Loss of ß-catenin reveals a role for glutathione in regulating oxidative stress during cholestatic liver disease.

BACKGROUND: Cholestasis is an intractable liver disorder that results from impaired bile flow. We have previously shown that the Wnt/ß-catenin signaling pathway regulates the progression of cholestatic liver disease through multiple mechanisms, including bile acid metabolism and hepatocyte proliferation. To further explore the impact of these functions during intrahepatic cholestasis, we exposed mice to a xenobiotic that causes selective biliary injury. METHODS: α-naphthylisothiocyanate (ANIT) was administered to liver-specific knockout (KO) of ß-catenin and wild-type mice in the diet. Mice were killed at 6 or 14 days to assess the severity of cholestatic liver disease, measure the expression of target genes, and perform biochemical analyses. RESULTS: We found that the presence of ß-catenin was protective against ANIT, as KO mice had a significantly lower survival rate than wild-type mice. Although serum markers of liver damage and total bile acid levels were similar between KO and wild-type mice, the KO had minor histological abnormalities, such as sinusoidal dilatation, concentric fibrosis around ducts, and decreased inflammation. Notably, both total glutathione levels and expression of glutathione-S-transferases, which catalyze the conjugation of ANIT to glutathione, were significantly decreased in KO after ANIT. Nuclear factor erythroid-derived 2-like 2, a master regulator of the antioxidant response, was activated in KO after ANIT as well as in a subset of patients with primary sclerosing cholangitis lacking activated ß-catenin. Despite the activation of nuclear factor erythroid-derived 2-like 2, KO livers had increased lipid peroxidation and cell death, which likely contributed to mortality. CONCLUSIONS: Loss of ß-catenin leads to increased cellular injury and cell death during cholestasis through failure to neutralize oxidative stress, which may contribute to the pathology of this disease.

Pirfenidone ameliorates ANIT-induced cholestatic liver injury via modulation of FXR, NF-кB/TNF-α, and Wnt/GSK-3ß/ß-catenin signaling pathways.

Cholestasis is a hepatobiliary disorder characterized by the excessive accumulation of toxic bile acids in hepatocytes, leading to cholestatic liver injury (CLI) through multiple pathogenic inflammatory pathways. Currently, there are limited therapeutic options for the management of cholestasis and associated CLI; therefore, new options are urgently needed. Pirfenidone (PF), an oral bioavailable pyridone analog, is used for the treatment of idiopathic pulmonary fibrosis. PF has recently demonstrated diverse potential therapeutic activities against different pathologies. Accordingly, the present study adopted the α-naphthyl isothiocyanate (ANIT)-induced CLI model in mice to explore the potential protective impact of PF and investigate the underlying mechanisms of action. PF intervention markedly reduced the serum levels of ALT, AST, LDH, total bilirubin, and total bile acids, which was accompanied by a remarkable amelioration of histopathological lesions induced by ANIT. PF also protected the mice against ANIT-induced redox imbalance in the liver, represented by reduced MDA levels and elevated GSH and SOD activities. Mechanistically, PF inhibited ANIT-induced downregulated expressions of the farnesoid X receptor (FXR), as well as the bile salt export pump (BSEP) and the multidrug resistance-associated protein 2 (MRP2) bile acid efflux channels. PF further repressed ANIT-induced NF-κB activation and TNF-α and IL-6 production. These beneficial effects were associated with its ability to dose-dependently inhibit Wnt/GSK-3ß/ß-catenin/cyclin D1 signaling. Collectively, PF protects against ANIT-induced CLI in mice, demonstrating powerful antioxidant and anti-inflammatory activities as well as an ability to oppose BA homeostasis disorder. These protective effects are primarily mediated by modulating the interplay between FXR, NF-κB/TNF-α/IL-6, and Wnt/ß-catenin signaling pathways.

Emodin alleviates cholestatic liver injury by modulating Sirt1/Fxr signaling pathways.

Emodin (EMO) has the effect of anti-cholestasis induced by alpha-naphthylisothiocyanate (ANIT). But its mechanism is still unclear. The farnesoid X receptor (Fxr) is the master bile acid nuclear receptor. Recent studies have reported that Sirtuin 1 (Sirt1) can regulate the activities of Fxr. The purpose of the current study was to investigate the mechanism of EMO against ANIT-induced liver injury based on Sirt1/Fxr signaling pathway. The ANIT-induced cholestatic rats were used with or without EMO treatment. Serum biochemical indicators, as well as liver histopathological changes were examined. The genes expressions of Sirt1, Fxr, Shp, Bsep and Mrp2 were detected. The expressions of Sirt1, Fxr and their downstream related genes were investigated in vitro. The results showed that EMO significantly alleviated ANIT-induced liver injury in rats, and increased Sirt1, Fxr, Shp, Bsep and Mrp2 gene expression in liver, while decreased the expression of Cyp7a1. EMO significantly activated Fxr, while Sirt1 inhibitor and Sirt1 gene silencing significantly reduced Fxr activity in vitro. Collectively, EMO in the right dose has a protective effect on liver injury induced by ANIT, and the mechanism may be through activation of Fxr by Sirt1, thus regulating bile acid metabolism, and reducing bile acid load in hepatocytes.

