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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.

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.

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.

Objective: To 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., Methods: The 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., Results: The 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., Conclusion: ERS 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.

Objective: To evaluate the roles of N-terminal lectin-like domain of thrombomodulin (TM-N) and receptor for advanced glycation end products (RAGE) in acute hepatic failure using a mouse model system., Methods: Acute hepatic failure was induced in Kunming mice by intraperitoneal injection of D-galactosamine (D-Galn at 600 mg/kg) and lipopolysaccharide (LPS at 5 mug/kg) and mice were divided into groups for injection with saline, recombinant (r)TM-N protein, or recombinant soluble (rs)RAGE protein. Unmanipulated model mice served as the negative controls. Effects on liver expression of high mobility group box-1 (HMGB1) were detected by immunohistochemistry and real time RT-PCR. Effects on serum levels of tumor necrosis factor-alpha (TNFa) and interleukin-1 beta (IL)-1b were quantified by ELISA., Results: Treatment with rTM-N and rsRAGE both alleviated the acute liver damage induced by D-Galn/LPS exposure, and decreased the hepatic expression of HMGB1 as well as the serum levels of TNFa and IL-1b., Conclusion: Intraperitoneal delivery of rTM-N and rsRAGE can alleviate acute liver damage by modulating the expression of necrosis- and inflammation-related factors.

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.

Objective: To 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., Methods: BALB/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., Results: The 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)., Conclusions: Liver-specific miR-122 supposed be a new marker molecule for early diagnosis of liver cells injury in the acute liver failure.

Objective: To 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., Methods: Immunological 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., Results: Most 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., Conclusion: We 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.