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Linarin, a flavone glycoside, is considered to be a promising natural product due to its diverse pharmacological activities. Recently, it has been brought into focus for its potential to treat liver failure. In this study, a rapid and sensitive liquid chromatography electrospray-ionization tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of linarin and its three metabolites (acacetin, apigenin, and p -hydroxy benzaldehyde) in plasma and liver tissue samples of normal rats and rats with d-galactosamine (d-GalN)-induced liver injury. After liquid-liquid extraction (LLE) with ethyl acetate, chromatographic separation of the four analytes was achieved using an ACQUITY UPLC BEH-C18 (1.7 μm, 2.1 × 50 mm) with a mobile phase of 0.01% formic acid in methanol and 0.01% formic acid at a flow rate of 0.3 mL/min. The detection was accomplished on a tandem mass spectrometer via an electrospray ionization (ESI) source by multiple reaction monitoring (MRM) in the negative ionization mode. The method had a good linearity over the concentration range of 1.00-200 ng/mL for linarin and its metabolites. The validated method was successfully applied to the pharmacokinetic and liver tissue distribution study of linarin and its metabolites after a single oral administration of linarin (90 mg/kg) to rats.

Geraniol (GOH), a natural component of plant essential oils, exhibits potent antioxidant and anti-inflammatory properties. The aim of this study was to assess the protective effects and mechanisms of GOH on lipopolysaccharide (LPS)/d-galactosamine (D-GalN)-induced fulminant hepatic failure (FHF). Mice were treated with GOH (12.5, 25, and 50 μg/kg) 1 h before challenging LPS (60 mg/kg) and D-GalN (800 mg/kg). 8 h later LPS/D-GlaN treatment, mice were sacrificed and the serum and the liver tissues were collected for testing. The liver pathological changes were assessed by H & E staining. MPO activity, MDA level in liver tissues, and AST, ALT levels in serum were detected by specific detection kits. The levels of TNF-α and IL-1β were detected by ELISA. The expression of NF-κB and PPARγ were detected by western blot analysis and qRT-PCR. The results showed that GOH had a protective effect on LPS/D-GalN-induced FHF, as evidence by the attenuation of liver pathological injury, MPO activity, MDA level, and serum AST and ALT levels. GOH reduced liver TNF-α and IL-1β levels through inhibiting NF-κB signaling pathway activation. Furthermore, GOH increased PPARγ expression in FHF induced by LPS/D-GalN. In conclusion, the present study proved that GOH protects against LPS/D-GalN-induced FHF through inhibiting inflammatory response and increasing PPARγ expression., (Copyright © 2018. Published by Elsevier Ltd.)

Alpinetin, a type of novel plant flavonoid derived from Alpinia katsumadai Hayata, has been reported to have anti-inflammatory effects. The aim of this investigation was designed to reveal the protective effects of alpinetin on Lipopolysaccharide (LPS)/d-galactosamine (D-Gal)-induced liver injury in mice. Alpinetin (12.5, 25, 50 mg/kg) were given 1 h before LPS and D-Gal treatment. 12 h after LPS and D-Gal treatment, the liver tissues and serum were collected. Our results showed that alpinetin treatment improved liver histology, indicating a marked decrease of inflammatory cell infiltration and restore hepatic lobular architecture. Alpinetin also inhibited liver myeloperoxidase (MPO) activity and malondialdehyde (MDA) level. Furthermore, LPS/D-Gal-induced tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β) production were dose-dependently inhibited by alpinetin. Alpinetin also attenuated LPS/D-Gal-induced expression of phospho-NF-κB p65 and phospho-IκBα. In addition, alpinetin was found to increase the expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). In conclusion, these findings suggested that alpinetin inhibited liver injury through inhibiting NF-κB and activating the Nrf2 signaling pathway., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study has evaluated the effects of a supplementation with taurine (TAU) on the actions of fish oil (FO) against the hypoglycemia, hypoproteinemia, and hepatic accumulation of lipids and liver damage caused by D-galactosamine (GAL) in the rat. To this end, male Sprague-Dawley rats (200-225 g), in groups of 6, were orally treated with physiological saline (2.5 mL, control group), FO (60 mg/kg), TAU (2.4 mmol/kg) or FO-TAU for three consecutive days and before a single oral dose of GAL (400 mg/kg) given on day 3. In parallel, rats receiving only GAL on day 3 or N-acetylcysteine (NAC, 2.4 mmol/kg) for 3 days before GAL served as controls. On day 4 blood samples were collected by cardiac puncture and used to either measure glucose (GLC) or to obtain plasma fractions. Immediately thereafter, the livers were excised, made into a homogenate in phosphate buffered saline pH 7.4, and centrifuged to obtain clear supernatant. Plasma samples were assayed for their total protein (TP), triglycerides (TG), cholesterol (CHOL), phospholipids (PLP), free fatty acids (FFA) and total bilirubin (TB) and direct bilirubin (DB) contents, and for the activities of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP). The liver homogenates were used to measure TG, CHOL, PLP and total lipids (TL) contents. Without exceptions, GAL was found to markedly affect (p < 0.001) all of the experimental parameters examined, with increases occurring in all instances except for the values of the plasma GLC, TP and PLP which were decreased. A pretreatment with either FO or TAU led to significant attenuation of the effects of GAL and which, in most cases, were of similar magnitude. On the other hand, a combined pretreatment with FO plus TAU usually resulted in a greater protection than with either agent alone (p ≤ 0.05). NAC, serving as a reference treatment, was, in most instances, equipotent with FO alone and. in addition, was the only agent that significantly attenuated the increases in both liver weight and liver weight to body weight ratio caused by GAL.

Acute liver failure (ALF), an inflammation-mediated hepatocellular injury process, is a life-threatening and fatal clinical syndrome. Daphnetin (Daph) has been reported to exhibit various pharmacological activities, particularly its antiinflammatory property. The present study was designed to evaluate the protective effects and underlying mechanisms of Daph against lipopolysaccharide and d-galactosamine (LPS/GalN)-induced ALF in mice. Our findings suggested that Daph treatment efficiently protected against LPS/GalN-induced ALF by lessening the lethality, decreasing the alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 secretion, malondialdehyde (MDA) formation and myeloperoxidase (MPO) level, inhibiting nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) protein expression, and increasing the glutathione (GSH) and superoxide dismutase (SOD) contents. Moreover, Daph treatment effectively inhibited LPS/GalN-induced nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathway activation, and significantly induced autophagy activation by enhancing the level of pro-autophagy proteins expression. Taken together, these findings suggested that Daph plays a pivotal role in liver protection by suppressing inflammatory responses which may be closely associated with the inhibition of NLRP3 inflammasome, MAPK and NF-κB activation, as well as the induction of autophagy., (Copyright © 2018. Published by Elsevier B.V.)

It has been described that Humulus japonicus has potential antituberculosis and anti-inflammatory effects. The antiaging activity of H. japonicus extract (HJE) was also examined not only in yeast but also in human fibroblast cells. We evaluated the protective effect of HJE on hepatotoxicity in this study. We demonstrated the expression of antioxidative enzyme and cytokines in plasma. The vehicle control group received orally administered normal saline. Acute hepatotoxicity rat model was induced by lipopolysaccharide (LPS) (30 μg/kg) and d-galactosamine (D-GalN) (500 mg/kg) by intraperitoneal injection. The positive control group received orally administered silymarin (10 mg/kg). Three HJE groups received 8, 16, and 32 mg/kg. The blood samples were prepared to evaluate malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) level to examine oxidative stress, and hepatic tissue was assayed for myeloperoxidase (MPO). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and tumor necrosis factor-α (TNF-α) activities were also assayed to examine liver cell viability. Pretreatment with HJE decreased levels of AST, ALT, MDA, MPO, and TNF-α and increased levels of SOD and CAT compared with the LPS/D-GalN-treated group. These results suggested that HJE has the potential to reduce oxidative damage and inflammatory reactions in LPS/D-GalN-induced liver injury in the rat model. It can also increase survival rate in LPS/D-GalN-induced hepatotoxicity rat models.