Targeting SIRT1/AMPK/Nrf2/NF-кB by sitagliptin protects against oxidative stress-mediated ER stress and inflammation during ANIT-induced cholestatic liver injury.

Intrahepatic cholestasis is a common clinical form of hepatobiliary injury characterized by the intrahepatic accumulation of toxic bile acids. Besides its antidiabetic activity, the dipeptidyl peptidase IV inhibitor sitagliptin (SG) has been recently assigned diverse pharmacological activities and therapeutic potential against different disorders owing to its emerging antioxidant and anti-inflammatory properties. The current study explored the potential hepatoprotective effect of SG on α-naphthyl isothiocyanate (ANIT)-induced cholestatic liver injury (CLI) in mice and investigate its possible targeted signaling pathways. Mice received SG (10 and 20 mg/kg) for four consecutive days, two days before and after a single oral administration of ANIT (75 mg/kg). Our results revealed that SG administration remarkably prevented ANIT-induced histopathological lesions in the liver and maintained hepatic functions and oxidative/antioxidant balance. Ultimately, SG counteracted the inflammatory response in the liver, as indicated by the marked suppression of hepatic expression of NF-κB, TNF-α, and IL-6. Moreover, it inhibited the endoplasmic reticulum (ER) stress response in the liver. These beneficial effects of SG were accompanied by upregulation of SIRT1, p-AMPK, and Nrf2 expressions while downregulating keap1 expression in the liver. In conclusion, this study is the first to demonstrate the ability of SG to protect against ANIT-induced CLI through modulating multiple signaling cascades, including SIRT1/AMPK, Nrf2/keap1, and NF-кB, which resulted in enhanced antioxidant capacity and repressed inflammatory and ER stress responses in the liver.

The contribution of small heterodimer partner to the occurrence and progression of cholestatic liver injury.

BACKGROUND AND AIM: Small heterodimer partner (SHP, encoded by NR0B2) plays an important role in maintaining bile acid homeostasis. The loss of the hepatic farnesoid X receptor (FXR)/SHP signal can cause severe cholestatic liver injury (CLI). FXR and SHP have overlapping and nonoverlapping functions in bile acid homeostasis. However, the key role played by SHP in CLI is unclear. METHODS: In this study, an alpha-naphthylisothiocyanate (ANIT)-induced cholestasis mouse model was established. The effect of SHP knockout (SHP-KO) on liver and ileal pathology was evaluated. 16S rRNA gene sequencing analysis combined with untargeted metabolomics was applied to reveal the involvement of SHP in the pathogenesis of CLI. RESULTS: The results showed that ANIT (75 mg/kg) induced cholestasis in WT mice. No significant morphological changes were found in the liver and ileal tissue of SHP-KO mice. However, the serum metabolism and intestinal flora characteristics were significantly changed. Moreover, compared with the WT + ANIT group, the serum levels of ALT and AST in the SHP-KO + ANIT group were significantly increased, and punctate necrosis in the liver tissue was more obvious. The ileum villi showed obvious shedding, thinning, and shortening. In addition, SHP-KO-associated differential intestinal flora and differential biomarkers were significantly associated. CONCLUSION: In this study, we elucidated the serum metabolic characteristics and intestinal flora changes related to the aggravation of CLI in SHP-KO mice induced by ANIT.

