Decreased Expression and Uncoupling of Endothelial Nitric Oxide Synthase in the Cerebral Cortex of Rats with Thioacetamide-Induced Acute Liver Failure.

Acute liver failure (ALF) is associated with deregulated nitric oxide (NO) signaling in the brain, which is one of the key molecular abnormalities leading to the neuropsychiatric disorder called hepatic encephalopathy (HE). This study focuses on the effect of ALF on the relatively unexplored endothelial NOS isoform (eNOS). The cerebral prefrontal cortices of rats with thioacetamide (TAA)-induced ALF showed decreased eNOS expression, which resulted in an overall reduction of NOS activity. ALF also decreased the content of the NOS cofactor, tetrahydro-L-biopterin (BH4), and evoked eNOS uncoupling (reduction of the eNOS dimer/monomer ratio). The addition of the NO precursor L-arginine in the absence of BH4 potentiated ROS accumulation, whereas nonspecific NOS inhibitor L-NAME or EDTA attenuated ROS increase. The ALF-induced decrease of eNOS content and its uncoupling concurred with, and was likely causally related to, both increased brain content of reactive oxidative species (ROS) and decreased cerebral cortical blood flow (CBF) in the same model.

Liver-specific Bid silencing inhibits APAP-induced cell death in mice.

Acetaminophen (APAP)-induced acute liver failure (ALF) is a life-threatening disease with only a few treatment options available. Though extensive research has been conducted for more than 40 years, the underlying pathomechanisms are not completely understood. Here, we studied as to whether APAP-induced ALF can be prevented in mice by silencing the BH3-interacting domain death agonist (Bid) as a potential key player in APAP pathology. For silencing Bid expression in mice, siRNABid was formulated with the liver-specific siRNA delivery system DBTC and administered 48 h prior to APAP exposure. Mice which were pre-treated with HEPES (vehicleHEPES) and siRNALuci served as siRNA controls. Hepatic pathology was assessed by in vivo fluorescence microscopy, molecular biology, histology and laboratory analysis 6 h after APAP or PBS exposure. Application of siRNABid caused a significant decrease of mRNA and protein expression of Bid in APAP-exposed mice. Off-targets, such as cytochrome P450 2E1 and glutathione, which are known to be consumed under APAP intoxication, were comparably reduced in all APAP-exposed mice, underlining the specificity of Bid silencing. In APAP-exposed mice non-sterile inflammation with leukocyte infiltration and perfusion failure remained almost unaffected by Bid silencing. However, the Bid silencing reduced hepatocellular damage, evident by a remarkable decrease of DNA fragmented cells in APAP-exposed mice. In these mice, the expression of the pro-apoptotic protein Bax, which recently gained importance in the cell death pathway of regulated necrosis, was also significantly reduced, in line with a decrease in both, necrotic liver tissue and plasma transaminase activities. In addition, plasma levels of HMGB1, a marker of sterile inflammation, were significantly diminished. In conclusion, the liver-specific silencing of Bid expression did not protect APAP-exposed mice from microcirculatory dysfunction, but markedly protected the liver from necrotic cell death and in consequence from sterile inflammation. The study contributes to the understanding of the molecular mechanism of the APAP-induced pathogenic pathway by strengthening the importance of Bid and Bid silencing associated effects.

Acute liver failure after sevoflurane anesthesia in a pediatric patient. / Fallo hepático agudo tras anestesia con sevoflurano en un paciente pediátrico.

Sevoflurane is a volatile anesthetic characterized by low toxicity and is the most used in pediatric age. Unlike other halogenated anesthetic, sevoflurane is not metabolized to reactive intermediates that lead to the formation of hepatotoxic proteins. However, a few cases of hepatotoxicity have been associated with its use. We report a case of an 11-year-old boy who developed acute liver failure after neurosurgical intervention, resection of a posterior fossa mass, under sevoflurane anesthesia and other drugs. Postoperatively, he presented abdominal pain and the laboratory tests showed markedly elevated aminotransferase levels, coagulopathy and thrombocytopenia. He had no fever, hypoglycemia or evidence of encephalopathy. The clinical evolution was favorable and after 7 days, laboratory values were completely normalized. All the possible causes of acute liver failure were evaluated and the differential diagnosis is exposed.

Mesenchymal stem cells increase heme oxygenase 1-activated autophagy in treatment of acute liver failure.

In recent years, transplantation of mesenchymal stem cells (MSCs) has attracted much attention as a potential cell-based therapy for acute liver failure (ALF). As an inducible enzyme, heme oxygenase 1 (HO-1) has been reported to have cytoprotective, anti-apoptotic and immunoregulatory effects. Autophagy, a conserved catabolic process in cells, may be an important pathway for MSCs to treat ALF. In this study, we aimed to explore whether MSCs treat ALF by regulating autophagy and whether HO-1 was involved in the same pathway. Bone marrow-derived MSCs were isolated from Sprague-Dawley rats and cultured according to an established protocol. Co-culture systems of MSCs and hepatocytes were used to assess autophagy in the treatment of ALF. Meanwhile, MSCs were transplanted into rats with d-galactosamine (Gal)-induced ALF. Autophagy inhibitor (3-methyladenine, 3-MA), HO-1 inhibitor (zinc protoporphyrin, ZnPP) and PI3K specific inhibitor (LY294002) were employed in the study. Blood samples and liver tissues were collected before euthanasia. Survival rate, liver function, inflammatory factors, histology, Ki67 and TUNEL staining were determined. MSCs transplantation alleviated ALF both in vivo and in vitro. Autophagy and autophagy-related proteins were significantly up-regulated during MSCs treatment. 3-MA attenuated the therapeutic effect of MSCs. Administration of LY294002 before ALF induction inhibited hepatocyte autophagy. During the MSCs treatment, the HO-1 expression was increased, while inhibiting HO-1 attenuated the therapeutic effect of MSCs as well as hepatocyte autophagy. These findings suggested MSCs could alleviate ALF by increasing the HO-1 expression, which played an important role in activating autophagy through PI3K/AKT signaling pathway.

