Effects of in vivo targeted carboxylesterase 1f gene knockdown on the Kupffer cells polarization activity in mice with acute liver failure / 中华肝脏病杂志

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.

Liver fibrosis inhibits lethal injury through D-galactosamine/lipopolysaccharide-induced necroptosis / 中华肝脏病杂志

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.

Dexmedetomidine alleviates LPS/D-Gal-induced acute liver injury via up-regulation of LC3-II expression in mice / 生理学报

The aim of the present study was to investigate the effects of dexmedetomidine (DEX) on acute liver injury induced by lipopolysaccharide (LPS)/D-galactosamine (D-Gal) and the underlying mechanism. Male BALB/c mice were intraperitoneally injected with LPS/D-Gal to induce acute liver injury model, and pretreated with DEX or in combination with the autophagy inhibitor, 3-methyladenine (3-MA) 30 min before injection. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, as well as myeloperoxidase (MPO) activity in liver tissue were determined with the corresponding kits. Serum tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) levels were determined by ELISA. The protein expression levels of LC3-II and P62 in liver tissue were determined by Western blot. Liver histopathological changes were detected by HE staining. The results showed that, compared with control group, LPS/D-Gal enhanced ALT and AST activity, increased TNF-α and IL-6 levels, as well as MPO activity, up-regulated LC3-II and P62 protein expression levels, and significantly induced pathological damage in liver tissue. DEX reversed the above changes in the LPS/D-Gal group, whereas these protective effects of DEX were blocked by 3-MA. The above results suggest that DEX alleviates LPS/D-Gal-induced acute liver injury, which may be associated with the up-regulation of LC3-II protein expression and the activation of autophagy.

Effects of saikosaponin b_2 on inflammation and energy metabolism in mice with acute liver injury induced by LPS/GalN / 中国中药杂志

To study the effects of saikosaponin b2( SS-b2) on inflammatory factors and energy metabolism against lipopolysaccharide/galactosamine( LPS/Gal N) induced acute liver injury in mice. Mice were randomly divided into normal group( equal amount of normal saline),model group( 100 g·kg~(-1) LPS and 400 mg·kg~(-1) Gal N),low,medium,high dose group of SS-b2( SS-b25,10,20 mg·kg~(-1)·d-1) and positive control group( dexamethasone,10 mg·kg~(-1)). All of the groups except for the normal group were treated with LPS/Gal N though intraperitoneally injection to establish the acute liver injury model. The organ indexes were calculated. The levels of serum transaminases( ALT and AST) and the activities of ATPase( Na+-K+-ATPase,Ca2+-Mg2+-ATPase) in liver were detected. The activity of tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β) and interleukin-6( IL-6) were determined by the enzyme-linked immunosorbent assay( ELISA). The contents of lactate dehydrogenase( LDH) in liver were determined by micro-enzyme method. HE staining was used to observe the histopathological changes of the liver. Histochemical method was used to investigate the protein expression of liver lactate dehydrogenase-A( LDH-A). The protein expressions of Sirt-6 and NF-κB in the liver were detected by Western blot. According to the results,compared with the model group,there were significant changes in organ indexes in the high-dose group of SS-b2( P<0. 05). The level of ALT,AST,TNF-α,IL-1β,IL-6 and the activities of LDH in serum of mice with liver injury were significantly reduced in the medium and high dose groups of SS-b2( P<0. 01). With the increase of the concentration of SS-b2,the range of hepatic lesions and the damage in mice decreased. The activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in liver of mice were significantly enhanced in each dose group( P<0. 01). The expression of NF-κB in liver tissues was significantly down-regulated in the medium and high dose group( P<0. 01). Meanwhile,the expression of Sirt-6 protein in the liver of mice with acute liver injury was significantly increased in each dose group( P<0. 01).In summary,SS-b2 has a significant protective effect on LPS/Gal N-induced acute liver injury in mice,which may be related to the down-regulation of NF-κB protein expression and up-regulation of Sirt-6 protein expression to improve inflammatory injury and energy metabolism.