Mechanistic studies on the alleviation of ANIT-induced cholestatic liver injury by Polygala fallax Hemsl. polysaccharides.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygala fallax Hemsl. is a traditional folk medicine commonly used by ethnic minorities in the Guangxi Zhuang Autonomous Region, and has a traditional application in the treatment of liver disease. Polygala fallax Hemsl. polysaccharides (PFPs) are of interest for their potential health benefits. AIM OF THIS STUDY: This study explored the impact of PFPs on a mouse model of cholestatic liver injury (CLI) induced by alpha-naphthyl isothiocyanate (ANIT), as well as the potential mechanisms. MATERIALS AND METHODS: A mouse CLI model was constructed using ANIT (80 mg/kg) and intervened with different doses of PFPs or ursodeoxycholic acid. Their serum biochemical indices, hepatic oxidative stress indices, and hepatic pathological characteristics were investigated. Then RNA sequencing was performed on liver tissues to identify differentially expressed genes and signaling pathways and to elucidate the mechanism of liver protection by PFPs. Finally, Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to verify the differentially expressed genes. RESULTS: Data analyses showed that PFPs reduced the levels of liver function-related biochemical indices, such as ALT, AST, AKP, TBA, DBIL, and TBIL. PFPs up-regulated the activities of SOD and GSH, down-regulated the contents of MDA, inhibited the release of IL-1ß, IL-6, and TNF-α, or promoted IL-10. Pathologic characterization of the liver revealed that PFPs reduced hepatocyte apoptosis or necrosis. The RNA sequencing indicated that the genes with differential expression were primarily enriched for the biosynthesis of primary bile acids, secretion or transportation of bile, the reactive oxygen species in chemical carcinogenesis, and the NF-kappa B signaling pathway. In addition, the results of qRT-PCR and Western blotting analysis were consistent with those of RNA sequencing analysis. CONCLUSIONS: In summary, this study showed that PFPs improved intrahepatic cholestasis and alleviated liver damage through the modulation of primary bile acid production, Control of protein expression related to bile secretion or transportation, decrease in inflammatory reactions, and inhibition of oxidative pressure. As a result, PFPs might offer a hopeful ethnic dietary approach for managing intrahepatic cholestasis.

JiaGaSongTang improves chronic cholestasis via enhancing FXR-mediated bile acid metabolism.

BACKGROUND: Bile acid (BA) enterohepatic circulation disorders are a main feature of chronic cholestatic diseases. Promoting BA metabolism is thus a potential method of improving enterohepatic circulation disorders, and treat enterohepatic inflammation, oxidative stress and fibrosis due to cholestasis. PURPOSE: To investigate the effect of JiaGaSongTang (JGST) and its blood-absorbed ingredient 6-gingerol on α-naphthylisothiocyanate (ANIT)-induced chronic cholestasis, as well as elucidate the underlying regulatory mechanism. METHODS: Chronic cholestasis was induced in mice via subcutaneous injection of ANIT (50 mg/kg) every other day for 14 d. Treatment groups were administered JGST orally daily. Damage to the liver and intestine was observed using histopathological techniques. Biochemical techniques were employed to assess total BA (TBA) levels in the serum, liver, and ileum samples. Liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze fecal BA components. Bioinformatic methods were adopted to screen the core targets and pathways. The blood-absorbed ingredients of JGST were scrutinized via LC-MS/MS. The effects of the major JGST ingredients on farnesoid X receptor (FXR) transactivation were validated using dual luciferase reporter genes. Lastly, the effects of the FXR inhibitor, DY268, on JGST and 6-gingerol pharmacodynamics were observed at the cellular and animal levels. RESULTS: JGST ameliorated pathological impairments in the liver and intestine, diminishing TBA levels in the serum, liver and gut. Fecal BA profiling revealed that JGST enhanced the excretion of toxic BA constituents, including deoxycholic acid. Bioinformatic analyses indicated that JGST engaged in anti-inflammatory mechanisms, attenuating collagen accumulation, and orchestrating BA metabolism via interactions with FXR and other pertinent targets. LC-MS/MS analysis identified six ingredients absorbed to the bloodstream, including 6-gingerol. Surface plasmon resonance (SPR) and dual luciferase reporter gene assays confirmed the abilities of 6-gingerol to bind to FXR and activate its transactivation. Ultimately, in both cellular and animal models, the therapeutic efficacy of JGST and 6-gingerol in chronic cholestasis was attenuated in the presence of FXR inhibitors. CONCLUSION: The findings, for the first time, demonstrated that 6-gingerol, a blood-absorbed ingredient of JGST, can activate FXR to affect BA metabolism, and thereby attenuate ANIT-induced liver and intestinal injury in chronic cholestasis mice model via inhibition of inflammation, oxidative stress, and liver fibrosis, in part in a FXR-dependent mechanism.

