Hepatoprotective effect of bis(4‐methylbenzoyl) diselenide against CCl4‐induced oxidative damage in mice

From a pharmacological point of view, organoseleniums are compounds with important and interesting antioxidant and biological activities. The aim of this study was to evaluate the hepatoprotective effect of bis(4‐methylbenzoyl) diselenide (BMD) against carbon tetrachloride (CCl₄)–induced oxidative damage in mice. The animals received BMD (25 mg/kg p.o., for 3 days), and after 1 day, CCl₄ (1 mg/kg body weight) was administered by intraperitoneal route. One day after the CCl₄ exposure, the animals were euthanized for biochemical and histological analysis. Treatment with BMD (25 mg/kg p.o.) protected against aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma‐glutamyl transferase and lactate dehydrogenase activity increases induced by CCl₄ plasma exposure. Treatment with BMD (25 mg/kg) protected against increases in thiobarbituric reactive species and decreasing non‐protein thiols and ascorbic acid levels in liver of mice. Catalase and superoxide dismutase activity inhibition in the liver caused by CCl₄ were protected by treatment with BMD (25 mg/kg). Glutathione S‐transferase activity was inhibited by CCl₄ and remained unaltered even after treatment with BMD. Sections of liver from CCl₄‐exposed mice presented an intense infiltration of inflammatory cells and loss of the cellular architecture. BMD (25 mg/kg) attenuated CCl₄‐induced hepatic histological alterations. The results demonstrated the hepatoprotective effects of BMD in the mouse liver, possibly by modulating the antioxidant status. Copyright © 2012 John Wiley & Sons, Ltd.     

Elucidation of the role of COX-2 in liver fibrogenesis using transgenic mice

Hepatic COX-2 overexpression is sufficient to induce hepatitis, but its role on liver fibrosis remains unknown. We aim to elucidate possible biological effects of COX-2 in liver fibrosis using both gain-of-function and loss-of-function mouse models. COX-2 transgenic (TG) mice that specifically overexpress the human COX-2 cDNA in the liver, knockout (KO), and wild type (WT) mice were studied in two different murine fibrosis models induced by carbon tetrachloride (CCl₄) injection or methionine and choline-deficient (MCD) diet. Liver injury was assessed by serum ALT and bilirubin levels and histological examination. Hepatic collagen content was determined by picrosirius red stain morphometry assay and quantitation of hydroxyproline. Hepatic stellate cell (HSC) activation was determined by immunohistochemical analysis of α-smooth muscle actin (α-SMA). mRNA expression of fibrogenic genes was assayed by real-time quantitative PCR. COX-2 protein was overexpressed in the liver of TG mice compared with WT littermates. CCl₄ or MCD-induced liver fibrotic injury was equally severe in TG and WT mice, as demonstrated by similar elevated levels of hepatic collagen contents. Enhanced COX-2 expression in TG liver did not affect HSC activation and fibrogenic gene expression upon CCl₄ or MCD treatment. Importantly, CCl₄-treated KO mice did not show significant difference in liver fibrotic damage and fibrogenic gene expression compared with the WT counterparts. This is the first report on the effect of COX-2 in liver fibrosis based on genetic mouse models. The results suggest that COX-2 does not appear to mediate the development of liver fibrosis.     

