVES: Byler disease, originally described in Amish kindred, results from mutations in ATPase Class I Type 8b Member 1 (ATP8b1). Specific clinical reports of Amish Byler disease were last published 40 years ago. These investigations were directed at the present detailed clinical understanding of the early course of hepatic manifestations of Byler disease. METHODS: This study analyzed routine clinical practice and outcomes of children with Byler disease (defined by homozygous c.923G>T mutation in ATP8b1), who initially presented to Children's Hospital of Pittsburgh of UPMC between January 2007 and October 2014. Data were analyzed to the earlier of 24 months of age or partial external biliary diversion. RESULTS: Six children presented between 1 and 135 days of life: 2 presented with newborn direct hyperbilirubinemia, 2 had complications of coagulopathy, 1 had failure to thrive and rickets, and 1 sibling was identified by newborn genetic testing. Intensive fat-soluble vitamin supplementation was required to prevent insufficiencies in vitamins D, E, and K. Hyperbilirubinemia was variable both over time and between children. Serum bile acid levels were elevated, whereas γ-glutamyltranspeptidase levels were low normal. Scratching behavior (pruritus) was intractable in 4 of 6 children with onset between 6 and 12 months of age. Features of portal hypertension were not observed. Partial external biliary diversion was used during the second year of life in 4 children. CONCLUSIONS: Detailed analysis of Byler disease revealed varied disease presentation and course. Nutritional issues and pruritus dominated the clinical picture in the first 2 years of life.

Hepatic cytochrome P450 deficiency in mouse models for intrahepatic cholestasis predispose to bile salt-induced cholestasis.

Progressive familial intrahepatic cholestasis (PFIC) types 1 and 3 are severe cholestatic liver diseases caused by deficiency of ATB8B1 and ABCB4, respectively. Mouse models for PFIC display mild phenotypes compared with human patients, and this can be explained by the difference in bile salt pool composition. Mice, unlike humans, have the ability to detoxify hydrophobic bile salts by cytochrome P450-mediated (re)hydroxylation and thus have a less toxic bile salt pool. We have crossed mouse models for PFIC1 and PFIC3 with Hrn mice that have a reduced capacity to (re)hydroxylate bile salts. Double transgenes were obtained by backcrossing Atp8b1(G308V/G308V) and Abcb4(-/-) mice with Hrn mice that have a liver-specific disruption of the cytochrome P450 reductase gene and therefore have markedly reduced P450 activity. In these mice, a more hydrophobic bile salt pool was instilled by cholic acid supplementation of the diet, and bile formation and liver pathology was studied. As opposed to single transgenes, Atp8b1(G308V/G308V)/Hrn and Abcb4(-/-)/Hrn mice rapidly developed strong cholestasis that was evidenced by increased plasma bilirubin and bile salt levels. The bile salt pool was more toxic in both models; Atp8b1(G308V/G308V)/Hrn mice had a more hydrophobic plasma pool compared with the single transgene, whereas Abcb4(-/-)/Hrn mice had a more hydrophobic biliary pool compared with the single transgene. In line with these findings, liver damage was not aggravated in Atp8b1(G308V/G308V)/Hrn but was more severe in Abcb4(-/-)/Hrn mice. These data indicate that bile salt pool composition is a critical determinant in the initiation and progression of cholestasis and liver pathology in PFIC1 and PFIC3. Most importantly, our data suggest that the hydrophobicity of the plasma bile salt pool is an important determinant of the severity of cholestasis, whereas the hydrophobicity of the biliary bile salt pool is an important determinant of the severity of liver pathology.

Discovery that theonellasterol a marine sponge sterol is a highly selective FXR antagonist that protects against liver injury in cholestasis.

BACKGROUND: The farnesoid-x-receptor (FXR) is a bile acid sensor expressed in the liver and gastrointestinal tract. Despite FXR ligands are under investigation for treatment of cholestasis, a biochemical condition occurring in a number of liver diseases for which available therapies are poorly effective, mice harboring a disrupted FXR are protected against liver injury caused by bile acid overload in rodent models of cholestasis. Theonellasterol is a 4-methylene-24-ethylsteroid isolated from the marine sponge Theonella swinhoei. Here, we have characterized the activity of this theonellasterol on FXR-regulated genes and biological functions. PRINCIPAL FINDINGS: Interrogation of HepG2 cells, a human hepatocyte cell line, by microarray analysis and transactivation assay shows that theonellasterol is a selective FXR antagonist, devoid of any agonistic or antagonistic activity on a number of human nuclear receptors including the vitamin D receptor, PPARs, PXR, LXRs, progesterone, estrogen, glucorticoid and thyroid receptors, among others. Exposure of HepG2 cells to theonellasterol antagonizes the effect of natural and synthetic FXR agonists on FXR-regulated genes, including SHP, OSTα, BSEP and MRP4. A proof-of-concept study carried out to investigate whether FXR antagonism rescues mice from liver injury caused by the ligation of the common bile duct, a model of obstructive cholestasis, demonstrated that theonellasterol attenuates injury caused by bile duct ligation as measured by assessing serum alanine aminostrasferase levels and extent of liver necrosis at histopathology. Analysis of genes involved in bile acid uptake and excretion by hepatocytes revealed that theonellasterol increases the liver expression of MRP4, a basolateral transporter that is negatively regulated by FXR. Administering bile duct ligated mice with an FXR agonist failed to rescue from liver injury and downregulated the expression of MRP4. CONCLUSIONS: FXR antagonism in vivo results in a positive modulation of MRP4 expression in the liver and is a feasible strategy to target obstructive cholestasis.

Combination of retinoic acid and ursodeoxycholic acid attenuates liver injury in bile duct-ligated rats and human hepatic cells.