Gancao decoction attenuates hepatic necroptosis via activating caspase 8 in cholestatic liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Gancao Decoction (GCD) is widely used to treat cholestatic liver injury. However, it is unclear whether is related to prevent hepatocellular necroptosis. AIM OF THE STUDY: The purpose of this study is to clarify the therapeutic effects of GCD against hepatocellular necroptosis induced by cholestasis and its active components. MATERIALS AND METHODS: We induced cholestasis model in wild type mice by ligating the bile ducts or in Nlrp3-/- mice by intragastrical administering Alpha-naphthylisothiocyanate (ANIT). Serum biochemical indices, liver pathological changes and hepatic bile acids (BAs) were measured to evaluate GCD's hepatoprotective effects. Necroptosis was assessed by expression of hallmarkers in mice liver. Moreover, the potential anti-necroptotic effect of components from GCD were investigated and confirmed in ANIT-induced cholestasis mice and in primary hepatocytes from WT mouse stimulated with Tumor Necrosis Factor alpha (TNF-α) and cycloheximide (CHX). RESULTS: GCD dose-dependently alleviated hepatic necrosis, reduced serum aminotranferase activity in both BDL and ANIT-induced cholestasis models. More importantly, the expression of hallmarkers of necroptosis, including MLKL, RIPK1 and RIPK3 phosphorylation (p- MLKL, p-RIPK1, p-RIPK3) were reduced upon GCD treatment. Glycyrrhetinic acid (GA), the main bioactive metabolite of GCD, effectively protected against ANIT-induced cholestasis, with decreased expression of p-MLKL, p-RIPK1 and p-RIPK3. Meanwhile, the expression of Fas-associated death domain protein (FADD), long isoform of cellular FLICE-like inhibitory protein (cFLIPL) and cleaved caspase 8 were upregulated upon GA treatment. Moreover, GA significantly increased the expression of active caspase 8, and reduced that of p-MLKL in TNF-α/CHX induced hepatocytes necroptosis. CONCLUSIONS: GCD substantially inhibits necroptosis in cholestatic liver injury. GA is the main bioactive component responsible for the anti-necroptotic effects, which correlates with upregulation of c-FLIPL and active caspase 8.

Allocholic acid protects against α-naphthylisothiocyanate-induced cholestasis in mice by ameliorating disordered bile acid homeostasis.

Cholestasis is a pathological condition characterized by disruptions in bile flow, leading to the accumulation of bile acids (BAs) in hepatocytes. Allocholic acid (ACA), a unique fetal BA known for its potent choleretic effects, reappears during liver regeneration and carcinogenesis. In this research, we investigated the protective effects and underlying mechanisms of ACA against mice with cholestasis brought on by α-naphthylisothiocyanate (ANIT). To achieve this, we combined network pharmacology, targeted BA metabolomics, and molecular biology approaches. The results demonstrated that ACA treatment effectively reduced levels of serum AST, ALP, and DBIL, and ameliorated the pathological injury caused by cholestasis. Network pharmacology analysis suggested that ACA primarily regulated BA and salt transport, along with the signaling pathway associated with bile secretion, to improve cholestasis. Subsequently, we examined changes in BA metabolism using UPLC-MS/MS. The findings indicated that ACA pretreatment induced alterations in the size, distribution, and composition of the liver BA pool. Specifically, it reduced the excessive accumulation of BAs, especially cholic acid (CA), taurocholic acid (TCA), and ß-muricholic acid (ß-MCA), facilitating the restoration of BA homeostasis. Furthermore, ACA pretreatment significantly downregulated the expression of hepatic BA synthase Cyp8b1, while enhancing the expression of hepatic efflux transporter Mrp4, as well as the renal efflux transporters Mdr1 and Mrp2. These changes collectively contributed to improved BA efflux from the liver and enhanced renal elimination of BAs. In conclusion, ACA demonstrated its potential to ameliorate ANIT-induced liver damage by inhibiting BA synthesis and promoting both BA efflux and renal elimination pathways, thus, restoring BA homeostasis.

Detrimental Role of CXCR3 in α-Naphthylisothiocyanate- and Triptolide-Induced Cholestatic Liver Injury.

The chemokine receptor CXCR3 is functionally pleiotropic, not only recruiting immune cells to the inflamed liver but also mediating the pathological process of cholestatic liver injury (CLI). However, the mechanism of its involvement in the CLI remains unclear. Both alpha-naphthylisothiocyanate (ANIT) and triptolide are hepatotoxicants that induce CLI by bile acid (BA) dysregulation, inflammation, and endoplasmic reticulum (ER)/oxidative stress. Through molecular docking, CXCR3 is a potential target of ANIT and triptolide. Therefore, this study aimed to investigate the role of CXCR3 in ANIT- and triptolide-induced CLI and to explore the underlying mechanisms. Wild-type mice and CXCR3-deficient mice were administered with ANIT or triptolide to compare CLI, BA profile, hepatic recruitment of IFN-γ/IL-4/IL-17+CD4+T cells, IFN-γ/IL-4/IL-17+iNKT cells and IFN-γ/IL-4+NK cells, and the expression of ER/oxidative stress pathway. The results showed that CXCR3 deficiency ameliorated ANIT- and triptolide-induced CLI. CXCR3 deficiency alleviated ANIT-induced dysregulated BA metabolism, which decreased the recruitment of IFN-γ+NK cells and IL-4+NK cells to the liver and inhibited ER stress. After triptolide administration, CXCR3 deficiency ameliorated dysregulation of BA metabolism, which reduced the migration of IL-4+iNKT cells and IL-17+iNKT cells and reduced oxidative stress through inhibition of Egr1 expression and AKT phosphorylation. Our findings suggest a detrimental role of CXCR3 in ANIT- and triptolide-induced CLI, providing a promising therapeutic target and introducing novel mechanisms for understanding cholestatic liver diseases.