Adiponectin Agonist ADP355 Attenuates CCl4-Induced Liver Fibrosis in Mice

Liver fibrosis is a growing global health problem characterized by excess deposition of fibrillar collagen, and activation of hepatic stellate cells (HSCs). Adiponectin is known to possess anti-fibrotic properties; however a high physiological concentration and multiple forms circulating in blood prohibit clinical use. Recently, an adiponectin-like small synthetic peptide agonist (ADP355: H-DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH2) was synthesized for the treatment of murine breast cancer. The present study was designed to evaluate the efficacy of ADP355 as an anti-fibrotic agent in the in vivo carbon tetrachloride (CCl₄)-induced liver fibrosis model. Liver fibrosis was induced in eight-week old male C57BL/6J mice by CCl₄-gavage every other day for four weeks before injection of a nanoparticle-conjugated with ADP355 (nano-ADP355). Control gold nanoparticles and nano-ADP355 were administered by intraperitoneal injection for two weeks along with CCl₄-gavage. All mice were sacrificed after 6 weeks, and serum and liver tissue were collected for biochemical, histopathologic and molecular analyses. Biochemical studies suggested ADP355 treatment attenuates liver fibrosis, determined by reduction of serum aspartate aminotransferase (AST), alanine aminotransferase ALT) and hydroxyproline. Histopathology revealed chronic CCl₄-treatment results in significant fibrosis, while ADP355 treatment induced significantly reversed fibrosis. Key markers for fibrogenesis–α-smooth muscle actin (α-SMA), transforming growth factor-beta1 (TGF-β1), connective tissue growth factor (CTGF), and the tissue inhibitor of metalloproteinase I (TIMP1) were also markedly attenuated. Conversely, liver lysates from ADP355 treated mice increased phosphorylation of both endothelial nitric oxide synthase (eNOS) and AMPK while AKT phosphorylation was diminished. These findings suggest ADP355 is a potent anti-fibrotic agent that can be an effective intervention against liver fibrosis.     

Oroxylin A Accelerates Liver Regeneration in CCI4-Induced Acute Liver Injury Mice

Introduction

Based on the previous research that oroxylin A can suppress inflammation, we investigated the hepatoprotective role of oroxylin A against CCl₄-induced liver damage in mice and then studied the possible alteration of the activities of cytokine signaling participating in liver regeneration. Wild type (WT) mice were orally administrated with oroxylin A (60 mg/kg) for 4 days after CCl₄ injection, the anti-inflammatory effects of oroxylin A were assessed directly by hepatic histology and indirectly by measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Albumin. Proliferating cell nuclear antigen (PCNA) staining was performed to evaluate the role of oroxylin A in promoting hepatocyte proliferation. Serum IL-1β, TNF-α, IL-6 and IL-1Ra levels were measured by enzyme-linked immunosorbent assay (ELISA) and liver HGF, EGF, TNF-α, IL-6, IL-1Ra and IL-1β gene expression was determined by quantitative real-time PCR. The data indicated that the IL-6 and TNF-α mRNA of oroxylin A administered group significantly increased higher than the control within 12 hours after CCl₄ treatment. Meanwhile, oroxylin A significantly enhanced the expression of IL-1Ra at the early phase, which indicated that oroxylin A could facilitate the initiating events in liver regeneration by increasing IL-1Ra which acts as an Acute-Phase Protein (APP). In addition, a lethal CCl₄-induced acute liver failure model offers a survival benefit in oroxylin A treated WT mice. However, oroxylin A could not significantly improve the percent survival of IL-1RI^−/− mice with a lethal CCl₄-induced acute liver failure.

Conclusions

Our study confirmed that oroxylin A could strongly promote liver structural remodeling and functional recovery through IL-1Ra/IL-1RI signaling pathway. All these results support the possibility of oroxylin A being a therapeutic candidate for acute liver injury.     

Effects of indoleamine 2,3‐dioxygenases in carbon tetrachloride‐induced hepatitis model of rats

Indoleamine 2,3‐dioxygenase (IDO) converts tryptophan to l ‐kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, to investigate the effects of IDO in carbon tetrachloride (CCl₄)–induced hepatitis model, the levels of IDO enzymic activities in the mock group, the control group and the 1‐methyl‐d ‐tryptophan (1‐MT)–treated group were confirmed by determination of l ‐kynurenine concentrations. Serum alanine aminotransferase levels in 1‐MT‐treated rats after CCl₄ injection significantly increased compared with those in mock and control groups. In CCl₄‐induced hepatitis models, tumour necrosis factor‐α (TNF‐α) is critical in the development of liver injury. The mRNA expression and secretion levels of TNF‐α in the liver from 1‐MT‐treated rats were more enhanced compared with those in the mock and the control groups. Moreover, the levels of cytokine and chemokine from mock, control group and 1‐MT‐treated rats after treated with CCl₄ were analyzed by ELISA, and the level of interleukin‐6 was found to increase in 1‐MT‐treated rats. It was concluded that the deficiency of IDO exacerbated liver injury in CCl₄‐induced hepatitis and its effect may be connected with TNF‐α and interleukin‐6. Copyright © 2012 John Wiley & Sons, Ltd.     

