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Objective: To investigate the beneficial effects of Rhein (RH) on hepatic progression in hepatitis B virus (HBV)-transgenic mice with nonalcoholic steatohepatitis induced by a high-fat (HF) diet., Methods: A mice model of HBV chronic infection concomitant with liver steatosis was induced by a HF diet in 4-week old HBV-transgenic mice for 16 weeks (n = 130). Thereafter, the mice were divided randomly into control group (back to normal chow), model group (continuing HF diet), RH group [continuing HF diet and administering with 120 mg/(kg x d) RH by gavage] and Essentiale group [continuing HF diet and administering with 69.2 mg/(kg x d) Essentiale by gavage] with 30 mice in each, and were sacrificed at the end of 24-week and 48-week respectively. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG) and fasting plasma glucose (FPG) were measured by an automatic biochemical analyzer, and serum HBV-DNA was determined with qPCR. Hepatic histology was evaluated by HE staining with a light microscope., Results: (1) An histological change composed of steatosis, lymphocytes intralobular infiltration and ballooning was observed after 48 weeks feeding of HF diet, in part mimicking that of NASH patients as evidenced by a NAFLD activity score (NAS) of 3.58 +/- 1.44 points. (2) Histologically, the NAS of model group was higher than that of control group at both time points. RH failed to lessen NAS whereas Essentiale improved the NAS at 48-week. (3) Serum levels of TC, TG and FPG were significantly different between 4 groups at 24-week, with a comparable low value in both RH and Essentiale group. A similar change was evident at 48-week. (4) In terms of HBV viral load, a significantly lower level in Essentiale group than the others was observed at both time points., Conclusion: HF diet feeding is able to induce a mouse model of HBV chronic infection concomitant with NASH. RH is effective in alleviating the glucose and lipid metabolism but ineffective in improving the hepatic histology in this model, in contrast, backing to normal chow achieved a better effect in this aspect.

Objective: The aim of this study is to investigate the possible associations of chemokines IP-10, Rantes and oxidative stress in chronic hepatits B (CHB)., Methods: 70 CHB patients and 10 healthy controls were enrolled in the study. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of IFN-gamma-inducible protein-10 (IP-10) and regulated on activation normal T-cell-expressed and secreted (Rantes) and oxidative stress parameters (glutathione, GSH; glutathione disulfide, GSSG). Correlationship were analyzed by Spearman's rank correlation., Result: The levels of IP-10 and Rantes were higher in CHB patients than healthy controls, and strong positive associations were found between IP-10/Rantes and alanine aminotransferase (ALT). The levels of GSH and GSH/GSSG were lower in CHB patients than healthy controls, and GSH and GSH/GSSG were negatively correlated with ALT. The levels of IP-10 and Rantes were negatively correlated with GSH and GSH/GSSG respectively., Conclusion: Strong associations were found between chemokines and oxidative stress which participated in the pathogenesis of CHB.

Objective: The present study was designed to investigate the possible epigenetic alteration in the promoter of TNF-alpha in the patients with acute-on-chronic hepatitis B liver failure (ACHBLF)., Methods: The methylation of TNF-alpha promoter in peripheral blood mononuclear cells (PBMCs) was measured by methylation specific PCR (MSP). The level of serum TNF-alpha was determined by enzyme-linked immunosorbent assay (ELISA). Model for End-stage Liver Disease (MELD) was performed for the evaluation of liver failure., Results: The serum level of TNF-alpha in patients with ACHBLF(44.9260 +/- 26.48523) was higher than that in CHB (18.92505 +/- 9.04461) and healthy controls (11.9172 +/- 5.04612) (P < 0.05). Moreover, the serum TNF-alpha level was significantly decreased in methylation group as compared to unmethylaiton group in patients with ACHBLF (P < 0.05). MELD was not significantly different between methylated and unmethylated group of ACHBLF patients (P > 0.05). In addition, the serum level of TNF-alpha was found to be positively correlated with serum total bilirubin (r = 0.891, P < 0.01) and MELD score (r = 0.792, P < 0.01), but to be negatively correlated with prothrombin activity (r = - 0.511, P < 0.05) in patients with ACHBLF., Conclusion: The TNF-alpha methylation patten is stable for the liver failure, suggesting the effect of environment on methylation.