Luteolin and luteolin-7-O-glucoside protect against acute liver injury through regulation of inflammatory mediators and antioxidative enzymes in GalN/LPS-induced hepatitic ICR mice

BACKGROUND/OBJECTIVES: Anti-inflammatory and antioxidative activities of luteolin and luteolin-7-O-glucoside were compared in galactosamine (GalN)/lipopolysaccharide (LPS)-induced hepatitic ICR mice. MATERIALS/METHODS: Male ICR mice (6 weeks old) were divided into 4 groups: normal control, GalN/LPS, luteolin, and luteolin-7-O-glucoside groups. The latter two groups were administered luteolin or luteolin-7-O-glucoside (50 mg/kg BW) daily by gavage for 3 weeks after which hepatitis was induced by intraperitoneal injection of GalN and LPS (1 g/kg BW and 10 µg/kg BW, respectively). RESULTS: GalN/LPS produced acute hepatic injury by a sharp increase in serum AST, ALT, and TNF-α levels, increases that were ameliorated in the experimental groups. In addition, markedly increased expressions of cyclooxygenase (COX)-2 and its transcription factors, nuclear factor (NF)-κB and activator protein (AP)-1, were also significantly attenuated in the experimental groups. Compared to luteolin-7-O-glucoside, luteolin more potently ameliorated the levels of inflammatory mediators. Phase II enzymes levels and NF-E2 p45-related factor (Nrf)-2 activation that were decreased by GalN/LPS were increased by luteolin and luteolin-7-O-glucoside administration. In addition, compared to luteolin, luteolin-7-O-glucoside acted as a more potent inducer of changes in phase II enzymes. Liver histopathology results were consistent with the mediator and enzyme results. CONCLUSION: Luteolin and luteolin-7-O-glucoside protect against GalN/LPS-induced hepatotoxicity through the regulation of inflammatory mediators and phase II enzymes.

Evaluating the best time to intervene acute liver failure in rat models induced by d-galactosamine

ABSTRACT PURPOSE: To describe an animal model for acute liver failure by intraperitoneal d-galactosamine injections in rats and to define when is the best time to intervene through King's College and Clichy´s criteria evaluation. METHODS: Sixty-one Wistar female rats were distributed into three groups: group 1 (11 rats received 1.4 g/kg of d-galactosamine intraperitoneally and were observed until they died); group 2 (44 rats received a dose of 1.4 g/kg of d-galactosamine and blood and histological samples were collected for analysis at 12 , 24, 48 , 72 and 120 hours after the injection); and the control group as well (6 rats) . RESULTS: Twelve hours after applying d-galactosamine, AST/ALT, bilirubin, factor V, PT and INR were already altered. The peak was reached at 48 hours. INR > 6.5 was found 12 hours after the injection and factor V < 30% after 24 hours. All the laboratory variables presented statistical differences, except urea (p = 0.758). There were statistical differences among all the histological variables analyzed. CONCLUSION: King's College and Clichy´s criteria were fulfilled 12 hours after the d-galactosamine injection and this time may represent the best time to intervene in this acute liver failure animal model.

Association between protective effect of Liuwei Wuling tablets against acute liver injury and its inhibitory effect on cytoplasmic translocation of high-mobility group box-1 in hepatocytes in mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of Liuwei Wuling tablets on the cytoplasmic translocation and release of high-mobility group box-1 (HMGB1) in hepatocytes in mice with acute live injury induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS).</p><p><b>METHODS</b>A Balb/c mouse model of acute liver injury was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (5 ug/kg). A total of 24 healthy mice were randomly and equally divided into acute liver injury control group and Liuwei Wuling tablet treatment group. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in both groups at each time point within one week. Liver tissues were collected at 36 hours to perform pathological examination. The serum levels of HMGB1, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), complement 3a (C3a), and complement 5a (C5a) were measured. Immunohistochemistry was used to determine the expression and cytoplasmic translocation of HMGB1 in hepatocytes.</p><p><b>RESULTS</b>At 6, 12, and 24 hours, the Liuwei Wuling tablet treatment group had significantly lower serum levels of ALT than the control group (225.33±181.64 U/L vs 471.17±174.72 U/L, t = 3.38, P < 0.01; 1509.53±182.51 U/L vs 7149.52±734.25 U/L, t = 25.82, P < 0.01; 162.89±86.51 U/L vs 1318.16±557.71 U/L, t = 7.09, P < 0.01), as well as significantly lower serum levels of AST than the control group (179.22±94.57 U/L vs 561.91±209.6 U/L, t = 5.76, P < 0.01; 590.92±190.92 U/L vs 2266.48±705.64 U/L, t = 7.94, P < 0.01; 231.24±87.7 U/L vs 444.32±117.01 U/L, t = 5.05, P < 0.01). The treatment group had significantly lower levels of HMGB1 than the control group at 6 and 12 hours (54.21±11.89 ng/ml vs 72.07±13.65 ng/ml, t = 3.41, P < 0.01; 49.23±5.97 ng/ml vs 68.71±13.07 ng/ml, t = 4.70, P < 0.01). The treatment group had significantly lower levels of TNF-α, IL-1β, and IL-6 than the control group at 12 hours (163.62±9.12 pg/ml vs 237.09±51.47 pg/ml, t = 4.86, P < 0.01; 15.66±13.13 pg/ml vs 37.43±18.68 pg/ml, t = 3.30, P < 0.01; 7.10±3.06 pg/ml vs 21.42±8.23 pg/ml, t = 5.65, P < 0.01). The treatment group had significantly lower levels of C3a and C5a than the control group at 12 hours (2.52±1.27 pg/ml vs 9.83±2.96 ng/ml, t = 7.86, P < 0.01; 2.16±1.03 ng/ml vs 7.23±1.55 ng/ml, t = 9.67, P < 0.01). Compared with the control group, the treatment group had significantly reduced liver inflammation and necrosis, and a significantly lower cytoplasmic translocation rate of HMGB1 in hepatocytes (38.76%±7.37% vs 8.15%±2.11%, P < 0.01).</p><p><b>CONCLUSION</b>Liuwei Wuling tablets can reduce the cytoplasmic translocation of HMGB1 in hepatocytes and relieve liver inflammation in mice with acute liver injury.</p>