Methane alleviates carbon tetrachloride induced liver injury in mice: anti-inflammatory action demonstrated by increased PI3K/Akt/GSK-3β-mediated IL-10 expression

The inflammatory response plays an important role in carbon tetrachloride (CCl₄)-induced acute liver injury and methane has been shown to exert beneficial effects on inflammation-associated diseases. Thus, we investigated the potential protective effects of methane-rich saline (MS) on CCl₄-induced acute liver injury and explored the underlying mechanism. A CCl₄-induced acute liver injury model was established by injection of CCl₄ (0.6 ml/kg, ip) in mice followed by treatment with MS (16 ml/kg, ip), 24 h later. All groups of mice were sacrificed and blood and liver tissues were collected. Serum aminotransferase, necrotic areas, and inflammatory cell infiltration in liver slices were enhanced after CCl₄ treatment but decreased with MS treatment. IL-6, TNF-α, IL-1β, IFN-γ, ICAM-1, CXCL1, MPO, NF-κB p65, ERK, JNK, and MAPK P38, expression in serum or liver homogenate were greater after CCl₄ treatment but comparatively less after MS treatment. Only IL-10 increased after MS treatment. Anti-IL10 blockade (1.5 mg/kg) restored MS-mediated attenuated phosphorylation of NF-?bB/MAPK and the protective effect of MS was abolished for all indices examined. The PI3K inhibitor, wortmannin had the same effects on MS as anti-IL-10 antibody. MS also induced phosphorylation of GSK-3β and AKT in CCl₄-treated mice. After pre-treatment with wortmannin (0.7 mg/kg), phosphorylation of GSK-3β and AKT proteins were reduced compared to its solvent control group-DMSO-treated animals. Thus, the data provide evidence that MS may activate the PI3K–AKT–GSK-3β pathway to induce IL-10 expression and produce anti-inflammatory effects via the NF-κB and MAPK pathways. The findings provide a new pharmacological strategy for management of inflammatory response after acute liver injury.     

FACTORS INFLUENCING THE EFFICIENCY OF LIPOPLEXES MEDIATED GENE TRANSFER IN LUNG AFTER INTRAVENOUS ADMINISTRATION*

The objectives of this study were to test the influence of different parameters on the in vivo cationic lipid mediated gene transfer in lung after intravenous administration. Luciferase activity was evaluated in lung tissue 24 hours after intravenous administration of different types of lipoplexes. These included lipoplexes prepared using cationic phosphonolipids or DOTAP and various amounts of plasmid DNA. Using two different plasmids we tested the influence of plasmid size on transfection efficiency in vivo. In a last series of experiments, lipoplexes were prepared using different excipients (water, NaCl or 5% glucose solution) and three injection volumes were tested. We demonstrate that chemical structure modifications such as cation substitution and increment of the aliphatic chain length significantly improve transfection efficiency. High luciferase levels are obtained by increasing lipid to DNA charge ratio and plasmid DNA dose and decreasing plasmid size. Lipoplexes prepared in physiological NaCl solution and injected using a volume of 800μl are significantly the most effective.

Cationic lipid mediated gene transfer in lung tissue after intravenous administration is influenced by factors including cationic lipid chemical structure, lipid to DNA ratio and plasmid dose. Nevertheless, plasmid size, injection volume and the excipient, used for the lipoplexes preparation, are also important factors and must be considered for an optimization of in vivo gene delivery using intravenous administration.     

Small interfering RNA delivery into the liver by cationic cholesterol derivative-based liposomes

Purpose: Previously, we reported that the cationic liposomes composed of a cationic cholesterol derivative, cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (termed LP-C), could deliver small interfering RNAs (siRNAs) with high transfection efficiency into tumor cells. In this study, to develop a liposomal vector for siRNA delivery in vivo, we prepared the poly(ethyleneglycol) (PEG)-modified cationic liposomes (LP-C-PEG) and evaluated their transfection efficiency in vitro and in vivo.

Materials and methods: We prepared LP-C-PEG/siRNA complexes (LP-C-PEG lipoplexes) formed in water or 50?mM NaCl solution, and evaluated their siRNA biodistribution and gene silencing effect in mice after intravenous injection.

