RESUMO
AIM: To investigate the expression and activity of leukotriene C4 (LTC4) synthesis enzymes and their underlying relationship with cysteinyl leukotriene (cys-LT) generation in a rat fulminant hepatic failure (FHF) model induced by D-galactosamine/lipopolysaccharide (D-GalN/ LPS). METHODS: Rats were treated with D-GalN (300 mg/kg) plus LPS (0.1 mg/kg) for 1, 3, 6, and 12 h. Enzyme immunoassay was used to determine the hepatic cys-LT content. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot or immunohistochemical assay were employed to assess the expression or location of LTC4 synthesis enzymes, which belong to membrane associated proteins in eicosanoid and glutathione (MAPEG) metabolism superfamily. Activity of LTC4 synthesis enzymes was evaluated by determination of the products of LTA4 after incubation with liver microsomes using high performance liquid chromatography (HPLC). RESULTS: Livers were injured after treatment with D-GalN/LPS, accompanied by cys-LT accumulation at the prophase of liver injury. Both LTC4 synthase (LTC4S) and microsomal glutathione-S-transferase (mGST) 2 were expressed in the rat liver, while the latter was specifically located in hepatocytes. Their mRNA and protein expressions were up-regulated at an earlier phase after treatment with D-GalN/LPS. Meantime, a higher activity of LTC4 synthesis enzymes was detected, although the activity of LTC4S played the main role in this case. CONCLUSION: The expression and activity of both LTC4S and mGST2 are up regulated in a rat FHF model, which are, at least, partly responsible for cys-LT hepatic accumulation.
Assuntos
Cisteína/metabolismo , Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Leucotrieno C4/metabolismo , Leucotrienos/metabolismo , Falência Hepática Aguda/enzimologia , Fígado/enzimologia , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Western Blotting , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Galactosamina , Glutationa Transferase/genética , Imuno-Histoquímica , Isoenzimas/genética , Lipopolissacarídeos , Falência Hepática Aguda/induzido quimicamente , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Regulação para CimaRESUMO
OBJECTIVE: To explore the gene expressions of LTC4 synthase homologs in concanavalin A (Con A)-induced mouse hepatitis and regulation role of cyclosporine A (Cs A) treatment. METHODS: Male Balb/c mouse liver injury model was developed by iv injection of Con A (20 mg/kg) and protected by Cs A pretreatment (150 mg/kg) before Con A administration. Blood samples were collected at indicated times after Con A treatment with or without Cs A pretreatment. Liver damage was assessed by serum transaminase ALT and AST measurement and histological evaluation. Meantime, three LTC4 synthase homolog gene expressions were determined by RT-PCR. RESULTS: Serum ALT and AST upregulation were accompanied with histological damage at 2 h after Con A administration, and further aggravated at 8 h. mGST2 gene expression increased 1.7 fold at 2 h and 1.9 fold at 8 h, while the expression of LTC4 S and mGST3 changed little. Pretreatment with Cs A prevented mouse liver from injury by Con A and partly inhibited the mGST2 gene expression upregulation. CONCLUSIONS: Administration of Con A in mouse lead to a significant increase of mGST2 gene expression without any significant effect on LTC4 S and mGST3 mRNA levels. Cs A pretreatment results in protection of liver damage, whereas fails to fully inhibit the increase of mGST2 gene expression.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Concanavalina A/toxicidade , Ciclosporina/farmacologia , Glutationa Transferase/genética , Hepatite Animal/prevenção & controle , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Concanavalina A/administração & dosagem , Regulação Enzimológica da Expressão Gênica/genética , Hepatite Animal/induzido quimicamente , Hepatite Animal/enzimologia , Imunossupressores/farmacologia , Injeções Intravenosas , Isoenzimas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To investigate the protective effects and mechanism of triterpenoids on primarily cultured rat hepatocytes injured by D-galactosamine (D-GalN) or carbon tetrachloride (CCl4). METHODS: Rat hepatocytes were isolated by two-step collagenase perfusion and cultured in RPMI 1640 medium. Protective effects of asiatic acid (AA) and beta-glycyrrhetinic acid (GA) were evaluated on hepatocytes injured by D-GalN (2 mmol/L) or CCl4 (10 mmol/L). Cell morphology was observed by light microscope, cell viability was measured by MTT assay, AST and LDH were determined by an automatic analyzer. Fluorescence assay was applied to test reactive oxygen species (ROS), nitric oxide end products (NOx) and reduced glutathione (GSH), and JC-1 staining was used to determine mitochondria membrane potential (DeltaPsim). RESULTS: AST and LDH in medium were decreased when treated with AA and GA after D-GalN injury (P<0.05), furthermore AA enhanced the hepatocyte viability (P<0.05). Moreover, AA and GA significantly reduced ROS and NOx generation, and ameliorated DeltaPsim lost induced by D-GalN. AA also inhibited GSH decrease due to D-GalN and CCl4 treatment. CONCLUSION: Both AA and GA could protect hepatocytes from D-GalN and CCl4 injuries, which is associated with reducing intracellular ROS and NOx, reversing GSH depression and ameliorating DeltaPsim lost.