It is reported that sesquiterpenoids from Panax ginseng (SPG) possess various pharmacological activities, for example, antidepressant, antioxidative, and anti-inflammatory activities. The purpose of this study was to examine the hepatoprotective effects of SPG (2.5 and 10 mg/kg, i.g.) on fulminant liver injury induced by d-galactosamine (d-GalN) and lipopolysaccharide (LPS) and discuss its mechanisms of action. After 24 h of d-GalN (400 mg/kg, i.p.) and LPS (25 μg/kg, i.p.) exposure, the serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), hepatic malondialdehyde (MDA) level, hepatic activities of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), and hepatic tissue histology were measured. Expression levels of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Moreover, the nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), sirtuin type 1 (Sirt 1), and heme oxygenase 1 (HO-1) were determined by western blotting. The results indicated that SPG evidently restrained the increase of serum ALT and AST levels induced by d-GalN/LPS. SPG obviously downregulated TNF-α and IL-1β levels and their mRNA expression in liver. In addition, d-GalN/LPS injection induced severe oxidative stress in liver by boosting the MDA level as well as decreasing CAT, GSH, and SOD capacities, and SPG reversed these changes. Meanwhile, SPG inhibited NF-κB activation induced by d-GalN/LPS and upregulated Sirt 1, Nrf2, and HO-1 expression levels. Therefore, SPG might protect against the fulminant liver injury induced by d-GalN/LPS via inhibiting inflammation and oxidative stress. The protective effect of SPG on fulminant liver injury induced by d-GalN/LPS might be mediated by the Sirt 1/Nrf2/NF-κB signaling pathway. All of these results implied that SPG might be a promising food additive and therapeutic agent for fulminant liver injury.

Acute liver injury is a life-threatening syndrome that often caused by hepatocyte damage. In this study, we investigated the protective effects of maslinic acid (MA) on lipopolysaccharide (LPS)/d-galactosamine (D-gal)-induced acute liver injury and clarified its mechanism. Mice acute liver injury model was induced by given LPS and D-gal and MA was given intraperitoneally 1 h before LPS and D-gal. Our results showed that MA protected against liver injury by attenuating liver histopathologic changes, serum AST and ALT levels. The increased inflammatory cytokines TNF-α and IL-6 in serum and liver tissues were also inhibited by MA. The level of MDA and the activity of MPO in liver tissues were up-regulated by LPS/D-gal and dose-dependently inhibited by MA. Furthermore, MA attenuated hepatic NF-κB protein expression and increased hepatic Nrf2 and HO-1 protein expression. Taken together, MA offers a protective role against LPS/D-gal-induced liver injury through suppressing NF-κB and activating Nrf2 signaling pathways., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the protective effects and mechanism of isovitexin, a glycosylflavonoid isolated from rice hulls of Oryza sativa, on Lipopolysaccharide (LPS)/d-galactosamine (D-Gal)-induced acute liver injury. The mice were randomly divided into five groups: control group, LPS/D-Gal group, and LPS/D-Gal + isovitexin groups. The mice of LPS/D-Gal group were received of LPS (50 μg/kg) and D-gal (800 mg/kg) intraperitoneal. The mice of LPS/D-Gal + isovitexin groups were received isovitexin (25, 50, 100 mg/kg) 1 h before LPS/D-Gal treatment. The results showed that the severity of liver injury was attenuated by treatment of isovitexin, as confirmed by the decreased liver histopathologic changes, as well as serum AST and ALT levels. Furthermore, the levels of TNF-α in serum and liver tissues, MPO activity and MDA content were significantly inhibited by isovitexin. In addition, isovitexin significantly attenuated NF-κB phosphorylation induced by LPS/D-Gal. The expression of Nrf2 and HO-1 were significantly up-regulated by isovitexin. In conclusion, isovitexin could protect against LPS/D-Gal-induced liver injury by inhibiting inflammatory and oxidative responses. Isovitexin also had protective effects against carbon tetrachloride (CCl 4 )-induced liver injury. Isovitexin may used as a potential agent for the treatment of liver injury., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Tenuigenin (TEN), a major active component of polygala tenuifolia root, has been reported to have a number of biological properties, such as anti-oxidative and anti-inflammatory activities. However, the protective effect of TEN on acute liver injury has not yet been reported. This research aims to detect the protective effect of TEN on lipopolysaccharide (LPS) and d-galactosamine (D-GalN)-induced acute liver injury in mice and to investigate the molecular mechanisms. TEN was administered intraperitoneally 1 h before LPS/D-GalN treatment. The levels of TNF-α, IL-1β, ALT, and AST were measured. The expression of NF-κB, ASK1, MAPKs, Nrf2, and HO-1 were detected by western blot analysis. The results showed that TEN significantly inhibited LPS/D-GalN-induced serum ALT and AST levels. TEN also inhibited LPS/D-GalN-induced TNF-α and IL-1β production. Furthermore, LPS/D-GalN-induced hepatic MDA and MPO activities were also inhibited by TEN. In addition, TEN was found to inhibit LPS/D-GalN-induced ASK1 expression, NF-κB and MAPKs activation and up-regulate the expression of Nrf2 and HO-1. In conclusion, TEN protected against LPS/GalN-induced acute liver injury by suppressing inflammatory and oxidative responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The gut microbiota is altered in liver diseases, and several probiotics have been shown to reduce the degree of liver damage. We hypothesized that oral administration of specific Bifidobacterium strains isolated from healthy guts could attenuate liver injury. Five strains were tested in this study. Acute liver injury was induced by D-galactosamine after pretreating Sprague-Dawley rats with the Bifidobacterium strains, and liver function, liver and ileum histology, plasma cytokines, bacterial translocation and the gut microbiome were assessed. Two strains, Bifidobacterium pseudocatenulatum LI09 and Bifidobacterium catenulatum LI10, conferred liver protection, as well as alleviated the increase in plasma M-CSF, MIP-1α and MCP-1 and bacterial translocation. They also ameliorated ileal mucosal injury and gut flora dysbiosis, especially the enrichment of the opportunistic pathogen Parasutterella and the depletion of the SCFA-producing bacteria Anaerostipes, Coprococcus and Clostridium XI. Negative correlations were found between MIP-1α / MCP-1 and Odoribacter (LI09 group) and MIP-1α / M-CSF and Flavonifractor (LI10 group). Our results indicate that the liver protection effects might be mediated through gut microbiota modification, which thus affect the host immune profile. The desirable characteristics of these two strains may enable them to serve as potential probiotics for the prevention or adjuvant treatment of liver injury.

Nymphaea candida was used to treat hepatitis in Ugyhur medicine, and nicotiflorin (kaempferol 3-O-β-rutinoside) is the main characteristic component in this plant [...].

Targeting myeloid differentiation protein 2 (MD-2) or Toll-like receptor 4 (TLR4) with small molecule inhibitor rescues the systemic inflammatory response syndrome (SIRS) in sepsis due to infection with Gram-negative bacteria but not other microbes. Herein, we provided IκB kinase β (IKKβ) in innate immune process as a molecular target of caffeic acid cyclohexylamide (CGA-JK3) in the treatment of polymicrobial TLR agonists-induced lethal inflammation. CGA-JK3 ameliorated E. coli lipopolysaccharide (LPS, MD-2/TLR4 agonist)-induced endotoxic shock, cecal ligation and puncture (CLP)-challenged septic shock or LPS plus D-galactosamine (GalN)-induced acute liver failure (ALF) in C57BL/6J mice. As a molecular basis, CGA-JK3 inhibited IKKβ-catalyzed kinase activity in a competitive mechanism with respect to ATP, displaced fluorescent ATP probe from the complex with IKKβ, and docked at the ATP-binding active site on the crystal structure of human IKKβ. Furthermore, CGA-JK3 inhibited IKKβ-catalyzed IκB phosphorylation, which is an axis leading to IκB degradation in the activating pathway of nuclear factor-κB (NF-κB), in macrophages stimulated with TLR (1/2, 2/6, 4, 5, 7, 9) agonists from Gram-positive/negative bacteria and viruses. CGA-JK3 consequently interrupted IKKβ-inducible NF-κB activation and NF-κB-regulated expression of TNF-α, IL-1α or HMGB-1 gene, thereby improving TLRs-associated redundant inflammatory responses in endotoxemia, polymicrobial sepsis and ALF., Competing Interests: The authors declare no competing financial interests.