Results: LP-C-PEG lipoplexes strongly exhibited in vitro gene silencing effects in human breast tumor MCF-7 cells as well as LP-C lipoplexes. In particular, formation of LP-C and LP-C-PEG lipoplexes in the NaCl solution increased the cellular association. When LP-C-PEG lipoplexes with Cy5.5-labeled siRNA formed in water or NaCl solution were injected into mice, accumulation of the siRNA was observed in the liver. Furthermore, injection of LP-C-PEG lipoplexes with ApoB siRNA could suppress ApoB mRNA levels in the liver and reduce very-low-density lipoprotein/low-density lipoprotein levels in serum compared with that after Cont siRNA transfection, although the presence of NaCl solution in forming the lipoplexes did not affect gene silencing effects in vivo.

Conclusions: LP-C-PEG may have potential as a gene vector for siRNA delivery to the liver.     

Hepatoprotective Effect of Manganese Chloride Against CCl4-Induced Liver Injury in Rats

The aim of the present study is to evaluate the protective effect of manganese chloride against carbon tetrachloride (CCl₄)-induced liver injury in rats. Manganese chloride (0.001, 0.01, 0.05 and 0.1 g/kg bw) was administered intragastrically for 28 consecutive days to male CCl₄-treated rats. The hepatoprotective activity was assessed using various biochemical parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and superoxide dismutase (SOD). Histopathological changes in the liver of different groups were also studied. Administration of CCl₄ increased the serum ALT, AST, ALP and GGT but decreased SOD levels in rats. Treatment with manganese chloride significantly attenuated these changes to nearly normal levels. The animals treated with manganese chloride have shown decreased necrotic zones and hepatocellular degeneration when compared to the liver exposed to CCl₄ intoxication alone. Thus, the histopathalogical studies also supported the protective effect of manganese chloride. Therefore, the results of this study suggest that manganese chloride exerts hepatoprotection via promoting antioxidative properties against CCl₄-induced oxidative liver damage.     

Differential effect of schisandrin B and dimethyl diphenyl bicarboxylate (DDB) on hepatic mitochondrial glutathione redox status in carbon tetrachloride intoxicated mice

The effects of schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, and dimethyl diphenyl bicarboxylate (DDB), a synthetic intermediate of schisandrin C (also a dibenzocyclooctadiene derivative), on hepatic mitochondrial glutathione redox status in control and carbon tetrachloride (CCl₄)-intoxicated mice were examined. Treating mice with Sch B or DDB at a daily oral dose of 1 mmol/kg for 3 d did not produce any significant alterations in plasma alanine aminotransferase (ALT) and sorbital dehydrogenase (SDH) activities. CCl₄ treatment caused drastic increases in both plasma ALT and SDH activities in mice. Pretreating mice with Sch B or DDB at the same dosage regimen significantly suppressed the CCl₄-induced increase in plasma ALT activity, with the inhibitory effect of Sch B being much more potent. Sch B, but not DDB, pretreatment could also decrease the plasma SDH activity in CCl₄-intoxicated mice. The lowering of plasma SDH activity, indicative of hepatoprotection against CCl₄ toxicity, by Sch B pretreatment was associated with an enhancement in hepatic mitochondrial glutathione redox status as well as an increase in mitochondrial glutathione reductase (mtGRD) activity in both non-CCl₄ and CCl₄-treated mice. DDB pretreatment, though enhancing both hepatic mitochondrial glutathione redox status and mtGRD activity in control animals, did not produce any beneficial effect in CCl₄-treated mice. The difference in hepatoprotective action against CCl₄ toxicity between Sch B and DDB may therefore be related to their ability to maintain hepatic mitochondrial glutathione redox status under oxidative stress condition.     

Immunological study of carbon tetrachloride-mediated induction of tyrosine aminotransferase in rat liver

Administration of CCl₄ (1.0 ml/kg) to rats resulted in a rise of liver tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) activity to a maximum of about 3.6 times the normal level 6 hr later. An immunological titration study proved that the phenomenon was due to increased enzyme content. Using an isotopic-immunochemical procedure the half-life of liver tyrosine aminotransferase at 3.5 hr after CCl₄ administration was shown to be 11.9 hr in contrast to 2.1 hr in the normal liver. Immunochemical analysis revealed that enzyme synthesis was decreased by CCl₄. Thus, in the early stage of CCl₄ poisoning, enzyme synthesis proceeded at a moderate rate while degradation was markedly impaired, resulting in the rise of tyrosine aminotransferase in the liver tissue.Several hours after administration of hydrocortisone to adrenalectomized rats, induced tyrosine aminotransferase reached its peak activity and then subsided to the basal level. At any time following hydrocortisone administration, administration of CCl₄ consistently caused an elevation of the enzyme activity above the level in controls not treated with CCl₄. Actinomycin D (5 mg/kg) also increased the enzyme at an early period of induction cycle but failed to do so at a later period.The CCl₄-mediated “superinduction” of hormonally preinduced tyrosine aminotransferase, like the induction of this enzyme by CCl₄ at a basal level, was found to be caused by the differential inhibitory effect of CCl₄ on the synthesis and degradation of this enzyme.     

