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We have previously shown that the ethanol-mediated elevation of lipocaline-2 (LCN2) is closely associated with the development of alcoholic fatty liver disease (AFLD) in mice. Herein, we aimed to understand the functional significance of LCN2 induction by ethanol and to explore its underlying mechanisms. We evaluated the effects of LCN2 in an in vitro cellular alcoholic steatosis model and in an animal study using wild-type and LCN2 knockout mice fed for 4 weeks with an ethanol-supplemented Lieber-DeCarli diet. In the cellular model of alcoholic steatosis, recombinant LCN2 or overexpression of LCN2 exacerbated ethanol-induced fat accumulation, whereas knocking down LCN2 prevented steatosis in hepatocytes exposed to ethanol. Consistently, removal of LCN2 partially but significantly alleviated alcoholic fatty liver injury in mice. Mechanistically, LCN2 mediates detrimental effects of ethanol in the liver via disrupted multiple signaling pathways, including aberrant nicotinamide phosphoribosyltransferase–sirtuin 1 axis, perturbed endocrine metabolic regulatory fibroblast growth factor 15/19 signaling, and impaired chaperone-mediated autophagy. Finally, compared with healthy human livers, liver samples from patients with AFLD had lower gene expression of several LCN2-regualted molecules. Our study demonstrated a pivotal and causal role of LCN2 in the development of AFLD and suggested that targeting the LCN2 could be of great value for the treatment of human AFLD.

Background Hyperhomocysteinemia plays an important role in the development of hepatic steatosis, and studies indicate that homocysteine-lowering treatment inhibits the development of fatty liver. Objective We evaluated the effects of L-serine on alcoholic fatty liver and homocysteine metabolism. Methods In a binge ethanol study, male C57BL/6 mice were divided into 4 groups: control, ethanol + vehicle, and ethanol + 20 or 200 mg/kg L-serine. Mice were gavaged with ethanol (5 g/kg body weight) 3 times every 12 h with or without L-serine which was given twice 30 min before the last 2 ethanol doses. Control mice were fed isocaloric dextran-maltose. In a chronic ethanol study, male Wistar rats were divided into 3 groups: control, ethanol, and ethanol + L-serine. Rats were fed a standard Lieber-DeCarli ethanol diet (36% ethanol-derived calories) for 4 wk with or without dietary L-serine supplementation (1%; wt:vol) for the last 2 wk. In control rats, the ethanol-derived calories were replaced with dextran-maltose. The effects of L-serine were also tested in AML12 cells manipulated to have high homocysteine concentrations by silencing the genes involved in homocysteine metabolism. Results Binge ethanol treatment increased serum homocysteine and hepatic triglyceride (TG) concentrations by >5-fold vs. controls, which were attenuated in the 200-mg/kg L-serine treatment group by 60.0% and 47.5%, respectively, compared with the ethanol group. In the chronic ethanol study, L-serine also decreased hepatic neutral lipid accumulation by 63.3% compared with the ethanol group. L-serine increased glutathione and S-adenosylmethionine by 94.0% and 30.6%, respectively, compared with the ethanol group. Silencing betaine homocysteine methyltransferase, cystathionine β-synthase, or methionine increased intracellular homocysteine and TG concentrations by >2-fold, which was reversed by L-serine when L-serine-independent betaine homocysteine methyltransferase was knocked down. Conclusion These results demonstrate that L-serine ameliorates alcoholic fatty liver by accelerating L-serine-dependent homocysteine metabolism.

PBDEs (polybrominated diphenyl ethers) are environmental pollutants that have been linked to the development of type 2 diabetes, however, the precise mechanisms are not clear. Particularly, their direct effect on insulin secretion is unknown. In this study, we show that two PBDE congeners, BDE-47 and BDE-85, potentiate glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 cells. This effect of BDE-47 and BDE-85 on GSIS was dependent on thyroid receptor (TR). Both BDE-47 and BDE-85 (10μM) activated Akt during an acute exposure. The activation of Akt by BDE-47 and BDE-85 plays a role in their potentiation of GSIS, as pharmacological inhibition of PI3K, an upstream activator of Akt, significantly lowers GSIS compared to compounds alone. This study shows that BDE-47 and BDE-85 directly act on pancreatic β-cells to stimulate GSIS, and that this effect is mediated by the thyroid receptor (TR) and Akt activation.

