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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 GdCl

SP600125 is a classic inhibitor of c‑Jun‑N‑terminal kinase (JNK) that is widely used in numerous medicinal studies, but its administration regimen has yet to be optimized. In the present study, intraperitoneal (i.p.) and intragastric (i.g.) injections of 15 mg/kg SP600125 was performed in mice to compare the inhibitory effect against JNK signalling in cholestasis induced by α‑naphthylisothiocyanate (ANIT). SP600125 at a dose of 15 mg/kg administered by i.p. substantially decreased ANIT‑induced liver injury as observed by biochemical and histopathological examinations. The adaptation of bile acid synthesis was inhibited in the A‑SP‑i.p. group compared with that in the A‑SP‑i.g. group, as indicated by the expression analysis of CYP7A1 and CYP8B1. The transcription of the pro‑inflammatory factors IL‑6, IL‑1β, ICAM‑1 and IL‑10 supported the differential toxic responses. Western blot analysis revealed that JNK signalling activated by ANIT was inhibited more markedly in the A‑SP‑i.p. group than in the A‑SP‑i.g. group. The peak concentration and the AUC0‑24 of SP600125 in the A‑SP‑i.p. group were 5‑fold and 1.56‑fold higher, respectively, compared with those in the A‑SP‑i.g. group. These data indicated that i.p. administration of SP600125 produced a high plasma exposure profile, which directly determined its efficacy of blocking the JNK signalling. This effect of SP600125 on the JNK pathway may provide an optimized design for future in vivo investigations.

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

Gemfibrozil is a fibrate drug used widely for dyslipidemia associated with atherosclerosis. Clinically, both gemfibrozil and its phase II metabolite gemfibrozil 1-O-β-glucuronide (gem-glu) are involved in drug-drug interaction (DDI). But the DDI risk caused by gem-glu between human and mice has not been compared. In this study, six volunteers were recruited and took a therapeutic dose of gemfibrozil for 3 days for examination of the gemfibrozil and gem-glu level in human. Male mice were fed a gemfibrozil diet (0.75%) for 7 days, following which a cocktail-based inhibitory DDI experiment was performed. Plasma samples and liver tissues from mice were collected for determination of gemfibrozil, gem-glu concentration and cytochrome p450 enzyme (P450) induction analysis. In human, the molar ratio of gem-glu/gemfibrozil was 15% and 10% at the trough concentration and the concentration at 1.5 h after the 6th dose. In contrast, this molar ratio at steady state in mice was 91%, demonstrating a 6- to 9-fold difference compared with that in human. Interestingly, a net induction of P450 activity and in vivo inductive DDI potential in mice was revealed. The P450 activity was not inhibited although the gem-glu concentration was high. These data suggested species difference of relative gem-glu exposure between human and mice, as well as a net inductive DDI potential of gemfibrozil in mouse model.

Fenofibrate was reported to be beneficial for cholestasis in combination with ursodeoxycholic acid. However, its therapeutic action as single therapy for chronic cholestasis and the underlying mechanism are not known. In the present study, wild-type (WT) mice were administered a 0.05% ANIT diet to mimic chronic cholestatic liver injury. Mice were dosed fenofibrate 25 mg/kg twice every day for 10 days to investigate the therapeutic action of fenofibrate on chronic cholestatic liver injury. Ppara-null (KO) mice were used to explore PPARα’s role in the therapeutic outcome. Fenofibrate, administered at 25 mg/kg twice daily, substantially reversed ANIT-induced chronic cholestatic liver injury shown by biochemical and pathological end points. The modifications of bile acid metabolism were found to be adaptive responses. The JNK–AP1–CCL2/CXCL2 axis was activated in all the mice administered ANIT which developed chronic cholestatic liver injury. But it was substantially decreased by fenofibrate in WT mice rather than that in KO mice. Low-dose fenofibrate reversed chronic cholestatic liver injury in mice. The therapeutic action was dependent on PPARα activation and occurred by inhibiting chemotaxis via the JNK–AP1–CCL2/CXCL2 signaling. These data provided an exciting basis for optimization of therapeutic fenofibrate regimen in the clinic. Additionally, they suggested anti-chemotaxis of low-dose fenofibrate in single therapy to treat cholestatic liver diseases.