Zonation of Nitrogen and Glucose Metabolism Gene Expression upon Acute Liver Damage in Mouse

Zonation of metabolic activities within specific structures and cell types is a phenomenon of liver organization and ensures complementarity of variant liver functions like protein production, glucose homeostasis and detoxification. To analyze damage and regeneration of liver tissue in response to a toxic agent, expression of liver specific enzymes was analyzed by in situ hybridization in mouse over a 6 days time course following carbon tetrachloride (CCl₄) injection. CCl₄ mixed with mineral oil was administered to BALB/c mice by intraperitoneal injection, and mice were sacrificed at different time points post injection. Changes in the expression of albumin (Alb), arginase (Arg1), glutaminase 2 (Gls2), Glutamine synthetase (Gs), glucose-6-phosphatase (G6pc), glycogen synthase 2 (Gys2), Glycerinaldehyd-3-phosphat-Dehydrogenase (Gapdh), Cytochrom p450 2E1 (Cyp2e1) and glucagon receptor (Gcgr) genes in the liver were studied by in situ hybridization and qPCR. We observed significant changes in gene expression of enzymes involved in nitrogen and glucose metabolism and their local distribution following CCl₄ injury. We also found that Cyp2e1, the primary metabolizing enzyme for CCl₄, was strongly expressed in the pericentral zone during recovery. Furthermore, cells in the damaged area displayed distinct gene expression profiles during the analyzed time course and showed complete recovery with strong albumin production 6 days after CCl₄ injection. Our results indicate that despite severe damage, liver cells in the damaged area do not simply die but instead display locally adjusted gene expression supporting damage response and recovery.     

Hepatoprotective effect of a protein-enriched fraction from the maggots (Musca domestica) against CCl4-induced hepatic damage in rats

The hepatoprotective potential of a protein-enriched fraction (PEF) isolated from the maggots of housefly (Musca domestica) was evaluated in rats against carbon tetrachloride (CCl₄)-induced acute hepatic damage. Activities of serum aspartate aminotransferase and alanine aminotransferase increased by 4- and 13-fold induced by CCl₄, were significantly inhibited by pretreatment with 50, 100 and 200 mg PEF/kg. The formation of malondialdehyde was also significantly decreased in PEF-treated group compared with CCl₄-treated group. The treatments with PEF also elevated total protein levels significantly. These results were further supplemented by histopathological examination of liver sections. Hyperplasia of kupffer cells was observed after treatment with PEF (100 and 200 mg/kg). We conclude that PEF is effective in this model of liver damage.     

The Role of Interferon-γ Inducible Protein-10 in a Mouse Model of Acute Liver Injury Post Induced Pluripotent Stem Cells Transplantation

Background

Liver injuries are important medical problems that require effective therapy. Stem cell or hepatocyte transplantation has the potential to restore function of the damaged liver and ameliorate injury. However, the regulatory factors crucial for the repair and regeneration after cell transplantation have not been fully characterized. Our study investigated the effects and the expression of the regulatory factors in mouse models of acute liver injury either transplanted with the induced pluripotent stem cells (iPS) or the hepatocytes that differentiated from iPS cells (iHL).