5-Hydroxymethylfurfural protects against ER stress-induced apoptosis in GalN/TNF-α-injured L02 hepatocytes through regulating the PERK-eIF2α signaling pathway / 中国天然药物

5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine (GalN) and tumor necrosis factor-α (TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by GalN/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that GalN/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca(2+) concentration, the activation of protein kinase RNA (PKR)-like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with GalN/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the GalN/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect GalN/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERK-eIF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy.

Role of the Notch signaling pathway in development of acute liver failure in a mouse model / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of the Notch signaling pathway, and the underlying mechanism, in development of acute liver failure (ALF) in a mouse model.</p><p><b>METHODS</b>For in vivo analysis of the role of Notch signaling in ALF, a mouse model of ALF was generated by intraperitoneal injection of 3.0 g/kg D-galactosamine. Histological specimens were stained by hematoxylin-eosin, and then studied microscopically.Expression level of Jaggedl, Notchl, NICD, and Hes5 was measured by western blotting (for protein) and real time-PCR (for mRNA). The level of CD68 protein was detected by immunohistochemical staining. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-10, high mobility group box 1 (HMGB1) chromatin protein, and lipopolysaccharide (LPS) were measured by standard methods. For in vitro analysis of the molecular mechanism, the RAW264.7 macrophage cell line was cultured with LPS in the absence or presence of the Notch inhibitor DAPT, and the intracellular levels of Notch1, NICD, and Hes5 were measured by western blotting and real time-PCR and the extracellular levels of IL-10 and HMGB1 were detected in the supematant.</p><p><b>RESULTS</b>Compared with unmodeled (normal control) mice, the ALF modeled mice showed higher levels of serum ALT (848.40+/-94.83 U/L vs. 38.99+/-9.63 U/L), AST (911.49+/-67.65 U/L vs. 55.28+/-7.50 U/L), HMGB1 (101.91+/-12.43 µg/L vs. 20.73+/-5.37 µg/L), 1L-10 (4 627.88+/-842.45 pg/mL vs. 1 064.92+/-238.46 pg/mL) and LPS (11.80+/-0.89 EU/mL vs. 0.58+/-0.12 EU/mL), as well as higher expression of Jagged1 (mRNA: 7.63+/-1.41 vs. 1.00+/-0.00; protein: 0.71+/-0.07 vs. 0.34+/-0.07), Notch1 (mRNA: 7.10+/-0.66 vs. 1.00+/-0.00; protein: 0.66+/-0.11 vs. 0.27+/-0.08), NICD (protein: 0.76+/-0.08 vs. 0.27+/-0.08), Hes5 (mRNA: 7.95+/-0.71 vs. 1.00+/-0.00; protein: 1.20+/-0.07 vs. 0.76+/-0.07), and CD68 (protein: 7 685.05+/-417.34 vs. 2 294.01+/-392.93) (all P<0.01). In vitro, LPS increased the extracellular levels of HMGB1 (7.44+/-0.63 vs. 0.21+/-0.05), IL-10 (315.19+/-79.13 vs. 59.19+/-23.30) and the intracellular expression of Notch1 (mRNA: 6.49+/-0.73 vs. 1.00+/-0.00), NICD (protein: 0.65+/-0.10 vs. 0.23+/-0.07), and Hes5 (mRNA: 7.30+/-0.85 vs. 1.00+/-0.00; protein: 0.96+/-0.10 vs. 0.54+/-0.07) (all P<0.01). DAPT treatment led to a decrease above the index serum levels of HMGB1 (6.22+/-0.71) and IL-10 (252.06+/-57.63), and of expression of Notch 1 (mRNA: 3.20+/-0.68), NICD (protein: 0.42+/-0.05), and Hes5 (mRNA: 4.72+/-0.67; protein: 0.84+/-0.09) (P<0.01 or <0.05).</p><p><b>CONCLUSION</b>The Notch signaling pathway may plan an important role in the development of ALF upon activation of the pathway in macrophages by LPS and leading to promoted secretion of HMGB 1 and IL-10, with a greater effect on the former.</p>

