[Pathogenesis of liver fibrosis in patients with chronic hepatitis B].

OBJECTIVE: To explore the role of sinusoidal endothelial cell in the development of liver fibrosis, and to dissect the relationship among hepatic microcirculation disorders, hepatic sinusoidal capilarization and liver fibrosis. METHODS: Liver biopsy was performed in fifty-six patients with chronic hepatitis B. The liver tissues were observed under light microscope and transmitted electronic microscope. RESULTS: Of 56 cases, 39 cases were mild hepatitis, 10 were moderate hepatitis, and 7 were severe hepatitis. The morphology of hepatic stellate cells (HSCs) was similar to that of fibroblasts in the tissues of the patients with chronic hepatitis B. Collagenous fibers were deposited around the hepatic stellate cells. Electron-dense materials were deposited between sinusoidal endothelial cell and hepatic stellate cell. The size and amount of fenestraes of sinusoidal endothelial cells were reduced in 53 of 56 cases. The consecutive or inconsecutive membrane-like materials were observed along sinusoidal endothelial cells in 20 cases. Collagen fibers were observed in the space of Disse in 15 cases. Even in the patients with normal hepatic functions, red blood cells aggregation and microthrombi could be observed in the liver tissues. CONCLUSION: Sinusoidal endothelial cells are involved in development of liver fibrosis by interacting with hepatic stellate cells. Hepatic microcirculation disorders and sinusoidal capillarization are important changes in the early stage of liver fibrosis.

Effects of endogenous nitric oxide induced by 5-fluorouracil and L-Arg on liver carcinoma in nude mice.

AIM: To study the effects of endogeous nitric oxide induced by 5-fluorouracil (5-FU) and L-arginine (L-Arg) on the human liver carcinoma model in nude mice. METHODS: The human liver carcinoma model in nude mice was established with BEL-7402 cells and normal saline (NS), 5-FU and 5-FU + L-Arg injected intraperitoneally. The tumor size was measured. The necrotic degree and range were observed under microscope. The apoptosis of cancer cell was detected by turmina deoxynucleotidyl transferanse mediated dUTP nick end labeling (TUNEL) method. Immunohistochemical method was performed to determine the expression of iNOS, P16, BAX. The chemical colorimetry was used to test the activity and nitrate reductase method was adopted to test the concentration of nitric oxide (NO) in the tumor tissue. The BI2000 pathological image analyzer was used to analyze the result of immunohistochemistry. RESULTS: 5-FU combined with L-Arg could inhibit the tumor growth apparently. In NS, 5-FU and 5-FU+L-Arg groups, the changes of tumor volumes were 257.978 +/- 59.0, 172.232 +/- 66.0 and 91.523 +/- 26.7 mm(3), respectively (P < 0.05 5-FU vs 5-FU + L-Arg group; P < 0.05 NS vs 5-FU + L-Arg group; P < 0.05, NS vs 5-FU group). The necrotic range and apoptosis index were significantly increased after the drug injection. The necrotic range was biggest in 5-FU + L-Arg group (c2 = 15.963, P < 0.05). The apoptosis indexes were as follows: NS, 17.4% +/- 6.19%; 5-FU, 31.3% +/- 12.3%; and 5-FU + L-Arg, 46% +/- 15.24% (P < 0.05, 5-FU vs 5-FU + L-Arg; P < 0.05, NS vs 5-FU + L-Arg; P < 0.05, NS vs 5-FU). The expression and activity of iNOS were increased in the tumor tissue. The concentration of NO was also increased. F of optical density of iNOS, iNOS activity and NO concentration are 31.693, 21.949, and 33.909, respectively, P < 0.05. The concentration of NO was related to the expression of P16 and BAX. The correlation coefficient was 0.764 and 0.554. CONCLUSION: 5-FU combined with L-Arg can inhibit the growth of tumor in nude mice. The effect may be related to inducing the synthesis and increasing the activity of iNOS. The production of NO is increased, and it can enhance the expression of apoptosis-related gene and antioncogene.