Protective role of c-Jun N-terminal kinase-2 (JNK2) in ibuprofen-induced acute liver injury.

Ibuprofen is a worldwide used non-steroidal anti-inflammatory drug which may cause acute liver injury (ALI) requiring liver transplantation. We aimed to unveil the molecular pathways involved in triggering ibuprofen-induced ALI, which, at present, remain elusive. First, we investigated activation of essential pathways in human liver sections of ibuprofen-induced ALI. Next, we assessed the cytotoxicity of ibuprofen in vitro and developed a novel murine model of ibuprofen intoxication. To assess the role of JNK, we used animals carrying constitutive deletion of c-Jun N-terminal kinase 1 (Jnk1-/- ) or Jnk2 (Jnk2-/- ) expression and included investigations using animals with hepatocyte-specific Jnk deletion either genetically (Jnk1Δhepa ) or by siRNA (siJnk2Δhepa ). We found in human and murine samples of ibuprofen-induced acute liver failure that JNK phosphorylation was increased in the cytoplasm of hepatocytes and other non-liver parenchymal cells (non-LPCs) compared with healthy tissue. In mice, ibuprofen intoxication resulted in a significantly stronger degree of liver injury compared with vehicle-treated controls as evidenced by serum transaminases, and hepatic histopathology. Next, we investigated molecular pathways. PKCα, AKT, JNK and RIPK1 were significantly increased 8 h after ibuprofen intoxication. Constitutive Jnk1-/- and Jnk2-/- deficient mice exhibited increased liver dysfunction compared to wild-type (WT) animals. Furthermore, siJnk2Δhepa animals showed a dramatic increase in biochemical markers of liver function, which correlated with significantly higher serum liver enzymes and worsened liver histology, and MAPK activation compared to Jnk1Δhepa or WT animals. In our study, cytoplasmic JNK activation in hepatocytes and other non-LPCs is a hallmark of human and murine ibuprofen-induced ALI. Functional in vivo analysis demonstrated a protective role of hepatocyte-specific Jnk2 during ibuprofen ALI. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Liver maximum capacity (LiMAx) test as a helpful prognostic tool in acute liver failure with sepsis: a case report.

BACKGROUND: Acute liver failure (ALF) is a life-threatening entity particularly when infectious complications worsen the clinical course. Urgent liver transplantation (LT) is frequently the only curative treatment. However, in some cases, recovery is observed under conservative treatment. Therefore, prognostic tools for estimating course of the disease are of great clinical interest. Since laboratory parameters sometimes lack sensitivity and specificity, enzymatic liver function measured by liver maximum capacity (LiMAx) test may offer novel and valuable additional information in this setting. CASE PRESENTATION: We here report the case of a formerly healthy 20-year old male caucasian patient who was admitted to our clinic for ALF of unknown origin in December 2017. Laboratory parameters confirmed the diagnosis with an initial MELD score of 28 points. Likewise, enzymatic liver function was significantly impaired with a value of 147 [> 315] µg/h/kg. Clinical and biochemical analyses for viral-, autoimmune-, or drug-induced hepatitis were negative. Liver synthesis parameters further deteriorated reaching a MELD score of 40 points whilst clinical course was complicated by septic pneumonia leading to severe hepatic encephalopathy grade III-IV, finally resulting in mechanical ventilation of the patient. Interestingly, although clinical course and laboratory data suggested poor outcome, serial LiMAx test revealed improvement of the enzymatic liver function at this time point increasing to 169 µg/h/kg. Clinical condition and laboratory data slowly improved likewise, however with significant time delay of 11 days. Finally, the patient could be dismissed from our clinic after 37 days. CONCLUSION: Estimating prognosis in patients with ALF is challenging by use of the established scores. In our case, improvement of enzymatic liver function measured by the LiMAx test was the first parameter predicting beneficial outcome in a patient with ALF complicated by sepsis.

Interfering RNA against PKC-α inhibits TNF-α-induced IP3R1 expression and improves glomerular filtration rate in rats with fulminant hepatic failure.

