Precision targeting in hepatocellular carcinoma: Exploring ligand-receptor mediated nanotherapy.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and poses a major challenge to global health due to its high morbidity and mortality. Conventional chemotherapy is usually targeted to patients with intermediate to advanced stages, but it is often ineffective and suffers from problems such as multidrug resistance, rapid drug clearance, nonspecific targeting, high side effects, and low drug accumulation in tumor cells. In response to these limitations, recent advances in nanoparticle-mediated targeted drug delivery technologies have emerged as breakthrough approaches for the treatment of HCC. This review focuses on recent advances in nanoparticle-based targeted drug delivery systems, with special attention to various receptors overexpressed on HCC cells. These receptors are key to enhancing the specificity and efficacy of nanoparticle delivery and represent a new paradigm for actively targeting and combating HCC. We comprehensively summarize the current understanding of these receptors, their role in nanoparticle targeting, and the impact of such targeted therapies on HCC. By gaining a deeper understanding of the receptor-mediated mechanisms of these innovative therapies, more effective and precise treatment of HCC can be achieved.

Prohibitin 1 inhibits cell proliferation and induces apoptosis via the p53-mediated mitochondrial pathway in vitro.

BACKGROUND: Prohibitin 1 (PHB1) has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed, and it participates in a variety of essential cellular functions, including apoptosis, cell cycle regulation, proliferation, and survival. Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma (HCC). However, the role of PHB1 in HCC is controversial. AIM: To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro. METHODS: HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria; then, PHB1 levels in the sera and liver tissues of these participates were determined using ELISA, RT-PCR, and immunohistochemistry. Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA (shRNA-PHB1) for 24-72 h. Cell proliferation was analysed with an MTT assay. Cell cycle progression and apoptosis were analysed using flow cytometry (FACS). The mRNA and protein expression levels of the cell cycle-related molecules p21, Cyclin A2, Cyclin E1, and CDK2 and the cell apoptosis-related molecules cytochrome C (Cyt C), p53, Bcl-2, Bax, caspase 3, and caspase 9 were measured by real-time PCR and Western blot, respectively. RESULTS: Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals, and decreased PHB1 was positively correlated with low differentiation, TNM stage III-IV, and alpha-fetoprotein ≥ 400 µg/L. Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner. FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis. The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells. The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41% ± 1.06%, which was significantly greater than that of apoptotic control cells (3.65% ± 0.85%, P < 0.01) and empty vector-transfected cells (4.21% ± 0.52%, P < 0.01). Similar results were obtained with SMMC-7721 cells. Furthermore, the mRNA and protein expression levels of p53, p21, Bax, caspase 3, and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2, Cyclin E1, CDK2, and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells. However, when PHB1 was upregulated in human HCC cells, Cyt C expression levels were increased in the cytosol and decreased in the mitochondria, which indicated that Cyt C had been released into the cytosol. Conversely, these effects were reversed when PHB1 was knocked down. CONCLUSION: PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.

VCAN, expressed highly in hepatitis B virus-induced hepatocellular carcinoma, is a potential biomarker for immune checkpoint inhibitors.