Collaborative regulation of liver X receptor (LXR) and sterol regulatory element binding protein (SREBP)-1 are main determinants in hepatic steatosis, as shown in both animal models and human patients. Recent studies indicate that selective intervention of overly functional LXRα in the liver shows promise in treatment of fatty liver disease. In the present study, we evaluated the effects of meso-dihydroguaiaretic acid (MDGA) on LXRα activation and its ability to attenuate fatty liver in mice. MDGA inhibited activation of the LXRα ligand-binding domain by competitively binding to the pocket for agonist T0901317 and decreased the luciferase activity in LXRE-tk-Luc-transfected cells. MDGA significantly attenuated hepatic neutral lipid accumulation in T0901317- and high fat diet (HFD)-induced fatty liver. The effect of MDGA was so potent that treatment with 1mg/kg for 2 weeks completely reversed the lipid accumulation induced by HFD feeding. MDGA reduced the expression of LXRα co-activator protein RIP140 and LXRα target gene products associated with lipogenesis in HFD-fed mice. These results demonstrate that MDGA has the potential to attenuate nonalcoholic steatosis mediated by selective inhibition of LXRα in the liver in mice.

Background & Aims Sirtuin (SIRT1) is a nicotinamide adenine dinucleotide−dependent protein deacetylase that regulates hepatic lipid metabolism by modifying histones and transcription factors. Ethanol exposure disrupts SIRT1 activity and contributes to alcoholic liver disease in rodents, but the exact pathogenic mechanism is not clear. We compared mice with liver-specific deletion of Sirt1 (Sirt1LKO) mice with their LOX littermates (controls). Methods We induced alcoholic liver injury in male Sirt1LKO and control mice, placing them on Lieber-DeCarli ethanol-containing diets for 10 days and then administering a single dose of ethanol (5 g/kg body weight) via gavage. Liver and serum samples were collected. We also measured messenger RNA levels of SIRT1, SFRS10, and lipin-1β and lipin-1α in liver samples from patients with alcoholic hepatitis and individuals without alcoholic hepatitis (controls). Results On the ethanol-containing diet, livers of Sirt1LKO mice accumulated larger amounts of hepatic lipid and expressed higher levels of inflammatory cytokines than control mice; serum of Sirt1LKO mice had increased levels of alanine aminotransferase and aspartate aminotransferase. Hepatic deletion of SIRT1 exacerbated ethanol-mediated defects in lipid metabolism, mainly by altering the function of lipin-1, a transcriptional regulator of lipid metabolism. In cultured mouse AML-12 hepatocytes, transgenic expression of SIRT1 prevented fat accumulation in response to ethanol exposure, largely by reversing the aberrations in lipin-1 signaling induced by ethanol. Liver samples from patients with alcoholic hepatitis had reduced levels of SIRT1 and a higher ratio of Lpin1β/α messenger RNAs than controls. Conclusions In mice, hepatic deletion of Sirt1 promotes steatosis, inflammation, and fibrosis in response to ethanol challenge. Ethanol-mediated impairment of hepatic SIRT1 signaling via lipin-1 contributes to development of alcoholic steatosis and inflammation. Reagents designed to increase SIRT1 regulation of lipin-1 can be developed to treat patients with alcoholic fatty liver disease.