Background The antiepileptic drug carbamazepine (CBZ) is a typical inducer of cytochrome P450 (CYP) 3A and 2C in the clinic. It is considered a strong constitutive androstane receptor activator, however both CBZ and its main metabolite CBZ 10, 11-epoxide have been reported to be pregnane X receptor (PXR) activators whose maximal efficacy and potency are comparable with the human PXR ligand rifampicin. It is unknown whether or not PXR plays a substantially important role in in vivo induction of CYP by CBZ administration. Methods In this study, wild type and Pxr −/− mice were administered with CBZ for 5 days. Serum and liver samples were collected and subjected to hepatotoxicity assessment and CYP induction analysis. Results CYP2b, 2c and 3a were induced similarly in terms of transcription level, enzyme activity and protein abundance in both wild type and Pxr −/− mice. Inductive profile of CYPs in mice by CBZ administration accorded with those reported in rats, but differed from clinically reported data. Conclusions These data suggest that in vivo induction of CYP in mice by multiple administration of CBZ is independent of PXR. Knowledge of the featured CYP induction profile in mice helps us understand species related CYP induction profiles among rodents and humans resulting from administration of CBZ.

Gemfibrozil, a peroxisome proliferator-activated receptor α (PPARα) agonist, is widely used for hypertriglyceridaemia and mixed hyperlipidaemia. Drug-drug interaction of gemfibrozil and other PPARα agonists has been reported. However, the role of PPARα in cytochrome P450 (CYP) induction by fibrates is not well known. In this study, wild-type mice were first fed gemfibrozil-containing diets (0.375%, 0.75% and 1.5%) for 14 days to establish a dose-response relationship for CYP induction. Then, wild-type mice and Pparα-null mice were treated with a 0.75% gemfibrozil-containing diet for 7 days. CYP3a, CYP2b and CYP2c were induced in a dose-dependent manner by gemfibrozil. In Pparα-null mice, their mRNA level, protein level and activity were induced more than those in wild-type mice. So, gemfibrozil induced CYP, and this action was inhibited by activated PPARα. These data suggested that the induction potential of CYPs was suppressed by activated PPARα, showing a potential role of this receptor in drug-drug interactions and metabolic diseases treated with fibrates.

TiO2 nano-particle (TiO2 NP) is widely used in industrial, household necessities, as well as medicinal products. However, the effect of TiO2 NP on liver metabolic function has not been reported. In this study, after mice were orally administered TiO2 NP (21nm) for 14days, the serum and liver tissues were assayed by biochemical analysis, real time quantitative polymerase chain reaction, western blot and transmission electron microscopy. The serum bilirubin was increased in a dose dependent manner. Deposition of TiO2 NP in hepatocytes and the abnormality of microstructures was observed. Expression of metabolic genes involved in the endogenous and exogenous metabolism was modified, supporting the toxic phenotype. Collectively, oral administration of TiO2 NP (21nm) led to deposition of particles in hepatocytes, mitochondrial edema, and the disturbance of liver metabolism function. These data suggested oral administration disrupts liver metabolic functions, which was more sensitive than regular approaches to detect material hepatotoxicity. This study provided useful information for risk analysis and regulation of TiO2 NPs by administration agencies.

Perfluorodecanoic acid (PFDA) is widely used in production of many daily necessities based on their surface properties and stability. It was assigned as a Persistent Organic Pollutant in 2009 and became a public concern partly because of its potential for activation of the peroxisome proliferator-activated receptor alpha (PPARα). In this study, wild-type and Ppara-null mice were administered PFDA (80 mg/kg). Blood and liver tissues were collected and subjected to systemic toxicological and mechanistic analysis. UPLC-ESI-QTOFMS-based metabolomics was used to explore the contributing components of the serum metabolome that led to variation between wild-type and Pparα-null mice. Bile acid homeostasis was disrupted, and slight hepatocyte injury in wild-type mice accompanied by adaptive regulation of bile acid synthesis and transport was observed. The serum metabolome in wild-type clustered differently from that in Pparα-null, featured by sharp increases in bile acid components. Differential toxicokinetic tendency was supported by regulation of UDP-glucuronosyltransferases dependent on PPARα, but it did not contribute to the hepatotoxic responses. Increase in Il-10 and activation of the JNK pathway indicated inflammation was induced by disruption of bile acid homeostasis in wild-type mice. Inhibition of p-p65 dependent on PPARα activation by PFDA stopped the inflammatory cascade, as indicated by negative response of Il-6, Tnf-α, and STAT3 signaling. These data suggest disruptive and protective role of PPARα in hepatic responses induced by PFDA.