Objective: To investigate the clinical effect of astragaloside IV in the early treatment of mice with acute liver failure and possible mechanisms. Methods: A mouse model of acute liver failure induced by D-galactosamine/lipopolysaccharide (D-GalN/LPS) was established, and the mice were given astragaloside IV at different doses. The survival rate of mice, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver histopathological changes, apoptosis of hepatocytes, and the expression of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured in each group. The least significant difference test was used for data with homogeneity of variances, the Dunnett's T3 test was used for data with heterogeneity of variance, and the Kaplan-Meier method was used for survival analysis. Results: Compared with the model group, the high-dose astragaloside IV group had a significant increase in the 48-hour survival rate [60% (9/15) vs 13.3% (2/15), P < 0.05], significant reductions in the serum ALT and AST levels ( P < 0.01), and significant reductions in liver histopathological indices and the degree of apoptosis of hepatocytes ( P < 0.01), as well as a significant reduction in the content of MDA in liver homogenate ( P < 0.01) and a significant increase in the activity of SOD ( P < 0.05). Conclusion: High-dose astragaloside IV has a significant protective effect against D-GalN/LPS-induced acute liver injury in mice, and its mechanisms may be associated with its effects against cell apoptosis and oxidative damage.

Adiponectin is an antidiabetic and antiatherogenic adipokine, which plays distinct roles in the balance of energy homoeostasis. As an insulin sensitizing hormone, adiponectin exerts multiple biological effects by the specific receptors (AdipoR1 and AdipoR2), through activation of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR)α pathways. AdipoRon, an orally active synthetic small-molecule AdipoR agonist, shows very similar effects to adiponectin in vitro and in vivo, which could be a promising therapeutic approach for obesity-related disorders. In view of the regulatory effects of adiponectin or AdipoRon on inflammatory response and energy metabolism, they might be endowed a curative potential for tissue damage. Hence, its effects and possible mechanism were investigated. In vitro studies on hepatocytes (L02) and macrophages (RAW264.7) suggested a protective and anti-inflammatory potential of AdipoRon. The effects were verified in acute hepatic injury mice induced by d-galactosamine (D-GalN): hepatic lesions were restored by AdipoRon or bicyclol (positive reference drug) pretreatment, which were characterized by a significant increase in serological and hepatic biomarkers (AST, ALT, MDA and NOSs). Besides, AdipoRon attenuated the inflammation in the liver, characterized by the dwindling proinflammatory macrophage infiltration, as well as the shrinkage of tumor necrosis factor-α (TNF-α), transforming growth factor beta 1 (TGF-β1), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6); meanwhile conversely promoted AMPK activation by phosphorylation. Combined with liver histopathology, these results demonstrated the hepatoprotective effects of AdipoRon against D-GalN-induced damage, which might be ascribed to the attenuation of inflammation, inhibition of free radical reactions, as well as enhancement of liver energy metabolism., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: To investigate the therapeutic effect of bone marrow mesenchymal stromal cell (BMSC) transplantation on D-galactosamine-induced acute liver failure in Sprague-Dawley (SD) rats, as well as the mechanism of neutrophils in this process. Methods: A total of 39 male SD rats were divided into control group (8 rats, intraperitoneal injection of isotonic saline), model group (10 rats, intraperitoneal injection of D-galactosamine), solvent group (9 rats, tail vein injection of isotonic saline at 2 hours after intraperitoneal injection of D-galactosamine), and treatment group (12 rats, tail vein injection of MSCs at 2 hours after intraperitoneal injection of D-galactosamine). The rats were sacrificed at 24 hours after the model of D-galactosamine-induced acute liver failure was established, and the blood and liver tissue were harvested. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and total bilirubin (TBil) were measured, and blood analysis was performed to measure the number and percentage of neutrophils in peripheral blood. Immunofluorescence assay was used to measure the expression of the neutrophil marker Ly6g in the liver, the myeloperoxidase (MPO) kit was used to measure the activity of MPO in liver, and RT-PCR was used to measure the mRNA expression of inflammatory cytokines and chemokines in the liver, i.e., tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interferon-γ(IFN-γ), interleukin-10 (IL-10), CXC chemokine ligand 1 (CXCL1), and CXC chemokine ligand 2 (CXCL2). Another 64 male SD rats were randomly divided into groups, and the survival rates of rats in each group were observed for 7 days. The independent samples t-test was used for comparison between any two groups (Levene homogeneity test of variance, and the corrected t-test was used for a P value of < 0.05), and the log-rank test was used for comparison of survival rates between any two groups. Results: At 24 hours after acute liver failure was induced by D-galactosamine in the SD rats, there were significant increases in the liver function parameters (ALT: 2884.1±541.0 U/L vs 45.4±11.0 U/L, P < 0.001; AST: 3634.9±755.9 U/L vs 143.9±23.7 U/L, P < 0.001; TBil: 44.4±8.4μmmol/L vs 0.9±0.2μmmol/L, P < 0.001) and the number and percentage of peripheral blood neutrophils [number: (4.7±1.1)×109 vs (1.4±0.4)×109, P < 0.001; percentage: 44.9%±8.0% vs 18.3%±4.4%, P < 0.001]. A large number of neutrophils aggregated in the liver tissue, and there were significant increases in the MPO activity (4.72±1.09 U/g vs 1.13±0.24 U/g, P < 0.001), inflammatory cytokines, and chemokines. Compared with the model group, the treatment group showed significant improvements in liver function (ALT: 1 823.9±389.2 U/L vs 2 884.1±541.0 U/L, P < 0.001; AST: 2173.0±567.3 U/L vs 3634.9±755.9 U/L, P < 0.001; TBil: 30.9±6.5μmmol/L vs 44.4±8.4μmmol/L, P < 0.001) and survival rate (50% vs 12.5%, P = 0.023). Meanwhile, the treatment group also showed significant reductions in the number and percentage of peripheral blood neutrophils [number: (3.5±1.0)×109 vs (4.7±1.1)×109, P = 0.012; percentage: 35.9%±8.9% vs 44.9%±8.0%, P = 0.021], number of neutrophils in the liver, and MPO activity (3.52±1.03 U/g vs 4.72±1.09 U/g, P = 0.040), as well as significantly inhibited expression of inflammatory cytokines and chemokines (TNF-α: 2.458±0.762 vs 3.778±1.046, P = 0.005; IL-1β: 2.498±0.547 vs 4.065 ± 0.953, P = 0.002; IFN-γ: 3.977±1.039 vs 5.418±1.255, P = 0.025; IL-10: 6.056±1.542 vs 3.368±0.952, P = 0.001; CXCL1: 7.988±1.911 vs 10.366±1.239, P = 0.010; CXCL2: 3.441±1.005 vs 4.847±1.113, P = 0.019). Conclusion: BMSC transplantation has a therapeutic effect on D-galactosamine-induced acute liver failure in rats, and this process is accompanied by reduced aggregation and activity of neutrophils in peripheral blood and liver. Inflammatory cytokines and chemokines may be involved in the mechanism of regulation of these two aspects.

Objective: To investigate the expression and role of autophagy in the progression of acute liver failure (ALF) using the mouse model of ALF induced by D-galactosamine/LPS (D-GalN/LPS). Methods: The C57BL/6 mice were used, and intraperitoneal injection of D-galactosamine (D-GalN) and lipopolysaccharide (LPS) was performed to establish the mouse model of ALF. The mice were divided into control group and 2-, 4-, and 6-hour D-GalN/LPS-induced ALF model groups. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess liver function, and the pathological changes in liver tissue were observed to evaluate the status of liver injury. Quantitative real-time PCR was used to measure the expression of autophagy-related genes, Western blot was used to measure the expression of autophagy-related proteins in liver tissue, and a fluorescence microscope was used to observe the expression of autophagosome in the progression of liver failure. A one-way ANOVA was used for comparison of means of multiple samples between any two groups (LSD-t test for data with homogeneity of variance and Games-Howell method for data with heterogeneity of variance). P < 0.05 was considered statistically significant. Results: The ALF model groups showed gradual liver impairment over the time of D-GalN/LPS stimulation. There were significant increases in ALT and AST after 4 hours; the pathological injury of liver tissue gradually aggravated over the time of D-GalN/LPS stimulation and fulfilled the criteria for ALF at 6 hours. The mRNA and protein expression of autophagy-related genes (ATG-7, ATG-5, Beclin-1, Lamp-1, and LC3a) increased in the early and medium stages of ALF (2 and 4 hours) and decreased after ALF progressed to liver failure (6 hours). As was observed via the fluorescence microscope, the 4-hour D-GalN/LPS-induced ALF model group showed the highest expression of autophagosome. Conclusion: The expression of autophagy gradually increases in the early and medium stages of ALF and decreases when ALF progresses to liver failure. Therefore, autophagy plays an important role in the pathogenesis of ALF.