Lipin-1 regulates lipid metabolism by way of its function as an enzyme in the triglyceride synthesis pathway and as a transcriptional coregulatory protein and is highly up-regulated in alcoholic fatty liver disease. In the present study, using a liver-specific lipin-1-deficient (lipin-1LKO) mouse model, we aimed to investigate the functional role of lipin-1 in the development of alcoholic steatohepatitis and explore the underlying mechanisms. Alcoholic liver injury was achieved by pair feeding wild-type and lipin-1LKO mice with modified Lieber-DeCarli ethanol-containing low-fat diets for 4 weeks. Surprisingly, chronically ethanol-fed lipin-1LKO mice showed markedly greater hepatic triglyceride and cholesterol accumulation, and augmented elevation of serum liver enzymes accompanied by increased hepatic proinflammatory cytokine expression. Our studies further revealed that hepatic removal of lipin-1 in mice augmented ethanol-induced impairment of hepatic fatty acid oxidation and lipoprotein production, likely by way of deactivation of peroxisome proliferator-activated receptor γ coactivator-1alpha, a prominent transcriptional regulator of lipid metabolism. Conclusions: Liver-specific lipin-1 deficiency in mice exacerbates the development and progression of experimental alcohol-induced steatohepatitis. Pharmacological or nutritional modulation of hepatic lipin-1 may be beneficial for the prevention or treatment of human alcoholic fatty liver disease. (Hepatology 2013; 58:1953–1963)

Alcohol consumption synergistically increases the risk and severity of liver damage in obese patients. To gain insight into cellular or molecular mechanisms underlying the development of fatty liver caused by ethanol-obesity synergism, we have carried out animal experiments that examine the effects of ethanol administration in genetically obese mice. Lean wild-type (WT) and obese (ob/ob) mice were subjected to ethanol feeding for 4 wk using a modified Lieber-DeCarli diet. After ethanol feeding, the ob/ob mice displayed much more pronounced changes in terms of liver steatosis and elevated plasma levels of alanine aminotransferase and aspartate aminotransferase, indicators of liver injury, compared with control mice. Mechanistic studies showed that ethanol feeding augmented the impairment of hepatic sirtuin 1 (SIRT1)-AMP-activated kinase (AMPK) signaling in the ob/ob mice. Moreover, the impairment of SIRT1-AMPK signaling was closely associated with altered hepatic functional activity of peroxisome proliferator-activated receptor γ coactivator-α and lipin-1, two vital downstream lipid regulators, which ultimately contributed to aggravated fatty liver observed in ethanol-fed ob/ob mice. Taken together, our novel findings suggest that ethanol administration to obese mice exacerbates fatty liver via impairment of the hepatic lipid metabolism pathways mediated largely by a central signaling system, the SIRT1-AMPK axis.

Human cadmium (Cd) exposure is associated with cancers of the lung and kidney. Using cDNA microarray analysis, we have recently reported that the expression of E2F1 is reduced by Cd in human lung fibroblasts, indicating the possibility of G1-phase arrest. To test this hypothesis, we investigated the effects of Cd on the cyclin-dependent kinase (CDK2) and retinoblastoma protein (Rb) regulatory pathways in WI38 human lung fibroblasts. We demonstrate here that G1-phase accumulation was induced by Cd in WI38 (wild-type for p53 and Rb), but not in the SV40 large T antigen-transformed variant WI38-VA13 (p53- and Rb-defective). Cd-induced cell-cycle arrest was associated with a decrease in CDK2 protein and with increase in p21 expression and p53 phosphorylation. Cd treatment caused a distinct increase in the formation of p21-cyclin E-CDK2 complex, as revealed by immunoprecipitation. The level of Rb-E2F1 complexes was increased, and the translocation of E2F1 to the nucleus was decreased by Cd treatment. Consequently, the transcriptional activity of E2F1 and the expression of the E2F1 target genes were also decreased by Cd. These results clearly demonstrate that Cd-mediated G1 arrest in WI38 cells is associated with the suppression of Rb phosphorylation and with the inhibition of E2F1 transcriptional activity.

