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.

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.

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.

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.

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.

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.

Ginsenosides Restore Lipid and Redox Homeostasis in Mice with Intrahepatic Cholestasis through SIRT1/AMPK Pathways.

Intrahepatic cholestasis (IC) occurs when the liver and systemic circulation accumulate bile components, which can then lead to lipid metabolism disorders and oxidative damage. Ginsenosides (GS) are pharmacologically active plant products derived from ginseng that possesses lipid-regulation and antioxidation activities. The purpose of this study was to evaluate the possible protective effects of ginsenosides (GS) on lipid homeostasis disorder and oxidative stress in mice with alpha-naphthylisothiocyanate (ANIT)-induced IC and to investigate the underlying mechanisms. A comprehensive strategy via incorporating pharmacodynamics and molecular biology technology was adopted to investigate the therapeutic mechanisms of GS in ANIT-induced mice liver injury. The effects of GS on cholestasis were studied in mice that had been exposed to ANIT-induced cholestasis. The human HepG2 cell line was then used in vitro to investigate the molecular mechanisms by which GS might improve IC. The gene silencing experiment and liver-specific sirtuin-1 (SIRT1) knockout (SIRT1LKO) mice were used to further elucidate the mechanisms. The general physical indicators were assessed, and biological samples were collected for serum biochemical indexes, lipid metabolism, and oxidative stress-related indicators. Quantitative PCR and H&E staining were used for molecular and pathological analysis. The altered expression levels of key pathway proteins (Sirt1, p-AMPK, Nrf2) were validated by Western blotting. By modulating the AMPK protein expression, GS decreased hepatic lipogenesis, and increased fatty acid ß-oxidation and lipoprotein lipolysis, thereby improving lipid homeostasis in IC mice. Furthermore, GS reduced ANIT-triggered oxidative damage by enhancing Nrf2 and its downstream target levels. Notably, the protective results of GS were eliminated by SIRT1 shRNA in vitro and SIRT1LKO mice in vivo. GS can restore the balance of the lipid metabolism and redox in the livers of ANIT-induced IC models via the SIRT1/AMPK signaling pathway, thus exerting a protective effect against ANIT-induced cholestatic liver injury.

IL-25 ameliorates acute cholestatic liver injury via promoting hepatic bile acid secretion.

Cholestasis caused by bile secretion and excretion disorders is a serious manifestation of hepatopathy. Interleukin (IL)-25 is a member of the IL-17 cytokine family, which involves in mucosal immunity and type 2 immunity via its receptor-IL-17RB. Our previous studies have shown that IL-25 improves non-alcoholic fatty liver via stimulating M2 macrophage polarization and promotes development of hepatocellular carcinoma via alternative activation of macrophages. These hepatopathy are closely associated with cholestasis. However, whether IL-25 play an important role in cholestasis remains unclear. IL-25 treatment and IL-25 knockout (Il25-/-) mice were injected intragastrically with α-naphthyl isothiocyanate (ANIT) to determine the biological association between IL-25 and cholestasis. Here, we found that IL-25 and IL-17RB decreased in ANIT-induced cholestatic mice. Il25-/- mice showed exacerbated ANIT-induced parenchymal injury and IL-25 treatment significantly alleviated cholestatic liver injury induced by ANIT. We found that IL-25 reduced the level of hepatic total bile acids and increased the expression of multidrug resistance-associated protein 2 (MRP2) and multidrug resistance-associated protein 3 (MRP3) in liver. In conclusion, IL-25 exhibited a protective effect against ANIT-induced cholestatic liver injury in mice, which may be related to the regulation on bile acids secretion. These results provide a theoretical basis for the use of IL-25 in the treatment of cholestatic hepatopathy.

Combination of resveratrol and luteolin ameliorates α-naphthylisothiocyanate-induced cholestasis by regulating the bile acid homeostasis and suppressing oxidative stress.

Cholestasis is a common liver injury without any effective therapeutic drugs so far. Resveratrol (RES) and luteolin (LUT) are natural polyphenols that exert protective effects on multiple liver injuries. Coadministration of RES and LUT could significantly improve the bioavailability of LUT and increase the systemic exposure to RES, and the combined treatment could also benefit from their multi-component and multi-target characteristics. Our current aim is to study the protective effects of coadministration of RES and LUT on α-naphthylisothiocyanate (ANIT)-induced cholestasis. Serum biochemical indices and liver histopathology in rats indicated that coadministration of RES and LUT could improve liver function by suppressing oxidative stress. Dysregulated bile acid (BA) homeostasis is a significant pathological feature of cholestasis, which was determined to explore the potential biomarkers and to clarify the protection mechanism of coadministration of RES and LUT. The levels of cholic acid, chenodeoxycholic acid, taurine conjugates and glycine conjugates, and the ratios of taurine conjugates to their free forms could be used as diagnosis indicators for cholestasis in rats. Furthermore, the coadministration of RES and LUT could restore the BA levels and exert better protective effects than administration alone. This study suggested that the coadministration of RES and LUT could protect against ANIT-induced cholestasis and the mechanism was closely related to regulating BA homeostasis and suppressing oxidative stress.

