OBJECTIVETo establish a practical and reproducible animal model of human acute-on-chronic liver failure for further study of the pathophysiological mechanism of acute-on-chronic liver failure and for drug screening and evaluation in its treatment.

METHODSImmunological hepatic fibrosis was induced by human serum albumin in Wistar rats. In rats with early-stage cirrhosis (fibrosis stage IV), D-galactosamine and lipopolysaccharide were administered. Mortality and survival time were recorded in 20 rats. Ten rats were sacrificed at 4, 8, and 12 hours. Liver function tests and plasma cytokine levels were measured after D-galactosamine/lipopolysaccharide administration and liver pathology was studied. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay.

RESULTSMost of the rats treated with human albumin developed cirrhosis and fibrosis, and 90% of them died from acute liver failure after administration of D-galactosamine/lipopolysaccharide, with a mean survival time of (16.1+/-3.7) hours. Liver histopathology showed massive or submassive necrosis of the regenerated nodules, while fibrosis septa were intact. Liver function tests were compatible with massive necrosis of hepatocytes. Plasma level of TNFalpha increased significantly, parallel with the degree of the hepatocytes apoptosis. Plasma IL-10 levels increased similarly as seen in patients with acute-on-chronic liver failure.

CONCLUSIONWe established an animal model of acute-on-chronic liver failure by treating rats with human serum albumin and later with D-galactosamine and lipopolysaccharide. TNFalpha-mediated liver cell apoptoses plays a very important role in the pathogenesis of acute liver failure.

Animals ; Disease Models, Animal ; Female ; Galactosamine ; adverse effects ; Humans ; Lipopolysaccharides ; adverse effects ; Liver Failure, Acute ; chemically induced ; Rats ; Rats, Wistar ; Serum Albumin ; adverse effects

OBJECTIVETo apply an orthogonal design optimization strategy to a mouse model of acute liver failure induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS) exposure.

METHODSA 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.

RESULTSThe 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.

CONCLUSIONThe 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.

Animals ; Apoptosis ; Disease Models, Animal ; Galactosamine ; adverse effects ; Lipopolysaccharides ; adverse effects ; Liver Failure, Acute ; chemically induced ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVETo study the role of endoplasmic reticulum stress (ERS) in acute liver failure (ALF) using a mouse model of D-Galactosamine/lipopolysaccharide (D-GalN/LPS)-induced ALF.

METHODSThe ALF model was established by administering intraperitoneal (i.p.) injections of D-Ga1N (700 mg/kg) and LPS (10 mug/kg) to six C57BL/6 mice. Three of the modeled mice were also administered 4-phenylbutyrate (4-PBA; 100 mg/kg i.p.) at 6 hours before the onset of ALF and served as the intervention group. Non-modeled mice served as controls. All mice were analyzed by western blotting and qRT-PCR to determine the expression levels of ERS-related proteins in liver tissue. Liver function was assessed by measuring levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. Extent of injury to the liver tissue was assessed by hematoxylin-eosin staining and histological analysis. qRT-PCR was also used to detect differences in expression of inflammation-related genes, and western blotting was also used to detect differences in expression of the apoptosis related protein Caspase-3.The extent of apoptosis in liver tissue was assessed by TUNEL assay.

RESULTSThe ERS markers GRP78 and GRP94 showed increased expression at both the gene and protein levels which followed progression of ALF. The ERS effector proteins XBP-1, ATF-6 and IRE 1 a involved in the unfolded protein response were activated in the early stages of ALF, and the ERS-induced apoptosis regulators Caspase-12 and CHOP were activated in the late stage of ALF. Inhibition of ERS by 4-PBA intervention protected against injury to liver tissue and function, as evidenced by significantly lower levels of serum ALT and AST and a remarkably decreased extent of histological alterations. Furthermore, the inhibition of ERS suppressed expression of the proinflammatory cytokines TNFa, IL-6 and IL-1 β, and reduced the extent of hepatocyte apoptosis.

CONCLUSIONERS is activated in the mouse model of D-GalN/LPS-induced ALF. Inhibition of ERS may be protective against liver injury and the mechanism of action may involve reductions in inflammatory and apoptotic factors and/or signaling. Therefore, inhibiting ERS may represent a novel therapeutic approach for treating ALF.

Animals ; Apoptosis ; Disease Models, Animal ; Endoplasmic Reticulum Stress ; Galactosamine ; adverse effects ; Lipopolysaccharides ; adverse effects ; Liver Failure, Acute ; chemically induced ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVETo compare the effects of different approaches to establishing rat models of acute liver failure (ALF).