Methods/Principal Findings

Mice received CCl₄ injection and were randomized to receive vehicle, iPS, or iHL transfusions vial tail veins and were observed for 24, 48 or 72 hours. The group of mice with iPS transplantation performed better than the group of mice receiving iHL in reducing the serum alanine aminotransferase, aspartate aminotransferase, and liver necrosis areas at 24 hours after CCl₄ injury. Moreover, iPS significantly increased the numbers of proliferating hepatocytes at 48 hours. Cytokine array identified that chemokine IP-10 could be the potential regulatory factor that ameliorates liver injury. Further studies revealed that iPS secreted IP-10 in vitro and transfusion of iPS increased IP-10 protein and mRNA expressions in the injured livers in vivo. The primary hepatocytes and non-parenchyma cells were isolated from normal and injured livers. Hepatocytes from injured livers that received iPS treatment expressed more IP-10 mRNA than their non-hepatocyte counter-parts. In addition, animal studies revealed that administration of recombinant IP-10 (rIP-10) effectively reduced liver injuries while IP-10-neutralizing antibody attenuated the protective effects of iPS and decreased hepatocyte proliferation. Both iPS and rIP-10 significantly reduced the 72-hour mortality rate in mice that received multiple CCl₄-injuries.

Conclusions/Significance

These findings suggested that IP-10 may have an important regulatory role in facilitating the repair and regeneration of injured liver after iPS transplantation.     

Anti-apoptotic and antioxidant effect of leptin on CCl4-induced acute liver injury in rats

The aim of this study is to investigate the effect of leptin in rats on carbon tetrachloride (CCl₄) induced acute liver damage using immunohistochemical methods for apoptosis and biochemical parameters. In this experimental study, 18 Spraque-Dawley rats were divided into three groups viz; control, CCl₄ and CCl₄+leptin treatment. 0.8 ml/kg olive oil was administered intraperitoneally (i.p.) to the control group and 0.8 ml/kg CCl₄ (1:1 dissolved in olive oil) was administered i.p. to the CCl₄ and CCl₄+leptin treatment groups, respectively. After 6 h of administrating CCl₄, CCl₄+leptin treatment group was given i.p. leptin (10 μg/kg). Twenty-four hours after administrating CCl₄ all of the groups were euthanized. Biochemical assessments were performed using serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), plasma tumor necrosis factor alpha (TNF-α) levels and tissue malondialdehyde (MDA), and TNF-α levels. Histological assessments were then performed using Hematoxylin&Eosin (H&E) staining in light microscope and apoptosis assessment using Terminal Transferase dUTP Nick End Labeling (TUNEL)-staining. Serum AST, ALT, ALP and plasma TNF-α levels, tissue MDA and TNF-α levels had all increased in CCl₄ group, but were found to be significantly decreased in CCl₄+leptin treatment group. Moreover, TUNEL-positive cell counts in liver had significantly increased in CCl₄ group, but decreased in CCl₄+leptin treatment group (P < 0.05). The results of our study the biochemical, histological and TUNEL-staining showed that leptin has treatment effects on liver CCl₄ induced injury. It plays a role as a potent free radical scavenger, a powerful antioxidant and it also has anti-apoptotic effects.     

Ascorbate promotes carbon tetrachloride-induced hepatic injury in senescence marker protein 30-deficient mice by enhancing inflammation

The genetic deletion of the senescence marker protein 30 (SMP30) gene results in ascorbate deficiency and the premature aging processes in mice. Apparent liver injury of SMP30^−/− mice was less severe than those of wild type (WT) mice, upon chronic CCl₄ injection. The purpose of this study was to investigate the pathophysiology underlying the mild CCl₄ toxicity in SMP30^−/− mice. Along with the lower level of serum alanine aminotransferase, the livers of SMP30^−/− mice revealed a lesser glycogen depletion, a decrease in c-Jun N-terminal kinase (JNK)-mediated inflammatory signaling in parallel with tumor necrosis factor-alpha and interleukin-1 beta, inducible nitric oxide synthase and glutathione peroxidase, and the lower lipid peroxidation as compared to those of WT mice. CCl₄-induced proliferation, measured by the expression of proliferating cell nuclear antigen, was low in SMP30^−/− mice as compared with that of WT mice whereas the levels of p21 and Bax were comparable to those of the CCl₄-treated WT mice. Moreover, CCl₄ toxicity in ascorbate-fed SMP30^−/− mice was comparable to that of the CCl₄-alone treated WT mice, accompanied by an increase in the above mentioned factors. Conversely, ascorbate partly compensated for the CCl₄-induced oxidative stress in WT mice, indicating that sufficient ascorbate may be required for an antioxidant function under severe levels of oxidative stress. Our data suggest that the restoration of ascorbate-deficiency reverses a sluggish immune system into an activated condition by an increase in JNK-mediated inflammation and free radical cascade; thus leading to accelerated hepatic damage in SMP30^−/− mice.     