We have reported that tumor necrosis factor-α (TNF-α) is critical for reduction of glomerular filtration rate (GFR) in rats with fulminant hepatic failure (FHF). The present study aims to evaluate the underlying mechanisms of decreased GFR during acute hepatic failure. Rats with FHF induced by d-galactosamine plus lipopolysaccharide (GalN/LPS) were injected intravenously with recombinant lentivirus harboring short hairpin RNA against the protein kinase C-α ( PKC-α) gene (Lenti-shRNA-PKC-α). GFR, serum levels of aminotransferases, creatinine, urea nitrogen, potassium, sodium, chloride, TNF-α, and endothelin-1 (ET-1), as well as type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) expression in renal tissue were assessed. The effects of PKC-α silencing on TNF-α-induced IP3R1, specificity protein 1 (SP-1), and c-Jun NH2-terminal kinase (JNK) expression, as well as cytosolic calcium content were determined in glomerular mesangial cell (GMCs) with RNAi against PKC-α. Renal IP3R1 overexpression was abrogated by pre-treatment with Lenti-shRNA-PKC-α. The PKC-α silence significantly improved the compromised GFR, reduced Cr levels, and reversed the decrease in glomerular inulin space and the increase in glomerular calcium content in GalN/LPS-exposed rats. TNF-α treatment increased expression of PKC-α, IP3R1, specificity protein 1 (SP-1), JNK, and p-JNK in GMCs and increased Ca2 + release and binding activity of SP-1 to the IP3R1 promoter. These effects were blocked by transfection of siRNA against the PKC-α gene, and the PKC-α gene silence also restored cytosolic Ca2+ concentration. RNAi targeting PKC-α inhibited TNF-α-induced IP3R1 overexpression and in turn improved compromised GFR in the development of acute kidney injury during FHF in rats.

Acute liver failure enhances oral plasma exposure of zidovudine in rats by downregulation of hepatic UGT2B7 and intestinal P-gp.

HIV infection is often associated with liver failure, which alters the pharmacokinetics of many drugs. In this study we investigated whether acute liver failure (ALF) altered the pharmacokinetics of the first-line anti-HIV agent zidovudine (AZT), a P-gp/BCRP substrate, in rats. ALF was induced in rats by injecting thioacetamide (TAA, 300 mg·kg-1·d-1, ip) for 2 days. On the second day after the last injection of TAA, the pharmacokinetics of AZT was investigated following both oral (20 mg/kg) and intravenous (10 mg/kg) administration. ALF significantly increased the plasma concentrations of AZT after both oral and intravenous doses of AZT, but without affecting the urinary excretion of AZT. AZT metabolism was studied in rat hepatic microsomes in vitro, which revealed that hepatic UGT2B7 was the main enzyme responsible for the formation of AZT O-glucuronide (GAZT); ALF markedly impaired AZT metabolism in hepatic microsomes, which was associated with the significantly decreased hepatic UGT2B7 expression. Intestinal absorption of AZT was further studied in rats via in situ single-pass intestinal perfusion. Intestinal P-gp function and intestinal integrity were assessed with rhodamine 123 and FD-70, respectively. We found that ALF significantly downregulated intestinal P-gp expression, and had a smaller effect on intestinal BCRP. Further studies showed that ALF significantly increased the intestinal absorption of both rhodamine 123 and AZT without altering intestinal integrity, thus confirming an impairment of intestinal P-gp function. In conclusion, ALF significantly increases the oral plasma exposure of AZT in rats, a result partly attributed to the impaired function and expression of hepatic UGT2B7 and intestinal P-gp.

Action of vitamin e on experimental severe acute liver failure / Composição corporal de pacientes com doença de Crohn e colite ulcerativa

ABSTRACT BACKGROUND Severe Acute Liver Failure (ALF) is a life-threatening clinical syndrome characterized by hepatocyte necrosis, loss of hepatic architecture, and impairment of liver functions. One of the main causes of ALF is hepatotoxicity from chemical agents, which damage hepatocytes and result in increase of reactive oxygen species. The vitamin E isoform is the one with the strongest biological antioxidant activity. OBJECTIVE To evaluate the antioxidant effect of vitamin E in this ALF model. METHODS We used 56 rats (mean weight of 300 g) divided into eight groups, four groups assessed at 24 hours and 4 assessed at 48 hours after induction: control group (CO); Vitamin E (Vit. E); Thioacetamide (TAA) and Thioacetamide + Vitamina E (TAA+Vit.E). Rats were submitted to injections of thioacetamide (400 mg/kg i.p.) at baseline and 8 hours later. Vitamin E (100 mg/kg ip) was administered 30 minutes after the second dose of thioacetamide. The 48-hour group rats received two additional doses of vitamin E (24h and 36h). At 24h or 48 hours after the administration of the first dose of TAA, rats were weighed and anesthetized and their blood sampled for evaluation of liver integrity through enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Liver tissue was sampled for assessment of lipid peroxidation (LPO) by the technique TBARS, antioxidant enzymes SOD, CAT, GPx and GST activity, levels of the NO 2 /NO 3 and histology by H&E in two times. The results were expressed as mean ± standard deviation and statistically analyzed by ANOVA followed by Student-Newman-Keuls, with P <0.05 considered as significant. RESULTS After treatment with vitamin E, we observed a reduction in liver enzymes AST (U/L) (101.32±19.45 in 24 hours and 97.85±29.65 in 48 hours) related to the TAA group (469.56± 0.69 in 24 hours and 598.23±55.45 in 48 hours) and ALT (U/L) (76.59±8.56 in 24 hours and 68.47±6.49 in 48 hours) compared to the TAA group (312.21±10.23 in 24 hours and 359.15±17.58 in 48 hours). There was a reduction of LPO (nmol/mg Prot) in the TAA+Vit.E group (0.77±0.07 in 24 hours and 0.95±0.08 in 48 hours) compared to the TAA group (1.50±0.07 in 24 hours e 1.65±0.16 in 48 hours). SOD decreased in the TAA+Vit.E group (49.48±9.47 in 24 hours and 62.45±18, 47 in 48 hours), related to the TAA group (98.46±15.48 in 24 hours and 154.13±21.46 in 48 hours), as well as GST (nmol/min/mg Prot) in the TAA+Vit.E group (350.57±36.93 in 24 hours and 453.29±13.84 in 48 hours) compared to the TAA group (561.57±64.56 in 24 hours and 673.43±38.13 in 48 hours). There was an increase in CAT (pmol/min/mg Prot) in the TAA+Vit.E group (3.40±0.44 in 24 hours and 3.0±0.35 in 48 hours) compared to the TAA group (1.65±0.21 in 24 hours and 1.86±0.42 in 48 hours). The GPx (nmol/min/mg Prot) increased in 24 hours in the TAA+Vit.E group (1.01±0.16) compared to the TAA group (0.41±0.04) and decreased in 48 hours (1.19±0.17) compared to the TAA group (1.76±0.21). There was a reduction in NO2/NO3 (mmol/L) levels in the TAA+Vit.E group (31.47±4.26 in 24 hours and 38.93±5.20 in 48 hours) compared to the TAA group (49.37±5.12 in 24 hours and 53.53±5.97 in 48 hours). The histopathological evaluation showed a decrease in liver injury (necrosis and inflammation) in both studied times. CONCLUSION These results suggest that vitamin E was able to protect the liver from lesions caused by thioacetamide.