Objective: To observe p53 expression in liver tissue of patients with chronic hepatitis B and its influencing factors., Methods: 17 cases HBeAg-negative chronic hepatitis B patients and 31 cases HBeAg-positive chronic hepatitis B patients were divided into 2 groups., Results: (1) HBeAg-negative chronic hepatitis B patients were older, mostly male and HBV DNA lower. These three indicators between two groups patients appeared statistical difference. Serum markers were no statistical difference between two groups patients except Glo. (2) Pathological inflammation and fibrosis Staging were no statistical difference between two groups patients. p53 expression positive rate and p53 expression semi-quantitative scoring in liver tissue were no statistical difference between the two groups. (3) Logistic regression analysis showed that only liver fibrosis staging (S) is a risk factor for p53 expression. Compared with the S0-1, p53 expression increased by 3.9 times the rate of positive in S > or = 2., Conclusion: Liver fibrosis staging in patients with chronic hepatitis B is a risk factor for p53 positive expression in liver.

Objective: To explore the histopathological features of chronic hepatitis B virus (HBV) carriers and chronic hepatitis B (CHB) patients with mildly elevated serum alanine aminotransferase (ALT)., Methods: 105 patients were divided into three groups according to serum ALT levels: Group A [ALT level < or = 0.5 x upper limits of normal (ULN)], Group B (0.5 x ULN < ALT level < or = 1 x ULN) and Group C(1 x ULN < ALT level < 2 x ULN). Grade of liver inflammation and stage of liver fibrosis in the three groups were compared. The changes in clinical parameters were then observed in patients who had liver histopathological changes., Results: Among 40.95% of the patients, hepatitis degree went to G2 or even worse; and among 30.43% of the patients whose ALT level were normal, the hepatitis degree reached G2 or even worse. In 26.67% of the patients, degree of fibrosis went to S2 or even worse, and for the 17.39% patients whose ALT level were normal, degree of fibrosis went to S2 or even worse. The aggravation of liver inflammation and fibrosis was correlated with ALT and hyaluronic acid increasing (all P < 0.05)., Conclusions: Frequent monitoring of serum ALT and hyaluronic acid may help to understand histopathological changes in the liver. Liver biopsy applied to CHB should be regarded as a main basis if antiviral therapy should be conducted.

Objective: To investigate oxidative stress in chronic hepatitis B (CHB) patients with elevated serum total bilirubin (TBIL)., Methods: 75 CHB patients with elevated serum TBIL were enrolled in the present study. A, B, C, D and E group were defined. Serum Malondialdehyde (MDA), Xanthine Oxidase (XOD), Vitamin C (V(C)) and Vitamin E (V(E)) were determined. The control group contained 11 healthy donors and the carrier group contained 16 Hepatitis B surface antigen (HBsAg) carriers., Results: The concentrations of MDA and XOD were significantly higher in each group of patients than in the control (P < 0.05), while V(C) and V(E) were significantly lower (P < 0.05). The concentration of XOD was significantly higher in the carrier group than in the control (P < 0.05), while MDA, V(C) and V(E) were not significantly different (P > 0.05). The concentrations of MDA and XOD were significantly positively correlated with TBIL (r = 0.670, P < 0.01; r = 0.737, P < 0.01, respectively) in the patients, while V(C) and V(E) were significantly negatively correlated with TBIL (r = -0.463, P < 0.01; r = -0.247, P < 0.05, respectively). The concentration of MDA was significantly different among all the groups in the patients except the comparison between group A and group B. The concentration of XOD was significantly different between group A, B, C and group D, E (P < 0.05). The concentration of V(C) was significantly different between group A and group D, E and between group B, C, D and group E (P < 0.05). The concentration of V(E) was significantly different between group A, B and group E (P < 0.05)., Conclusion: There was a disturbance between oxidative stress and anti-oxidative ability in CHB patients with elevated serum TBIL. Oxidative stress became more serious along with the increasing of serum TBIL. In HBsAg carriers, oxidative stress level was low. The results suggest antioxidant treatment for CHB patients with elevated serum TBIL may help to improve the effect of therapy.