Effects of sodium phenylbutyrate on differentiation and induction of the P21WAF1/CIP1 anti-oncogene in human liver carcinoma cell lines.

OBJECTIVES: To explore the effects of sodium phenylbutyrate on the proliferation, differentiation, cell cycle arrest and induction of the P(21WAF1/CIP1) anti-oncogene in human liver carcinoma cell lines Bel-7402 and HepG2. METHODS: Bel-7402 and HepG2 human liver carcinoma cells were treated with sodium phenylbutyrate at different concentrations. Light microscopy was used to observe morphological changes in the carcinoma cells. Effects on the cell cycle were detected by using flow cytometry. P(21WAF1/CIP1) expression was determined by both reverse transcription-polymerase chain reaction and western blotting. Statistical analysis was performed by using one-way anova and Student's t-test. RESULTS: Sodium phenylbutyrate treatment caused time- and dose-dependent growth inhibition of Bel-7402 and HepG2 cells. This treatment also caused a decline in the proportion of S-phase cells and an increase in the proportion of G(0)/G(1) cells. Sodium phenylbutyrate increased the expression of P(21WAF1/CIP1). CONCLUSIONS: Sodium phenylbutyrate inhibits the proliferation of human liver carcinoma cells Bel-7402 and HepG2, induces partial differentiation, and increases the expression of P(21WAF1/CIP1).

[Methylated oligonucleotide inhibiting expression of human MRP2 and reversing multidrug resistance in hepatocellular carcinoma Cell HepG2].

BACKGROUND & OBJECTIVE: Accumulating evidences showed that the overexpression of multidrug resistance-associated protein 2 (MRP2) was the main cause of multidrug resistance in hepatocellular carcinoma (HCC); and the methylated cytosine at cytosine-guanine (CpG) dinucleotides might silence the gene expression. This study was to evaluate the inhibiting effect of methylated oligonucleotide (MON) on MRP2 gene transcription. METHODS: MON complementary to human MRP2 promoter region was designed. The non- methylated oligonucleotide (NON) carried same nucleotide acid sequence with MON, but the cytosines were not methylated. Cells from human HCC cell line HepG2 were divided into 3 groups: control group, NON group, and MON group. HepG2 cells were transfected with liposome-encapsulated oligonucleotide, cytotoxicity was determined by MTT assay. Reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry (FCM) were used to analyze the expression of MRP2. RESULTS: MON specifically inhibited MRP2 gene transcription and down-regulated the expression of MRP2 in a concentration- dependent manner in vitro, whereas NON had no effect. The positive rates of MRP2 protein were 18.0%, 9.15%, 5.73%, 3.73%, and 2.56% respectively after transfected with 1, 2, 4, 8, and 16 micromol/L MON. It was 24.5% in NON group. The 50% inhibitory concentration (IC50) of epirubicin, hydroxyl- camptothecin, and carboplatin were reduced in MON group. CONCLUSIONS: MON can inhibit MRP2 gene transcription, enhance chemosensitivity, and reverse multidrug resistance of HCC cells,it may become a new gene therapeutic agent for HCC.

Long-term effect of stent placement in 115 patients with Budd-Chiari syndrome.