RESUMO CONTEXTO A Insuficiência Hepática Aguda Grave (IHAG) é uma síndrome clínica potencialmente fatal, na qual ocorre necrose dos hepatócitos, perda da arquitetura hepática e deterioração de suas funções. Dentre as principais causas da IHAG está a hepatotoxicidade decorrente de agentes químicos, que lesam os hepatócitos e acarretam aumento das espécies reativas de oxigênio. A vitamina E tem alta atividade antioxidante biológica e é amplamente distribuída nos tecidos. OBJETIVO Avaliar o efeito antioxidante da Vitamina E no modelo de IHAG. MÉTODOS Foram utilizados 56 ratos, com peso médio de 300 g, divididos em oito grupos, quatro grupos avaliados em 24 horas e quatro em 48 horas após a indução: grupo controle (CO); Vitamina E (Vit.E); Tioacetamida (TAA) e Tioacetamida + Vitamina E (TAA+Vit.E). Os ratos foram submetidos a injeções de tioacetamida, na dose de 400 mg/Kg de peso i.p., no início do experimento e, posteriormente, após 8 horas. A vit E (100 mg//Kg i.p.) foi administrada 30 minutos após a segunda dose de tioacetamida. Os animais do tempo 48 horas receberam mais duas doses de vit. E (24h e 36h). Transcorridas 24 ou 48 horas após a administração da primeira dose de TAA, os animais foram pesados, anestesiados e o sangue retirado para a avaliação da integridade hepática através das enzimas Aspartatoaminotransferase (AST) e Alanina aminotransferase (ALT). O tecido hepático foi retirado para avaliação da lipoperoxidação através da técnica de TBARS, atividade das enzimas antioxidantes SOD, CAT, GPx, e GST, avaliação de NO 2 /NO 3 e avaliação histológica pela coloração de hematoxilina e eosina nos dois tempos. Os resultados foram expressos como média ± erro padrão e a análise estatística utilizada foi ANOVA, seguido de teste de Student-Newman-Keuls, considerado significativo P <0,05. RESULTADOS Após o tratamento com a vit. E, observamos uma redução nas enzimas de integridade hepática AST (U/L) (101,32±19,45 em 24h e 97,85±29,65 em 48h) relacionado ao grupo TAA (469,56±20,69 em 24h e 598,23±55,45 em 48h) e ALT (U/L) (76,59±8,56 em 24h e 68,47±6,49 em 48h) comparado ao grupo TAA (312,21±10,23 em 24h e 359,15±17,58 em 48h). Houve uma redução da LPO (nmol/mg Prot), no grupo TAA+Vit.E (0,77±0,07 em 24h e 0,95±0,08 em 48h) comparado ao grupo TAA (1,50±0,07 em 24h e 1,65±0,16 em 48h). A SOD (USOD/min/mg Prot) diminuiu no grupo TAA+Vit.E (49,48±9,47 em 24h e 62,45±18,47 em 48h) relacionado ao grupo TAA (98,46±15,48 em 24h e 154,13±21,46 em 48h), assim como a GST (nmol/min/mg Prot) no grupo TAA+Vit.E (350,57±36,93 em 24h e 453,29±13,84 em 48h) comparado ao grupo TAA (561,57±64,56 em 24h e 673,43±38,13 em 48h). Houve aumento da CAT (pmol/min/mg Prot) no grupo TAA+Vit.E (3,40±0,44 em 24h e 3,01±0,35 em 48h) em relação ao grupo TAA (1,65±0,21 em 24h e 1,86±0,42 em 48h). A GPx (nmol/min/mg Prot) aumentou em 24h no grupo TAA+Vit.E (1,01±0,16) comparado ao grupo TAA (0,41±0,04) e diminuiu em 48h (1,19±0,17) em relação ao grupo TAA (1,76±0,21). Verificou-se redução nos níveis de NO 2 /NO 3 (mmol/L) no grupo TAA+Vit.E (31,47±4,26 em 24h e 38,93±5,20 em 48h) em relação ao grupo TAA (49,37±5,12 em 24h e 53,53±5,97 em 48h). A avaliação histopatológica mostrou diminuição da lesão hepática (necrose e inflamação) em ambas os tempos estudados. CONCLUSÃO Estes resultados sugerem que a vitamina E foi capaz de proteger o fígado de lesões causadas por tioacetamida.