Edaravone protects endotoxin-induced liver injury by inhibiting apoptosis and reducing proinflammatory cytokines

Studies have shown that edaravone may prevent liver injury. This study aimed to investigate the effects of edaravone on the liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in female BALB/c mice. Edaravone was injected into mice 30 min before and 4 h after GalN/LPS injection. The survival rate was determined within the first 24 h. Animals were killed 8 h after GalN/LPS injection, and liver injury was biochemically and histologically assessed. Hepatocyte apoptosis was measured by TUNEL staining; proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in the liver were assayed by ELISA; expression of caspase-8 and caspase-3 proteins was detected by Western blot assay; and caspase-3 activity was also determined. Results showed that GalN/LPS induced marked elevations in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Edaravone significantly inhibited elevation of serum AST and ALT, accompanied by an improvement in histological findings. Edaravone lowered the levels of TNF-α and IL-6 and reduced the number of TUNEL-positive cells. In addition, 24 h after edaravone treatment, caspase-3 activity and mortality were reduced. Edaravone may effectively ameliorate GalN/LPS-induced liver injury in mice by reducing proinflammatory cytokines and inhibiting apoptosis.

Anti-inflammatory and hepatoprotective effects of total flavonoid C-glycosides from Abrus mollis extracts / 中国天然药物

The aim of this study was to evaluate the anti-inflammatory and hepatoprotective effects of the total flavonoid C-glycosides isolated from Abrus mollis extracts (AME). In the anti-inflammatory tests, xylene-induced ear edema model in mice and carrageenan-induced paw edema model in rats were applied. The hepatoprotective effects of AME were evaluated with various in vivo models of acute and chronic liver injury, including carbon tetrachloride (CCl4)-induced hepatitis in mice, D-galactosamine (D-GalN)-induced hepatitis in rats, as well as CCl4-induced hepatic fibrosis in rats. In the acute inflammation experiment, AME significantly suppressed xylene-induced ear edema and carrageenan-induced paw edema, respectively. In the acute hepatitis tests, AME significantly attenuated the excessive release of ALT and AST induced by CCl4 and D-GalN. In CCl4-induced hepatic fibrosis model, AME alleviated liver injury induced by CCl4 shown by histopathological sections of livers and improved liver function as indicated by decreased liver index, serum ALT, AST, TBIL, and ALP levels and hydroxyproline contents in liver tissues, and increased serum ALB and GLU levels. These results indicated that AME possesses potent anti-inflammatory activity in acute inflammation models and hepatoprotective activity in both acute and chronic liver injury models. In conclusion, AME is a potential anti-inflammatory and hepatoprotective agent and a viable candidate for treating inflammation, hepatitis, and hepatic fibrosis.

