RESUMO
To investigate the different expression profiles of MAPK pathway genes and their corresponding functions during liver regeneration, we used a CCl4 induced mouse liver regeneration model in this study. Mouse was injected with CCl4 in the abdominal cavity to cause damage in the liver and followed by liver histology examination and measurement of serum ALT levels in blood sample collected at 0, 0.5, 1.5, 4.5, and 7 d after CCl4 injection. Differentially expressed genes in the MAPK pathway during liver regeneration were analyzed using mouse cDNA microarray method (Affymetrix). The results obtained were further subjected to hierarchical clustering study and were validated with real-time PCR. Microarray hybridization identified 31 out of the 93 MAPK pathway component genes, which have significantly altered their expression levels during liver regeneration. Among them, both up- and down-regulated genes were classified into various groups according to clustering studies and functional analysis. At the initial stage of liver regeneration, the number of up-regulated genes was greater than the down-regulated genes, while at the late stage the situation was reversed. Our results suggest that MAPK pathway might play different regulatory roles in responding to different stages of liver regeneration.
Assuntos
Regeneração Hepática , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
AIM: To investigate the hepatoprotective effects of phycocyanobilin (PCB) in reducing hepatic injury and accelerating hepatocyte proliferation following carbon tetrachloride (CCl4) treatment. METHODS: C57BL/6 mice were orally administered PCB 100 mg/kg for 4 d after CCl4 injection, and then the serum and liver tissue of the mice were collected at days 1, 2, 3, 5 and 7 after CCl4 treatment. A series of evaluations were performed to identify the curative effects on liver injury and recovery. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin and superoxide dismutase (SOD) were detected to indirectly assess the anti-inflammatory effects of PCB. Meanwhile, we detected the expressions of hepatocyte growth factor, transforming growth factor alpha (TGF-α), TGF-ß, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), the factors which are associated with inflammation and liver regeneration. The protein expressions of proliferating cell nuclear antigen (PCNA), TNF-α and cytochrome C were detected by western blot. Furthermore, the survival rates were analyzed of mice which were administered a lethal dose of CCl4 (2.6 mg/kg) with or without PCB. RESULTS: In our research, PCB showed a strongly anti-inflammatory effect on CCl4-induced liver injury in mice. The ALT was significantly decreased after CCl4 treatment from day 1 (P < 0.01) and the AST was significantly decreased from day 2 (P < 0.001). Both albumin and liver SOD were increased from day 2 (P < 0.001 and P < 0.01), but serum SOD levels did not show a significant increase (P > 0.05). PCB protected the structure of liver from the injury by CCl4. TUNEL assay showed that PCB dramatically reduced the number of apoptotic cells after CCl4 treatment compared to the control (101.0 ± 25.4 vs 25.7 ± 6.4, P < 0.01). The result of western blotting showed that PCB could increase PCNA expression, decrease TNF-α and cytochrome C expression. Furthermore, data shows that PCB could improve the survival rate of acute liver failure (ALF) mice which were injected with a lethal dose of CCl4 (60.0% vs 20.0%). CONCLUSION: Our study indicated that PCB could be an ideal candidate for reversing acute liver injury or ALF.
Assuntos
Anti-Inflamatórios/farmacologia , Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Falência Hepática Aguda/tratamento farmacológico , Regeneração Hepática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Ficobilinas/farmacologia , Ficocianina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Fígado/enzimologia , Fígado/patologia , Falência Hepática Aguda/sangue , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/patologia , Masculino , Camundongos Endogâmicos C57BL , Necrose , Fatores de TempoRESUMO
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.