ACTION OF VITAMIN E ON EXPERIMENTAL SEVERE ACUTE LIVER FAILURE.

BACKGROUND: Severe Acute Liver Failure (ALF) is a life-threatening clinical syndrome characterized by hepatocyte necrosis, loss of hepatic architecture, and impairment of liver functions. One of the main causes of ALF is hepatotoxicity from chemical agents, which damage hepatocytes and result in increase of reactive oxygen species. The vitamin E isoform is the one with the strongest biological antioxidant activity. OBJECTIVE: To evaluate the antioxidant effect of vitamin E in this ALF model. METHODS: We used 56 rats (mean weight of 300 g) divided into eight groups, four groups assessed at 24 hours and 4 assessed at 48 hours after induction: control group (CO); Vitamin E (Vit. E); Thioacetamide (TAA) and Thioacetamide + Vitamina E (TAA+Vit.E). Rats were submitted to injections of thioacetamide (400 mg/kg i.p.) at baseline and 8 hours later. Vitamin E (100 mg/kg ip) was administered 30 minutes after the second dose of thioacetamide. The 48-hour group rats received two additional doses of vitamin E (24h and 36h). At 24h or 48 hours after the administration of the first dose of TAA, rats were weighed and anesthetized and their blood sampled for evaluation of liver integrity through enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Liver tissue was sampled for assessment of lipid peroxidation (LPO) by the technique TBARS, antioxidant enzymes SOD, CAT, GPx and GST activity, levels of the NO 2 /NO 3 and histology by H&E in two times. The results were expressed as mean ± standard deviation and statistically analyzed by ANOVA followed by Student-Newman-Keuls, with P <0.05 considered as significant. RESULTS: After treatment with vitamin E, we observed a reduction in liver enzymes AST (U/L) (101.32±19.45 in 24 hours and 97.85±29.65 in 48 hours) related to the TAA group (469.56± 0.69 in 24 hours and 598.23±55.45 in 48 hours) and ALT (U/L) (76.59±8.56 in 24 hours and 68.47±6.49 in 48 hours) compared to the TAA group (312.21±10.23 in 24 hours and 359.15±17.58 in 48 hours). There was a reduction of LPO (nmol/mg Prot) in the TAA+Vit.E group (0.77±0.07 in 24 hours and 0.95±0.08 in 48 hours) compared to the TAA group (1.50±0.07 in 24 hours e 1.65±0.16 in 48 hours). SOD decreased in the TAA+Vit.E group (49.48±9.47 in 24 hours and 62.45±18, 47 in 48 hours), related to the TAA group (98.46±15.48 in 24 hours and 154.13±21.46 in 48 hours), as well as GST (nmol/min/mg Prot) in the TAA+Vit.E group (350.57±36.93 in 24 hours and 453.29±13.84 in 48 hours) compared to the TAA group (561.57±64.56 in 24 hours and 673.43±38.13 in 48 hours). There was an increase in CAT (pmol/min/mg Prot) in the TAA+Vit.E group (3.40±0.44 in 24 hours and 3.0±0.35 in 48 hours) compared to the TAA group (1.65±0.21 in 24 hours and 1.86±0.42 in 48 hours). The GPx (nmol/min/mg Prot) increased in 24 hours in the TAA+Vit.E group (1.01±0.16) compared to the TAA group (0.41±0.04) and decreased in 48 hours (1.19±0.17) compared to the TAA group (1.76±0.21). There was a reduction in NO2/NO3 (mmol/L) levels in the TAA+Vit.E group (31.47±4.26 in 24 hours and 38.93±5.20 in 48 hours) compared to the TAA group (49.37±5.12 in 24 hours and 53.53±5.97 in 48 hours). The histopathological evaluation showed a decrease in liver injury (necrosis and inflammation) in both studied times. CONCLUSION: These results suggest that vitamin E was able to protect the liver from lesions caused by thioacetamide.

Dual Role of Epidermal Growth Factor Receptor in Liver Injury and Regeneration after Acetaminophen Overdose in Mice.