Protective effect of baicalin solid dispersion on D-galactosamine induced acute hepatic injury in mice / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the protective effect of baicalin solid dispersion (BSD) on D-galactosamine (D-GalN) induced acute hepatic injury in mice, and to compare it with baicalin alone.</p><p><b>METHODS</b>Sixty mice were randomly divided into six groups, i.e., the normal control group, the D-GalN model group, the bifendate group (at the daily dose of 200 mg/kg), the baicalin group (at the daily dose of 50 mg/kg), the low dose BSD group (at the daily dose of 50 mg/kg), and the high dose BSD group (at the daily dose of 100 mg/kg), 10 in each group. 0.5% CMC-Na at 20 mL/kg was administered to mice in the normal group and the model group by gastrogavage, while corresponding medication was administered to mice in the other three groups by gastrogavage. Seven days after administration, acute hepatic injury model was induced by intraperitoneal injection of D-GalN. The liver index and the spleen index were calculated. The serum activities of alanine aminotransferase (ALT) and asparate aminotransferase (AST), the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in the liver homogenate were measured. The pathological changes of the liver tissue were observed by HE staining.</p><p><b>RESULTS</b>Compared with the normal control group, widespread inflammation and necrosis was significant in the liver tissue of the D-GalN model group; the liver index, serum ALT and AST levels and hepatic MDA content obviously increased, hepatic SOD activity decreased, showing statistical difference (P < 0.05). Compared with the model group, the liver index, the serum levels of ALT and AST, and hepatic MDA decreased, hepatic SOD increased, the degree of hepatic tissue injury was significantly improved in the low dose and high dose BSD groups. Besides, better effects were obtained in the low dose BSD group than in the baicalin group with statistical difference (P < 0.05).</p><p><b>CONCLUSION</b>BSD could significantly protect D-GalN induced acute hepatic injury of mice, and its effect was superior to that of baicalin alone.</p>

Role of endoplasmic reticulum stress in D-GalN/LPS-induced acute liver failure / 中华肝脏病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Establishment of a rat model of acute liver failure by a modified 90% bloodless hepatectomy and by D-galactosamine and lipopolysaccharide injection / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the effects of different approaches to establishing rat models of acute liver failure (ALF).</p><p><b>METHODS</b>Sixty-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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Both 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.</p>

Expression of intestinal defensin-5, soluble phospholipase A2 and lysozyme and the relation to bacterial translocation in rat models of acute liver failure / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the intestinal expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in acute liver failure (ALF) using rat models, and to determine the relation of these expressions to intestinal bacterial translocation.</p><p><b>METHODS</b>Forty-eight healthy male Sprague-Dawley rats were divided into a control group (n=8) and a model group (n=40; intraperitoneal injection of 10% D-galactosamine). The model group was further divided into five subgroups according to the time lapse after model establishment (8, 16, 24, 48, and 72 hours). At the end of the experiments, homogenates of mesenteric lymph nodes, liver and spleen were cultured in agar for bacterial outgrowth.Hematoxylin-eosin stained sections of liver and terminal ileum were examined under an optical microscope to assess pathological changes. mRNA expression of RD-5, sPLA2 and lysozyme in the terminal ileum was determined by reverse transcription-polymerase reaction (RT-PCR), and protein expression of sPLA2 and lysozyme from the same anatomic location was determined by western blotting and immunohistochemistry. Means between groups were compared with one-way analysis of variance.</p><p><b>RESULTS</b>ALF was successfully induced in the D-galactosamine injected rats. No bacteria grew in the organ cultures from the control group, while 8.3%, 37.5% and 58.3% of the rats in the 24-, 48-and 72-hour model groups showed positive cultures. Despite this, the structure of the terminal ileum from the rats in the 72-hour model group was nearly intact, without obvious necrosis of mucosal epithelial cells. Expression of RD-5 and sPLA2 mRNA in the model groups gradually increased at early time points and peaked at 16 hours after induction of ALF (1.291+/-0.153 and 1.131+/-0.128), which was significantly higher than that detected in the control group (0.725+/-0.116 and 0.722+/-0.112, t=69.25, 95.71, all P<0.01). After that, the expression of RD-5 and sPLA2 mRNA progressively decreased, and by 72 hours after the induction of ALF, the expression (0.415+/-0.104 and 0.425+/-0.076) was significantly lower than that of the control group (t=31.55 and 44.98, all P<0.01). Lysozyme mRNA expression in the model group peaked at 8 hours after ALF induction (1.211+/-0.107), which was higher than that of the control group at this time point (0.853+/-0.093), and by 72 hours after ALF induction it declined to 0.704+/-0.103, which was significantly lower than that of the control group (t=9.224; all P=0.009). In addition, at 72 hours after ALF induction the protein expression of both lysozyme and sPLA2 was significantly lower in the model group (0.327+/-0.086 and 0.382+/-0.057) than in the control group (0.583+/-0.121 and 0.650+/-0.093, t=12.28 and 15.83, P=0.004 and 0.001). Similar results were obtained with immunohistochemical staining.</p><p><b>CONCLUSION</b>The function of the ileal mucosal immune barrier in the rat model of acute liver failure decreased, along with decreases in expression of RD-5, sPLA2 and lysozyme in the Paneth cells.At the same time, the rate of organ bacterial translocation increased without obvious injury to the intestinal mucosa structure.</p>