BACKGROUND: As a proteoglycan, VCAN exists in the tumor microenvironment and regulates tumor proliferation, invasion, and metastasis, but its role in hepatocellular carcinoma (HCC) has not yet been elucidated. AIM: To investigate the expression and potential mechanism of action of VCAN in HCC. METHODS: Based on The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset, we explored the correlation between VCAN expression and clinical features, and analyzed the prognosis of patients with high and low VCAN expression. The potential mechanism of action of VCAN was explored by Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and gene set enrichment analysis. We also explored immune cell infiltration, immune checkpoint gene expression, and sensitivity of immune checkpoint [programmed cell death protein 1 (PD-1)/cytotoxic T lymphocyte antigen 4 (CTLA4)] inhibitor therapy in patients with different VCAN expression. VCAN mRNA expression and VCAN methylation in peripheral blood were tested in 100 hepatitis B virus (HBV)-related patients (50 HCC and 50 liver cirrhosis). RESULTS: VCAN was highly expressed in HCC tissues, which was associated with a poor prognosis in HCC patients. No significant difference was found in VCAN mRNA expression in blood between patients with HBV-related cirrhosis and those with HCC, but there was a significant difference in VCAN methylation between the two groups. The correlation between VCAN and infiltrations of several different tumor immune cell types (including B cells, CD8+ T cells, and eosinophils) was significantly different. VCAN was strongly related to immune checkpoint gene expression and tumor mutation burden, and could be a biomarker of sensitivity to immune checkpoint (PD1/CTLA4) inhibitors. In addition, VCAN mRNA expression was associated with hepatitis B e antigen, HBV DNA, white blood cells, platelets, cholesterol, and coagulation function. CONCLUSION: High VCAN level could be a possible biomarker for poor prognosis of HCC, and its immunomodulatory mechanism in HCC warrants investigation.

DNA methylation and single-nucleotide polymorphisms in DDX58 are associated with hand, foot and mouth disease caused by enterovirus 71.

BACKGROUND: This research aimed to explore the association between the RIG-I-like receptor (RIG-I and MDA5 encoded by DDX58 and IFIH1, respectively) pathways and the risk or severity of hand, foot, and mouth disease caused by enterovirus 71 (EV71-HFMD). In this context, we explored the influence of gene methylation and polymorphism on EV71-HFMD. METHODOLOGY/PRINCIPAL FINDINGS: 60 healthy controls and 120 EV71-HFMD patients, including 60 mild EV71-HFMD and 60 severe EV71-HFMD patients, were enrolled. First, MiSeq was performed to explore the methylation of CpG islands in the DDX58 and IFIH1 promoter regions. Then, DDX58 and IFIH1 expression were detected in PBMCs using RT-qPCR. Finally, imLDR was used to detect DDX58 and IFIH1 single-nucleotide polymorphism (SNP) genotypes. Severe EV71-HFMD patients exhibited higher DDX58 promoter methylation levels than healthy controls and mild EV71-HFMD patients. DDX58 promoter methylation was significantly associated with severe HFMD, sex, vomiting, high fever, neutrophil abundance, and lymphocyte abundance. DDX58 expression levels were significantly lower in mild patients than in healthy controls and lower in severe patients than in mild patients. Binary logistic regression analysis revealed statistically significant differences in the genotype frequencies of DDX58 rs3739674 between the mild and severe groups. GeneMANIA revealed that 19 proteins displayed correlations with DDX58, including DHX58, HERC5, MAVS, RAI14, WRNIP1 and ISG15, and 19 proteins displayed correlations with IFIH1, including TKFC, IDE, MAVS, DHX58, NLRC5, TSPAN6, USP3 and DDX58. CONCLUSIONS/SIGNIFICANCE: DDX58 expression and promoter methylation were associated with EV71 infection progression, especially in severe EV71-HFMD patients. The effect of DDX58 in EV71-HFMD is worth further attention.

Real life efficacy and safety of direct-acting antiviral therapy for treatment of patients infected with hepatitis C virus genotypes 1, 2 and 3 in northwest China.