Assuntos
Anti-Inflamatórios/farmacologia , Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Flavonóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Falência Hepática Aguda/tratamento farmacológico , Fígado/efeitos dos fármacos , Animais , Biomarcadores/sangue , Inibidores de Caspase/farmacologia , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Citocromos c/metabolismo , Modelos Animais de Doenças , Mediadores da Inflamação/sangue , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Fígado/enzimologia , Fígado/patologia , Falência Hepática Aguda/sangue , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/enzimologia , Falência Hepática Aguda/patologia , Regeneração Hepática/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/sangueRESUMO
AIM: To investigate the effects of dihydromyricetin (DHM) on the migration and invasion of human hepatic cancer cells. METHODS: The hepatoma cell lines SK-Hep-1 and MHCC97L were used in this study. The cells were cultured in RPIM-1640 medium supplemented with 10% fetal bovine serum at 37â °C in a humidified 5% CO2 incubator. DHM was dissolved in dimethyl sulfoxide and diluted to various concentrations in medium before applying to cells. MTT assays were performed to measure the viability of the cells after DHM treatment. Wound healing and Boyden transwell assays were used to assess cancer cell motility. The invasive capacity of cancer cells was measured using Matrigel-coated transwell chambers. Matrix metalloproteinase (MMP)-2/9 activity was examined by fluorescence analysis. Western blot was carried out to analyze the expression of MMP-2, MMP-9, p-38, JNK, ERK1/2 and PKC-δ proteins. All data were analyzed by Student's t tests in GraphPad prism 5.0 software and are presented as mean ± SD. RESULTS: DHM was found to strongly inhibit the migration of the hepatoma cell lines SK-Hep-1 (without DHM, 24 h: 120 ± 8 µmol/L vs 100 µmol/L DHM, 24 h: 65 ± 10 µmol/L, P < 0.001) and MHCC97L (without DHM, 24 h: 126 ± 7 µmol/L vs 100 µmol/L DHM, 24 h: 74 ± 6 µmol/L, P < 0.001). The invasive capacity of the cells was reduced by DHM treatment (SK-Hep-1 cells without DHM, 24 h: 67 ± 4 µmol/L vs 100 µmol/L DHM, 24 h: 9 ± 3 µmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 117 ± 8 µmol/L vs 100 µmol/L DHM, 24 h: 45 ± 2 µmol/L, P < 0.001). MMP2/9 activity was also inhibited by DHM exposure (SK-Hep-1 cells without DHM, 24 h: 600 ± 26 µmol/L vs 100 µmol/L DHM, 24 h: 100 ± 6 µmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 504 ± 32 µmol/L vs 100 µmol/L DHM 24 h: 156 ± 10 µmol/L, P < 0.001). Western blot analysis showed that DHM decreased the expression level of MMP-9 but had little effect on MMP-2. Further investigation indicated that DHM markedly reduced the phosphorylation levels of p38, ERK1/2 and JNK in a concentration-dependent manner but had no impact on the total protein levels. In addition, PKC-δ protein, a key protein in the regulation of MMP family protein expression, was up-regulated with DHM treatment. CONCLUSION: These findings demonstrate that DHM inhibits the migration and invasion of hepatoma cells and may serve as a potential candidate agent for the prevention of HCC metastasis.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular/enzimologia , Movimento Celular/efeitos dos fármacos , Flavonóis/farmacologia , Neoplasias Hepáticas/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Carcinoma Hepatocelular/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/patologia , Metaloproteinase 2 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Fosforilação , Proteína Quinase C-delta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
AIM: To investigate the hepatoprotective effect of baicalein against carbon tetrachloride (CCl4)-induced liver damage in mice. METHODS: Mice were orally administered with baicalein after CCl4 injection, and therapeutic baicalein was given twice a day for 4 d. The anti-inflammation effects of baicalein were assessed directly by hepatic histology and serum alanine aminotranferease and aspartate aminotransferase measurement. Proliferating cell nuclear antigen was used to evaluate the effect of baicalein in promoting hepatocyte proliferation. Serum interleukin (IL)-6, IL-1ß and tumor necrosis factor-α (TNF-α) levels were measured by enzyme-linked immunosorbent assay and liver IL-6, TNF-α, transforming growth factor-α (TGF-α), hepatocyte growth factor (HGF) and epidermal growth factor (EGF) genes expression were determined by quantitative real-time polymerase chain reaction. RESULTS: CCl4-induced acute liver failure model offers a survival benefit in baicalein-treated mice. The data indicated that the mRNA levels of IL-6 and TNF-α significantly increased within 12 h after CCl4 treatment in baicalein administration groups, but at 24, 48 and 72 h, the expression of IL-6 and TNF-α was kept at lower levels compared with the control. The expression of TGF-α, HGF and EGF was enhanced dramatically in baicalein administration group at 12, 24, 48 and 72 h. Furthermore, we found that baicalein significantly elevated the serum level of TNF-α and IL-6 at the early phase, which indicated that baicalein could facilitate the initiating events in liver regeneration. CONCLUSION: Baicalein may be a therapeutic candidate for acute liver injury. Baicalein accelerates liver regeneration by regulating TNF-α and IL-6 mediated pathways.
