The therapeutic effect of CORM-3 on acute liver failure induced by lipopolysaccharide/D-galactosamine in mice.

BACKGROUND: Acute liver failure (ALF) is a severe and life-threatening clinical syndrome resulting in a high mortality and extremely poor prognosis. Recently, a water-soluble CO-releasing molecule (CORM-3) has been shown to have anti-inflammatory effect. The present study was to investigate the effect of CORM-3 on ALF and elucidate its underlying mechanism. METHODS: ALF was induced by a combination of LPS/D-GalN in mice which were treated with CORM-3 or inactive CORM-3 (iCORM-3). The efficacy of CORM-3 was evaluated based on survival, liver histopathology, serum aminotransferase activities (ALT and AST) and total bilirubin (TBiL). Serum levels of inflammatory cytokines (TNF-alpha, IL-6, IL-1beta and IL-10) and liver immunohistochemistry of NF-kappaB-p65 were determined; the expression of inflammatory mediators such as iNOS, COX-2 and TLR4 was measured using Western blotting. RESULTS: The pretreatment with CORM-3 significantly improved the liver histology and the survival rate of mice compared with the controls; CORM-3 also decreased the levels of ALT, AST and TBiL. Furthermore, CORM-3 significantly inhibited the increased concentration of pro-inflammatory cytokines (TNF-alpha, IL-6 and IL-1beta) and increased the anti-inflammatory cytokine (IL-10) productions in ALF mice. Moreover, CORM-3 significantly reduced the increased expression of iNOS and TLR4 in liver tissues and inhibited the nuclear expression of NF-kappaB-p65. CORM-3 had no effect on the increased expression of COX-2 in the ALF mice. An iCORM-3 failed to prevent acute liver damage induced by LPS/D-GalN. CONCLUSION: These findings provided evidence that CORM-3 may offer a novel alternative approach for the management of ALF through anti-inflammatory functions.

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.

Antitumor and immunomodulating activity of a polysaccharide from Sophora flavescens Ait.

The immunostimulatory activity of Sophora flavescens polysaccharide (SFPW1) was evaluated by using in vitro cell models and in vivo animal models. The results demonstrated that SFPW1 could effectively inhibit the tumor growth in H22 tumor-bearing mice and promote the splenocyte proliferation, thus resulting in a prolonged life survival. For assay in vitro, SFPW1 significantly strengthened peritoneal macrophages to devour H22 tumor cells and stimulated macrophages to produce nitric oxide (NO) via up-regulation of inducible NO synthase (iNOS) activity. However, no direct cytotoxicity against H22 tumor cells was observed in vitro. These results suggest that SFPW1 might be a strong natural immunomodulator and the antitumor effect of this polysaccharide is associated with its potent immunostimulating effect.

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.

[Observations on clinic curative effect of oxymatrine to cure hemorrhagic fever with renal syndrome (HFRS)].

OBJECTIVE: To observe the curative effect of oxymatrine to cure hemorrhagic fever with renal syndrome. METHODS: Randomly divide 418 patients suffering from hemorrhagic fever with renal syndrome into a treatment group and a control group, use oxymatrine to give an intravenous drip to the patients in the treatment group, one time a day, each time 600 mg, lasting for 7 days as one period of treatment and meanwhile take a traditional equilibrium salt treatment for the patients in the control group. Observe the change of the illness in both groups and occurrence of complications, dynamically test the magnitude of serum urination-regulated protein (THP) and beta(2) microglobulin (beta(2)M) of the patients, in the meantime observe the change in magnitude of serum white blood cell medium 15 (IL-15) and soluble intercellular adhesion molecules (sICAM-1). RESULTS: Shorten the course of the disease of the patients in the oxymatrine treatment group, make an improvement after the recovery and obviously reduce the magnitude of serum urination-regulated protein (THP) and beta(2) microglobulin and show a striking difference in magnitude of serum IL-15 and sICAM-1 after the treatment compared with that of the patients in the control group. CONCLUSION: Oxymatrine has a certainly true curative effect to hemorrhagic fever with renal syndrome and worth of furthering its widespread use in clinics.

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.

The roles of serum IL-18, IL-10, TNF-alpha and sIL-2R in patients with chronic hepatitis C.

OBJECTIVE: To identify the roles of serum IL-18, IL-10, TNF-alpha and sIL-2R in the pathogenesis of chronic hepatitis C and the effects of interferon on the mentioned serum cytokines. METHODS: The levels of IL-18, IL-10, TNF-alpha and sIL-2R were detected in 10 healthy controls, 24 asymptomatic HCV carriers, and 27 patients with chronic hepatitis C (before and after IFN treatment) by enzyme linked immunosorbent assay (ELISA). RESULTS: The levels of IL-18, IL-10, TNF-alpha and sIL-2R in the patients of chronic hepatitis C were higher than those in the healthy controls (P<0.05) and in asymptomatic HCV carriers (P<0.05). The values of the mentioned cytokines showed a significant positive correlation to GPT. The levels of the mentioned cytokines decreased obviously after IFN treatment (P<0.05), while the serum levels of IL-10 and sIL-2R reduced in sequence in no-response group, partial-response group and complete-response group. CONCLUSIONS: IL-18, IL-10, TNF-alpha and sIL-2R co-participate in the pathogenesis of chronic hepatitis C, and are used to evaluate the effect of IFN on the immune state of organisms, and IL-10 and sIL-2R are important for predicting the anti-viral efficacy of IFN.