Selenium Supplementation Decreases Hepatic Fibrosis in Mice After Chronic Carbon Tetrachloride Administration

Oxidative stress stimulates fibrogenesis, and selenium (Se) has antioxidant properties. This study determined whether Se supplementation affects CCl₄-induced liver injury and fibrosis. Mice were administered CCl₄ over 4 weeks, while controls received olive oil. Se was provided as sodium selenite in the drinking water. Se increased liver Se-dependent glutathione peroxidase activity and decreased liver malondialdehyde after CCl₄. Se decreased liver inflammation but not necrosis caused by CCl₄. Se increased hepatocyte apoptosis after CCl₄ and the pro-apoptotic BAX and Bcl Xs/l proteins. Stellate cell apoptosis occurred only after CCl₄ in Se-supplemented mice. Se decreased stellate cell number and fibrosis after CCl₄. Liver matrix metalloproteinase-9 increased after CCl₄ with Se supplementation. In conclusion, Se supplementation decreased hepatic fibrosis after CCl₄ in the setting of decreased inflammation but increased apoptosis. The principal mechanisms for the decreased fibrosis are a lower number of collagen-producing stellate cells and increased collagen degradation.     

Protective Effect of Cornuside against Carbon Tetrachloride-Induced Acute Hepatic Injury

This study elucidated the effects of cornuside on carbon tetrachloride (CCl₄)-induced hepatotoxicity. Rats were treated intraperitoneally with 0.5 mL/kg of CCl₄. Sixteen h after CCl₄ treatment, the levels of serum aminotransferases, tumor necrosis factor-α (TNF-α), and lipid peroxidation were significantly elevated, whereas the hepatic antioxidative enzyme activities were decreased. These changes were attenuated by cornuside. Histological studies also indicated that cornuside inhibited CCl₄-induced liver damage. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were elevated after CCl₄ treatment, while cytochrome P450 2E1 (CYP2E1) expression was suppressed. Cornuside treatment inhibited the formation of liver nitrite, and reduced the overexpression of iNOS and COX-2 proteins, but restored the liver CYP2E1 content as compared with the CCl₄-treated rats. Our data indicate that cornuside protects the liver from CCl₄-induced acute hepatotoxicity, perhaps due to its ability to restore the CYP2E1 function and suppress inflammatory responses, in combination with its capacity to reduce oxidative stress.     

Pathological Changes in Pulmonary Circulation in Carbon Tetrachloride (ccl4)-Induced Cirrhotic Mice

Rationale

Lack of an experimental model of portopulmonary hypertension (POPH) has been a major obstacle in understanding of pathophysiological mechanisms underlying the disease.

Objective

We investigated the effects of CCl₄-mediated cirrhosis on the pulmonary vasculature, as an initial step towards an improved understanding of POPH.

Methods And Results

Male C57BL/6 mice received intraperitoneal injection of either sterile olive oil or CCl₄ 3 times/week for 12 weeks. Cirrhosis and portal hypertension were confirmed by evidence of bridging fibrosis and nodule formation in CCl₄-treated liver determined by trichrome/picrosirius red staining and an increase in spleen weight/body weight ratio, respectively. Staining for the oxidative stress marker, 4-hydroxynonenal (4-HNE), was strong in the liver but was absent in the lung, suggesting that CCl₄ did not directly induce oxidative injury in the lung. Pulmonary acceleration time (PAT) and the ratio of PAT/pulmonary ejection time (PET) measured by echocardiography were significantly decreased in cirrhotic mice. Increase in right ventricle (RV) weight/body weight as well as in the weight ratio of RV/(left ventricle + septum) further demonstrated the presence of pathological changes in the pulmonary circulation in these mice. Histological examination revealed that lungs of cirrhotic mice have excessive accumulation of perivascular collagen and thickening of the media of the pulmonary artery.

Conclusion

Collectively, our data demonstrate that chronic CCl₄ treatment induces pathological changes in pulmonary circulation in cirrhotic mice. We propose that this murine cirrhotic model provides an exceptional tool for future studies of the molecular mechanisms mediating pulmonary vascular diseases associated with cirrhosis and for evaluation of novel therapeutic interventions.