Effects of microsampling on toxicity evaluation of 1-naphthylisothiocyanate (ANIT), a hepatotoxic substance, in a mouse toxicity study.

ICH S3A Q&A focused on microsampling (MS) was published to help accelerate the use of MS and states that MS is useful because toxicokinetic (TK) evaluation with conventional blood sampling volume requires many animals for TK satellite groups; however, there are few reports of MS application in mice. We investigated the influence of MS on toxicity evaluation in mice by comparing the toxicity parameters with and without MS after a single oral administration of 1-naphthylisothiocyanate (ANIT), a hepatotoxic substance. Blood samples (50 µL/point) were collected from the tail vein of 3 mice per group at 2 or 3 time points during a 24-hr period, and toxicity was evaluated 2 days after administration. ANIT-related changes suggesting liver or gallbladder injury were noted in blood chemistry and histopathology. Some of these changes such as increases in focal hepatocyte necrosis and inflammatory cell infiltration in the liver as well as mucosal epithelium necrosis in the gallbladder were apparently influenced by MS. A tendency to anemia was noted in animals with MS but not without MS, which was also noted in the vehicle-treated controls, suggesting influence of blood loss. The current results indicate that ANIT hepatotoxicity could be evaluated in mice in which blood samples were collected by MS for most parameters; however, parameters in anemia and pathology in the liver and gallbladder were influenced by MS in this study condition with ANIT. Therefore, MS application in mice should be carefully considered.

Modulating intestinal barrier function by sphingosine-1-phosphate receptor 1 specific agonist SEW2871 attenuated ANIT-induced cholestatic hepatitis via the gut-liver axis.

Bile acid (BA) homeostasis throughout the enterohepatic circulation system is a guarantee of liver physiological functions. BA circulation disorders is one of the characteristic clinical manifestations of cholestasis, and have a closely relationship with intestinal barrier function, especially ileum. Here, our in vivo and in vitro studies showed that intestinal tight junctions (TJs) were disrupted by α-naphthylisothiocyanate (ANIT), which also down-regulated the protein expression of sphingosine-1-phosphate receptor 1 (S1PR1) in the top of villus of mice ileum. Activating S1PR1 by specific agonist SEW2871 could improve TJs via inhibiting ERK1/2/LKB1/AMPK signaling pathway in the ileum of ANIT-treated mice and ANIT-cultured Caco-2 cells. SEW2871 not only regained ileum TJs by activating S1PR1 in the epithelial cells of ileum mucosa, but also recovered ileum barrier function, which was further verified by the recovered BA homeostasis in mice ileum (content and tissue) by using of high-performance liquid chromatographytandem mass spectrometry (LC-MS/MS). Subsequently, the improved intestinal injury and inflammation further strengthened that SEW2871 modulated intestinal barrier function in ANIT-treated mice. Finally, our data revealed that along with the down-regulated levels of serum lipopolysaccharides (LPS), SEW2871 improved liver function and relieved hepatitis via blocking TLR4/MyD88/NF-kB signaling pathway in ANIT-treated mice. In conclusion, these results demonstrated that activating intestinal S1PR1 by SEW2871 could modulate intestinal barrier function, leading to the improvement of cholestatic hepatitis in ANIT-treated mice via the "gut-liver" axis.

Molecular mechanisms of hepatoprotective effect of tectorigenin against ANIT-induced cholestatic liver injury: Role of FXR and Nrf2 pathways.