Epidermal growth factor receptor (EGFR) plays a crucial role in hepatocyte proliferation. Its role in acetaminophen (APAP)-mediated hepatotoxicity and subsequent liver regeneration is completely unknown. Role of EGFR after APAP-overdose in mice was studied using pharmacological inhibition strategy. Rapid, sustained and dose-dependent activation of EGFR was noted after APAP-treatment in mice, which was triggered by glutathione depletion. EGFR-activation was also observed in primary human hepatocytes after APAP-treatment, preceding elevation of toxicity markers. Treatment of mice with an EGFR-inhibitor (EGFRi), Canertinib, 1h post-APAP resulted in robust inhibition of EGFR-activation and a striking reduction in APAP-induced liver injury. Metabolic activation of APAP, formation of APAP-protein adducts, APAP-mediated JNK-activation and its mitochondrial translocation were not altered by EGFRi. Interestingly, EGFR rapidly translocated to mitochondria after APAP-treatment. EGFRi-treatment abolished mitochondrial EGFR activity, prevented APAP-mediated mitochondrial dysfunction/oxidative-stress and release of endonucleases from mitochondria, which are responsible for DNA-damage/necrosis. Treatment with N-acetylcysteine (NAC), 4h post-APAP in mice did not show any protection but treatment of EGFRi in combination with NAC showed decrease in liver injury. Finally, delayed treatment with EGFRi, 12-h post-APAP, did not alter peak injury but caused impairment of liver regeneration resulting in sustained injury and decreased survival after APAP overdose in mice. Impairment of regeneration was due to inhibition of cyclinD1 induction and cell cycle arrest. Our study has revealed a new dual role of EGFR both in initiation of APAP-injury and in stimulation of subsequent compensatory regeneration after APAP-overdose.

Hepatitis A virus genotype IA-infected patient with marked elevation of aspartate aminotransferase levels.

We describe a case of acute liver failure (ALF) without hepatic encephalopathy with marked elevation of aminotransferase due to hepatitis A, according to the revised Japanese criteria of ALF. This liver biopsy of the patient showed compatible to acute viral hepatitis and she immediately recovered without intensive care. She had no comorbid disorders. Of interest, phylogenetic tree analysis using almost complete genomes of hepatitis A virus (HAV) demonstrated that the HAV isolate from her belonged to the HAV subgenotype IA strain and was similar to the HAJFF-Kan12 strain (99% nucleotide identity) or FH1 strain (98% nucleotide identity), which is associated with severe or fulminant hepatitis A. Careful interpretation of the association between HAV genome variations and severity of hepatitis A is needed and the mechanism of the severe hepatitis should be explored.

Trichostatin A protects against intestinal injury in rats with acute liver failure.

BACKGROUND: Histone deacetylase (HDAC) inhibitors have been widely applied in the clinic as anticancer drugs against multiple neoplasms and proved their anti-inflammation under different pathology recently. Trichostatin A (TSA) is an HDAC inhibitor specific in class I and II HDAC enzymes. The aim of the present study was to elucidate the protective effects of TSA on acute liver failure (ALF) in rats and its potential mechanism. METHODS: A total of 18 female Sprague-Dawley rats were separated into control, model, and TSA groups. We used Western blotting to determine the expression of HDACs, inflammatory cytokines, and acetylation of histone in liver and small intestine. The gene expression of inflammatory factors and Cox-2 was detected by a polymerase chain reaction. Colonic motility was assessed by spatiotemporal mapping. Histologic analysis and immunohistochemistry were performed. Intestinal permeability examination and levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin were also observed. RESULTS: ALF procedure caused harm to histology of liver and small intestine, increased the intestinal permeability and serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin. It also interrupted the normal organization of colonic motor patterns by hurting enteric nervous system and pacemaker cells. Along with the decrease of inflammatory factors in ALF rats by TSA administration, all the damage to the liver, the small intestine, and the colon was repaired. CONCLUSIONS: TSA alleviates the lesion in liver, as well as in small intestine and colon in ALF rats by directly inhibiting inflammatory response.

Resolvin D1 attenuates CCl4-induced acute liver injury involving up-regulation of HO-1 in mice.

Acute hepatic failure involves in excessive oxidative stress and inflammatory responses, leading to a high mortality due to lacking effective therapy. Resolvin D1 (RvD1), an endogenous lipid mediator derived from polyunsaturated fatty acids, has been shown anti-inflammatory and anti-oxidative actions, however, whether RvD1 has protective effects on hepatic failure remains elusive. In this study, the roles and molecular mechanisms of RvD1 were explored in carbon tetrachloride (CCl4)-induced acute liver injury. Our results showed that RvD1 protected mice against CCl4-induced hepatic damage, as evaluated by reduced aminotransferase activities and malondialdehyde content, elevated glutathione and superoxide dismutase activities, and alleviated hepatic pathological damage. Moreover, RvD1 significantly attenuated serum tumor necrosis factor-α and interleukin-6 levels as well as hepatic myeloperoxidase activity, whereas enhanced serum IL-10 level in CCl4-administered mice. Further, RvD1 markedly up-regulated the expression and activity of heme oxygenase-1 (HO-1). However, inhibition of HO-1 activity reversed the protective effects of RvD1 on CCl4-induced liver injury. These results suggest that RvD1 could effectively prevent CCl4-induced liver injury by inhibition of oxidative stress and inflammation, and the underlying mechanism may be related to up-regulation of HO-1.