METHODSSixty-eight Sprague-Dawley rats were randomly divided into 3 groups for establishing ALF models using 3 different approaches, namely conventional hepatectomy for resecting 90% liver tissue as described by Higgins and Anderson, modified bloodless hepatectomy for resecting 90% liver tissue, and intraperitoneal injections of 700 mg/kg D-galactosamine (D-gal) and 5 µg/kg lipopolysaccharide (LPS). The mortality of the rats due to postoperative bleeding and survival rate at 7 days after the surgery were recorded. The levels of alanine aminotransferase (ALT), total bilimbin (Tbil), albumin (ALB), NH3, glucose (Glu) and prothrombin time (PT) were monitored, and histopathologies of the liver were examined at 24 and 72 h after the surgery.

RESULTSThe mortality rate due to postoperative bleeding was higher in conventional hepatectomy group than in the modified surgical group (15% vs 0). The survival rate at 7 days was 25%, 0%, 15% in conventional surgical group, modified surgical group and drug injection group, respectively. In the latter two groups, significant changes of ALT, Tbil, ALB, NH3, Glu, and PT were recorded at 24 and 72 h after the modeling (P<0.05), and these changes were the most obvious at 24 h in modified surgical group and at 72 h in the drug injection group; ALB in both groups declined to the lowest at 7 days and then increased gradually. Liver cell degeneration and necrosis were found in modified surgical group and drug injection group at 24 h and 72 h after the modeling.

CONCLUSIONBoth the modified 90% bloodless hepatectomy and injections of D-gal and LPS can be used to establish ideal rat models of ALF to suit different ALF-related researches.

Animals ; Disease Models, Animal ; Galactosamine ; adverse effects ; Hepatectomy ; Injections, Intraperitoneal ; Lipopolysaccharides ; adverse effects ; Liver Failure, Acute ; chemically induced ; pathology ; Rats ; Rats, Sprague-Dawley

Objective: To explore the new mechanism of liver fibrosis through D-galactosamine/lipopolysaccharide (D-GalN/LPS)-induced necroptosis as an entry point to inhibit lethal injury. Methods: The carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis was established. At 6 weeks of fibrosis, the mice were challenged with a lethal dose of D-GalN/LPS, and the normal mice treated with the same treatment were used as the control. The experiment was divided into four groups: control group (Control), acute injury group (D-GalN/LPS), liver fibrosis group (Fib), and liver fibrosis + acute challenge group (Fib + D-GalN/LPS). Quantitative PCR and immunofluorescence were used to analyze the expression of necroptosis key signal molecules RIPK1, RIPK3, MLKL and/or P-MLKL in each group. Normal mice were treated with inhibitors targeting key signaling molecules of necroptosis, and then given an acute challenge. The inhibitory effect of D-GalN/LPS-induced-necroptosis on acute liver injury was evaluated according to the changes in transaminase levels and liver histology. Liver fibrosis spontaneous ablation model was established, and then acute challenge was given. Necroptosis key signal molecules expression was analyzed in liver tissue of mice in each group and compared by immunohistochemistry. The differences between groups were compared with t-test or analysis of variance. Results: Quantitative PCR and immunofluorescence assays result showed that D-GalN/LPS-induced significant upregulation of RIPK1, RIPK3, MLKL and/or P-MLKL. Necroptosis key signal molecules inhibition had significantly reduced D-GalN/LPS-induced liver injury, as manifested by markedly reduced serum ALT and AST levels with improvement in liver histology. Necroptosis signaling molecules expression was significantly inhibited in fibrotic livers even under acute challenge conditions. Additionally, liver fibrosis with gradual attenuation of fibrotic ablation had inhibited D-GalN/LPS-induced necroptosis. Conclusion: Liver fibrosis may protect mice from acute lethal challenge injury by inhibiting D-GalN/LPS-induced necroptosis.
Animals ; Chemical and Drug Induced Liver Injury/pathology* ; Galactosamine/adverse effects* ; Lipopolysaccharides/adverse effects* ; Liver/pathology* ; Liver Cirrhosis/pathology* ; Liver Failure, Acute/chemically induced* ; Mice ; Necroptosis

OBJECTIVETo study the approaches for making acute-on-chronic hepatic failure model in rat.

METHODSSD rats were intraperitoneally injected with 50% CCl4 (Carbon tetrachloride) olive solution every three days for 6 or 10 weeks. Then they were divided into two groups randomly and injected with D-galactosamine (D-gal) at a dose of 2 g/kg, lipopolysaccharide (LPS) at a dose of 100 pg/kg and D-gal at a dose of 0.5 g/kg BW respectively. The levels of serum ALT, AST and TBil were detected,and histopathological changes were observed to evaluate these two models.