Assuntos
Anti-Inflamatórios/farmacologia , Tetracloreto de Carbono/toxicidade , Flavanonas/farmacologia , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/tratamento farmacológico , Fígado/lesões , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Proliferação de Células , Hepatócitos/citologia , Interleucina-1beta/sangue , Interleucina-6/sangue , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/sangueRESUMO
Recent studies indicate that the process of liver regeneration involves multiple signaling pathways and a variety of genes, cytokines and growth factors. Protein-protein interactions (PPIs) play a role in nearly all events that take place within the cell and PPI maps should be helpful in further understanding the process of liver regeneration. In this review, we discuss recent progress in understanding the PPIs that occur during liver regeneration especially those in the transforming growth factor beta signaling pathways. We believe the use of large-scale PPI maps for integrating the information already known about the liver regeneration is a useful approach in understanding liver regeneration from the standpoint of systems biology.
Assuntos
Regeneração Hepática/fisiologia , Mapeamento de Interação de Proteínas , Transdução de Sinais/fisiologia , Algoritmos , Animais , Proliferação de Células , Humanos , Camundongos , Modelos Animais , Fator de Crescimento Transformador beta/fisiologiaRESUMO
AIM: To evaluate the effects of positive regulation of recombinant human interleukin 1 receptor antagonist (rhIL-1Ra) on hepatic tissue recovery in acute liver injury in mice induced by carbon tetrachloride (CCl(4)). METHODS: Acute liver damage was induced by injecting 8-wk-old mice with CCl(4) 1 mL/kg (1:3 dilution in corn oil) intraperitoneally (ip). Survival after liver failure was assessed by injecting 8-wk-old mice with a lethal dose of CCl(4) 2.6 mL/kg (1:1 dilution in corn oil) ip. Mice were subcutaneously injected with 1 mg/kg recombinant human IL-1Ra twice a day after CCl(4) treatment for 5 d. Serum alanine amino transferase (ALT) and aspartate aminotransferase (AST) levels were determined with a commercial assay kit. Serum IL-1beta, IL-1Ra levels were measured by enzyme-linked immunosorbent assay kit. Quantitative real-time polymerase chain reaction was used to determine liver IL-1beta, IL-1Ra and IL-6 expression during CCl(4)-induced acute liver injury. Liver sections were stained with hematoxylin-eosin. A histology-injury grading system was used to evaluate the degree of necrosis after acute liver injury. Proliferating cell nuclear antigen (PCNA) staining was used to evaluate the role of rhIL-1Ra in promoting hepatocyte proliferation. RESULTS: Quantitative analysis showed a higher level of IL-6 mRNA expression and reduced serum AST and ALT levels in the livers of the rhIL-1Ra-treated group at the early phase of CCl(4)-induced acute liver injury. Histological examination indicated a decrease in centrilobular necrotic areas in mice treated with rhIL-1Ra, and a novel role of rhIL-1Ra in promoting hepatocyte proliferation was also supported by an increase of PCNA staining. All these results, accompanied by a strong survival benefit in rhIL-1Ra-treated vs PBS-treated groups, demonstrated that rhIL-1Ra administration ameliorated the histological damage and accelerated the regeneration and recovery process of the liver. CONCLUSION: rhIL-1Ra could be further developed as a novel therapeutic agent for the treatment of acute liver injury because of its ability to reduce hepatocellular damage and facilitate liver regeneration.