Carvacrol suppresses the expression of inflammatory marker genes in D-galactosamine-hepatotoxic rats

OBJECTIVE@#To unravel the mechanism of anti-inflammatory activity of carvacrol in D-galactosamine (D-GalN)-induced hepatotoxic rats.@*METHODS@#The mRNA and protein expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nuclear factor kappa-B (NF-κB) were assayed by semi-quantitative reverse transcriptase polymerase chain reaction (RTPCR) and western blot analysis.@*RESULTS@#We found that the mRNA and protein expressions of TNF-α, IL-6, iNOS, COX-2 and NF-κB were significantly up-regulated in D-galactosamine induced hepatotoxic rats and treatment with carvacrol significantly down-regulated the expressions of these genes showing the mechanism behind the anti-inflammatory activity of carvacrol.@*CONCLUSIONS@#All above results reveal that the carvacrol well known anti-inflammatory activities in D-galactosamine induced hepatotoxic rats.

Protective effect of soyasaponins on acute liver injury induced by D-galactosamine and lipopolysaccharide in mice / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the protective effect of soyasaponins on acute liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in mice.</p><p><b>METHOD</b>The mice were randomly divided into five groups: the normal control, the model group, the silymarin (positive control) group, and soyasaponins high and low-dose groups. They were administered with drugs once every day for 7 days. At the end of the experiment, GalN and LPS were injected intraperitoneally to all of the groups except for the normal group to establish the acute liver injury model. The pathological changes were detected with hematoxylin & eosin (HE) staining, tumor necrosis factor-alpha (TNF-alpha) was detected by ELISA method, and the alanine aminotransferase (ALT), aspartate aminotransferase (AST), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), reduced glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), and the activation of Caspase-3 and Caspase-8 were detected by the colorimetric method.</p><p><b>RESULT</b>Soyasaponins could reduce the activities of serum ALT and AST, the acute hepatic injury induced by GalN/LPS, serum TNF-alpha level, hepatic NO and MDA contents, and the Caspase-3 and Caspase-8 activations of liver tissues, and increase the hepatic CAT, GPx, GST and GSH levels.</p><p><b>CONCLUSION</b>Soyasaponins shows the protective effect on acute liver injury induced by GalN and LPS in mice, which may be related to its antioxidative ability and anti-liver apoptosis.</p>

Orthogonal design based optimization of a mouse model of acute liver failure induced by D-galactosamine and lipopolysaccharide / 中华肝脏病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Protective effect of recombinant TM-N and recombinant soluble RAGE in a mouse model of acute hepatic failure / 中华肝脏病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Intraperitoneal delivery of rTM-N and rsRAGE can alleviate acute liver damage by modulating the expression of necrosis- and inflammation-related factors.</p>

Lycopene stabilizes lipoprotein levels during D-galactosamine/lipopolysaccharide induced hepatitis in experimental rats

<p><b>OBJECTIVE</b>To investigate the effect of lycopene on lipoprotein metabolism during D-galactosamine/lipopolysaccharide (D-Gal/LPS) induced hepatitis in experimental rats.</p><p><b>METHODS</b>The efficacy of lycopene was validated during D-Gal/LPS induced hepatitis by analyzing the activity of lipid metabolizing enzymes such as lipoprotein lipase (LPL), lecithin-cholesterol acyl transferase (LCAT) and hepatic triglyceride lipase (HTGL). Lipo protein analyses were done by the estimation of very low density lipoprotein cholesterol (VLDL), low density lipoprotein cholesterol (LDL) and high density lipoprotein cholesterol (HDL).</p><p><b>RESULTS</b>The toxic insult of D-galactosamine/lipopolysaccharide (D-Gal/LPS) in experimental group of animals reduces the normal values of lipid metabolizing enzymes due to liver injury. The significant drop in the levels of HDL and concomitant increase in the values of VLDL and LDL were observed. The pretreatment of lycopene restore these altered values to near normal level in experimental group of animals.</p><p><b>CONCLUSIONS</b>In the light of results, it can be concluded that administration lycopene stabilizes the lipoprotein levels by regulating the lipid metabolizing enzymes through its antioxidant defense and helps to maintain the normal lipid metabolism during toxic injury in liver.</p>