Metformin is a widely‑used antidiabetic drug with hypoglycemic activity and previously described anti‑inflammatory properties. Previous studies have demonstrated that metformin attenuates endotoxic hepatitis, however the mechanisms remain unclear. Inflammation and coagulation are closely associated pathological processes, therefore the potential effects of metformin on key steps in activation of the coagulation system were further investigated in endotoxic hepatitis induced by lipopolysaccharide/D‑galactosamine (LPS/D‑Gal). The current study demonstrated that treatment with metformin significantly suppressed the upregulation of tissue factor and plasminogen activator inhibitor‑1 in LPS/D‑Gal‑exposed mice. In addition, a reduction in the expression of interleukin 6 and inhibition of nuclear translocation of nuclear factor‑κB were observed. These data indicate that the LPS/D‑Gal‑induced elevation of the stable protein level of hypoxia inducible factor 1α, the mRNA level of erythropoietin, vascular endothelial growth factor and matrix metalloproteinase‑3, and the hepatic level of lactic acid were also suppressed by metformin. The current study indicates that the suppressive effects of metformin on inflammation‑induced coagulation may be an additional mechanism underlying the hepatoprotective effects of metformin in mice with LPS/D‑Gal‑induced fulminant hepatitis.

Acute liver failure (ALF) is a potentially life-threatening disorder without any effective treatment strategies. d-Galactosamine (GalN)/lipopolysaccharide (LPS)-induced ALF is a widely used animal model to identify novel hepato-protective agents. In the present study, we investigated the potential of a cannabinoid receptor 2 (CB2) agonist, JWH-133 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran], in the amelioration of GalN/LPS-induced ALF. JWH-133 treatment protected the mice from ALF-associated mortality, mitigated alanine transaminase and proinflammatory cytokines, suppressed histopathological and apoptotic liver damage, and reduced liver infiltration of mononuclear cells (MNCs). Furthermore, JWH-133 pretreatment of M1/M2-polarized macrophages significantly increased the secretion of anti-inflammatory cytokine interleukin-10 (IL-10) in M1 macrophages and potentiated the expression of M2 markers in M2-polarized macrophages. In vivo, JWH-133 treatment also suppressed ALF-triggered expression of M1 markers in liver MNCs, while increasing the expression of M2 markers such as Arg1 and IL-10. microRNA (miR) microarray analysis revealed that JWH-133 treatment altered the expression of only a few miRs in the liver MNCs. Gene ontology analysis of the targets of miRs suggested that Toll-like receptor (TLR) signaling was among the most significantly targeted cellular pathways. Among the altered miRs, miR-145 was found to be the most significantly decreased. This finding correlated with concurrent upregulated expression of its predicted target gene, interleukin-1 receptor-associated kinase 3, a negative regulator of TLR4 signaling. Together, these data are the first to demonstrate that CB2 activation attenuates GalN/LPS-induced ALF by inducing an M1 to M2 shift in macrophages and by regulating the expression of unique miRs that target key molecules involved in the TLR4 pathway., (U.S. Government work not protected by U.S. copyright.)

Acute liver failure (ALF) is a life threatening disease for which only few treatment options exist. The molecular pathways of disease progression are not well defined, but the death receptor Fas (CD95/Apo-1) appears to play a pivotal role in hepatocyte cell death and the development of ALF. Here, we explored posttranscriptional gene silencing of Fas by RNAi to inhibit pathophysiological gene expression. For targeting Fas expression in mice, Fas siRNA was formulated with the liver-specific siRNA delivery system DBTC. Treatment of mice with DBTC/siRNA(Fas) reduced Fas expression in the liver, but not in the spleen, lung, kidney or heart. Furthermore, silencing of Fas receptor was effective in blocking or reducing several aspects of ALF when it was tested in mice exposed to galactosamine/lipopolysaccharide (G/L), a well-known model of ALF. The application of DBTC/siRNA(Fas) 48 h prior G/L exposure resulted in amelioration of hepatic perfusion, reduction of hepatocellular death and increase of survival rate. The administration of DBTC/siRNA(Fas) formulation further diminished the inflammatory response upon G/L challenge, as indicated by a marked decrease of TNFα mRNA expression. However, IL-6 plasma concentration remained unaffectedly by DBTC/siRNA(Fas) formulation. Since the specific silencing of hepatic Fas expression only partially protected from inflammation, but completely attenuated apoptotic and necrotic cell death as well as microcirculatory dysfunction, the development of therapeutic strategies with DBTC lipoplex formulations to treat ALF should be combined with anti-inflammatory strategies to reach maximal therapeutic efficacy.

Nanostructure-mediated drug delivery is known to have a potential to improve drug bioavailability, apart from fostering release deviation of drug molecules and enabling precision drug targeting. Solid lipid nanoparticles (SLNs) have drawn great deal of the attention of scientists in finding a solution to minimize pharmaceutic limitations of the drugs used. Silymarin (Sm) has so far been used for treating diverse liver and gallbladder disorders, such as cirrhosis, hepatitis, and jaundice, and for protecting the liver against poisoning from chemical and environmental toxins on account of its antihepatotoxic and antioxidative properties. The present study aims to develop a novel silymarin-loaded solid lipid nanoparticle (Sm-loaded SLN) system with enhanced bioavailability and with an ability to provide excellent hepatic protection for poorly water-soluble drugs. Based upon our investigation results with apoptotic markers, PCNA and lightmicroscopic findings, it can be concluded that Sm-loaded SLN significantly reduced D-GaIN/TNF-α-induced hepatotoxicity, which suggested improved bioactivity compared to Sm. In conclusion, Sm-loaded SLN could be a useful system for the delivery of poorly water-soluble Sm, apart from providing favourable hepatic protection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Intestinal endotoxemia-induced liver injury is a common clinical disease which leads to liver failure and death. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, could be used for suppressing autophagy in vitro and in vivo. Autophagy is an evolutionarily conserved and lysosome dependent protein degradation pathway, which participates in various physiological and pathological processes. The present study aims to explore the effect of pretreatment with wortmannin on acute liver injury and the autophagy in acute liver injury. We demonstrated that wortmannin could downregulate the expression of phosphorylated extracellular regulated protein kinase and p65, decrease the production and release of hepatic inflammatory cytokines, and then reduce hepatocytes apoptosis and necrosis. More importantly, we found that autophagy was induced to increase in LPS/D-GalN-induced acute liver injury, and pretreatment with wortmannin could effectively inhibit increased autophagy in acute liver injury. In conclusion, these results indicate that wortmannin plays a protective role in LPS/D-GalN induced hepatocytotoxity maybe by inhibiting autophagy and could be acted as a target for the treatment of acute liver injury., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Fulminant hepatic failure (FHF) is a life-threatening clinical syndrome, with liver transplantation being the only effective therapy. Sulforaphane (SFN) is a natural compound that is extracted from cruciferous vegetables and possesses potent anti-inflammatory, antioxidant, and anticancer activities. This study was designed to test the hypothesis that SFN (3 mg/kg) may protect against FHF induced in rats by administering a combination of D-galactosamine (GalN; 300 mg/kg) and lipopolysaccharide (LPS; 30 μg/kg). The rats were given a single intraperitoneal injection of SFN, 1 hour before the FHF induction. Sulforaphane reduced the mortality and alleviated the pathological liver injury. In addition, SFN significantly reduced the increase in serum aminotransferase activities and lipid peroxidation. The glutathione content decreased in the GalN/LPS group, and this decrease was attenuated by SFN. Increases in serum tumor necrosis factor α, interleukin-6, and interleukin-10, which were observed in GalN/LPS-treated rats, were significantly reduced after using SFN. The GalN/LPS treatment increased the expression of superoxide dismutase-1, glutathione peroxidase 2, catalase, and heme oxygenase-1 genes. Sulforaphane inhibited the induction of reactive oxygen species scavenging proteins. Moreover, SFN inhibited GalN/LPS-induced caspase-3 activation and suppressed FAS and FASL expression. These findings suggest that SFN alleviates GalN/LPS-induced liver injury, possibly by exerting antioxidant, anti-inflammatory, and antiapoptotic effects and modulating certain antioxidant defense enzymes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Curcumin, a naturally occurring antioxidant, has various beneficial effects in the treatment of human diseases. However, little information regarding the protection it provides against acute liver injury is available. The present study investigated the protective effects of curcumin against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver injury in mice. A total of 40 male Kunming mice were randomly assigned to 5 groups: 1) mice administered saline vehicle injection (control), 2) mice administered 200 mg/kg body weight (BW) curcumin by i.p. injection (CUR), 3) mice administered D-GalN/LPS (700 mg and 5 μg/kg BW) via i.p. injection (GL), 4) mice administered 200 mg/kg BW curcumin i.p. 1 h before D-GalN/LPS injection (CUR-GL), and 5) mice administered 200 mg/kg BW curcumin i.p. 1 h after D-GalN/LPS injection (GL-CUR). Twenty h after D-GalN/LPS injection, serum alanine aminotransferase activities were 18.5% and 13.5% lower (P < 0.05) and aspartate aminotransferase (AST) activities were 26.6% and 9.6% lower (P < 0.05) in the CUR-GL and GL-CUR groups, respectively, than in the GL group. The CUR-GL and GL-CUR groups had 64.4% and 15.0% higher (P < 0.05) mitochondrial membrane potentials, respectively, and the CUR-GL group had a 44.7% lower reactive oxygen species concentration than the GL group (P < 0.05). Mitochondrial manganese superoxide dismutase activities were 111% and 77.9% higher (P < 0.05) and the percentages of necrotic cells were 47.0% and 32.4% lower (P < 0.05) in the CUR-GL and GL-CUR groups, respectively, than in the GL group. Liver mRNA levels of sirtuin 1 (Sirt1) were 56.4% lower (P < 0.05) in the CUR-GL group than in the GL group. Moreover, compared with the GL-CUR group, the CUR-GL group had an 18.7% lower serum AST activity, a 31.7% lower mitochondrial malondialdehyde concentration, a 36.0% lower hepatic reactive oxygen species concentration, and a 43.0% higher mitochondrial membrane potential. These results suggested that curcumin protects against D-GalN/LPS-induced liver damage by the enhancing antioxidant defense system, attenuating mitochondrial dysfunction and inhibiting apoptosis. This was especially true for curcumin pretreatment, which highlighted its promise as a preventive treatment for acute liver injury in clinical settings., (© 2014 American Society for Nutrition.)

