Association of Upregulated HMGB1 and c-IAP2 Proteins With Hepatocellular Carcinoma Development and Progression.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most important health problems in China. OBJECTIVES: This study analyzed expression of high-mobility group protein B1 (HMGB1) and inhibitor of apoptosis protein-2 (c-IAP2) proteins in HCC compared to paired para-tumor tissue samples to assess the association with HCC pathogenesis and progression. MATERIALS AND METHODS: Sixty-eight HCC and para-tumor tissue samples were collected for Western blot, qRT-PCR and immunohistochemical analyses of HMGB1 and c-IAP2. RESULTS: HMGB1 and c-IAP2 proteins were highly expressed in HCC tissue samples [85.3% (58/68) and 82.4% (56/68), respectively] compared to para-tumor tissue samples [32.3% and 27.9%, respectively]. Furthermore, expression of HMGB1 was significantly associated with enhanced c-IAP2 expression in HCC tissue samples (r = 0.878, P < 0.01). Expression of HMGB1 was associated with tumor multiplicity and size, alpha-fetoprotein (AFP) level and advanced TNM stage, while expression of c-IAP2 was associated with tumor size, AFP level and advanced TNM stage. CONCLUSIONS: Expression of HMGB1 and c-IAP2 proteins was associated with HCC development and progression, and the expression of HMGB1 and c-IAP2 proteins in HCC were significantly associated with each other. Additionally, these proteins may show promise as biomarkers to predict HCC progression.

Hepatoprotective effects of cathepsin B inhibitor on acute hepatic failure induced by lipopolysaccharide/D-galactosamine in mice.

BACKGROUND: Increasing evidence suggests that the inactivation of cathepsin B attenuates hepatocyte apoptosis and liver damage. This study aimed to investigate the protective effects of a cathepsin B inhibitor (CA-074me) on lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute hepatic failure (AHF) in mice. METHODS: Mice were intraperitoneally injected with a combination of LPS/D-GalN to induce AHF with or without CA-074me pretreatment. The cumulative survival rates were calculated 48 hours after the induction of AHF. As well as changes in biochemical indicators and liver histology, hepatocyte apoptosis was assessed using a TUNEL method. Serum tumor necrosis factor-alpha (TNF-alpha) production, caspase-3, caspase-8, and caspase-9 activity was evaluated. Cytosolic cytochrome c and Bcl-2 expression were measured by Western blotting. RESULTS: The marked elevation in serum aminotransferase activity and prothrombin time found in LPS/D-GalN-treated mice was significantly improved by pretreatment with CA-074me. The efficacy of CA-074me was also confirmed by histological analysis and TUNEL assay. The survival rate significantly improved in LPS/D-GalN-induced mice given CA-074me compared with untreated mice. LPS/D-GalN-induced caspase-3 and caspase-9 activation was remarkably suppressed by CA-074me. However, the increased levels of serum TNF-alpha and elevated caspase-8 activity in AHF mice were not significantly reduced by CA-074me. Moreover, CA-074me sharply reduced the increased expression of cytosolic cytochrome c and markedly augmented Bcl-2 expression. CONCLUSION: These results suggest that CA-074me has a protective effect in acute hepatic failure induced by LPS/D-GalN.

Regulatory T-cell responses in chronic hepatitis B patients treated with nucleos(t)ide analogs compared with healthy subjects and untreated infected individuals.

BACKGROUND/AIMS: The purpose of this prospective case-control study was to evaluate the clinical effects and host immune response in patients with chronic hepatitis B (CHB) treated with either entecavir (ETV)or adefovir dipivoxil (ADV). METHODOLOGY: Forty-two patients diagnosed with CHB were recruited and randomly assigned to receive either ADV (n=19) or ETV(n=18) and were followed for a minimum of 96 weeks.Serum hepatitis B virus (HBV) DNA, hepatitis B e antigen and antibody (HBeAg, HBeAb), alanine amino-transferase (ALT) and aspartate aminotransferase(AST) were measured at baseline and every 24 weeks until study completion. After 96 weeks of therapy, regulatory T-cells (Tregs) were measured in the patients treated with ETV or ADV. RESULTS: Significant decreases in serum ALT, AST and HBV DNA, but not in HBeAgor HbeAb, were noted in the treatment group. The ra-tios of CD4+CD25+ and CD4+CD25+CD45RO+CD125+in CD4+ T-cells were significantly higher in the untreated group compared to those in the ETV and ADV groups. Treg profiles were significantly altered in CHB patients after 96 weeks of nucelos(t)ide therapy HBV-infected individuals. CONCLUSIONS: Study results support the hypothesis that Tregs play a role in regulating the immune response in patients with CHB.

Role of cathepsin B-mediated apoptosis in fulminant hepatic failure in mice.