Silver nanoparticles (AgNP) are among the fastest growing product categories in the nanotechnology industry. Despite the importance of AgNP in consumer products and clinical applications, relatively little is known regarding AgNP toxicity and its associated risks. We investigated the effects of AgNP on gene expression in the mouse brain using Affymetrix Mouse Genome Arrays. C57BL/6 mice were exposed to AgNP (geometric mean diameter, 22.18 ± 1.72 nm; 1.91 × 107 particles/cm3) for 6 h/day, 5 days/week using the nose-only exposure system for 2 weeks. Total RNA isolated from the cerebrum and cerebellum was subjected to hybridization. From over 39,000 probe sets, 468 genes in the cerebrum and 952 genes in the cerebellum were identified as AgNP-responsive (one-way analysis of variance; p < 0.05). The largest groups of gene products affected by AgNP exposure included 73 genes in the cerebrum and 144 genes in the cerebellum. AgNP exposure modulated the expression of several genes associated with motor neuron disorders, neurodegenerative disease, and immune cell function, indicating potential neurotoxicity and immunotoxicity associated with AgNP exposure. Real-time PCR data for five genes analyzed from whole blood showed good correlation with the observed changes in the brain. Following rigorous validation and substantiation, these genes may assist in the development of surrogate markers for AgNP exposure and/or toxicity.

Tanshinone IIA is one of the most abundant constituents of the root of Salvia miltiorrhiza BUNGE which exerts antioxidant and anti-inflammatory actions in many experimental disease models. In the present study, we demonstrated that the standardized fraction of S. miltiorrhiza (Sm-SF) was able to protect RAW 264.7 cells from ethanol-and lipopolysaccharide (LPS)-induced production of superoxide radical, activation of NADPH oxidase and subsequently death of the cells. Among four main components of Sm-SF, tanshinone IIA was the most potent in protecting cells from LPS-and ethanol-induced cytotoxicity. LPS or ethanol induced the expression of CD14, iNOS, and SCD1 and decreased RXR-alpha, which was completely reversed by tanshinone IIA. In H4IIEC3 cells, 10 microM tanshinone IIA effectively blocked ethanol-induced fat accumulation as evidenced by Nile Red binding assay. These results indicate that tanshinone IIA may have potential to inhibit alcoholic liver disease by reducing LPS-and ethanol-induced Kupffer cell sensitization, inhibiting synthesis of reactive oxygen/nitrogen species, inhibiting fatty acid synthesis and stimulating fatty acid oxidation.

Ethanol induces cumulative liver damage including steatosis, steatohepatitis and cirrhosis. The aim of this study is to investigate the global intrahepatic gene expression profile in the mouse liver treated with ethanol. A single oral dose of 0.5 or 5 g/kg ethanol was administered to male ICR mice, and liver samples were obtained after 6, 24 and 72 h. Histopathological evaluation showed typical fatty livers in the high-dose group at 24 h. Microarray analysis identified 28 genes as being ethanol responsive (two-way ANOVA; p0.05), after adjustment by the Benjamini-Hochberg multiple testing correction; these genes displayedor=2-fold induction or repression. The expression of genes that are known to be involved in fatty acid synthesis was examined. The transcript for lipogenic transcription factor, sterol regulatory element (SRE)-binding factor 1 (Srebf1), was upregulated by acute ethanol exposure. Of the genes known to contain SRE or SRE-like sequences and to be regulated by SRE-binding protein 1 (SREBP1), those encoding malic enzyme (Mod1), ATP-citrate lyase (Acly), fatty acid synthase (Fasn) and stearyl-CoA desaturase (Scd1) were induced by ethanol. Quantitative real-time PCR confirmed the changes in the expression levels of the selected genes. The change in the Srebf1 mRNA level correlates well with that of the SREBP1 protein expression as well as its binding to the promoters of the target genes. The present study identifies differentially expressed genes that can be applied to the biomarkers for alcohol-binge-induced fatty liver. These results support the hypothesis by which ethanol-induced steatosis in mice is mediated by the fatty acid synthetic pathway regulated by SREBP1.