Combined Activities of JNK1 and JNK2 in Hepatocytes Protect Against Toxic Liver Injury.

BACKGROUND & AIMS: c-Jun N-terminal kinase (JNK) 1 and JNK2 are expressed in hepatocytes and have overlapping and distinct functions. JNK proteins are activated via phosphorylation in response to acetaminophen- or carbon tetrachloride (CCl4)-induced liver damage; the level of activation correlates with the degree of injury. SP600125, a JNK inhibitor, has been reported to block acetaminophen-induced liver injury. We investigated the role of JNK in drug-induced liver injury (DILI) in liver tissue from patients and in mice with genetic deletion of JNK in hepatocytes. METHODS: We studied liver sections from patients with DILI (due to acetaminophen, phenprocoumon, nonsteroidal anti-inflammatory drugs, or autoimmune hepatitis) or patients without acute liver failure (controls) collected from a DILI Biobank in Germany. Levels of total and activated (phosphorylated) JNK were measured by immunohistochemistry and Western blotting. Mice with hepatocyte-specific deletion of Jnk1 (Jnk1(Δhepa)) or combination of Jnk1 and Jnk2 (Jnk(Δhepa)), as well as Jnk1-floxed C57BL/6 (control) mice, were given injections of CCl4 (to induce fibrosis) or acetaminophen (to induce toxic liver injury). We performed gene expression microarray and phosphoproteomic analyses to determine mechanisms of JNK activity in hepatocytes. RESULTS: Liver samples from DILI patients contained more activated JNK, predominantly in nuclei of hepatocytes and in immune cells, than healthy tissue. Administration of acetaminophen to Jnk(Δhepa) mice produced a greater level of liver injury than that observed in Jnk1(Δhepa) or control mice, based on levels of serum markers and microscopic and histologic analysis of liver tissues. Administration of CCl4 also induced stronger hepatic injury in Jnk(Δhepa) mice, based on increased inflammation, cell proliferation, and fibrosis progression, compared with Jnk1(Δhepa) or control mice. Hepatocytes from Jnk(Δhepa) mice given acetaminophen had an increased oxidative stress response, leading to decreased activation of adenosine monophosphate-activated protein kinase, total protein adenosine monophosphate-activated protein kinase levels, and pJunD and subsequent necrosis. Administration of SP600125 before or with acetaminophen protected Jnk(Δhepa) and control mice from liver injury. CONCLUSIONS: In hepatocytes, JNK1 and JNK2 appear to have combined effects in protecting mice from CCl4- and acetaminophen-induced liver injury. It is important to study the tissue-specific functions of both proteins, rather than just JNK1, in the onset of toxic liver injury. JNK inhibition with SP600125 shows off-target effects.

Inhibition of sphingosine kinase 1 ameliorates acute liver failure by reducing high-mobility group box 1 cytoplasmic translocation in liver cells.

AIM: To determine the therapeutic potential of sphingosine kinase 1 (Sphk1) inhibition and its underlying mechanism in a well-characterized mouse model of D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver failure (ALF). METHODS: Balb/c mice were randomly assigned to different groups, with ALF induced by intraperitoneal injection of D-GaIN (600 mg/kg) and LPS (10 µg/kg). The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at different time points within one week were determined using a multi-parametric analyzer. Serum high-mobility group box 1 (HMGB1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, IL-10, and sphingosine-1-phosphate were detected by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after acute liver injury induction were assessed by hematoxylin and eosin staining. HMGB1 expression in hepatocytes and cytoplasmic translocation were detected by immunohistochemistry. Expression of Sphk1 in liver tissue and peripheral blood mononuclear cells (PBMCs) was analyzed by Western blot. RESULTS: The expression of Sphk1 in liver tissue and PBMCs was upregulated in GalN/LPS-induced ALF. Upregulated Sphk1 expression in liver tissue was mainly caused by Kupffer cells, the resident macrophages of the liver. The survival rates of mice in the N,N-dimethylsphingosine (DMS, a specific inhibitor of SphK1) treatment group were significantly higher than that of the control group (P < 0.001). DMS treatment significantly decreased the levels of serum ALT and AST at 6, 12, and 24 h compared with that of the control group (P < 0.01 for all). Serum HMGB1 levels at 6, 12, and 24 h, as well as serum TNF-α, IL-6, and IL-1ß levels at 12 h, were significantly lower in the DMS treatment group than in the control group (P < 0.01 for all). Furthermore, hepatic inflammation, necrosis, and HMGB1 cytoplasm translocation in liver cells were significantly decreased in the DMS treatment group compared to the control group (43.72% ± 5.51% vs 3.57% ± 0.83%, χ(2) = 12.81, P < 0.01). CONCLUSION: Inhibition of SphK1 ameliorates ALF by reducing HMGB1 cytoplasmic translocation in liver cells, and so might be a potential therapeutic strategy for this disease.

Liver Regeneration Induced By Extracorporeal Portal Vein Arterialization in a Swine Model of Carbon Tetrachloride Intoxication.