Aims: Acute liver failure (ALF) is a fatal syndrome attributed to massive hepatocyte death. Hydrogen sulfide (H2S) has been reported to exert cytoprotective or cytotoxic effects. Here, we examined the role of cystathionine γ-lyase (CSE, an enzyme produces H2S) in ALF induced by D-Galactosamine (GalN) and lipopolysaccharide (LPS)., Results: Wild-type (WT) mice exhibited high mortality rate, prominent liver injury, and increased plasma alanine aminotransferase levels after GalN/LPS challenge. Congenital deficiency or chemical inhibition of CSE by DL-propargylglycine attenuated GalN/LPS-induced liver injury. CSE deficiency markedly improved survival rate and attenuated GalN/LPS-induced upregulation of inflammatory cytokines and activation of caspase 3 and poly (ADP-ribose) polymerase (PARP) in the liver. CSE deficiency protected primary hepatocytes from GalN/tumor necrosis factor-α (TNF-α)-induced cell death without affecting LPS-induced TNF-α production from primary peritoneal macrophages. Beneficial effects of CSE deficiency were associated with markedly elevated homocysteine and thiosulfate levels, upregulation of NF-E2 p45-related factor 2 (Nrf2) and antioxidant proteins, activation of Akt-dependent anti-apoptotic signaling, and inhibition of GalN/LPS-induced JNK phosphorylation in the liver. Finally, administration of sodium thiosulfate (STS) attenuated GalN/LPS-induced liver injury via activation of Akt- and Nrf2-dependent signaling and inhibition of GalN/LPS-induced JNK phosphorylation in WT mice., Innovation: These results suggest that inhibition of CSE or administration of STS prevents acute inflammatory liver failure by augmenting thiosulfate levels and upregulating antioxidant and anti-apoptotic defense in the liver., Conclusion: Congenital deficiency or chemical inhibition of CSE increases thiosulfate levels in the liver and prevents ALF at least in part by augmentation of antioxidant and anti-apoptotic mechanisms.

D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5'-monophosphate (5'-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5'-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5'-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5'-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5'-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury.

Cordyceps sinensis (C. sinensis) has long been considered to be an herbal medicine and has been used in the treatment of various inflammatory diseases. The present study examined the cytoprotective properties of C. sinensis on D(+)-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were randomly assigned into control, GalN/LPS, CS 20 mg and CS 40 mg groups (C. sinensis, oral gavage, five days/week, four weeks). After receiving saline or C. sinensis, mice were intraperitoneally given GalN (800 mg/kg)/LPS (10 μg/kg). The effects of C. sinensis on TNF-α, IL-10, AST, NO, SOD, and apoptoticrelated proteins after the onset of endotoxin intoxication were determined. Data demonstrated that GalN/LPS increased hepatocyte degeneration, circulating AST, TNF-α, IL-10, and hepatic apoptosis and caspase activity. C. sinensis pre-treatment reduced AST, TNF-α, and NO and increased IL-10 and SOD in GalN/LPS induced fulminant hepatic failure. C. sinensis attenuated the apoptosis of hepatocytes, as evidenced by the TUNEL and capase-3, 6 activity analyses. In summary, C. sinensis alleviates GalN/LPS-induced liver injury by modulating the cytokine response and inhibiting apoptosis.

Objective: To apply an orthogonal design optimization strategy to a mouse model of acute liver failure induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS) exposure., Methods: A four-level orthogonal array design (L16(45)) was constructed to test factors with potential impact on successful establishment of the model (D-GalN and LPS dosages, and dilution rate of the D-GalN/LPS mixture). The mortality rate of mice within 24 hours of D-GalN/LPS administration was determined by the Kaplan-Meier method. The model outcome was verified by changes in serum alanine transferase level, liver histology, and hepatocyte apoptosis., Results: The orthogonal array identified the optimal model technique as intraperitoneal injection of a combination of D-GalN and LPS at dosages of 350 mg/kg and 30 mug/kg, respectively, and using a dilution rate of 3. The dosages tested had no effect on survival. The typical signs of liver failure appeared at 6 hrs after administration of the D-GalN/LPS combination., Conclusion: The orthogonal design optimization strategy provided a procedure for establishing a mouse model of acute liver failure induced by D-GalN and LPS that showed appropriate disease outcome and survival, and which will serve to improve future experimental research of acute liver failure.

Grape pomace is generated in the production process of wine and grape juices and is an industrial waste. This study investigated whether an intake of grape pomace was able to suppress chronic inflammation induced by lipopolysaccharide (LPS) and galactosamine (GalN) in vivo. When Sprague-Dawley rats were orally given methanolic extracts from red and white grape pomace, the extracts inhibited the LPS/GalN-evoked activation of nuclear factor-κB (NF-κB) dose-dependently, and red grape pomace exerted a stronger effect than white grape one. Next, rats were fed an AIN93 M-based diet containing 5% red grape pomace for 7 days, followed by the intraperitoneal injection of LPS and GalN. The intake of the red grape pomace-supplemented diet was found to suppress the LPS/GalN-induced activation of NF-κB and expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins. These results suggest that red grape pomace may contain an abundance of effective compound(s) for anti-inflammatory action.

The purpose of this study was to investigate the bioactive phytochemicals of leaf essential oils of Cinnamomum osmophloeum on lipopolysaccharide/D-galactosamine (LPS/D-GalN)-induced acute hepatitis. The results revealed that post-treatment with 100 μmol/kg trans-cinnamaldehyde, (-)-aromadendrene, T-cadinol, or α-cadinol significantly decreased the aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) levels in serum. Moreover, both T-cadinol and α-cadinol treatments decreased the expressions of cleaved caspase-3 and cleaved poly-ADP ribose polymerase (PARP) in the liver tissues when compared with the LPS/D-GalN group. Liver histopathology also showed that silymarin, trans-cinnamaldehyde, (-)-aromadendrene, T-cadinol, or α-cadinol significantly reduced the incidence of liver lesions induced by LPS/D-GalN. These results suggest that the above phytochemicals exhibit potent hepatoprotection against LPS/D-GalN-induced liver damage in mice, and their hepatoprotective effects may be due to the modulation of anti-inflammatory activities.

Only liver transplantation is currently available therapy for the patients with acute liver failure (ALF). This study was designed to determine whether administration of granulocyte colony-stimulating factor (G-CSF) has therapeutic efficacy in animals with ALF. Female Sprague-Dawley (SD) rats were intraperitoneally injected with a single dose of d-galactosamine (d-GalN, 1.4g/kg) to induce ALF. After 2h, the rats were randomized to receive G-CSF (50μg/kg/day), or saline vehicle injection for 5 days. Rats were observed for survival and assessed for liver injury by serum alanine transaminase (ALT) measurement and histological analysis. CD34+ cells in bone marrow were assessed by flow cytometry. CD34+ cells and Ki-67+ hepatocytes in liver tissue were evaluated by immunohistochemistry. In the ALF model, 5-day survival after d-GalN injection was 33.3% (10/30), while G-CSF administration following d-GalN resulted in 53.3% (16/30) survival (p=0.027). G-CSF treated rats had lower ALT level and less hepatic injury compared with saline vehicle rats. The increases of CD34+ cells in bone marrow and liver tissue and Ki-67+ cells in liver tissue in G-CSF treated rats were higher than those in saline rats. No correlation was observed between CD34+ cells and Ki-67+ hepatocytes in liver tissue in both G-CSF and vehicle rats. It is suggested that G-CSF increases survival rate, decreases liver injury and enhances hepatocyte proliferation in rats with d-GalN-induced ALF possibly through actions including but not limiting to CD34+ cell mobilization, and that G-CSF may be of potential value in treating ALF., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: Protein kinase D (PKD) is a newly described serine/threonine protein kinase that plays a pivotal role in inflammatory response. In the present study, we examined the protective effect of Gö6976, a PKD inhibitor, on lipopolysaccharide (LPS) and D: -galactosamine (D: -GalN)-induced acute liver injury in mice., Materials and Methods: Mice were pretreated intraperitoneally with Gö6976 30 min before LPS/D: -GalN administration . The mortality and degree of hepatic injury was subsequently assessed., Results: The results indicated that LPS/D: -GalN administration markedly induced hepatic PKD activation, lethality and liver injury, while pretreatment of the PKD inhibitor Gö6976 significantly inhibited LPS-induced PKD activation, improved the survival of LPS/D: -GalN-administered mice and attenuated LPS/D: -GalN-induced liver injury, as evidenced by reduced levels of serum aminotransferases as well as reduced histopathological changes. In addition, the protective effects of Gö6976 were paralleled by suppressed activation of mitogen-activated protein kinases (MAPKs), decreased expression of tumor necrosis factor-α (TNF-α) and adhesion molecules, and reduced apoptosis and myeloperoxidase (MPO) activity in liver., Conclusions: Our experimental data indicated that Gö6976, a PKD inhibitor, could effectively prevent LPS/D: -GalN-induced acute liver injury by inhibition of MAPKs activation to reduce TNF-α production. This suggests the potential pharmacological value of PKD inhibitors in the intervention of inflammation-based liver diseases.

Unlabelled: Normal hepatocytes do not express endogenous uncoupling protein 2 (UCP2) in adult liver, although Kupffer cells do, and it is strikingly induced in hepatocytes in steatotic liver and obese conditions. However, the direct link of UCP2 with the pathogenic development of liver diseases and liver injury remains elusive. Here we report that targeted expression of UCP2 to mouse liver increases susceptibility to acute liver injury induced by lipopolysaccharide (LPS) and galactosamine (GalN). UCP2 appears to enhance proton leak, leading to mild uncoupling in a guanosine diphosphate-repressible manner. Indeed, mitochondria from the genetically manipulated mouse liver have increased state 4 respiration, lower respiratory control ratio, and reduced adenosine triphosphate (ATP) levels, which altered mitochondrial physiology. To address the underlying mechanism of how UCP2 and the reduced energy coupling efficiency enhance cell death in mouse liver, we show that the reduced ATP levels lead to activation of 5'AMP-activated protein kinase (AMPK) and its downstream effector, c-Jun N-terminal kinase; thus, the increased sensitivity toward LPS/GalN-induces apoptosis. Importantly, we show that inhibition of UCP2 activity by its pharmacological inhibitor genipin prevents LPS/GalN-induced ATP reduction, AMPK activation, and apoptosis. Also, inhibition of ATP production by oligomycin promotes LPS/GalN-induced cell death both in vivo and in vitro., Conclusion: Our results clearly show that targeted expression of UCP2 in liver may result in compromised mitochondrial physiology that contributes to enhanced cell death and suggests a potential role of UCP2 in the development of liver diseases.

The present study was conducted to investigate effect of pentoxifylline (PTX) on acute liver injury caused by galactosamine (D-Gal) in rats and the underlying mechanism involved in this setting. Moreover, we attempted to compare its effect to the well-established hepatoprotective agent, silymarin (SYM). The rats were randomly assigned 5 groups, control, PTX-treated (100 mg/kg, 3 weeks), SYM-treated (100 mg/kg, 3 weeks) and their combination. Hepatic injury was induced by intraperitoneal single dose injection of D-Gal (800 mg/kg). Hepatic functions parameters, including serum albumin and alkaline phosphatase (ALP) levels were determined. Antioxidants enzyme activities such as superoxide dismutase (SOD), catalase (CAT) as well as lipid peroxides and hepatic total nitrites were measured. Besides, histopathological examination was also performed using portions of liver tissues. Results showed that the liver injury induced by D-Gal was improved in the three pretreated groups to variable extents. Pretreatment with PTX prevented D-Gal-induced reduction of antioxidant enzyme activities, SOD and CAT, and attenuated the elevated malonaldahyde (MDA) level in hepatic tissue as marker of lipid peroxidation. In addition, pretreatment with PTX resulted in an increase in hepatic triglycerides, normalization of nitric oxide level, and lowering serum ALP activity as well as inhibited the decreased serum albumin level caused by D-Gal. These biochemical changes were reflected on attenuation the structural alterations of the liver integrity. Collectively, our data suggest that PTX exhibits a potential hepatoprotective effect against D-Gal-induced hepatotoxicity and this effect might be attributed to its antioxidant properties.

Objective: To reproduce a Kunming murine endotoxin shock model suitable for the anti-endotoxin pharmaceutical research., Methods: Kunming mice were challenged with an intraperitoneal (i. p.) injection of different doses of D-galactosamine (D-Gal) and endotoxin (LPS) and divided into 10 groups: i.e, group 1 [with injection of isotonic saline solution (NS) and LPS]; group 2 (with injection of NS and 90mg/kg LPS), group 3 (with injection of NS and 500mg/kg D-Gal), group 4 (with injection of 500mg/kgD-Gal and 25 microg /kg LPS), group 5 (with injection of 500mg/kg D-Gal and 50 microg/kg LPS), group 6(with injection of 500mg/kg D-Gal and 250 microg/kg LPS), group 7( with injection of NS and 600mg/ kg D-Gal), group 8 (with injection of 600mg/kg D-Gal and 10 microg/kg LPS), group 9( with injection of 600mg/kg D-Gal and 25 microg/kg LPS), group 10 (with injection of 600mg/kg D-Gal and 50 microg/kg LPS). The death of the mice were observed and the mortality rate was recorded at 48 post-injection hour (PIH). The dose of D-Gal and LPS which caused 100% lethality was chosen for the subsequent experiment to serve as control group (with injection of NS and 600mg/kg D-Gal), LPS group (with injection of 600mg/kg D-Gal and 580mg/kg LPS for later experiment). The venous blood of the mice were collected for the detection of serum content of TNF-alpha with ELISA method at 30, 75 and 120 post-injection minutes (PIM). The tissues of lung, liver, intestine were also harvested at 5 PIH for the pathological examination., Results: The lethality of mice was 100% in the groups 2, 6 and 10 (P < 0.01). The serum content of TNF-alpha was maintained in a low level in control group, but it increased remarkably in LPS group, and it reached peak at 75 PIM (6365 +/- 2087ng/L, P < 0.01). Obvious inflammatory reaction was observed in the lung, liver and intestine in LPS group, while only mild inflammatory reaction was observed in liver in control group., Conclusion: The Kunming mice showed signs of endotoxin shock after D-galactosamine presensitizing and endotoxin challenge, and it is suitable for anti-endotoxin pharmaceutical research.