Tetracycline is one of a group of drugs known to induce microvesicular steatosis. In the present study, we investigated the effects of tetracycline on gene expression in mouse liver, using Applied Biosystems Mouse Genome Survey Microarrays. A single oral dose of 0.1 or 1 g/kg tetracycline was administered to male ICR mice, and liver samples were obtained after 6, 24, or 72 h. Histopathological evaluation showed microvesicular steatosis in the high-dose group at 24 h. In total, 96 genes were identified as tetracycline responsive. Their level of expression differed significantly from controls (two-way analysis of variance; p0.05), after adjustment by the Benjamini-Hochberg multiple testing correction, and displayed a twofold or greater induction or repression. The largest groups of gene products affected by tetracycline exposure were those involved in signal transduction, nucleic acid metabolism, developmental processes, and protein metabolism. The expression of genes known to be involved in lipid metabolism was examined, using two-sample Student's t-test for each treatment group versus a corresponding control group. The overall net effects on expression of lipid metabolism genes indicated an increase in cholesterol and triglyceride biosynthesis and a decrease in beta-oxidation of fatty acids. Our data support a proposed mechanism for tetracycline-induced steatogenic hepatotoxicity that involves these processes. Moreover, we demonstrated global changes in hepatic gene expression following tetracycline exposure; many of these genes have the potential to be used as biomarkers of exposure to steatogenic hepatotoxic agents.

We have found in the previous study that 6-methoxydihydrosanguinarine (6ME), a benzophenanthridine alkaloid isolated from Hylomecon species, may have potential as a chemotherapeutic agent. However, the mechanisms of 6ME-induced cell death have not been investigated. The purpose of the present study was to determine the apoptosis-inducing potential of 6ME in human hepatocarcinoma HepG2 cells and the role of reactive oxygen species in 6ME-induced apoptosis. It can be concluded from the results that 6ME inhibits the growth of HepG2 cells in a concentration- and time-dependent manner (IC 50 =3.8 ± 0.2 μM following 6 h incubation). Treatment of HepG2 cells with 6ME resulted in the release of mitochondrial cytochrome c followed by the activation of caspase proteases, and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. 6ME increased the expression of p53 and bax and decreased the expression of bcl-2. The cytotoxic effect of 6ME is mediated by the time-dependent generation of reactive oxygen species. Our results also show that preincubation of HepG2 cells with vitamin C decreased the expression of p53 and bax and inhibited the release of cytochrome c , activation of downstream caspase and the cleavage of poly(ADP-ribose) polymerase, thus inhibiting the apoptosis inducing effect of 6ME.

6-methoxydihydrosanguinarine (6ME), a benzophenanthridine alkaloid derived from the methanol extracts of Hylomecon hylomeconoides, showed a dose-dependent effect at 1-10 microM on causing apoptotic cell death in HT29 colon carcinoma cells (IC50 = 5.0+/-0.2 microM). Treatment of HT-29 cells with 6ME resulted in the formation of internucleosomal DNA fragmentation. Treatment of the cells with 6ME caused activation of caspase-3, -8 and 9 protease and subsequent proteolytic cleavage of poly(ADP-ribose)polymerase. 6ME increased the expression of p53 and Bax and decreased the expression of Bid. These results indicate that p53 and proapoptotic Bcl-2 family proteins might participate in the antiproliferative activity of 6ME in HT29 cells.

The effect of an extract of Dalbergiae Lignum and four components that were isolated from the extract on the anticarcinogenic phase II marker enzyme, quinone reductase (QR), was investigated. Of the solvent extracts of Dalbergiae Lignum, the CH2Cl2 fraction was the most potent in inducing QR activity, with a CD value (the concentration required to double the QR activity) of 29.5 microg/mL. The CH2Cl2 extract was further separated into six compounds, four of which were identified as 4-methoxydalbergione, latifolin, 4',6-dihydroxy-7-methoxyflavanone, and obtusafuran. Obtusafuran [CD = 1.1 microM; chemopreventive index (CI) = 101.9] and latifolin (CD = 1.7 microM; CI = 154.6) displayed potent QR inducing activity and high chemopreventive indices. Latifolin and 4-methoxydalbergione were identified as strong DPPH-scavengers with half-maximal free radical scavenging concentrations of 15.9 and 17.2 microM, respectively.