Cholestatic liver injury is caused by toxic action or allergic reaction, resulting in abnormality of bile formation and excretion. Few effective therapies have become available for the treatment of cholestasis. Herein, we found that tectorigenin (TG), a natural isoflavone, showed definite protective effects on alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury, significantly reversing the abnormality of plasma alanine/aspartate aminotransferase, total/direct bilirubin and alkaline phosphatase, as well as hepatic reactive oxygen species, catalase and superoxide dismutase. Importantly, the targeted metabolomic determination found that BA homeostasis could be well maintained in TG-treated cholestatic mice, especially the levels of glycocholic acid, tauromuricholic acid, taurocholic acid, taurolithocholic acid, tauroursodeoxycholic acid and taurodeoxycholic acid. Overall, primary/secondary and amidated/unamidated bile acid (BA) levels were significantly altered upon ANIT stimulation but could be restored by TG intervention to certain extents. In addition, TG boosted the expression of farnesoid x receptor (FXR), which in turn upregulated multidrug resistance protein 2 (MRP2) and bile salt export pump (BSEP) to accelerate the excretion of BA. Meanwhile, TG enhanced the expression of Nrf2 and its upstream genes PI3K/Akt and downstream target genes HO-1, NQO1, GCLC and GCLM to strengthen the antioxidant capacity. Taken together, TG plays a vital role in maintaining BA homeostasis and ameliorating cholestatic liver injury through regulating FXR-mediated BA efflux and Nrf2-mediated antioxidative pathways.

The choleretic role of tauroursodeoxycholic acid exacerbates alpha-naphthylisothiocyanate induced cholestatic liver injury through the FXR/BSEP pathway.

The aim of this study was to determine the effect of tauroursodeoxycholic acid (TUDCA) on the alpha-naphthylisothiocyanate (ANIT)-induced model of cholestasis in mice. Wild-type and farnesoid X receptor (FXR)-deficient (Fxr-/- ) mice were used to generate cholestasis models by gavage with ANIT. Obeticholic acid (OCA) was used as a positive control. In wild-type mice, treatment with TUDCA for 7 days resulted in a dramatic increase in serum levels of alanine aminotransferase (ALT), with aggravation of bile infarcts and hepatocyte necrosis with ANIT-induction. TUDCA activated FXR to upregulate the expression of bile salt export pump (BSEP), increasing bile acids (BAs)-dependent bile flow, but aggravating cholestatic liver injury when bile ducts were obstructed resulting from ANIT. In contrast, TUDCA improved the liver pathology and decreased serum ALT and alkaline phosphatase (ALP) levels in ANIT-induced Fxr-/- mice. Furthermore, TUDCA inhibited the expression of cleaved caspase-3 and reduced the area of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in the model mice. TUDCA also upregulated anion exchanger 2 (AE2) protein expression, protecting cholangiocytes against excessive toxic BAs. Our results showed that TUDCA aggravated cholestatic liver injury via the FXR/BSEP pathway when bile ducts were obstructed, although TUDCA inhibited apoptotic activity and protected cholangiocytes against excessive toxic BAs.

7, 8-Dihydroxy-4-methyl coumarin alleviates cholestasis via activation of the Farnesoid X receptor in vitro and in vivo.

Cholestasis is primarily caused by bile acid homeostasis dysregulation, resulting in retention, aggregation, and accumulation of the toxic cholate in the hepatocytes. Existing therapies for cholestasis are limited, demanding the urgent development of novel drugs. As a result, targeting FXR specifically promises a unique treatment strategy for cholestasis. The current study aims to evaluate the influence of 7, 8-dihydroxy-4-methyl coumarin (DMC) against alpha-naphthyl isothiocyanate (ANIT)-induced liver injury in mice. The "Computer-Aided Drug Design" (CADD) and molecular docking study anticipated that DMC would proficiently bind and activate the FXR. Accordingly, the hepatoprotective activity of DMC against ANIT-induced hepatotoxicity and cholestasis was investigated in ANIT-treated HepaRG cells and the ANIT-induced cholestatic mouse model. Outcomes indicated the protective effects of DMC against ANIT toxicity in HepaRG cells after 24 h of intervention and animals after seven days of treatment. DMC partially blocks ANIT-induced increases in serum markers of hepatocellular injury, liver and gall bladder enlargement, and hepatic necrosis. Western blotting revealed that DMC alleviates ANIT-induced hepatotoxicity and cholestasis via activating the FXR receptor and regulating CYP7A1, the enzyme responsible for bile acid synthesis. DMC exhibited protective activity against cholestasis through activating FXR, suggesting it might be a promising strategy for preventing and treating cholestatic liver disease.

Hepatocytic AP-1 and STAT3 contribute to chemotaxis in alphanaphthylisothiocyanate-induced cholestatic liver injury.