BACKGROUND: Regimens involving direct-acting antiviral agents (DAAs) are recommended for the treatment of infection with hepatitis C virus (HCV) genotypes 1, 2 and 3. But real-world data is still not enough, especially in Asia. AIM: To investigate the efficacy and safety of DAA-based regimens in a real-life setting in China. METHODS: This study included 366 patients infected with HCV genotypes 1, 2 and 3, with or without cirrhosis, who were observed between May 2015 and December 2018. They were treated with ledipasvir and sofosbuvir (SOF) (genotype 1) with or without ribavirin (RBV), SOF and RBV (genotype 2), or SOF and daclatasvir (genotype 3), with or without RBV, for 12 or more wk. The participants' sustained virological responses (SVR) at post-treatment week 12 (SVR12) was the primary endpoint. The occurrence of adverse events and drug-drug interactions were recorded. RESULTS: In the 366 patients, genotype 1 (59.0%) was the most common genotype, followed by genotypes 2 (34.4%) and 3 (6.6%). Liver cirrhosis was diagnosed in 154 (42.1%) patients. Fifty (13.7%) patients were treatment-experienced. Intention-to-treat analysis revealed that SVR12 was 86.3% (316/366). For modified intention-to-treat analysis, SVR12 was achieved in 96.6% of overall patients (316/327), 96.3% in patients with genotype 1, 97.5% in those with genotype 2, and 95.0% in those with genotype 3. Most of the treatment failures were due to lack of follow-up (3 cases had non-responses, 1 had virological breakthrough, 11 relapsed and 36 did not participate in the follow-up). There was no significant difference in SVR between different genotypes and liver statuses (P < 0.05). Patients with lower alanine aminotransferase levels at baseline who achieved an end of treatment response were more likely to achieve SVR12 (P < 0.05). High SVR was observed regardless of age, gender, liver status, alpha-fetoprotein, HCV RNA levels or history of antiviral therapy (P > 0.05 for all). The cumulative hepatocellular carcinoma occurrence and recurrence rate after using the DAAs was 0.9%. Most of the adverse events were mild. We found two cases of special adverse events. One case involved facial and bilateral lower extremity edema, and the other case showed an interesting change in lipid levels while on medication. No severe adverse events were noted. CONCLUSION: The DAA-based regimens tested in this study have excellent effectiveness and safety in all patients infected with HCV genotypes 1, 2 and 3, including those with cirrhosis.

Caffeic acid phenethyl ester up-regulates antioxidant levels in hepatic stellate cell line T6 via an Nrf2-mediated mitogen activated protein kinases pathway.

AIM: To investigate the antioxidant effect of caffeic acid phenethyl ester (CAPE) in hepatic stellate cell-T6 (HSC-T6) cells cultured in vitro and the potential mechanisms. METHODS: HSC-T6 cells were cultured in vitro and treated with various concentrations of CAPE for 24, 48 and 72 h, respectively. Cell proliferation was investigated using the MTT assay, and cell ultrastructural alterations were observed by transmission electron microscopy. Flow cytometry was employed to investigate the effects of CAPE on apoptosis and the levels of reactive oxygen species in HSC-T6 cells cultured in vitro. An enzyme immunoassay instrument was used to evaluate antioxidant enzyme expression. The effect on α-smooth muscle actin was shown using immunofluorescence. Gene and protein levels of Nrf2, related factors, and mitogen activated protein kinases (MAPKs), in HSC-T6 cells were detected using RT-PCR and Western blot, respectively. RESULTS: CAPE inhibited the proliferation and activation of HSC-T6 cells cultured in vitro. CAPE increased the antioxidant levels and the translocation of Nrf2 from the cytoplasm to the nucleus in HSC-T6 cells. Moreover, the phosphorylation of MAPKs in cells decreased in response to CAPE. Interestingly, CAPE-induced oxidative stress in the cells was significantly attenuated by pretreatment with MAPKs inhibitors. CONCLUSION: CAPE inhibits cell proliferation and up-regulates the antioxidant levels in HSC-T6 cells partly through the Nrf2-MAPKs signaling pathway.

[Oestrogen inhibits invasion and metastasis of hepatocellular carcinoma MHCC97H cells by regulating the activity of AKT signaling pathway].

OBJECTIVE: To explore the inhibitory effect of estrogen against metastasis of human hepatocellular carcinoma MHCC97H cells and explore the molecular mechanism. METHODS: The inhibitory effect of estrogen on the migration and invasion of MHCC97H cells was evaluated with wound healing assay and Transwell assay. Western blotting was used for investigating the expression of MMP-2, MMP-9, AKT and p-AKT in the cells treated with estrogen. RESULTS: Estrogen treatment significantly inhibited the migration and invasion of MHCC97H cells in a dose-dependent manner. Estrogen significantly down-regulated the protein expressions of MMP-2 and MMP-9 and lowered the phosphorylation level of AKT. CONCLUSION: The anti-metastatic effect of estrogen involves inhibition of MMP-2 and MMP-9 in MHCC97H cells probably by regulating AKT signal pathway.