INTRODUCTION: This study aimed to determine whether a controlled portal blood arterialization by a liver extracorporeal device (L.E.O2 NARDO) is effective in treating acute hepatic failure (AHF) induced in swine by carbon tetrachloride (CCl4) administration. MATERIALS AND METHODS: Sixteen swine with AHF induced by intraperitoneal injection of CCl4 in oil solution were randomly divided into 2 groups: animals that received L.E.O2 NARDO treatment 48 hours after the intoxication (study group; n = 8); and animals that were sham operated 48 hours after the intoxication (control group; n = 8). Blood was withdrawn from the iliac artery and reversed in the portal venous system by an interposed extracorporeal device. Each treatment lasted 6 hours. The survival was assessed at 5 days after L.E.O2 NARDO treatment or sham operation. In both groups blood samples were collected for biochemical analysis at different study time and liver biopsies were performed 48 hours after intoxication and at humane killing. RESULTS: In the study group decreased transaminases levels and a more rapid international normalized ratio (INR) recover were detected as compared with the control group. Six animals of the study group (75%) versus 1 animal (12.5%) of the control group survived at 5 days after surgery with a statistically significant difference (P < .05). Liver biopsies performed at humane killing showed damaged areas of the livers reduced in the study group compared with biopsies of the control group. CONCLUSIONS: Arterial blood supply in the portal system through the L.E.O2 NARDO device is easily applicable, efficacious, and safe in a swine model of AHF induced by CCl4 intoxication.

Dihydromyricetin alleviates carbon tetrachloride-induced acute liver injury via JNK-dependent mechanism in mice.

AIM: To assess the effects of dihydromyricetin (DHM) as a hepatoprotective candidate in reducing hepatic injury and accelerating hepatocyte proliferation after carbon tetrachloride (CCl4) treatment. METHODS: C57 BL/6 mice were used in this study. Mice were orally administered with DHM (150 mg/kg) for 4 d after CCl4 treatment. Serum and liver tissue samples were collected on days 1, 2, 3, 5 and 7 after CCl4 treatment. The anti-inflammatory effect of DHM was assessed directly by hepatic histology detection and indirectly by serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, and superoxide dismutase (SOD). Inflammatory cytokines, such as interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α (TNF-α), were detected using ELISA kits. Proliferating cell nuclear antigen (PCNA) staining was used to evaluate the role of DHM in promoting hepatocyte proliferation. Hepatocyte apoptosis was measured by TUNEL assay. Furthermore, apoptosis proteins Caspases-3, 6, 8, and 9 were detected by Western blot. SP600125 were used to confirm whether DHM regulated liver regeneration through JNK/TNF-α pathways. RESULTS: DHM showed a strong anti-inflammatory effect on CCl4-induced liver injury in mice. DHM could significantly decrease serum ALT, AST, IL-1ß, IL-6 and TNF-α and increase serum albumin, SOD and liver SOD compared to the control group after CCl4 treatment (P < 0.05). PCNA results indicated that DHM could significantly increase the number of PCNA positive cells compared to the control (348.9 ± 56.0 vs 107.1 ± 31.4, P < 0.01). TUNEL assay showed that DHM dramatically reduced the number of apoptotic cells after CCl4 treatment compared to the control (365.4 ± 99.4 vs 90.5 ± 13.8, P < 0.01). Caspase activity detection showed that DHM could reduce the activities of Caspases- 8, 3, 6 and 9 compared to the control (P < 0.05). The results of Western blot showed that DHM increased the expression of JNK and decreased TNF-α expression. However, DHM could not affect TNF-α expression after SP600125 treatment. Furthermore, DHM could significantly improve the survival rate of acute liver failure (ALF) mice (73.3% vs 20.0%, P < 0.0001), and SP600125 could inhibit the effect of DHM. CONCLUSION: These findings demonstrate that DHM alleviates CCl4-induced liver injury, suggesting that DHM is a promising candidate for reversing liver injury and ALF.

Changes of the thioredoxin system, glutathione peroxidase activity and total antioxidant capacity in rat brain cortex during acute liver failure: modulation by L-histidine.

Glutathione and thioredoxin are complementary antioxidants in the protection of mammalian tissues against oxidative-nitrosative stress (ONS), and ONS is a principal cause of symptoms of hepatic encephalopathy (HE) associated with acute liver failure (ALF). We compared the activities of the thioredoxin system components: thioredoxin (Trx), thioredoxin reductase (TrxR) and the expression of the thioredoxin-interacting protein, and of the key glutathione metabolizing enzyme, glutathione peroxidase (GPx) in the cerebral cortex of rats with ALF induced by thioacetamide (TAA). ALF increased the Trx and TrxR activity without affecting Trip protein expression, but decreased GPx activity in the brains of TAA-treated rats. The total antioxidant capacity (TAC) of the brain was increased by ALF suggesting that upregulation of the thioredoxin may act towards compensating impaired protection by the glutathione system. Intraperitoneal administration of L-histidine (His), an amino acid that was earlier reported to prevent acute liver failure-induced mitochondrial impairment and brain edema, abrogated most of the acute liver failure-induced changes of both antioxidant systems, and significantly increased TAC of both the control and ALF-affected brain. These observations provide further support for the concept of that His has a potential to serve as a therapeutic antioxidant in HE. Most of the enzyme activity changes evoked by His or ALF were not well correlated with alterations in their expression at the mRNA level, suggesting complex translational or posttranslational mechanisms of their modulation, which deserve further investigations.