The development of cholestatic liver injury (CLI) involves inflammation, but the dominant pathway mediating the chemotaxis is not yet established. This work explored key signaling pathway mediating chemotaxis in CLI and the role of Kupffer cells in the inflammatory liver injury. Probe inhibitors T-5224 (100 mg/kg) for AP-1 and C188-9 (100 mg/kg) for STAT3 were used to validate key inflammatory pathways in alpha-naphthylisothiocyanate (ANIT, 100 mg/kg)-induced CLI. Two doses of GdCl3 (10 mg/kg and 40 mg/kg) were used to delete Kupffer cells and explore their role in CLI. Serum and liver samples were collected for biochemical and mechanism analysis. The liver injury in ANIT-treated mice were significantly increased supported by biochemical and histopathological changes, and neutrophils gathering around the necrotic loci. Inhibitor treatments down-regulated liver injury biomarkers except the level of total bile acid. The chemokine Ccl2 increased by 170-fold and to a less degree Cxcl2 by 45-fold after the ANIT treatment. p-c-Jun and p-STAT3 were activated in the group A but inhibited by the inhibitors in western blot analysis. The immunofluorescence results showed AP-1 not STAT3 responded to inhibitors in ANIT-induced CLI. With or without GdCl3, there was no significant difference in liver injury among the CLI groups. In necrotic loci in CLI, CXCL2 colocalized with hepatocyte biomarker Albumin, not with the F4/80 in Kupffer cells. Conclusively, AP-1 played a more critical role in the inflammation cascade than STAT3 in ANIT-induced CLI. Hepatocytes, not the Kupffer cells released chemotactic factors mediating the chemotaxis in CLI.

Identification of Yinchenwuling fang's active components and hepatoprotective effects against cholestatic liver damage induced by alpha-naphthyl isothiocyanate in mice.

Yinchenwuling Fang (YCWLF), a famous traditional Chinese medicine, has been used clinically for cholestatic liver disease treatment. However, quantification analysis for YCWLF components and their pharmacological effects remains largely unknown. Therefore, we aimed to determine the YCWLF components and their activities. Quantification analysis of 12 YCWLF components was performed using a comprehensive ultra-performance liquid chromatography (UPLC) coupled with the triple-quadrupole mass spectrometry method. Then, the anti-cholestasis effect and potential mechanism of YCWLF were performed in a mouse model induced by alpha-naphthyl isothiocyanate (ANIT). YCWLF decreased serum biochemical indicators (ALT, AST, ALP, TBA, TBIL, and DBIL) and ameliorated liver tissue damage in cholestatic mice. Mechanically, YCWLF increased the expression of the farnesoid X receptor (FXR) and its downstream efflux transporters and metabolic enzyme genes, reversed the disordered homeostasis of bile acids, and decreased cholestatic liver injury. Based on the important role of FXR in YCWLF amelioration on cholestasis, a dual-luciferase assay was used to screen the potential agonist of FXR from 12 YCWLF components. Chlorogenic acid, 4-hydroxyacetophenone, scoparone, atractylenolide Ⅰ, atractylenolide Ⅱ, and alisol B 23-acetate exhibited an activity effect of FXR. This study provides novel a therapeutic mechanism and potential active compounds of YCWLF on cholestatic liver injury.

Liquiritin alleviates alpha-naphthylisothiocyanate-induced intrahepatic cholestasis through the Sirt1/FXR/Nrf2 pathway.

Liquiritin (LQ) is an important monomer active component in flavonoids of licorice. The objective of this study was to evaluate the hepatoprotective effects of LQ in cholestatic mice. LQ (40 or 80 mg/kg) was intragastrically administered to mice once daily for 6 days, and mice were treated intragastrically with a single dosage of ANIT (75 mg/kg) on the 5th day. On the 7th day, mice were sacrificed to collect blood and livers. The mRNA and protein levels were determined by qRT-PCR and western blot assay. We also conducted systematical assessments of miRNAs expression profiles in the liver. LQ ameliorated ANIT-induced cholestatic liver injury, as evidenced by reduced serum biochemical markers and attenuated pathological changes in liver. Pretreatment of LQ reduced the increase of malondialdehyde, TNF-α, and IL-1ß induced by ANIT. Moreover, ANIT suppressed the expression of Sirt1 and FXR in liver tissue, which was weakened in the LQ pre-treatment group. LQ enhanced the nuclear expression of Nrf2, which was increased in the ANIT group. LQ also increased the mRNA expressions of bile acid transporters Bsep, Ntcp, Mrp3, and Mrp4. Furthermore, a miRNA deep sequencing analysis revealed that LQ had a global regulatory effect on the hepatic miRNA expression. Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis showed that the differentially expressed miRNAs were mainly related to metabolic pathways, endocytosis, and MAPK signaling pathway. Collectively, LQ attenuated hepatotoxicity and cholestasis by regulating the expression of Sirt1/FXR/Nrf2 and the bile acid transporters, indicating that LQ might be an effective approach for cholestatic liver diseases.

Silymarin protects the liver from α-naphthylisothiocyanate-induced cholestasis by modulating the expression of genes involved in bile acid homeostasis.