Gemfibrozil is the most myotoxic fibrate drug commonly used for dyslipidemia, but the mechanism is poorly understood. The current study revealed that gemfibrozil inhibits myoblast differentiation through the regulation of intracellular calcium ([Ca(2+)]i) as revealed in L6 myoblasts by use of laser scan confocal microscopy and flow cytometry using Fluo-4 AM as a probe. Gemfibrozil at 20–400 μM, could regulate [Ca(2+)]i in L6 cells in a biphasic manner, and sustained reduction was observed when the concentration reached 200 μM. Inhibition of L6 differentiation by gemfibrozil was concentration-dependent with maximal effect noted between 200 and 400 μM, as indicated by creatine kinase activities and the differentiation index, respectively. In differentiating L6 myoblasts, gemfibrozil at concentrations below 400 μM led to no significant signs of apoptosis or cytotoxicity, whereas differentiation, inhibited by 200 μM gemfibrozil, was only partially recovered. A good correlation was noted between gemfibrozil concentrations that regulate [Ca(2+)]i and inhibit L6 myoblasts differentiation, and both are within the range of total serum concentrations found in the clinic. These data suggest a potential pharmacodynamic effect of gemfibrozil on myogenesis as a warning sign, in addition to the complex pharmacokinetic interactions. It is also noteworthy that mobilization of [Ca(2+)]i by gemfibrozil may trigger complex biological responses besides myocyte differentiation. Information revealed in this study explores the mechanism of gemfibrozil-induced myotoxicity through the regulation of intracellular calcium.

Overdose of acetaminophen (APAP) can cause acute liver injury that is sometimes fatal, requiring efficient pharmacological intervention. The traditional Chinese herb Bupleurum falcatum has been widely used for the treatment of several liver diseases in eastern Asian countries, and saikosaponin d (SSd) is one of its major pharmacologically-active components. However, the efficacy of Bupleurum falcatum or SSd on APAP toxicity remains unclear. C57BL/6 mice were administered SSd intraperitoneally once daily for five days, followed by APAP challenge. Biochemical and pathological analysis revealed that mice treated with SSd were protected against APAP-induced hepatotoxicity. SSd markedly suppressed phosphorylation of nuclear factor kappa B (NF-kB) and signal transducer and activator of transcription 3 (STAT3) and reversed the APAP-induced increases in the target genes of NF-kB, such as pro-inflammatory cytokine Il6 and Ccl2, and those of STAT3, such as Socs3, Fga, Fgb and Fgg. SSd also enhanced the expression of the anti-inflammatory cytokine Il10 mRNA. Collectively, these results demonstrate that SSd protects mice from APAP-induced hepatotoxicity mainly through down-regulating NF-kB- and STAT3-mediated inflammatory signaling. This study unveils one of the possible mechanisms of hepatoprotection caused by Bupleurum falcatum and/or SSd.

Fibrates are a group of peroxisome proliferator-activated receptor α agonists used in the treatment of dyslipidemia; however, they have been reported to cause species-related hepatocarcinogenesis and clinical myotoxicity. Gemfibrozil is one of the most commonly used fibrates, and it shows the highest risk for myotoxicity among the fibrates. The inhibitory drug-drug interaction mechanism associated with gemfibrozil has been explored recently, and the induction of human P450 3A4 and 2C8 has been reported. In this study, in vivo induction of rat P450 by gemfibrozil was studied in Sprague-Dawley rats. After the rats were dosed with gemfibrozil by oral gavage, microsomes were prepared. The metabolic activities of P450 3A1/2, 2C6, and 2D2 were assayed using probe substrates, and the systemic concentration of gemfibrozil during its administration was determined. P450 3A1/2 and 2C6 activities were induced 32-77% in the rats by gemfibrozil when the exposure concentration was in the clinical range. These data indicate that the inducibility of homologous P450 isoforms by gemfibrozil is similar in Sprague-Dawley rats and in humans. Inductive drug-drug interactions and inhibitory actions are involved in the co-administration of gemfibrozil with other drugs, which suggests the relevance for a fibrate-toxicology investigation.