Previous studies in our laboratory demonstrated the repressive effect of seaweeds (Laminaria sp., Sargassum fulvellum, Eisenia bicyclis and Gelidium sp.) against D-galactosamine (D-GalN)-induced hepatopathy. However, the mechanism by which these four seaweeds attenuate the D-GalN-hepatopathy has not been completely clarified. This study was carried out to determine the constituents of these seaweeds that protect rats against the D-GalN-hepatopathy. Male Wistar rats were fed for 8 d diets containing 5% seaweeds with or without the antibiotic neomycin (NEO) in experiment 1, and typical seaweed dietary fibers (laminaran, fucoidan, alginate, agar and kappa-carrageenan) of these seaweeds in experiment 2. On the 7th day, the rats were treated with D-GalN (1,900 mg in experiment 1 and 800 mg/kg in experiment 2) and then sacrificed 24 h after the injection of D-GalN. Their serum transaminase (aspartate and alanine aminotransferases: AST and ALT) activities were then determined. In experiment 1, the serum AST and ALT levels in the rats fed the four kinds of seaweeds without NEO were significantly low in comparison to that of the control group, but those with NEO were not significantly different among the groups. In experiment 2, the serum AST and ALT levels in the rats fed fucoidan were significantly low in comparison to those of the other groups fed the dietary fibers and the control. These results suggest that the protective effect of the three kinds of brown seaweeds Laminaria sp., Sargassum fulvellum and Eisenia bicyclis against D-GalN-hepatopathy was caused at least in part by fucoidan.

Aim: To examine the effects of adiponectin on the functions of Kupffer cells, key modulators of lipopolysaccharide (LPS) -induced liver injury., Methods: D-galactosamine (GalN) and LPS were injected intraperitoneally into adiponectin-/- mice and wild type mice. Kupffer cells, isolated from Sprague-Dawley rats, were preincubated with or without adiponectin, and then treated with LPS., Results: In knockout mice, GalN/LPS injection significantly lowered the survival rate, significantly raised the plasma levels of alanine transaminase and tumor necrosis factor-alpha (TNF-alpha) and significantly reduced IL-10 levels compared with wild type mice. TNF-alpha gene expression in the liver was which higher and those of IL-10 were lower in knockout mice than in wild type mice. In cultured adiponectin-pre-treated Kupffer cells, LPS significantly lowered TNF-alpha levels and raised IL-10 levels in the culture media and their respective gene expression levels, compared with Kupffer cells without adiponectin-pre-treatment., Conclusion: Adiponectin supresses TNF-alpha production and induces IL-10 production by Kupffer cells in response to LPS stimulation, and a lack of adiponectin enhances LPS-induced liver injury.

Non-viral gene delivery systems utilise either amine lipids or polyamines and although non-viral gene delivery systems are said to have a superior safety profile to viruses, the polyamines such as poly(L-lysine) are toxic when used without derivatisation and usually require specific receptor mediated uptake and/or endosomolytic agents to be effective. However, the conversion of poly(L-lysine) and poly(L-ornithine) polyamino acids into amphiphilic vesicle forming polymers reduces the toxicity of the polyamino acids and enables the resulting polyamino acid vesicles to deliver genes both in vitro and in vivo in the absence of receptor specific ligands and endosomolytic agents. The incorporation of a distearoylphosphatidylethanolamine poly(ethylene glycol)-galactosamine conjugate (with the galactosamine unit at the distal end of the poly(ethylene glycol) moiety) into the polyamino acid formulations improved in vitro gene transfer in the case of the amphiphilic poly(L-ornithine) (POP) although no in vivo targeting was detected with the galactosamine formulations. We conclude that the conversion of poly(L-lysine) and poly(L-ornithine) into amphiphilic colloid forming molecules reduces their toxicity, thus allowing these systems to be used for gene transfer in vivo. It is possible that this approach may be extended to other polyamines.

Background: Plasma exchange may be useful for treating patients with fulminant hepatic failure but during the procedure growth factors that are important for hepatic regeneration are discarded. Addition of a selective plasma filter to the plasmapheresis circuit could eliminate protein bound toxic substances and retain growth factors for hepatic regeneration. This process is called selective plasma filtration., Aims: To determine if selective plasma filtration could be a useful treatment modality for fulminant hepatic failure., Methods: The system was tested in five groups of pigs with fulminant hepatic failure induced by galactosamine: group I, diseased control group (n=5); group II, sham control, (n=6); group III, plasma exchange (n=6); group IV, treatment with AC-1770 selective plasma filter (n=7); and group V, treatment with AC-1730 selective plasma filter which had a smaller pore size than AC-1770 (n=7). Fresh pig plasma was given to replace filtered plasma in pigs of groups III, IV, and V. Treatment was initiated 48 hours after administration of 0.75 g/kg galactosamine. The efficacy of selective plasma filtration was assessed by survival rate and improvement in haematological, biochemical, and immunohistological parameters., Results: Pigs treated with AC-1770 or AC-1730 selective plasma filters survived longer than the other groups (group I: 55 (10) hours; group II: 68 (7) hours; group III: 91 (10) hours; group IV: 269 (156) hours; group V: 950 (555) hours). One pig in group IV survived for 50 days; one pig in group V survived for 77 days and another pig in group V is still alive (>150 days). After treatment, plasma levels of aspartate aminotransferase, bilirubin, bile acid, ammonia, lactate dehydrogenase, and alpha-glutathione-S-transferase decreased. Substantial amounts of tumour necrosis factor alpha (TNF-alpha) and endotoxin were found in the filtrate. The selective plasma filtration groups retained significantly higher amounts of hepatocyte growth factor than plasma exchange alone. Similar TNF-alpha clearance was observed in the selective plasma filtration groups and the plasma exchange group. On day 4, significant improvement in liver function, as measured by the indocyanine green clearance test, was observed in groups IV and V but not in the other groups. A higher regeneration index of hepatocytes was also observed in the groups treated with AC-1770 and AC-1730 selective plasma filters., Conclusion: Selective plasma filtration improved survival time and expedited liver regeneration in pigs with fulminant hepatic failure.

Extracts of various types of tea and coffee significantly suppressed lipopolysaccharide (LPS)-induced liver injury, as assessed by the plasma enzyme activities, in D-galactosamine-sensitized rats when administered orally once before injecting the drugs. There was a significant negative correlation between the caffeine levels of these extracts and liver injury. Authentic caffeine also had a hepatoprotective effect. These results suggest that caffeine-containing beverages generally suppress LPS-induced liver injury according to their caffeine content.

In contrast to human carriers of microfilariae, filariae infected rodents generally tolerate an effective microfilaricidal treatment without obvious signs of adverse reactions. The study shows, however, that also the filariae (Litomosoides carinii, Brugia malayi) infected rodent Mastomys coucha can be rendered sensitive to side effects of the treatment by the administration of D-galactosamine (D-Gal), due to reduction of liver UTP levels. Independent of the drug (diethylcarbamazine, ivermectin, CGP 20376) and the parasite species, D-Gal-primed infected animals died within 4 days after a microfilaricidal treatment. Lethal effects did also occur in naive animals to which microfilariae had been transfused 18 h prior to the challenge with D-Gal and a microfilaricidal, provided the animals had received at least approximately 10(3) larvae/g body weight. Both infected animals and naive recipients of microfilariae could be protected from death by cyclosporin A, polyclonal antibodies to mouse TNF or suitable amounts of NG-monomethyl-L-arginine. Pentoxifylline was less protective. The results suggest that components play a role in adverse reactions after microfilaricidal treatment, which are released by dying/dead microfilariae and may interact with T lymphocytes independent of a specific state of immunity. In a sequela, TNF released by T cells seems to induce an excess synthesis of N-oxides which appear to be the final morbific agent.

Polyamine concentrations including putrescine, spermidine and spermine were documented in two brain areas of rats with mild and severe stages of hepatic encephalopathy (HE) due to fulminant hepatic failure induced by galactosamine HC1 injection (3 g kg-1 i.p.). In the mild stage of HE putrescine increased by 3-4 times whereas spermidine and spermine showed a slight increase. The scenario, however, was found to be changed going from the mild to the severe stage of HE, since in this last stage spermidine and spermine showed a further rise while putrescine was found to be significantly lower than in the mild stage of HE in both the brain areas studied. The changes in the ratio among the three polyamines with an enhanced prevalence in the severe stage of HE of spermidine and spermine are likely to be related to the exhaustion of the synthetic pathway of putrescine or to a reduction of the interconversion to this polyamine from spermidine and spermine. Considering that these last two polyamines potentiate the N-methyl-D-aspartate glutamate receptor mediated toxicity and that they might exert neurotoxic effects per se, there are clear reasons for suspecting an implication of the described changes of polyamines in the neurochemical mechanism which sustain HE and to surmise a potential therapeutic effect in this pathology of non-competitive antagonists of polyamine-site on N-methyl-D-aspartate glutamate receptors.