Ethanol-mediated injury, combined with gut-derived lipopolysaccharide (LPS), provokes generation of proinflammatory cytokines in Kupffer cells, causing hepatic inflammation. Among the mediators of these effects, miR-217 aggravates ethanol-induced steatosis in hepatocytes. However, the role of miR-217 in ethanol-induced liver inflammation process is unknown. Here, we examined the role of miR-217 in the responses to ethanol, LPS, or a combination of ethanol and LPS in RAW 264.7 macrophages and in primary Kupffer cells. In macrophages, ethanol substantially exacerbated LPS-mediated induction of miR-217 and production of proinflammatory cytokines compared with LPS or ethanol alone. Consistently, ethanol administration to mice led to increases in miR-217 abundance and increased production of inflammatory cytokines in isolated primary Kupffer cells exposed to the combination of ethanol and LPS. miR-217 promoted combined ethanol and LPS-mediated inhibition of sirtuin 1 expression and activity in macrophages. Moreover, miR-217–mediated sirtuin 1 inhibition was accompanied by increased activities of two vital inflammatory regulators, NF-κB and the nuclear factor of activated T cells c4. Finally, adenovirus-mediated overexpression of miR-217 led to steatosis and inflammation in mice. These findings suggest that miR-217 is a pivotal regulator involved in ethanol-induced hepatic inflammation. Strategies to inhibit hepatic miR-217 could be a viable approach in attenuating alcoholic hepatitis.

Ethanol-mediated inhibition of hepatic sirtuin 1 (SIRT1) plays a crucial role in the pathogenesis of alcoholic fatty liver disease. Here, we investigated the underlying mechanisms of this inhibition by identifying a new hepatic target of ethanol action, microRNA-217 (miR-217). The role of miR-217 in the regulation of the effects of ethanol was investigated in cultured mouse AML-12 hepatocytes and in the livers of chronically ethanol-fed mice. In AML-12 hepatocytes and in mouse livers, chronic ethanol exposure drastically and specifically induced miR-217 levels and caused excess fat accumulation. Further studies revealed that overexpression of miR-217 in AML-12 cells promoted ethanol-mediated impairments of SIRT1 and SIRT1-regulated genes encoding lipogenic or fatty acid oxidation enzymes. More importantly, miR-217 impairs functions of lipin-1, a vital lipid regulator, in hepatocytes. Taken together, our novel findings suggest that miR-217 is a specific target of ethanol action in the liver and may present as a potential therapeutic target for treating human alcoholic fatty liver disease.

The generally accepted hypothesis for the pathogenesis of alcoholic liver disease (ALD) is the two-hit model, which proposes that fat accumulation in the liver increases the sensitivity of the liver to a second hit that leads to inflammatory liver cell damage. In this study we evaluated the effects of Magnolia officinalis (MO), which contains honokiol and magnolol as the primary pharmacological components, to eradicate fatty liver in rats fed an ethanol diet. In vitro studies showed that MO was able to protect RAW 264.7 cells from ethanol-induced production of tumor necrosis factor-α, reactive oxygen species, and superoxide anion radicals; the activation of NADPH oxidase; and subsequent cell death. We also investigated the therapeutic effects of MO on alcoholic fatty liver in Lieber-DeCarli ethanol diet–fed rats. MO treatment of the rats for the last 2 weeks of ethanol feeding completely reversed all the serum, hepatic parameters, and fatty liver changes. The increased maturation of sterol regulatory element– binding protein-1c in the liver by ethanol treatment was completely inhibited by treatment with MO. Therefore, MO may be a promising candidate for development as a therapeutic agent for ALD.[Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.08182FP] Keywords:: Magnolia officinalis, alcoholic fatty liver, tumor necrosis factor-α (TNF-α), reactive oxygen species, sterol regulatory element–binding protein (SREBP)