AIM: To investigate the pathogenic role of cathepsin B and the protective effect of a cathepsin B inhibitor (CA-074Me) in fulminant hepatic failure in mice. METHODS: LPS/D-Gal N was injected into mice of the model group to induce fulminant hepatic failure; the protected group was administered CA-074me for 30 min before LPS/D-Gal N treatment; the normal group was given isochoric physiologic saline. Liver tissue histopathology was determined with HE at 2, 4, 6 and 8 h after Lps/D-Gal injection. Hepatocyte apoptosis was examined by TUNEL method. The expression of cathepsin B in liver tissues was investigated by immunohistochemistry, Western blot and RT-PCR. RESULTS: Compared with the normal group, massive typical hepatocyte apoptosis occurred in the model group; the number of apoptotic cells reached a maximum 6 h after injection. The apoptosis index (AI) in the protected group was clearly reduced (30.4 +/- 2.8 vs 18.1 +/- 2.0, P < 0.01 ). Cathepsin B activity was markedly increased in drug-treated mice compared with the normal group (P < 0.01). Incubation with LPS/D-Gal N at selected time points resulted in a time-dependent increase in cathepsin B activity, and reached a maximum by 8 h. The expression of cathepsin B was significantly decreased in the protected group (P < 0.01). CONCLUSION: Cathepsin B plays an essential role in the pathogenesis of fulminant hepatic failure, and the cathepsin B inhibitor CA-074me can attenuate apoptosis and liver injury.

Protective effect and mechanism of stronger neo-minophagen C against fulminant hepatic failure.

AIM: To investigate the protective effect of stronger neo-minophafen C (SNMC) on fulminant hepatic failure (FHF) and its underlying mechanism. METHODS: A mouse model of FHF was established by intraperitoneal injection of galactosamine (D-Gal N) and lipopolysaccharide (LPS). The survival rate, liver function, inflammatory factor and liver pathological change were obtained with and without SNMC treatment. Hepatocyte survival was estimated by observing the stained mitochondria structure with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method and antibodies against cytochrome C (Cyt-C) and caspase-3. RESULTS: The levels of plasma tumor necrosis factor alpha (TNF-alpha), nitric oxide (NO), ET-1, interleukin-6 (IL-6), and the degree of hepatic tissue injury were decreased in the SNMC-treated groups compared with those in the model group (P < 0.01). However, there were no differences after different dosages administered at different time points. There was a significant difference in survival rates between the SNMC-treated groups and the model group (P < 0.01). The apoptosis index was 32.3% at 6 h after a low dose of SNMC, which was considerably decreased from 32.3% +/- 4.7% vs 5% +/- 2.83% (P < 0.05) to 5% on d 7. The expression of Cyt-C and caspase-3 decreased with the prolongation of therapeutic time. Typical hepatocyte apoptosis was obviously ameliorated under electron microscope with the prolongation of therapeutic time. CONCLUSION: SNMC can effectively protect liver against FHF induced by LPS/D-Gal N. SNMC can prevent hepatocyte apoptosis by inhibiting inflammatory reaction and stabilizing mitochondria membrane to suppress the release of Cyt-C and sequent activation of caspase-3.

[Protective effect of SNMC on mice with fulminant liver failure].

OBJECTIVE: To investigate the protective effect of stronger neo-minophagen C (SNMC) on fulminant liver failure (FLF). METHODS: D-Gal N and LPS were injected once into the abdominal cavity of rats to establish an experimental model of FLF. The level of plasma ALT, Alb, TBil, TNFalpha, NO, ET-1, IL-6 and liver histopathology of the rats were examined. RESULTS: In the D-Gal N and LPS model of FLF, there was an obvious decline of plasma TNFalpha (F = 52.84), NO (F = 15.81), ET-1 (F = 15.68), IL-6 (F = 15.32) and there was less hepatic tissue damage in SNMC-treated groups using different doses (high dose, medium dose, low dose) and at different times (pre-protection, simultaneous protection, post-protection) compared with those not treated with SNMC. These results indicated that SNMC could be used to treat FLF. It was better to use a low dose of SNMC and use it at the same time as inducing the FLF. There were no differences in the results of those treated with SNMC of different dosages and treated at different times. CONCLUSION: SNMC can decrease the mortality of FLF by preventing hepatocyte apoptosis induced by D-Gal N and LPS and inhibit liver inflammation caused by all kinds of factors.

[Molecular mechanisms of the protection of SNMC in HepG2 cell apoptosis].

OBJECTIVE: Apoptosis of the cells of liver cancer cell line HepG2 could be induced by TNF alpha and actinomycin D (Act D). In the current study, the molecular mechanism of the apoptosis protection of stronger neo-minophagen C (SNMC) to HepG2 cells was investigated. METHODS: SNMC was added to the HepG2 cell culture medium when the cell concentration reached 0, 2, 20, 100, 200, 800 microg/ml 30 min before their apoptosis were inducted with TNF alpha and Act D. A flow cytometry assay was performed to detect the cell apoptosis rate; electromicroscopy was employed to visualize the subcellular structure after apoptosis. DNA ladder formation was checked with genomic DNA agarose electrophoresis. The expression pattern of apoptosis related protein Caspase-3, Bcl-2 and Bax was detected by Western blot. RESULTS: After pretreatment with various concentrations of SNMC and 12 hours after treatment with TNF alpha and Act D, the HepG2 cell apoptosis rate and DNA ladder formation decreased dramatically when the SNMC concentration was higher in the media; the intracellular inactive form of Caspase-3 increased while the 17*10(3) active Caspase-3 decreased gradually. In addition, the expression of Bcl-2 increased and the expression of Bax decreased. Under the electromicroscope, the typical nucleolus condensation of HepG2 induced by TNF alpha and Act D was not seen among the 100 microg/ml SNMC treated cells. CONCLUSION: SNMC inhibits TNF alpha and Act D induced HepG2 cell apoptosis. This protective action may be regulated by intracellular apoptosis related factors.