Nrf2-Dependent Protective Effect of Paeoniflorin on α-Naphthalene Isothiocyanate-Induced Hepatic Injury.

The pathological mechanism of cholestatic hepatic injury is associated with oxidative stress, hepatocyte inflammation, and dysregulation of hepatocyte transporters. Paeonia lactiflora Pall. and its compound can improve hepatic microcirculation, dilate bile duct, and promote bile flow, which is advantageous to ameliorate liver damage. Paeoniflorin (PEA), as the main efficacy component of Paeonia lactiflora Pall., has multiple pharmacological effects. PEA improves liver injury, but it remains obscure whether the protective action on [Formula: see text]-naphthalene isothiocyanate (ANIT)-induced cholestatic liver injury is dependent on the NF-E2 p45-related Factor 2 (Nrf2) signaling pathway. In this study, C57BL/6 mice were administrated with 80 mg⋅kg[Formula: see text]⋅d[Formula: see text] ANIT followed by PEA (75, 150, and 300 mg⋅kg[Formula: see text]⋅d[Formula: see text]) orally for 10 days, respectively. Tissue histology and liver function were detected, including serum enzymes, gallbladder (GB) weight, phenobarbital-induced sleeping time (PEN-induced ST), hepatic uridine di-phosphoglucuronosyltransferase (UDPG-T), malondialdehyde (MDA), and glutathione (GSH). The expressions of protein Nrf2, sodium taurocholate cotransporting polypeptide (Ntcp), and NADPH oxidase 4 (Nox4) were evaluated. Nrf2 plasmid or siRNA-Nrf2 transfection on LO2 cells and Nrf2-/- mice were used to explore the liver protective mechanism of PEA. Compared to ANIT-treated mice, PEA decreased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBA), and phenobarbital-induced sleeping time. The bile secretion, hepatic UDPG-T, MDA, GSH, and liver histology were improved. The expressions of protein Nrf2 and Ntcp in liver tissues increased, but Nox4 decreased. After Nrf2 plasmid or small interfering RNA (siRNA)-Nrf2 transfection, the protective effects of PEA on LO2 cells were, respectively, strengthened or weakened. Moreover, PEA had no significant effects on ANIT-treated Nrf2-/- mice. Our results suggest that Nrf2 is essential for PEA protective effects on ANIT-induced liver injury.

Oleanolic acid alleviates ANIT-induced cholestatic liver injury by activating Fxr and Nrf2 pathways to ameliorate disordered bile acids homeostasis.

BACKGROUND: Cholestasis is a clinical syndrome with high incidence and few effective treatments. Oleanolic acid (OA) is a triterpenoid compound with anti-cholestatic effects. Studies using bile duct ligation or lithocholic acid modeling have shown that the alleviating effect of OA on cholerosis is related to the regulation of nuclear factor erythroid 2 related factor (Nrf2) or farnesoid X receptor (Fxr). PURPOSE: This study aims to investigate the underlying mechanism of OA against alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury based on Nrf2 and Fxr dual signaling pathways. METHODS: The ANIT-induced rats model was used with or without OA treatment. Serum biochemical indexes, liver histopathological changes and glutathione level were examined. Bile acids (BAs) targeted metabolomics based on UHPLC-MS/MS were performed. siRNA, RT-qPCR and western blot analysis were used to prove the role of Fxr and Nrf2 pathway in OA's anti-cholestatic liver injury in vivo and in vitro. RESULTS: OA significantly alleviated ANIT-induced liver injury in rats, reduced primary bile acids, accelerated metabolism of BAs and reduced the intrahepatic accumulation of BAs. The expressions of bile salt export pump (Bsep), Na+-taurocholic cotransport polypeptide (Ntcp), UDP-glucuronyl transferase 1a1 (Ugt1a1) and Fxr in rat liver were markedly up-regulated, the activation of Nrf2 was promoted, and the expression of cholesterol 7α-hydroxylase (Cyp7a1) was decreased after OA treatment. Moreover, Fxr or Nrf2 silencing attenuated the regulation of OA on BAs homeostasis related transporters and enzymes in rat primary hepatocytes. CONCLUSION: OA may regulate BAs-related transporters and metabolic enzymes by activating Fxr and Nrf2 pathways, thus alleviating the cholestatic liver injury induced by ANIT.