Guggulsterone induces apoptosis of human hepatocellular carcinoma cells through intrinsic mitochondrial pathway.

AIM: To investigate the effects of guggulsterone on the proliferation and apoptosis of human hepatoma HepG2 cells in vitro and relevant mechanisms. METHODS: Human hepatocellular carcinoma HepG2 cells and normal human liver L-02 cells were treated with different concentrations of guggulsterone (5-100 µmol/L) for 24-72 h. Cell proliferation was tested by MTT assay. Cell cycle and apoptosis were investigated using flow cytometry (FACS). Bcl-2 and Bax mRNA and protein expression was detected by real-time PCR and Western blot, respectively. TGF-ß1, TNF-α, and VEGF contents were determined by ELISA. RESULTS: Guggulsterone significantly inhibited HepG2 cell proliferation in a dose- and time-dependent manner. FACS showed that guggulsterone arrested HepG2 cell cycle at G0/G1 phase. Guggulsterone induced apoptosis was also observed in HepG2 cells, with 24.91% ± 2.41% and 53.03% ± 2.28% of apoptotic cells in response to the treatment with 50 µmol/L and 75 µmol/L guggulsterone, respectively. Bax mRNA and protein expression was significantly increased and Bcl-2 mRNA and protein expression was decreased. ELISA analysis showed that the concentrations of TGF-ß1 and VEGF were significantly decreased and TNF-α concentration was increased. CONCLUSION: Guggulsterone exerts its anticancer effects by inhibiting cell proliferation and inducing apoptosis in HepG2 cells. Guggulsterone induces apoptosis by activation of the intrinsic mitochondrial pathway.

Cytokeratin 8 is increased in hepatitis C virus cells and its ectopic expression induces apoptosis of SMMC7721 cells.

AIM: To investigate cytokeratin 8 (CK8) overexpression during hepatitis C virus (HCV) infection and its pathogenesis, and the effect of ectopic CK8 expression on hepatoma cell lines. METHODS: We successfully established an in vitro HCV cell culture system (HCVcc) to investigate the different expression profiles of CK8 in Huh-7-HCV and Huh-7.5-HCV cells. The expression of CK8 at the mRNA level was determined by real-time polymerase chain reaction (RT-PCR). The expression of CK8 at the protein level was evaluated by Western blotting. We then constructed a eukaryotic expression combination vector containing the coding sequence of human full length CK8 gene. CK8 cDNA was amplified by reverse transcription-PCR and inserted into pEGFP-C1 and the positive clone pEGFP-CK8 was obtained. After confirming the sequence, the recombinant plasmid was transfected into SMMC7721 cells with lipofectamine2000 and CK8 expression was detected using inverted fluorescence microscopy, RT-PCR and Western blotting. Besides, we identified biological function of CK8 on SMMC7721 cells, including cell proliferation, cell cycle and apoptosis detection. RESULTS: RT-PCR showed that the expression level of CK8 in Huh-7-HCV and Huh-7.5-HCV cells was 2.88 and 2.95 times higher than in control cells. Western blot showed that CK8 expression in Huh-7-HCV and Huh-7.5-HCV cells was 2.53 and 3.26 times higher than that in control cells, respectively. We found that CK8 at mRNA and protein levels were both significantly increased in HCVcc. CK8 was up-regulated in SMMC7721 cells. CK8 expression at the mRNA level was significantly upregulated in SMMC7721/pEGFP-CK8 cells. CK8 expression in SMMC7721/ pEGFP-CK8 cells was 2.69 times higher than in SMMC7721 cells, and was 2.64 times higher than in SMMC7721/pEGFP-C1 cells. CK8 expression at the protein level in SMMC7721/pEGFP-CK8 cells was 2.46 times higher than in SMMC7721 cells, and was 2.29 times higher than in SMMC7721/pEGFP-C1 cells. Further analysis demonstrated that forced expression of CK8 slowed cell growth and induced apoptosis of SMMC7721 cells. CONCLUSION: CK8 up-regulation might have a functional role in HCV infection and pathogenesis, and could be a promising target for the treatment of HCV infection.