UNLABELLED: Cholestasis leads to liver cell death, fibrosis, cirrhosis, and eventually liver failure. Despite limited benefits, ursodeoxycholic acid (UDCA) is the only Food and Drug Administration-approved treatment for cholestatic disorders. Retinoic acid (RA) is a ligand for nuclear receptors that modulate bile salt homeostasis. RA also possesses immunomodulatory effects and is used to treat acute promyelocytic leukemia and inflammatory disorders such as psoriasis, acne, and rheumatoid arthritis. To test whether the supplementation of RA with UDCA is superior to UDCA alone for treating cholestasis, male Sprague-Dawley rats underwent common bile duct ligation (BDL) for 14 days and were treated with phosphate-buffered saline (PBS), UDCA, all-trans retinoic acid (atRA), or UDCA and atRA by gavage. Treatment with UDCA and atRA substantially improved animal growth rates, significantly reduced liver fibrosis and bile duct proliferation, and nearly eliminated liver necrosis after BDL. Reductions in the bile salt pool size and liver hydroxyproline content were also seen with treatment with atRA or atRA and UDCA versus PBS and UDCA. Furthermore, atRA and UDCA significantly reduced liver messenger RNA and/or protein expression of transforming growth factor ß1 (Tgf-ß1), collagen 1a1 (Col1A1), matrix metalloproteinase 2 (Mmp2), cytokeratin 19, α-smooth muscle actin (α-SMA), cytochrome P450 7A1 (Cyp7a1), tumor necrosis factor α, and interleukin-ß1. The molecular mechanisms of this treatment were also assessed in human hepatocytes, hepatic stellate cells, and LX-2 cells. atRA alone or in combination with UDCA greatly repressed CYP7A1 expression in human hepatocytes and significantly inhibited COL1A1, MMP2, and α-SMA expression and/or activity in primary human hepatic stellate cells and LX-2 cells. Furthermore, atRA reduced TGF-ß1-induced Smad2 phosphorylation in LX-2 cells. CONCLUSION: Our findings indicate that the addition of RA to UDCA reduces the bile salt pool size and liver fibrosis and might be an effective supplemental therapy with UDCA for cholestatic diseases.

Strain background modifies phenotypes in the ATP8B1-deficient mouse.

BACKGROUND: Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease.

Hypervitaminosis A inducing intra-hepatic cholestasis--a rare case report.

The use of over-the-counter supplements is commonplace in today's health conscious society. We present an unusual case of intrahepatic cholestasis caused by vitamin A intoxication. The patient consumed one Herbalife shake with two multivitamin tablets of the same brand for 12 years. When calculated this equated to more than the recommended daily allowance for vitamin A consumption. Deranged liver function tests were consistent with a cholestatic process. Liver biopsy was obtained and revealed features pathognomonic of vitamin A toxicity, without the usual fibrosis. When the supplements were ceased, his jaundice and alkaline phosphatase completely normalized. This case highlights the importance of health care providers documenting non-prescribed dietary supplements and considering them in the etiology of cholestatic liver disease.

[Drug-induced toxic hepatitis]. / Medikamentös-toxische Hepatitis.

A 55-year-old male patient was hospitalized with severe nausea, vomiting and icterus. Laboratory testing showed hepatocellular damage. After exhaustive testing, the exclusion diagnosis of a toxic hepatitis was reached. There was a strong temporal correlation with the ingestion of Hong Hua 29, a preparation from Traditional Chinese Medicine (TCM). This medication had been started twelve days prior to the first appearance of symptoms. The existing drug regimen included gabapentin (Neurontin), esomeprazole (Nexium) and prednisone (Prednison Streuli) for the therapy of an acute sensory and motor neuropathy of unknown aetiology. After cessation of Hong Hua 29, gabapentin and esomeprazole, transaminase levels started to declined and normalized within three months. According to the Swissmedic criteria of imputability, a causal correlation between the observed symptoms and the administration of Hong Hua 29 is possible.

Role of hepatic phospholipids in development of liver injury in Mdr2 (Abcb4) knockout mice.

BACKGROUND/AIMS: Multidrug resistance protein 2 (Abcb4) gene knockout mice (Mdr2(-/-)) lack phosphatidylcholine (PC) excretion into bile and spontaneously develop sclerosing cholangitis, biliary fibrosis and hepatocellular carcinomas. We therefore aimed to test whether formation and hepatic retention of abnormal PC metabolites contribute to the pathogenesis of liver injury in Mdr2(-/-) mice. METHODS: Mdr2(-/-) mice were either fed a diet supplemented with soybean lecithin 2.5% w/w [phosphatidylcholine-enriched diet (PCD), to increase hepatic PC content] or a choline-deficient diet (CDD, to reduce hepatic PC content) for 4 weeks; controls received chow with energy and nutrient content equivalent to PCD and CDD. Serum liver tests, liver histology, markers of fibrosis, cholangiocyte activation, cell proliferation and thin-layer chromatography for phospholipid (PL) composition were carried out. RESULTS: PCD decreased serum alkaline phosphatase and total bilirubin levels compared with controls, while liver histology as well as hepatic hydroxyproline content as markers of liver fibrosis did not differ among groups. Both PCD and CDD decreased hepatocellular proliferation compared with controls. Hepatic, serum and biliary PLs remained unchanged despite dietary manipulations and no potentially toxic PL metabolites were detected. CONCLUSIONS: Mdr2(-/-) mice maintain stable hepatic, serum and biliary PL metabolism in response to dietary PC manipulations. Our findings therefore suggest that liver injury in Mdr2(-/-) mice is not due to formation of toxic PL metabolites.