Rat liver regeneration was studied from 24 hours to 8 days after a single intraperitoneal injection of D-galactosamine (GalN). Morphological changes in the liver were analyzed in parallel with sequential changes in expression of histone-3 mRNA (a marker of cell proliferation), fetal alpha-fetoprotein (AFP) mRNA and gamma-glutamyl transpeptidase (GGT) (markers of fetal hepatocytes), and albumin mRNA and glucose-6-phosphatase (G6Pase) (markers of adult hepatocytes). Proliferation of nonparenchymal epithelial cells (NPC), detected in situ by [3H]thymidine labeling or histone-3 mRNA expression, began after 24 hours primarily in the portal area around the bile ducts. After 2 days, histone-3 labelling intensity increased in rows and clusters of NPC which expanded from the portal zone and invaded into the parenchyma. On days 3 and 5, NPC expressing his-3 mRNA expanded further, forming pseudo-ducts and islet-like structures (NPC structures). Proliferating NPC were positive for GGT. Some GGT positive cells were also positive for the fetal form of AFP mRNA, which lagged behind GGT by 24 hours and peaked on day 5. On day 3, some cells with the appearance of NPC expressed albumin mRNA. Double label in situ hybridization for fetal AFP and albumin mRNAs and dual histochemistry for GGT and G6Pase showed simultaneous expression of these markers in NPC on day 5. Other cells expressing fetal AFP mRNA or GGT on day 5 had a morphological appearance between NPC and hepatocytes (transitional cells). Proliferation of hepatocytes began on day 2, reached maximum on day 5 and then declined. Proliferating hepatocytes did not express fetal AFP mRNA or GGT. These findings indicate that after GalN injury, the liver responds by activation of progenitor cells that proliferate and then differentiate into mature hepatocytes. Adult hepatocytes can also proliferate after GAlN injury, but these hepatocytes do not undergo dedifferentiation/redifferentiation during regeneration of the hepatic lobule.

Arachidonate metabolism was examined in rats with experimentally induced acute and chronic liver injuries. Acute liver injury was induced by a single administration of D-galactosamine (D-Galn) and lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). Chronic liver injury was produced by several administrations of CCl4 for 5 weeks. Non-parenchymal liver cells from rats with D-Galn/LPS-induced acute liver injury produced prominently leukotriene B4 and 5-hydroxy-arachidonic acid which were hardly synthesized by the normal rat liver. No apparent changes were observed in the arachidonate metabolism of the non-parenchymal cells of the acute CCl4-injured liver. In chronic liver injury, the production of 6-ketoprostaglandin F1 alpha, a stable metabolite of prostaglandin I2, by the non-parenchymal cell fraction was significantly enhanced in contrast with the fixed amount of the other arachidonate metabolites. These results suggested the arachidonate metabolism by non-parenchymal liver cells might change according to the pathogenesis of the liver disease.

Galactosamine hydrochloride induces liver disease in rats that morphologically resembles drug-induced hepatitis in man. In this study we analyzed the character of the inflammatory reaction following the toxic damage resulting from the administration of galactosamine hydrochloride using a broad panel of monoclonal antibodies to lymphocyte subsets and macrophages. Fat-storing cells were identified with a polyclonal anti-desmin antibody. Cellular proliferation was assessed by labeling S-phase cells with the thymidine analog bromodeoxyuridine. Injection of galactosamine hydrochloride was associated with conspicuous hepatocyte necrosis and parenchymal granulocyte influx in the first 24 hr. Thereafter, mononuclear inflammatory cells predominated, mainly T lymphocytes and macrophages, with maximal numbers at 48 hr. The majority of T lymphocytes were CD8-positive cells and were located in the portal tracts and parenchyma. CD4-positive T cells were scarce and confined to the portal tracts. Proliferation of fat-storing cells paralleled hepatocyte regeneration with maximal values after 48 to 72 hr. The temporal relationship between infiltrating mononuclear cells, mainly T lymphocytes of CD8 phenotype and macrophages, fat-storing cell proliferation and hepatic regeneration suggests pathophysiological interactions between these cell types in liver injury in the rat after galactosamine hydrochloride administration.

The hypothesis was tested whether under the presence of a membrane defect of hepatocytes an activation of the complement system leads to massive liver cell necrosis. Low doses of galactosamine (50, 100, and 200 mg/kg) were administered to rats with and without an additional i.v. injection of sublethal doses of endotoxin (1.5 mg/kg). The latter was done in order to activate the complement system via the C3-bypath. Neither after doses of 50 mg/kg and 100 mg/kg nor after an additional administration endotoxin liver cell necrosis were observed. A dose of 200 mg/kg led to moderate liver cell necrosis, and when administered with endotoxin fulminant hepatic necrosis developed. The explanation was given that only after 200 mg/kg alterations of hepatocytes membranes were present, which prepare liver cells for complement mediated hepatocytolysis. In rats to which 1 g/kg galactosamine was given in addition to endotoxin it was demonstrated by immunohistology that the third component of complement was already fixed on hepatocyte plasma membranes at hour 3 and was accumulated within areas of necrotic liver parenchymal cells at hour 12. Thus, liver cell death is suggested as complement mediated if the membranes are altered. Clinical implications are given in concern of fulminant hepatic failure and an approach to effective treatment regims.

The morphological and biochemical studies were performed on the injured livers of female rats produced by chronic administration of D-galactosamine (GALN) (250 mg/kg, i.p.) for 7 months. Light microscopically, cirrhotic changes were observed in most of the animals characterized by the proliferation of the connective tissues from portal triads into hepatic lobules. The electron microscopic study demonstrated mitochondrial proliferation and irregularities with crenated membranes, focal hypertrophy of the smooth endoplasmic reticulum, decrease of the rough endoplasmic reticulum with partial detouchment of ribosomes, slight loss of compactness of nucleoli and no remarkable accumulation of lipid droplets in the cytoplasm. The proliferation of collagen fibers was observed around the hepatocytes and acid mucopolysaccharides were seen in the space of Disse and partly in the sinusoids histochemically using electron microscope. Biochemically chronic GALN administration, which increased hepatic weight significantly, resulted in a significant decrease in microsomal protein concentration, whereas cytochrome P450 content significantly increased. There was no change in phospholipid contents. After GALN, plasma albumin concentration was significantly decreased and the value of zinc turbidity test was increased. However, there was no change in plasma GPT level and total cholesterol concentration.

A study was conducted to elucidate the possible role of singlet oxygen in pathogenesis of D-galactosamine-induced liver injury. Tissue and plasma levels of singlet oxygen were determined with chemiluminescence analysis. Following results were obtained: 1) Chemiluminescence as well as malondialdehyde, which is regarded as one of terminal products of lipid peroxidation, significantly increased in the liver and plasma of rats treated with D-galactosamine. 2) Elevation of plasma GPT and total bilirubin was also observed in rats with D-galactosamine-induced liver injury. Histological examination of the liver revealed submassive hepatic necrosis. 3) Administration of vitamin E, a radical scavenger of singlet oxygen, significantly inhibited the increases of chemiluminescence and MDA in the liver and plasma as well as the elevations of GPT and total bilirubin in the plasma. Histological changes of the liver were also found to improve significantly by vitamin E administration. In conclusion, singlet oxygen seems to be definitely involved, at least in part, in pathogenesis of liver damage induced by D-galactosamine. In addition, inhibition of the liver injury is possible, to some extent, by administration of vitamin E, one of the potent radical scavengers of singlet oxygen.

Galactosamine caused changes in the livers of rats that were similar to those seen in human viral hepatitis. In hamsters the changes were characteristic of a typical toxic hepatitis. The response by the liver to galactosamine was very variable in both the rat and the hamster and there was a degree of correlation between the raised transaminase levels and the liver cell damage.

The effect of a nucleoside-nucleotide mixture on liver injury of rats induced by D-galactosamine was studied by examining changes in function and histopathology of the liver. Animals with liver damage received total parenteral nutrition with glucose and amino acids supplemented with a nucleoside-nucleotide mixture containing inosine, cytidine, GMP, uridine and thymidine, or with uridine which inhibits galactosamine injury, or with liver cell extract containing flavin adenine dinucleotide and nucleic acid derivatives. As control, animals with liver damage received total parenteral nutrition with glucose and amino acids only. The serum GOT and GPT concentrations were significantly lower in the group supplemented with nucleoside-nucleotide mixture than those in other groups. A large dose (1.2 g/kg) of uridine inhibited liver injury, but a lower dose (0.14 g/kg) did not have any effect, whereas nucleoside-nucleotide mixture containing the same amount of uridine inhibited the injury. Liver cell extract also did not improve liver function. Thus infusion of a physiological and balanced mixture of nucleosides or nucleotides may improve liver function in rats with liver injury.