Increased expression of chondroitin sulphate proteoglycans in rat hepatocellular carcinoma tissues.

AIM: To investigate the expression of chondroitin sulphate proteoglycans (CSPGs) in rat liver tissues of hepatocellular carcinoma (HCC). METHODS: Thirty male Sprague Dawley rats were randomly divided into two groups: control group (n = 10) and HCC model group (n = 20). Rats in the HCC model groups were intragastrically administrated with 0.2% (w/v) N-diethylnitrosamine (DEN) every 5 d for 16 wk, whereas 0.9% (w/v) normal saline was administered to rats in the control group. After 16 wk from the initiation of experiment, all rats were killed and livers were collected and fixed in 4% (w/v) paraformaldehyde. All tissues were embedded in paraffin and sectioned. Histological staining (hematoxylin and eosin and Toluidine blue) was performed to demonstrate the onset of HCC and the content of sulphated glycosaminoglycan (sGAG). Immunohistochemical staining was performed to investigate the expression of chondroitin sulphate (CS)/dermatan sulphate (DS)-GAG, heparan sulphate (HS)-GAG, keratan sulphate (KS)-GAG in liver tissues. Furthermore, expression and distribution of CSPG family members, including aggrecan, versican, biglycan and decorin in liver tissues, were also immunohistochemically determined. RESULTS: After 16 wk administration of DEN, malignant nodules were observed on the surface of livers from the HCC model group, and their hepatic lobule structures appeared largely disrupted under microscope. Toluidine blue staining demonstrated that there was an significant increase in sGAG content in HCC tissues when compared with that in the normal liver tissues from the control group [0.37 ± 0.05 integrated optical density per stained area (IOD/area) and 0.21 ± 0.01 IOD/area, P < 0.05]. Immunohistochemical studies demonstrated that this increased sGAG in HCC tissues was induced by an elevated expression of CS/DS (0.28 ± 0.02 IOD/area and 0.18 ± 0.02 IOD/area, P < 0.05) and HS (0.30 ± 0.03 IOD/area and 0.17 ± 0.02 IOD/area, P < 0.01) but not KS GAGs in HCC tissues. Further studies thereby were performed to investigate the expression and distribution of several CSPG components in HCC tissues, including aggrecan, versican, biglycan and decorin. Interestingly, there was a distinct distribution pattern for these CSPG components between HCC tissues and the normal tissues. Positive staining of aggrecan, biglycan and decorin was localized in hepatic membrane and/or pericellular matrix in normal liver tissues; however, their expression was mainly observed in the cytoplasm, cell membranes in hepatoma cells and/or pericellular matrix within HCC tissues. Semi-quantitative analysis indicated that there was a higher level of expression of aggrecan (0.43 ± 0.01 and 0.35 ± 0.03, P < 0.05), biglycan (0.32 ± 0.01 and 0.25 ± 0.01, P < 0.001) and decorin (0.29 ± 0.01 and 0.26 ± 0.01, P < 0.05) in HCC tissues compared with that in the normal liver tissues. Very weak versican positive staining was observed in hepatocytes near central vein in normal liver tissues; however there was an intensive versican distribution in fibrosis septa between the hepatoma nodules. Semi-quantitative analysis indicated that the positive rate of versican in hepatoma tissues from the HCC model group was much higher than that in the control group (33.61% and 21.28%, P < 0.05). There was no positive staining in lumican and keratocan, two major KSPGs, in either normal or HCC liver tissues. CONCLUSION: CSPGs play important roles in the onset and progression of HCC, and may provide potential therapeutic targets and clinical biomarkers for this prevalent tumor in humans.