Cultured bear bile powder ameliorates acute liver injury in cholestatic mice via inhibition of hepatic inflammation and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Natural bear bile powder (NBBP) is a traditional Chinese medicine used for treating liver dysfunction. Cultured bear bile powder (CBBP), which is produced using biotransformation of chicken bile, acts as an appropriate substitute for NBBP when treating cholestatic liver injury. AIM OF THE STUDY: To investigate the molecular mechanisms underlying the hepatoprotective effects of CBBP in an α-naphthylisothiocyanate (ANIT)-induced cholestatic mouse model. MATERIALS AND METHODS: Cholestatic mice were pretreated with CBBP or NBBP via oral gavage once a day for two weeks. Their blood biochemistry and liver histopathology were then evaluated using standard protocols. Western blot analyses, real-time polymerase chain reaction, and immunohistochemistry were used to evaluate changes in the protein levels and gene expression profiles of factors associated with hepatic inflammation and apoptosis in cholestatic mice. RESULTS: CBBP significantly decreased the serum indices of liver injury, and ameliorated neutrophil infiltration and hepatocyte necrosis within liver tissue of cholestatic mice. Expression of the inflammatory factors, such as tumor necrosis factor-α, interleukin-1ß (IL-1ß), IL-6, monocyte chemoattractant protein-1, and intercellular adhesion molecule 1, was significantly reduced in CBBP-treated cholestatic mice. Moreover, proteins involved in the toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B (TLR4/Myd88/NF-κB) signaling pathway, such as CD14, TLR4, Myd88, and NF-κB, that were increased in cholestatic mice, were downregulated by CBBP. Meanwhile, increased expression of the apoptosis-related proteins, caspase-3 and Bax, in cholestatic mice was reversed by CBBP treatment. CONCLUSION: CBBP treatment alleviates ANIT-induced cholestasis and liver injury by reducing hepatocyte inflammation and apoptosis.

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.

Allopurinol Protects Against Cholestatic Liver Injury in Mice Not Through Depletion of Uric Acid.

Cholestasis is one of the most severe manifestations of liver injury and has limited therapeutic options. Allopurinol (AP), an inhibitor of uric acid (UA) synthesis, was reported to prevent liver damage in several liver diseases. However, whether AP protects against intrahepatic cholestatic liver injury and what is the role of UA in the pathogenesis of cholestasis remain unknown. In this study, we reported that AP attenuated liver injury in a mouse model of intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT). AP showed no significant effect on glutathione depletion, inflammation, or bile acid metabolism in livers of ANIT-treated mice. Instead, AP significantly improved fatty acid ß-oxidation in livers of ANIT-treated mice, which was associated with activation of PPARα. The protective effect of AP on cholestatic liver injury was not attributable to the depletion of UA, because both exogenous and endogenous UA prevented liver injury in ANIT-treated mice via inhibition of NF-kB-mediated inflammation. In conclusion, the present study provides a new perspective for the therapeutic use of AP and the role of UA in cholestatic liver injury.

A Network Pharmacology Study of the Molecular Mechanisms of Hypericum japonicum in the Treatment of Cholestatic Hepatitis with Validation in an Alpha-Naphthylisothiocyanate (ANIT) Hepatotoxicity Rat Model.

BACKGROUND This network pharmacology study aimed to identify the active compounds and molecular mechanisms involved in the effects of Hypericum japonicum on cholestatic hepatitis. We validated the findings in an alpha-naphthylisothiocyanate (ANIT) rat model of hepatotoxicity. MATERIAL AND METHODS The chemical constituents and targets of H. japonicum and target genes previously associated with cholestatic hepatitis were retrieved from public databases. A network was constructed using Cytoscape 3.7.2 software and the STRING database and potential protein functions were analyzed based on the public platform of bioinformatics. ANIT was used to induce cholestatic hepatitis in a rat model using 36 Sprague-Dawley rats, and this model was used to investigate intervention with 3 doses of quercetin (low-dose, 50 mg/kg; medium-dose, 100 mg/kg; and high-dose, 200 mg/kg), the main active component of H. japonicum. Levels of serum biochemical indexes were measured by commercial kits, and the messenger RNA (mRNA) levels of markers of liver and mitochondrial function and oxidative stress were detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS The main active ingredients of H. japonicum were quercetin, kaempferol, and tetramethoxyluteolin, and their key targets included prostaglandin G/H synthase 2 (PTGS2), B-cell lymphoma-2 (BCL2), cholesterol 7-alpha hydroxylase (CYP7A1), and farnesoid X receptor (FXR). Quercetin intervention promoted recovery from cholestatic hepatitis. CONCLUSIONS The findings from this research provide support for future research on the roles of quercetin, kaempferol, and tetramethoxyluteolin in human liver disease and the roles of the PTGS2, BCL2, CYP7A1, and FXR genes in cholestatic hepatitis.

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.