AIM: To report the long-term effect of stent placement in 115 patients with Budd-Chiari syndrome (BCS). METHODS: One hundred and fifteen patients with BCS were treated by percutaneous stent placement. One hundred and two patients had IVC stent placement, 30 patients had HV stent placement, 17 of them underwent both IVC stent and HV stent. All the procedures were performed with guidance of ultrasound. RESULTS: The successful rates in placing IVC stent and HV stent were 94% (96/102) and 87% (26/30), respectively. Ninety-seven patients with 112 stents (90 IVC stents, 22 HV stents) were followed up. 96.7% (87/90) IVC stents and 90.9% (20/22) HV stents remained patent during follow up periods (mean 49 months, 45 months, respectively). Five of 112 stents in the 97 patients developed occlusion. Absence of anticoagulants after the procedure and types of obstruction (segmental and occlusive) before the procedure were related to a higher incidence of stent occlusion. CONCLUSION: Patients with BCS caused by short length obstruction can be treated by IVC stent placement, HV stent placement or both IVC and HV stent placement depending on the sites of obstruction. The long-term effect is satisfactory. Anticoagulants are strongly recommended after the procedure especially for BCS patients caused by segmental occlusion.

Effects of heparin on liver fibrosis in patients with chronic hepatitis B.

AIM: To evaluate the effects of heparin on liver fibrosis in patients with chronic hepatitis B. METHODS: Fifty-two cases under study were divided into two groups, group A and group B. The two groups were given regular treatment and heparin/low molecular weight heparin (LMWH) treatment respectively. Hepatic functions, serum hyaluronic acid (HA) and type IV collagen levels were measured before and after the treatment, and six cases were taken liver biopsy twice. RESULTS: After treatment, hepatic functions became significantly better in both groups. Serum HA and type IV collagen levels in group B compared with group A, decreased significantly after treatment. Collagen proliferation also decreased in group B after treatment. CONCLUSION: Heparin/LMWH can inhibit collagen proliferation in liver tissues with hepatitis B.

Resveratrol induces apoptosis in human esophageal carcinoma cells.

AIM: To investigate the apoptosis in esophageal cancer cells induced by resveratrol, and the relation between this apoptosis and expression of Bcl-2 and Bax. METHODS: In in vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscope and TUNEL staining method were used to quantitatively and qualitively detect the apoptosis status of esophageal cancer cell line EC-9706 before and after the resveratrol treatment. Immunohistochemical staining was used to detect the expression of apoptosis-regulated gene Bcl-2 and Bax. RESULTS: Resveratrol inhibited the growth of esophageal cancer cell line EC-9706 in a dose-and time-dependent manner. Resveratrol induced EC-9706 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation. TUNEL assay showed that after the treatment of EC-9706 cells with resveratrol (10 mmol/L) for 24 to 96 hours, the AIs were apparently increased with treated time (P<0.05). Immunohistochemical staining showed that after the treatment of EC-9706 cells with resveratrol (10 mmol/L) for 24 to 96 hours, the PRs of Bcl-2 proteins were apparently reduced with treated time (P<0.05) and the PRs of Bax proteins were apparently increased with treated time (P<0.05). CONCLUSION: Resveratrol is able to induce the apoptosis in esophageal cancer. This apoptosis may be mediated by down-regulating the apoptosis-regulated gene Bcl-2 and up-regulating the expression of apoptosis-regulated gene bax.

Paclitaxel induces apoptosis in human gastric carcinoma cells.

AIM: To investigate the apoptosis in gastric cancer cells induced by paclitaxel, and the relation between this apoptosis and expression of Bcl-2 and Bax. METHODS: In in vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscope and TUNEL staining method were used to quantitatively and qualitively detect the apoptosis status of gastric cancer cell line SGC-7901 before and after the paclitaxel treatment. Immunohistochemical staining was used to detect the expression of apoptosis-regulated gene Bcl-2 and Bax. RESULTS: Paclitaxel inhibited the growth of gastric cancer cell line SGC-7901 in a dose-and time-dependent manner. Paclitaxel induced SGC-7901 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation. Paclitaxel could reduce the expression of apoptosis-regulated gene Bcl-2, and improve the expression of apoptosis-regulated gene Bax. CONCLUSION: Paclitaxel is able to induce the apoptosis in gastric cancer. This apoptosis may be mediated by down-expression of apoptosis-regulated gene Bcl-2 and up-expression of apoptosis-regulated gene Bax.