Comparison of entecavir and adefovir for the treatment of chronic hepatitis B

The results of several new clinical trials that compared the effectiveness of entecavir (ETV) treatment with that of adefovir (ADV) treatment in patients with chronic hepatitis B (CHB) were published in recent years. However, the numbers of patients included in these clinical trials were too small to draw a clear conclusion as to whether ETV is more effective than ADV. Therefore, a new meta-analysis was needed to compare ETV with ADV for the treatment of CHB. A search of the Cochrane Central Register of Controlled Trials (CCTR), MEDLINE, the Science Citation Index, Embase, the China National Knowledge Infrastructure (CNKI), and the Wanfang Database for relevant studies published between 1966 and 2010 was performed. Trials comparing the use of ETV and ADV for the treatment of CHB were assessed. Of the 2,358 studies screened, 13 randomized controlled clinical trials comprising 1,230 patients (ETV therapy, 621; ADV therapy, 609) were analyzed. The serum hepatitis B virus (HBV) DNA clearance rate obtained in patients treated with ETV was significantly higher than that in patients treated with ADV at the 24th and 48th weeks of treatment (24 weeks: 59.6% vs. 31.8%, relative risk [RR], 1.82, 95% CI: 1.49-2.23; 48 weeks: 78.3% vs. 50.4%, RR, 1.61, 95% CI: 1.32-1.96). The serum HBeAg clearance rate, the HBeAg seroconversion rate, and the ALT normalization rate obtained for patients treated with ETV were also higher than the corresponding values for patients treated with ADV at the 48th week of treatment. The safety profiles were similar between patients treated with ETV and those treated with ADV. The evidence reviewed in this meta-analysis suggests that patients with hepatitis B have a greater likelihood of achieving a viral response and a biomedical response when treated with ETV than when treated with ADV.

Prohibitin is overexpressed in Huh-7-HCV and Huh-7.5-HCV cells harboring in vitro transcribed full-length hepatitis C virus RNA.

BACKGROUND: Currently, up-regulated proteins and apoptosis in hepatitis C is a hot topic in exploring the pathogenic mechanism of Heptitis C Virus(HCV). Some recent studies shows that prohibitin is overexpressed in cells expressing HCV core proteins, and up-regulated prohibitin is also found in human hepatoma cell line HCC-M, lung cancer, prostate cancer, and other cancers. Prohibitin is an important member of the membrane protein superfamily, and it plays a role of molecular chaperones in mitochondrial protein stability. Meanwhile, it has a permissive action on tumor growth or acts as an oncosuppressor. Based on our previously established the in vitro HCV cell-culture system (HCVcc), here we aimed to investigate the different expression profiles of prohibitin in Huh-7-HCV and Huh-7.5-HCV cells METHODS: The total cellular RNA of Huh-7, Huh-7.5, Huh-7-HCV and Huh-7.5-HCV cells were extracted, and then the first-strand cDNA was reversely transcribed. The expression of prohibitin at the mRNA level was assessed by real-time PCR with GAPDH as the control. Furthermore, the expression of prohibitin at the protein level was evaluated by western blot with GAPDH as an internal control. RESULTS: Our results of real-time PCR showed that the mRNA expression level of prohibitin in Huh-7-HCV cells was 2.09 times higher than that in Huh-7 cells, while, the mRNA level of prohibitin in Huh-7.5-HCV cells was 2.25 times higher than that in Huh-7.5 cells. The results of western blot showed that the protein expression level of prohibitin in Huh-7-HCV cells was 2.38 times higher than that in Huh-7 cells, while the protein expression of prohibitin in Huh-7.5-HCV cells was 2.29 times higher than that in Huh-7.5 cells. CONCLUSIONS: The expression of prohibitin was relatively high in Huh-7-HCV and Huh-7.5-HCV cells harboring in vitro transcribed full-length HCV RNA.

Apoptotic bone marrow CD34+ cells in cirrhotic patients.

AIM: To access the frequency and level of apoptotic CD34+ cells isolated from the marrow fluid of patients with post-hepatitis cirrhosis. METHODS: The frequency of bone marrow CD34+ cells and apoptotic bone marrow CD34+ cells in 31 in-patients with post-hepatitis cirrhosis (cirrhosis group), and 15 out-patients without liver or blood disorders (control group) was calculated by flow cytometry. Parameters were collected to evaluate liver functions of patients in cirrhosis group. RESULTS: The percentage of normal bone marrow CD34+ cells was 6.30% ± 2.48% and 1.87% ± 0.53% (t = 3.906, P < 0.01) while that of apoptotic marrow CD34+ cells was 15.00% ± 15.81% and 5.73% ± 1.57% (t = 2.367, P < 0.05) in cirrhosis and control groups, respectively. The percentage of apoptotic marrow CD34+ cells was 6.25% ± 3.30% and 20.92 ± 18.5% (t = 2.409, P < 0.05) in Child-Pugh A and Child-Pugh B + C cirrhotic patients, respectively. The percentage of late apoptotic marrow CD34+ cells was positively correlated with the total bilirubin and aspartate aminotransferase serum levels in patients with cirrhosis. CONCLUSION: The status of CD34+ marrow cells in cirrhotic patients may suggest that the ability of hematopoietic progenitor cells to transform into mature blood cells is impaired.

[Angiogenesis inhibitory effect of saikosaponin-d on chicken embryo].

OBJECTIVE: To investigate the inhibitory effects of saikosaponin-d (SSd) on angiogenesis in chicken embryos and its mechanism of action. METHODS: Chorioallantoic membrane (CAM) model was established successfully in 86 chicken embryos. They were divided into 4 groups after fenestration: the three SSd treated groups (A, B and C) treated with high (20 microg/mL, n = 16), middle (10 microg/mL, n = 19) and low (5 microg/mL, n = 25) dose of SSd respectively, and the control group treated with 0.01 mol/L PBS (n = 26). The drug or reagent was administered by grafting 20 microL onto the surface of CAM. After incubation for 3 days, the vessel growth was recorded by digital photography; inflammatory cells were counted under light microscope with HE staining, and the positive rate of angiogenesis reaction was calculated by Leica image analyzer. RESULTS: On the 6th day of the embryonic age, vessels in the chicken embryo CAM showed a radial growing in spok-wheel pattern around the gelatin sponges with lateral axis running through it. Whereas after 3 days of SSd treatment, the angiogenesis reduced significantly with vague microvessels around the sponge, and vascular truncation and absence revealed. Microscopic examinations showed that the number of microvessels and infiltrated inflammatory cells in the sponge and peripheral CAM mesenchyme in the SSd groups were less than those in the control group, especially on vessels of medium and small size (P < 0.05, P < 0.01, respectively), but was insignificant on great vessels (P > 0.05). Correlation analysis revealed no correlation between the number of the great vessels in CAM and the infiltrated inflammatory degrees (r = 0.117, P > 0.05), but the increase of small vessels in CAM was positively correlated with that of inflammatory cells (r = 0.971, P < 0.01). CONCLUSIONS: SSd could inhibit the physiological angiogenesis of chicken embryoe, especially for the medium and small vessels, while there was no significant effect on great vessels (P > 0.05). Its mechanism of action may be related to its inhibition on leukocyte migration and activation.

Identification of a novel protein binding to hepatitis C virus core protein.

BACKGROUND: Hepatitis C virus (HCV) core protein is a multi-functional viral protein that interacts with several target proteins of both viral and cellular origin. AIM AND METHODS: To gain insight into the mechanism of action of HCV core protein, we used a yeast two-hybrid system to identify the core protein-interacting cellular targets. RESULTS: A cDNA clone encoding an aspartoacylase was obtained, termed aspartoacylase 3 (ACY3). Interaction between ACY3 and HCV core protein was verified using a co-immunoprecipitation assay in vitro, and a mammalian two-hybrid system in vivo. Fluorescence microscopy showed green fluorescence protein-fused ACY3 localized in the cytoplasm. CONCLUSION: Our data suggest that ACY3 is an HCV core binding protein, which may play a role in the development of HCV-associated diseases.

Inhibitory effects of saikosaponin-d on CCl4-induced hepatic fibrogenesis in rats.

AIM: To investigate the suppressive effect of saikosaponin-d (SSd) on hepatic fibrosis in rats induced by CCl(4) injections in combination with alcohol and high fat, low protein feeding and its relationship with the expression of nuclear factor-kappaB (NF-kappaB), tumor necrosis factor-alpha (TNF-alpha) and interleukins-6 (IL-6). METHODS: Hepatic fibrosis models were induced by subcutaneous injection of CCl(4) at a dosage of 3 mL/kg in rats. At the same time, rats in treatment groups were injected intraperitoneally with SSd at different doses (1.0, 1.5 and 2.0 mg/kg) once daily for 6 wk in combination with CCl(4), while the control group received olive oil instead of CCl(4). At the end of the experiment, rats were anesthetized and killed (except for 8 rats which died during the experiment; 2 from the model group, 3 in high-dose group, 1 in medium-dose group and 2 in low-dose group). Hematoxylin and eosin (HE) staining and Van Gieson staining were used to examine the changes in liver pathology. The levels of alanine aminotransferase (ALT), triglyeride (TG), albumin (ALB), globulin (GLB), hyaluronic acid (HA) and laminin (LN) in serum and the content of hydroxyproline (HYP) in liver were measured by biochemical examinations and radioimmuneoassay, respectively. In addition, the expression of TNF-alpha and IL-6 in liver homogenate was evaluated by enzyme-linked immunosorbent assay (ELISA) and the levels of NF-kappaBp65 and I-kappaBalpha in liver tissue were analyzed by Western blotting. RESULTS: Both histological examination and Van Gieson staining demonstrated that SSd could attenuate the area and extent of necrosis and reduce the scores of liver fibrosis. Similarly, the levels of ALT, TG, GLB, HA, and LN in serum, and the contents of HYP, TNF-alpha and IL-6 in liver were all significantly increased in model group in comparison with those in control group. Whereas, the treatment with SSd markedly reduced all the above parameters compared with the model group, especially in the medium group (ALT: 412 +/- 94.5 IU/L vs 113.76 +/- 14.91 IU/L, TG: 0.95 +/- 0.16 mmol/L vs 0.51 +/- 0.06 mmol/L, GLB: 35.62 +/- 3.28 g/L vs 24.82 +/- 2.73 g/L, HA: 42.15 +/- 8.25 ng/mL vs 19.83 +/- 3.12 ng/mL, LN: 27.56 +/- 4.21 ng/mL vs 13.78 +/- 2.57 ng/mL, HYP: 27.32 +/- 4.32 mug/mg vs 16.20 +/- 3.12 mug/mg, TNF-alpha: 4.38 +/- 0.76 ng/L vs 1.94 +/- 0.27 ng/L, IL-6: 28.24 +/- 6.37 pg/g vs 12.72 +/- 5.26 pg/g, respectively, P < 0.01). SSd also decreased ALB in serum (28.49 +/- 4.93 g/L vs 37.51 +/- 3.17 g/L, P < 0.05). Moreover, the expression of NF-kappaB p65 in the liver of treated groups was lower than that in model groups while the expression of I-kappaBalpha was higher in treated group than in model group (P < 0.01). The expression of NF-kappaBp65 and TNF-alpha had a positive correlation with the level of HA in serum of rats after treatment with CCl(4) (r = 0.862, P < 0.01; r = 0.928, P < 0.01, respectively). CONCLUSION: SSd attenuates CCl(4)-induced hepatic fibrosis in rats, which may be related to its effects of hepato-protective and anti-inflammation properties, the down-regulation of liver TNF-alpha, IL-6 and NF-kappaBp65 expression and the increased I-kappaBalpha activity in liver.