RESULTSAfter injection of 50% CCl4 olive solution intraperitoneally for 6 weeks, liver fibrosis happened in SD rats, 10 weeks, cirrhosis happened. Pelleta necrosis and massive or submassive necrosis was seen administration of two reagents mentioned above.

CONCLUSIONBy injecting of 50% CCl4 olive solution intraperitoneally, acute-on-chronic liver failure model could be induced by D-gal, LPS/D-gal in rats.

Alanine Transaminase ; metabolism ; Animals ; Carbon Tetrachloride ; adverse effects ; Disease Models, Animal ; Galactosamine ; adverse effects ; Humans ; Lipopolysaccharides ; adverse effects ; Liver Failure, Acute ; chemically induced ; enzymology ; mortality ; pathology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Objective: To investigate the effect of targeted carboxylesterase 1f (Ces1f) gene knockdown on the polarization activity of Kupffer cells (KC) induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in mice with acute liver failure. Methods: The complex siRNA-EndoPorter formed by combining the small RNA (siRNA) carrying the Ces1f-targeting interference sequence and the polypeptide transport carrier (Endoporter) was wrapped in β-1, 3-D glucan shell to form complex particles (GeRPs). Thirty male C57BL/6 mice were randomly divided into a normal control group, a model group (LPS/D-GalN), a pretreatment group (GeRPs), a pretreatment model group (GeRPs+LPS/D-GalN), and an empty vector group (EndoPorter). Real-time fluorescent quantitative PCR and western blot were used to detect Ces1f mRNA and protein expression levels in the liver tissues of each mouse group. Real-time PCR was used to detect the expression levels of KC M1 polarization phenotypic differentiation cluster 86(CD86) mRNA and KC M2 polarization phenotypic differentiation cluster 163 (CD163) mRNA in each group. Immunofluorescence double staining technique was used to detect the expression of Ces1f protein and M1/M2 polarization phenotype CD86/CD163 protein in KC. Hematoxylin-eosin staining was used to observe the pathological damage to liver tissue. A one-way analysis of variance was used to compare the means among multiple groups, or an independent sample nonparametric rank sum test was used when the variances were uneven. Results: The relative expression levels of Ces1f mRNA/protein in liver tissue of the normal control group, model group, pretreatment group, and pretreatment model group were 1.00 ± 0.00, 0.80 ± 0.03/0.80 ± 0.14, 0.56 ± 0.08/0.52 ± 0.13, and 0.26 ± 0.05/0.29 ± 0.13, respectively, and the differences among the groups were statistically significant (F = 9.171/3.957, 20.740/9.315, 34.530/13.830, P < 0.01). The percentages of Ces1f-positive Kupffer cells in the normal control group, model group, pretreatment group, and pretreatment model group were 91.42%, ± 3.79%, 73.85% ± 7.03%, 48.70% ± 5.30%, and 25.68% ± 4.55%, respectively, and the differences between the groups were statistically significant (F = 6.333, 15.400, 23.700, P < 0.01). The relative expression levels of CD86 mRNA in the normal control group, model group, and pretreatment model group were 1.00 ± 0.00, 2.01 ± 0.04, and 4.17 ± 0.14, respectively, and the differences between the groups were statistically significant (F = 33.800, 106.500, P < 0.01). The relative expression levels of CD163 mRNA in the normal control group, the model group, and the pretreatment model group were 1.00 ± 0.00, 0.85 ± 0.01, and 0.65 ± 0.01, respectively, and the differences between the groups were statistically significant (F = 23.360, 55.350, P < 0.01). The percentages of (F4/80(+)CD86(+)) and (F4/80(+)CD163(+)) in the normal control group and model group and pretreatment model group were 10.67% ± 0.91% and 12.60% ± 1.67%, 20.02% ± 1.29% and 8.04% ± 0.76%, and 43.67% ± 2.71% and 5.43% ± 0.47%, respectively, and the differences among the groups were statistically significant (F = 11.130/8.379, 39.250/13.190, P < 0.01). The liver injury scores of the normal control group, the model group, and the pretreatment model group were 0.22 ± 0.08, 1.32 ± 0.36, and 2.17 ± 0.26, respectively, and the differences among the groups were statistically significant (F = 12.520 and 22.190, P < 0.01). Conclusion: Ces1f may be a hepatic inflammatory inhibitory molecule, and its inhibitory effect production may come from the molecule's maintenance of KC polarization phenotypic homeostasis.
Animals ; Male ; Mice ; Carboxylesterase/genetics* ; Galactosamine ; Gene Knockdown Techniques ; Kupffer Cells ; Lipopolysaccharides/adverse effects* ; Liver Failure, Acute/chemically induced* ; Mice, Inbred C57BL ; RNA, Messenger

OBJECTIVETo reproduce a Kunming murine endotoxin shock model suitable for the anti-endotoxin pharmaceutical research.