Cholestasis is characterized by impaired bile flow which results in inflammation, cirrhosis, and ultimately liver failure. The current study is aimed to evaluate the anti-cholestatic effect of silymarin against α-naphthylisothiocyanate (ANIT) induced cholestasis. Mice were gavaged with various doses of silymarin or ursodeoxycholic acid (UDCA) for 19 days. Then they were challenged with α-naphthylisothiocyanate (ANIT) and after 48 hours the animals were sacrificed to obtain blood and liver sections. Serum levels of bilirubin, aspartate transaminase (AST), alanine transaminase (ALP), and liver histology were analyzed. mRNA expression of selected transporters (Bile salt export pump (BSEP) and sodium taurocholate cotransporting polypeptide (NTCP)) and proteins (farnesoid x receptor (FXR) and Cytochrome P450 Family 7 Subfamily A Member 1 (Cyp7a1)) involved in bile acids biosynthesis, excretion and uptake were also evaluated by quantitative PCR. The results indicated that the serum levels of bilirubin, AST, and ALP were significantly higher in a cholestatic model group as compared to an untreated control group. However, in silymarin groups, the serum level of these parameters is significantly lower than in a cholestatic model group. Liver histology also showed that silymarin prevents ANIT-induced hepatic injury. mRNA expression of FXR, BSEP, and NTCP was downregulated and expression of Cyp7a1 was upregulated in a cholestatic model group as compared to an untreated control group. However, in silymarin treatment groups, the expression of FXR, BSEP and NTCP was upregulated and the expression of Cyp7a1 was downregulated as compared to the cholestatic model group. In conclusion, silymarin could alleviate hepatic injury by modulating the expression of genes involved in bile acid homeostasis.

[Study on protective effect of Chaihu Shugan Powder against liver injury in rats with intrahepatic cholestasis by regulating FXR/Nrf2/ARE pathway].

This study aims to investigate the effect of Chaihu Shugan Powder(CHSG) on liver injury in rats with intrahepatic cholestasis by regulating farnesoid X receptor(FXR)/nuclear factor erythroid-2-related factor(Nrf2)/antioxidant response element(ARE) pathway. Eighty-four SD rats were classified into normal group, model group, CHSG-L group(0.5 g·kg~(-1)), CHSG-H group(2.5 g·kg~(-1)), ursodeoxycholic acid group(UDCA group, 100 mg·kg~(-1)), CHSG-H+sh-NC group(2.5 g·kg~(-1) CHSG+subcutaneous injection of sh-NC lentivirus), CHSG-H+sh-FXR group(2.5 g·kg~(-1) CHSG+subcutaneous injection of sh-FXR lentivirus), with 12 rats in each group. Rats were treated with corresponding drugs except for the normal group and the model group, once a day, for 7 days. On 5 th day, rats, except the normal group, were given α-naphthalene isothiocyanate(ANIT) at a dose of 100 mg·kg~(-1), once a day for 3 days to induce intrahepatic cholestasis, and the normal group was given the same amount of normal saline. Rats were anesthetized 1 h after the last administration and the 2 h bile flow was measured. Aeroset chemistry analyzer was employed to detect the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL), and total bile acid(TBA) in rat serum. Based on hematoxylin and eosin(HE) staining, the pathological changes of rat liver tissue were observed. Glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), and malondialdehyde(MDA) in rat liver tissue homogenate were monitored with corresponding kits. Western blot was used to detect the expression of FXR, Nrf2, and heme oxygenase-1(HO-1) proteins in rat liver tissue. Compared with the normal group, the model group showed many spots or concentrated necrotic areas in the liver tissue, infiltration of a large number of inflammatory cells, swelling liver cells with nuclear shrinkage. The 2 h bile flow, levels of GSH-Px and SOD, and relative expression of FXR, Nrf2, and HO-1 proteins were significantly lower, and the levels of ALT, AST, TBIL, TBA and MDA were significantly higher in the model group than in the normal group. Compared with the model group, CHSG-L group, CHSG-H group, and UDCA group demonstrated significant alleviation of pathological damage of the liver tissue, significantly high 2 h bile flow, levels of GSH-Px and SOD, and expression of FXR, Nrf2 and HO-1 proteins, and significantly low levels of ALT, AST, TBIL, TBA and MDA. Compared with the CHSG-H group, the CHSG-H+sh-FXR group had worse liver pathological damage, significantly low levels of 2 h bile flow, levels of GSH-Px and SOD, and expression of FXR, Nrf2, and HO-1 proteins, and significantly high levels of ALT, AST, TBIL, TBA, and MDA. CHSG may protect against liver injury in rats with intrahepatic cholestasis by activating the FXR/Nrf2/ARE pathway.