Protective effects of n-Butanol extract and iridoid glycosides of Veronica ciliata Fisch. Against ANIT-induced cholestatic liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Veronica ciliata Fisch. is a traditional medical herb that present in more than 100 types of Tibetan medicine prescriptions, most of which are used for liver disease therapy. Iridoid glycosides have been identified as the major active components of V.ciliata with a variety of biological activities. AIMS OF THE STUDY: The aim of this study is to explore the protective effect and potential mechanism of n-Butanol extract (BE) and iridoid glycosides (IG) from V.ciliata against ɑ-naphthyl isothiocyanate (ANIT)-induced hepatotoxicity and cholestasis in mice. MATERIALS AND METHODS: Mice were intragastrically (i.g.) given BE and IG at different dose or positive control ursodeoxycholic acid (UCDA) once a day for 14 consecutive days, and were treated with ANIT to cause liver injury on day 12th. Serum levels of hepatic injury markers and cholestasis indicators, liver index and liver histopathology were measured to evaluate the effect of BE and IG on liver injury caused by ANIT. The protein levels of tumor necrosis factor-α (TNF-α), nuclear factor kappa B(NF-κB), interleukin-6 (IL-6), Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), and the levels of oxidative stress indicators in liver tissue were investigated to reveal the underlying protective mechanisms of BE and IG against ANIT-induced hepatotoxicity and cholestasis. RESULTS: The n-Butanol extract (BE) and iridoid glycosides (IG) isolated from V.ciliata significantly decreased serum level of cholestatic liver injury markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), total bile acid (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL) in ANIT-treated mice. Histopathology of the liver tissue showed that pathological damages were relieved upon BE and IG treatment. Meanwhile, the results indicated BE and IG notably restored relative liver weights, inhibited oxidative stress induced by ANIT through increasing hepatic level of superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT) and decreasing hepatic content of malondialdehyde (MDA). Western blot revealed that BE and IG inhibited the expression of pro-inflammatory factors TGF-α, IL-6 and NF-κB. Furthermore, the decreased protein expression of bile acid transporters NTCP, BSEP, MRP2 were upregulated by BE and IG in a dose-dependent manner. CONCLUSION: The results have demonstrated that BE and IG exhibited a dose-dependently protective effect against ANIT-induced liver injury with acute intrahepatic cholestasis in mice, which might be related to the regulation of oxidative stress, inflammatory response and bile acid transport. In addition, these findings pointed out that iridoid glycosides as main active components of V.ciliata play a critical role in hepatoprotective effect of V.ciliata.

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.

Picroside II protects against cholestatic liver injury possibly through activation of farnesoid X receptor.

BACKGROUD: Cholestasis, accompanied by the accumulation of bile acids in body, may ultimately cause liver failure and cirrhosis. There have been limited therapies for cholesteric disorders. Therefore, development of appropriate therapeutic drugs for cholestasis is required. Picroside II is a bioactive component isolated from Picrorhiza scrophulariiflora Pennell, its mechanistic contributions to the anti-cholestasis effect have not been fully elucidated, especially the role of picroside II on bile acid homeostasis via nuclear receptors remains unclear. PURPOSE: This study was designed to investigate the hepatoprotective effect of picroside II against alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury and elucidate the mechanisms in vivo and in vitro. METHODS: The ANIT-induced cholestatic mouse model was used with or without picroside II treatment. Serum and bile biochemical indicators, as well as liver histopathological changes were examined. siRNA, Dual-luciferase reporter, quantitative real-time PCR and Western blot assay were used to demonstrate the farnesoid X receptor (FXR) pathway in the anti-cholestasis effects of picroside II in vivo and in vitro. RESULTS: Picroside II exerted hepatoprotective effect against ANIT-induced cholestasis by impaired hepatic function and tissue damage. Picroside II increased bile acid efflux transporter bile salt export pump (Bsep), uptake transporter sodium taurocholate cotransporting polypeptide (Ntcp), and bile acid metabolizing enzymes sulfate transferase 2a1 (Sult2a1) and UDP-glucuronosyltransferase 1a1 (Ugt1a1), whereas decreased the bile acid synthesis enzymes cholesterol 7α-hydroxylase (Cyp7a1) and oxysterol 12α-hydroxylase (Cyp8b1). In addition, expression of FXR and the target gene Bsep was increased, whereas aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARα) and their corresponding target genes were not significantly influenced by picroside II under cholestatic conditions. Furthermore, regulation of transporters and enzymes involved in bile acid homeostasis by picroside II were abrogated by FXR silencing in mouse primary cultured hepatocytes. Dual-luciferase reporter assay performed in HepG2 cells demonstrated FXR activation by picroside II. CONCLUSION: Our findings demonstrate that picroside II exerts protective effect on ANIT-induced cholestasis possibly through FXR activation that regulates the transporters and enzymes involved in bile acid homeostasis. Picroside II might be an effective approach for the prevention and treatment of cholestatic liver diseases.