Chronic consumption of ethanol can cause cumulative liver damage that can ultimately lead to cirrhosis. To explore the mechanisms of alcoholic steatosis, we investigated the global intrahepatic gene expression profiles of livers from mice administered alcohol. Ethanol was administered by feeding the standard Lieber-DeCarli diet, of which 36% (high dose) and 3.6% (low dose) of the total calories were supplied from ethanol for 1, 2, or 4 weeks. Histopathological evaluation of the liver samples revealed fatty changes and punctate necrosis in the high-dose group and ballooning degeneration in the low-dose group. In total, 292 genes were identified as ethanol responsive, and several of these differed significantly in expression compared to those of control mice (two-way ANOVA; p

Alcoholic liver disease involves hepatocellular injury induced by the acute or chronic consumption of ethanol. Fatty infiltration is usually followed by inflammation and focal necrosis, which can lead to cirrhosis if not treated properly in the initial stage. There have been many attempts to develop effective therapies for the disease, using natural products derived from medicinal plants. In this study, we report that the standardized fraction of Salvia miltiorrhiza Bunge (Sm-SF) and its active component, cryptotanshinone, were able to protect hepatocytes from lipopolysaccharide- and ethanol-induced cell death. They also suppressed ethanol-induced lipid accumulation as evidenced by the Nile red binding assay. The ethanol-induced activation and nuclear translocation of sterol regulatory element-binding protein-1 and the consequent transactivation of the target genes involved in fatty acid biosynthesis were inhibited by Sm-SF and cryptotanshinone in a dose-dependent manner. Cryptotanshinone, an active component of S. miltiorrhiza, has the potential to ameliorate alcoholic liver disease by blocking hepatic cell death and fatty acid synthesis.

Chronic exposure to ethanol induces cumulative damage to the liver starting from fatty infiltration to cirrhosis depending on the dose and duration of exposure. The whole process leading to the development of alcoholic liver disease is very complex and the mechanisms involved are not fully understood. Among many experimental animal models, Lieber-DeCarli liquid diet provides moderate to severe pathophysiological outcome depending on the compositional changes. In the present study, we investigated the temporal changes in the early phase hepatic disease in rats fed with standard Lieber-DeCarli diet. Male Wistar rats were fed with Lieber-Decarli ethanol diet for 6 weeks and the liver samples were obtained after 2, 4 and 6 weeks. Mild fatty infiltration was observed in 2 weeks of feeding and it became evident in 4 and 6 week samples. The level of hepatic triglyceride showed a good agreement with the data obtained in the pathological analysis. Feeding mice with ethanol diet resulted in the maturation and translocation of SREBP-1 to nucleus in the liver. Western blot analysis of the pooled liver sample of control and ethanol fed animals showed a clear-cut time-dependent increase in the expression of nSREBP-1. These data provide important information for selecting proper time point in experimental intervention study in the field of drug development for alcoholic liver disease.

This research team found in previous studies, that the ginseng saponin metabolite IH901 induces apoptosis in HepG2 cells via a mitochondrial-mediated pathway, which resulted in the activation of caspase-9 and subsequently of caspase-3 and -8. Based on these results, the involvement of the Fas/Fas ligand (FasL) death-receptor pathway, in IH901-induced apoptosis in HepG2 cells, was investigated. Levels of Fas and the Fas ligand (FasL) mRNA or protein were not increased by IH901, rather they were decreased significantly at 18 h post treatment. Soluble FasL (sFasL) was detectable by immunoprecipitation analysis in the medium of HepG2 cells treated with IH901. Increased levels of sFasL were inversely correlated with the levels of FasL. Preincubation of HepG2 cells with antagonistic anti-Fas antibody showed little protective effect, if any, on IH901-induced cell death. At a 30 microM (24 and 48 h) and 40 microM (24 h) concentration of IH901, the cytotoxic effect of IH901 was less then 50%, anti-Fas antibody prevented IH901-induced cell death. However, at a 60 microM (24 and 48 h) and 40 microM (48 h) concentration of IH901, cell death rates were about 80% or more and most of the chemopreventive and chemotherapeutic effects of IH901 were manifested. Blocking the Fas receptor did not influence IH901-induced cell death. These results indicate that the Fas/FasL system is engaged, but not required for IH901-induced cell death, at pharmacologically significant concentrations.