METHODSKunming 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.

RESULTSThe 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.

CONCLUSIONThe Kunming mice showed signs of endotoxin shock after D-galactosamine presensitizing and endotoxin challenge, and it is suitable for anti-endotoxin pharmaceutical research.

Animals ; Disease Models, Animal ; Female ; Galactosamine ; adverse effects ; Male ; Mice ; Mice, Inbred Strains ; Serum ; chemistry ; Shock, Septic ; chemically induced ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVE: To investigate the effect of diazepam and modafinil on acute hepatic failure in mice. METHODS: Acute liver failure was induced in male Kunming strain mice by enterocoelia injecting the mice with D-GalN and LPS . The mice in the treatment groups were given corresponding drug 2 h before the administration of D-GalN and LPS, and the mice in the control group were given the same dose of distilled water. The 24-hour survival rate, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were compared. Serum levels of TNF-alpha and IL-1 and the levels of SOD, MDA, GR, GSH, NO and NOS in the liver were determined. RESULTS: Treatment with diazepam increased the survival rate and improved liver histological feature. Diazepam inhibited the serum levels of ALT, AST, TNF-alpha and IL-1, and reduced levels of MDA, NO and NOS and increased levels of GR and SOD in the liver. Modafinil decreased liver histological feature, increased the serum levels of ALT, AST, TNF-alpha and IL-1, increased level of MDA, and inhabited levels of SOD and GR in the liver. CONCLUSION Treatment with diazepam may suppress the D-GalN/LPS-induced acute hepatic failure and modafinil may facilitate the acute hepatic failure.
Animals ; Benzhydryl Compounds ; adverse effects ; therapeutic use ; Diazepam ; adverse effects ; therapeutic use ; Galactosamine ; Lipopolysaccharides ; Liver ; pathology ; Liver Failure, Acute ; chemically induced ; drug therapy ; pathology ; Male ; Mice ; Modafinil ; Random Allocation

OBJECTIVETo investigate the expression of miR-122 and its relationship with progression and development of acute liver failure in mice induced by D-GalN/LPS, and to explore new biomarker(s) for early diagnosis of acute liver failure.

METHODSBALB/C mice were randomly divided into four groups: the mice were given D-GalN (900 mg/kg body weight) and LPS (10 micog/kg body weight) intraperitoneally (i.p.) to construct the acute liver model; whereas the control groups were given D-GalN (900 mg/kg), LPS (10 microg/kg) and normal saline respectively. All biochemical and histological indexes were determined at 0, 1, 3, 5, 7 and 9 h respectively after administration. Real-time RT-PCR were used to detect the expression of miR-122 and pro-inflammatory cytokines, furthermore, the expression of miR-122 was verified by LNA (lock nucleic acid)-Northern-blot. ALT and AST levels were tested by biochemistry analyzer. Serum pro-inflammatory cytokine levels were tested by ELISA.

RESULTSThe mortality rate was about 80% at 24h after D-GalN/LPS treatment, but no mortality was observed in the other three control groups. Liver special miRNA miR-122 was highly expressed in liver tissue of normal mice (ct is approximately equal to 14), it was up-regulated significantly (P = 0.013) at first hour after treatment then down-regulated according to the development of acute liver failure, the change was more obvious at 9 h (ct is approximately equal to 15, P = 0.002). ALT and AST levels increased obviously at 3h after treatment and reached peak at 7 hours then they were declined sharply. It was found that the expression of miR-122 was faster and more durable than ALT. Pro-inflammatory cytokines related to acute liver failure including TNFa and IL-6 were all up-regulated in serum as well as liver tissue (P less than 0.05). Correlation analysis showed that miR-122 had a negative correlation with ALT (correlation coefficients -0.505) and positive correlations with TNFa and IL-6 (correlation coefficients were 0.493 and 0.674 respectively).

CONCLUSIONSLiver-specific miR-122 supposed be a new marker molecule for early diagnosis of liver cells injury in the acute liver failure.

Animals ; Galactosamine ; adverse effects ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; adverse effects ; Liver Failure, Acute ; chemically induced ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; MicroRNAs ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism