Protection by bicyclol derivatives against acetaminophen‐induced acute liver failure in mice and its active mechanism

Background/Aims: To find a novel drug against acute liver failure, a methionine derivative of bicyclol (WLP‐S‐10) was studied in acetaminophen‐injected mice. Methods: At first, 10 derivatives of bicyclol were tested in male KunMing strain mice injected with CCl₄, acetaminophen or d ‐galactosamine plus lipopolysaccharide (LPS), serum alanine aminotransferase (ALT) and mortality rate were determined. Among the 10 derivatives, a methionine derivative of bicyclol (WLP‐S‐10) was shown to be the most effective. A single dose of WLP‐S‐10 200 mg/kg was intraperitoneally injected 1 h before administration of a lethal dose of acetaminophen; the mortality rate, liver lesions, serum ALT, aspartate aminotransferase (AST) and liver glutathione (GSH) were determined. Mitochondrial GSH and adenosine triphosphate (ATP) levels, cytochrome C and apoptosis‐inducing factor (AIF) leakage, mitochondrial swelling and membrane potential were determined. Results: As a result, WLP‐S‐10 200 mg/kg significantly reduced liver injury induced by CCl₄ and decreased the mortality rate of mice because of acute liver failure caused by lethal dosage of acetaminophen or d ‐galactosamine plus LPS. WLP‐S‐10 200 mg/kg markedly reduced liver necrosis, serum ALT and AST elevation and GSH depletion after injection of acetaminophen. WLP‐S‐10 inhibited mitochondrial swelling, breakdown of membrane potential and depletion of mitochondrial ATP, and also reduced release of cytochrome C and AIF from mitochondria induced by acetaminophen. Conclusions: The results indicate that WLP‐S‐10 is a novel potential compound against acetaminophen‐induced acute liver failure in mice, and its active mechanism is mainly related to protection against necrosis and apoptosis of hepatocytes through inhibition of mitochondrial energy (ATP) depletion and AIF and cytochrome C release.     

Lipopolysaccharide‐induced cytokine and receptor expression and neutrophil infiltration in the liver of osteopetrosis (op/op) mutant mice

Abstract: Background/Aims: Mice homozygous for the osteopetrosis (op) mutation are genetically deficient in macrophage colony‐stimulating factor (M‐CSF/CSF‐1) and are characterized by defective differentiation and function of macrophages. The aim of this study is to assess the contribution of M‐CSF to lipopolysaccharide (LPS)‐induced cytokine expression and neutrophil infiltration in the liver. Methods: We investigated the effects of LPS administration in M‐CSF‐deficient op/op mutant mice. The expression of cytokines and receptors in the liver was studied by immunohistochemistry and RT‐PCR. Neutrophil infiltration in the liver was also examined. Results: After LPS administration, cytokine production and expression of LPS receptors, such as CD14 and scavenger receptor class A (MSR‐A), were induced at lower levels in op/op mice than those in littermate mice. Neutrophil infiltration in the liver of op/op mice did not differ significantly from that of littermate mice. Anti‐IL‐8 receptor homologue and anti‐C5a receptor antibody reduced the number of infiltrating neutrophils. Conclusions: These findings indicate that deficient macrophage activation following LPS injection in op/op mice is associated with decreased expression of CD14 and MSR‐A in the liver. Thus, M‐CSF plays a critical role in LPS‐induced macrophage activation but does not exert a dominant role in neutrophil infiltration in the liver.     

Dependence of interleukin‐1–induced arthritis on granulocyte–macrophage colony‐stimulating factor

Objective

To determine whether granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and macrophage CSF (M‐CSF or CSF‐1) are involved in the methylated bovine serum albumin/interleukin‐1 (mBSA/IL‐1)–induced arthritis model.

Methods

Following systemic injection, IL‐1 has been shown to augment a weak inflammatory response to mBSA in murine joints and to induce an acute erosive arthritis. GM‐CSF and M‐CSF have been implicated in inflammatory reactions, including those in joints, and have recently been shown to exacerbate murine arthritis. Since in vitro studies have found that IL‐1 can enhance GM‐CSF and M‐CSF production, we reasoned that they might be playing a part in IL‐1–mediated arthritis. GM‐CSF–deficient (GM‐CSF−/−) and M‐CSF–deficient (op/op) mice were injected intraarticularly with mBSA and subcutaneously with IL‐1. Arthritis was monitored histologically on day 7. Normal mice were also treated intraperitoneally with blocking monoclonal antibodies to GM‐CSF and M‐CSF, and to the M‐CSF receptor. Numbers of macrophages (Mac‐2 and F4/80 staining) were monitored, as was the number of cycling (bromodeoxyuridine‐positive) cells.

Results

GM‐CSF−/− mice and normal mice treated with anti–GM‐CSF antibody did not show IL‐1–induced arthritis progression. There was a dramatic reduction in synovial cellularity, including reduced numbers of macrophages and cycling cells. The op/op mice did not develop mBSA/IL‐1–induced disease, but blocking antibody to M‐CSF or to the M‐CSF receptor failed to diminish disease in normal mice.

Conclusion

GM‐CSF is involved in the IL‐1–induced arthritis that follows mBSA injection; M‐CSF involvement in the model is also suggested, since op/op mice did not develop arthritis. These studies provide the first in vivo evidence for a role of GM‐CSF, and possibly M‐CSF, in the proinflammatory actions of IL‐1.

     

Conditional knockout of heparin‐binding epidermal growth factor‐like growth factor in the liver accelerates carbon tetrachloride‐induced liver injury in mice

Aim: We previously demonstrated that heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is induced in response to several liver injuries. Because the HB‐EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver‐specific HB‐EGF conditional knockout mice using the interferon‐inducible Mx‐1 promoter driven cre recombinase transgene and investigated its role during acute liver injury. Methods: We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl₄) in HB‐EGF KO mice and wild‐type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB‐EGF‐dependent anti‐apoptosis and wound‐healing process of the liver in vitro. Results: HB‐EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl₄ injection. We also demonstrated that HB‐EGF treatment inhibited tumor necrosis factor‐α‐induced apoptosis of AML12 mouse hepatocytes and promoted the wound‐healing response of these cells. Conclusion: This study showed that HB‐EGF plays a protective role during acute liver injury.     

Salvage effect of E5564, Toll‐like receptor 4 antagonist on d‐galactosamine and lipopolysaccharide‐induced acute liver failure in rats

Background and Aims: The transmembrane protein Toll‐like receptor 4 (TLR4), which exists mainly in macrophages such as Kupffer cells of the liver, plays an important role in recognizing and mediating macrophage activation and pro‐inflammatory cytokine release. Activation of the pro‐inflammatory cytokine cascade, including tumor necrosis factor‐alpha (TNF‐α), has a pivotal role in the progression of severe liver injury. D‐galactosamine (GalN) and lipopolysaccharide (LPS)‐induced liver injury in rats is an experimental model of fulminant hepatic failure, where TNF‐α plays a central role in the progression of liver injury. E5564, a synthetic analogue of the lipid A component of endotoxin, inhibits endotoxin‐stimulated inflammation and is under study for patients with sepsis. In the present study, we sought to explore the salvage effect of TLR4 antagonist E5564 on GalN+LPS‐induced acute liver failure (ALF) in rats. Methods: ALF was induced in male Wistar rats by the intraperitoneal injection of GalN (500 mg/kg) and LPS (50 µg/kg). Immediately after GalN+LPS injection, rats were treated with intravenous injection of E5564 (3 mg/kg). The cumulative survival rates of GalN+LPS‐induced ALF rats were compared between those with and without E5564 treatment. Results: The intravenous injection of E5564 reduced the elevation of serum total bilirubin, aspartate aminotransferase, alanine aminotransferase and TNF‐α levels in rats at 3 h after GalN+LPS injection, and improved the survival rate of GalN+LPS‐induced ALF rats at 24 h (8% vs 43%). Conclusions: TLR4 antagonist E5564 reduced GalN+LPS‐induced acute liver injury in rats and improved the overall survival rate of GalN+LPS‐induced ALF rats. It may contribute to the treatment of ALF through blocking endotoxin‐induced TNF‐α overproduction of macrophages.     

Effects of salviainolic acid A (SA‐A) on liver injury: SA‐A action on hepatic peroxidation

Abstract: Background/Aims: This study aimed to investigate the actions of salviainolic acid A (SA‐A), an antiperoxidative component of Salvia miltiorrhiza (Sm), on rat liver injury and fibrosis. Methods: Acute and chronic rat liver injury models were established using carbon tetrachloride (CCl₄). After 48 h (acute) or during 6 weeks of CCl₄ injection, rats were further divided and treated with biphenyl dimethyl‐dicarboxylate (BDD) or colchicine, as a control antifibrotic treatment, with Sm, a herbal compound, or SA‐A, a water‐soluble extract of Sm. Liver function was investigated by assessing alanine transaminase (ALT) and aspartate transaminase (AST) activities, histological analysis, hydroxyproline (Hyp) and malondiadehyde (MDA) content. In vitro, isolated cultured hepatocytes were injured with CCl₄ gas for 24 h, followed by treatment with either vitamin E or various concentrations of SA‐A. The extent of hepatocyte injury was monitored by analyzing various lipid peroxidative parameters such as superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), lactase dehydrogenase (LDH), and glutathione peroxidase (GSH‐PX) levels in hepatocyte supernatants. Results: SA‐A significantly decreased abnormal serum ALT activity both in acutely and chronically injured rat livers, decreased abnormal serum AST activity, Hyp and MDA content and attenuated hepatic collagen deposition. After CCl₄ incubation and injury, the activities of AST, ALT CAT, GSH‐PX and LDH and MDA content in hepatocyte supernatants increased significantly, but GSH levels decreased significantly. SA‐A markedly improved these pathological changes in a dose‐dependent manner. 10^?4 mol/l SA‐A had stronger inhibitory action than vitamin E. Conclusions: Our studies suggest that SA‐A has antiperoxidative effects on injured hepatocytes in liver injury and fibrosis induced by CCl₄.     

Increased hepatic expression is a major determinant of serum alanine aminotransferase elevation in mice with nonalcoholic steatohepatitis

Background: Serum alanine aminotransferase (ALT) is a biomarker for hepatitis of various aetiologies including fatty liver disease. Increased serum ALT is thought to be related to its increased release from dying hepatocytes. Aim: We sought to understand the mechanisms by which serum ALT is elevated in a mouse model of experimental fatty liver disease where hepatocyte death is minimal. Methods: To induce fatty liver disease, female A/J mice were fed a methionine‐choline deficient (MCD) diet for up to 12 weeks. Serum and liver ALT expression and hepatic inflammation, necrosis and apoptosis were assessed and expressed relative to their expressions in control‐diet‐fed mice. Results: Feeding mice the MCD diet produced hepatic steatosis with minimal hepatic inflammation or necrosis. Liver cell apoptosis was not significantly increased by MCD diet treatment. Conversely, serum ALT activity was approximately four‐fold increased at 12 weeks of diet treatment, and ALT protein expressions in serum were correspondingly increased: ALT1 1.7‐fold and ALT2 1.9‐fold at 12 weeks. The expressions of ALT1 and ALT2 protein in liver increased over 2–12 weeks of MCD treatment. At 12 weeks, liver ALT1 protein was 2.27±0.31‐fold increased and ALT2 protein 4.72±0.48‐fold increased relative to their expressions in the mice fed a diet replete with methionine and choline. Liver ALT mRNA expressions were correspondingly increased: ALT1 mRNA 2.58‐fold and ALT2 mRNA 4.97‐fold at 12 weeks. Linear regression analysis showed a strong correlation between serum and liver tissue expressions for both ALT1 and ALT2. Conclusions: These findings suggest that induction of hepatic expression significantly contributes to increased serum ALT in this model of experimental fatty liver disease, whereas cell death appears not to.     

Transplantation of bone marrow cells reduces CCl4‐induced liver fibrosis in mice

Background: We investigated the reversibility of liver fibrosis induced with a CCl₄ injection and the role of stem cells in reversing the hepatic injury. Furthermore, the most effective cell fraction among bone marrow cells (BMCs) in the repair process was analysed. Methods: C57BL/6 mice were divided into four groups after 5 weeks of injection of CCl₄: control, sacrificed after 5 weeks, sacrificed at 10 weeks and sacrificed 5 weeks later after GFP‐donor BM transplantation. Liver function tests and real‐time polymerase chain reaction (PCR) of markers indicating liver fibrosis were compared between the groups. To identify the most effective BMC fraction that repairs liver injury, the mice were divided into three groups after the injection of CCl₄ for 2 days: granulocyte colony stimulating factor (G‐CSF) only, mononuclear cell (MNC) transplantation and Lin‐Sca‐1+c‐kit+haematopoietic stem cell (HSC) transplantation. Eight days after transplantation, the mice were harvested and morphometric, immunohistochemical analyses were performed to compare the expression of extracellular matrix and liver fibrosis‐related factors. Results: The liver fibrosis induced by CCl₄ was not spontaneously recovered but was persistent until 10 weeks, but the group injected with BMCs had less fibrosis and better liver function. Mobilization with G‐CSF increased the recovery of the injured liver and the best results were seen in those mice administered the MNC fraction and Lin‐Sca‐1+c‐kit+HSC fraction, with no difference between the two groups. Conclusion: BMC transplantation and stem cell mobilization with G‐CSF effectively treats liver injury in mice. These are promising techniques for autologous transplantation in humans with liver fibrosis.     

Vascular endothelial growth factor reduces Fas-mediated acute liver injury in mice

Background and Aim:  Fulminant hepatitis is still a fatal liver disease, and no specific treatment for it has been available. Vascular endothelial growth factor (VEGF) is the focus of attention because of its various actions. We investigated the effect of vascular endothelial growth factor (VEGF) on Fas-induced fulminant hepatic failure (FHF).
Method:  Male Balb/c mice were treated with an intraperitoneal injection of an anti-Fas antibody (Jo-2 Ab) with or without premedication with intraperitoneally administered human recombinant VEGF.
Results:  The serum level of alanine aminotransferase (ALT) was up to 300 times higher that of normal mice following the Jo-2 Ab injection, and histological analysis revealed hepatic injury and massive hepatocyte apoptosis. The VEGF significantly suppressed an elevation in serum ALT levels and hepatocyte apoptosis. Immunohistochemically, VEGF-treated mice showed that Bcl-xL in hepatocytes was strongly expressed.
Conclusions:  Since hepatocytes do not express VEGF receptors, we speculated that VEGF acts on sinusoidal endothelial cells (SECs) and promotes production of cytokines such as hepatocyte growth factor in SECs, resulting in reducing apoptosis through an increase expression of Bcl-xL in hepatocytes. We suggest that VEGF has a potent antiapoptotic effect on hepatocytes through cell–cell interaction between SECs and hepatocytes.     

Dietary fructose exacerbates hepatocellular injury when incorporated into a methionine‐choline‐deficient diet

Background: Methionine‐choline‐deficient (MCD) diets cause steatohepatitis in rodents and are used to model fatty liver disease in human beings. Recent studies have identified sucrose as a major contributor to MCD‐related liver disease through its ability to promote hepatic de novo lipogenesis. Aims: To determine whether glucose and fructose, the two constitutents of sucrose, differ in their capacity to provoke steatohepatitis when incorporated individually into MCD formulas. Materials & Methods: MCD and control formulas prepared with either glucose or fructose as the sole source of carbohydrate were fed to mice for 21 days. Liver injury was assessed biochemically and histologically together with hepatic gene expression and fatty acid analysis. Results: Mice fed MCD formulas developed similar degrees of hepatic steatosis whether they contained glucose or fructose. By contrast, mice fed MCD‐fructose developed significantly more hepatocellular injury than mice fed MCD‐glucose, judged by histology, apoptosis staining and serum alanine aminotransferase. Liver injury in MCD‐fructose mice coincided with an exaggerated rise in the ratio of long‐chain saturated to unsaturated fatty acids in the liver. Notably, hepatic inflammation was not enhanced in mice fed MCD‐fructose, correlating instead with hepatic lipid peroxidation, which was equivalent in the two MCD groups. Discussion: Fructose is more cytotoxic than glucose when used as the source of carbohydrate in MCD formulas. Conclusion: The data suggest the enhanced cytotoxicity of fructose in the MCD model is related to its ability to stimulate de novo lipogenesis, which yields harmful long‐chain saturated fatty acids.     

Adiponectin protects LPS-induced liver injury through modulation of TNF-alpha in KK-Ay obese mice

Adiponectin, an adipocytokine, has been identified in adipose tissue, and its receptors are widely distributed in many tissues, including the liver. The present study was performed to clarify the role of adiponectin in lipopolysaccharide (LPS)-induced liver injury using KK-Ay obese mice. We analyzed the effects of adiponectin pretreatment on liver injury induced by D-galactosamine/LPS (GalN/LPS) in KK-Ay obese mice. GalN/LPS treatment induced significant increases in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the blood, apoptotic and necrotic changes in hepatocytes, and/or showed a high degree of lethality. The GalN/LPS-induced liver injury was more pronounced in KK-Ay obese mice than in lean controls. Pretreatment with adiponectin ameliorated the GalN/LPS-induced elevation of serum AST and ALT levels and the apoptotic and necrotic changes in hepatocytes, resulting in a reduction in lethality. In addition, pretreatment with adiponectin attenuated the GalN/LPS-induced increases in serum and hepatic tumor necrosis factor alpha (TNF-alpha) levels and increased peroxisome proliferator-activated receptor (PPAR) alpha messenger RNA expression in the liver. Furthermore, abdominal macrophages from KK-Ay obese mice pretreated with adiponectin in vitro exhibited decreased LPS-induced TNF-alpha production compared with controls. Finally, adiponectin pretreatment also ameliorated TNF-alpha-induced liver injury. In conclusion, these findings suggest that adiponectin prevents LPS-induced hepatic injury by inhibiting the synthesis and/or release of TNF-alpha of KK-Ay obese mice.     

Etoposide prevents apoptosis in mouse liver with D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure resulting in reduction of lethality

D-Galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver injury is an experimental model of fulminant hepatic failure in which tumor necrosis factor alpha (TNF-alpha) plays a pivotal role. We examined the effects of etoposide on GalN/LPS-induced fulminant hepatic failure. Mice were given an intraperitoneal dose of GalN (800 microg/g body weight)/LPS (100 ng/g body weight) with and without intraperitoneal etoposide (10 microg/g body weight) treatment. Liver injury was assessed biochemically and histologically. TNF-alpha levels in the serum, and apoptosis of hepatocytes and CPP32/caspase-3 in the liver, were determined. GalN/LPS treatment caused lethal liver injury in 87% of animals (13 of 15). The effect was associated with significant increases in TNF-alpha and alanine transaminase (ALT) levels in serum, the number of apoptotic hepatocytes, CPP32/caspase-3 activity, and TNF receptor 1 (TNFR1) mRNA expression in the liver. Etoposide (10 microg/g body weight) was given 3 times (at 50, 26, and 4 hours before GalN/LPS administration). Treatment of GalN/LPS-treated mice with etoposide reduced apoptosis of hepatocytes, resulting in reduction of lethality (13% [2 of 15]), while another topoisomerase II inhibitor, IRCF-193, showed no significant effect. The antilethal effect of etoposide was also confirmed in GalN/TNF-alpha-induced fulminant hepatic failure. Etoposide treatment reduced CPP32/caspase-3 activity in the liver, although it did not alter the serum TNF-alpha levels or hepatic TNFR1 mRNA expressions. In addition, etoposide treatment enhanced the mRNA and protein expression of Bcl-xL, an antiapoptotic molecule in the liver. The present findings suggest that etoposide prevents endotoxin-induced lethal liver injury by up-regulation of Bcl-xL, and that etoposide could be useful for the treatment of TNF-alpha-mediated liver diseases.     

Inhibition of nuclear factor κB and phosphatidylinositol 3‐kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor α in mice

Background/aims: Tumor necrosis factor (TNF)‐α itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF‐α/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)‐κB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF‐α. Here, we focused on the roles of NF‐κB and phosphatidylinositol 3‐kinase (PI3K)‐regulated serine/threonine kinase Akt. Methods: TNF‐α was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IκBα (Ad5IκB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl‐2 family members and caspase activity were examined. Results: In the mice livers, treatment with Ad5IκB or the wortmannin suppressed the activation of NF‐κB or Akt, respectively. Suppression of either NF‐κB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF‐α administration. However, in mice treated with both Ad5IκB and wortmannin, TNF‐α administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IκB, wortmannin, and TNF‐α markedly increased the activation of caspase‐3 and ‐9, and activated caspase‐8 to a lesser degree, suggesting that TNF‐α‐induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF‐κB and PI3K/Akt pathways had no effect on expression of Bcl‐2 families. Conclusion: The inducible activation of NF‐κB and constitutive activation of Akt regulate hepatocyte survival against TNF‐α, which occurs independent of Bcl‐2 families.     

Hepatocyte growth factor protects against Fas‐mediated liver apoptosis in transgenic mice

Background: Apoptosis via the Fas/Fas ligand signalling system plays an important role in the development of various liver diseases. The administration of an agonistic anti‐Fas antibody to mice causes massive hepatic apoptosis and fulminant hepatic failure. Several growth factors including hepatocyte growth factor (HGF) have been found to prevent apoptosis. Methods: In this study, we demonstrated the overexpression of HGF to have a protective effect on Fas‐mediated hepatic apoptosis using a transgenic mice (Tg mice) model. Results: In HGF Tg mice, the elevation of alanine aminotransferase was dramatically inhibited at 12 and 24 h after the administration of 0.15 mg/kg anti‐Fas antibody. HGF Tg mice showed a significantly lower number of apoptotic hepatocytes at 12 h compared with wild‐type (WT) mice. Furthermore, 85% (six of seven) HGF Tg mice were able to survive after the administration of 0.3 mg/kg anti‐Fas antibody, while none of the WT mice survived. The Bcl‐xL expression was increased in HGF Tg mice, while there was no difference in the expression of Bax, Bid, Mcl‐1 and bcl‐2 between WT mice and HGF Tg mice. In addition, the HGF Tg mice showed more Akt phosphorylation than the WT mice both before and after the anti‐Fas antibody injection. Conclusions: Taken together, our findings suggest that HGF protects against Fas‐mediated liver apoptosis in vivo, and the upregulation of Bcl‐xL via Akt activation may also play a role in the protective effects of HGF.