Objective: To evaluate the relationship of the expression of bone morphogenefic protein-7 (BMP-7) in the liver and the liver fibrosis and inflammation in patients with chronic hepatitis B virus (HBV) infection, to explore the role of BMP-7 in the fibrogenesis of chronic hepatitis B., Methods: 81 patients chronically infected with HBV were enrolled. Liver biopsy were performed in all these patients. The hematoxylin staining and reticular fiber staining were performed for the grading of inflammation and staging of fibrosis, respectively. The patients were divided into different groups by the inflammatory grade or fibrosis stage (8, 14, 19, 22, 18 patients with inflammatory grade of G0, G1, G2, G3, G4, respectively; 8, 16, 21, 24, 12 patients with fibrosis stage of S0, S1, S2, S3, S4, respectively ). Then the immunohistochemical staining by BMP-7 were performed. The expression of BMP-7 in the liver was evaluated by digital image quantitative analysis system. We compared the expression of BMP-7 in the liver with different inflammatory grade and fibrosis stage., Results: The expression of BMP-7 in the liver tissue increased with the increase of hepatic inflammatory grade and fibrosis. When liver biopsy showed a severe intrahepatic inflammation, the expression of BMP-7 significantly increased, regardless the intrahepatic fibrosis stage. Similarly, when liver biopsy showed a severe intrahepatic fibrosis, the expression of BMP-7 also significantly increased, regardless the intrahepatic inflammatory grade., Conclusion: BMP-7 may play an important role as anti-inflammatory and anti-fibrogenic effect in the fibrogenesis of chronic hepatitis B.

Objective: To understand the level and clinical significance of soluble CD40 in patients with chronic hepatitis B., Methods: Detecting the concentration of sCD40 from 176 cases with chronic hepatitis B by ELISA and analyzing its relationship with different grades of inflammation and necrosis in liver tissue., Results: sCD40 from patients with chronic hepatitis B was significantly higher than those from healthy. And that the concentration of sCD40 was positive correlation with severe clinical disease and liver inflammation and necrosis. In patients whose ALT lower than 80 IU/L and sCD40 higher than 80 pg/ml, it showed that 65.85% cases have high grade of liver inflammation and necrosis, which was significantly higher than patients with sCD40 lower than 80 pg/ ml., Conclusion: The concentration of sCD40 is positively related with the grade of liver inflammation and necrosis. This information could help us to evaluate the status of chronic hepatitis B as an immunological index.

Objective: To detect the level of serum and liver tissue TGF-beta1 in patients with chronic hepatitis B, to study their relation to liver fibrosis and gain the evidence for diagnosis of liver fibrosis., Methods: The liver fibrosis grades (S0-S4) of 131 cases with chronic HBV infection were diagnosed after liver biopsy. Serum TGF-beta1 was detected by enzyme-linked immunosorbent assay, and the semiquantitative analysis was applied after detecting the expression of TGF-beta1 in liver tissue with immunohistochemistry. Their relations to liver fibrosis were analyzed., Results: Serum and tissue level of TGF-beta1 increased significantly with the development of fibrosis, and the same result was obtained between themselves (P < 0.01). There was very significant difference for serum level of TGF-beta1 among the groups with different fibrosis grades (P < 0.01). Serum levels of TGF-beta1 were decreased significantly comparing the Group S0 or S1 to S4 (P < 0.005). There were significant difference for serum level of TGF-beta1 among S0 and the others (P < 0.005). And there was significant difference between S1 and S3 (P < 0.005). The expression level of TGF-beta1 in liver tissue has no significant difference between group S3 and S4 (P > 0.05). However, the differences were significantly among the other comparisons (P < 0.01)., Conclusion: There is close relation between the level of TGF-beta1 and the different liver fibrosis grades due to chronic hepatitis B. The serum level of TGF-beta1 is a potential noninvasive maker for diagnosis of liver fibrosis.

Objective: To investigate the intrahepatic expression of inducible nitric oxide synthase (iNOS) in patients with chronic hepatitis B (CHB) and its relation to liver histopathology., Methods: The intensity and distribution of the immunohistochemical staining of intrahepatic iNOS were studied in the liver biopsy specimens obtained from 74 patients with CHB and statistical analyses were performed between intrahepatic iNOS and ALT, HbeAg, HBV DNA grading of liver inflammation and staging of fibrosis. Seven histologically normal liver sections were used as a control group., Results: Compared with the control group, the intrahepatic iNOS immunoexpression was significantly higher in patients with CHB (P < 0.05), iNOS immunoreactivity was observed mainly in hepatocytes showing a predominant cytoplasmic staining, with the positive liver cells distributed diffusely throughout the hepatic lobule. Immunopositive staining could also be detected in Kupffer cells, sinusoidal lining cells and vascular endothelial cells. Compared with patients with normal ALT, the hepatocellular iNOS immunoexpression was significantly higher in patients with elevated ALT (P < 0.05) and the iNOS immunoexpression was significantly correlated with the serum level of ALT (r=0.601, P=0.000). Statistical analysis also showed that the intrahepatic iNOS immunoexpression was positively correlated with the grading of liver inflammation and the staging of liver fibrosis (r=0.660, P=0.000; r=0.507, P=0.000). No significant correlation between iNOS and HBeAg and HBV DNA was detected. CONCLUSION The intrahepatic expression of iNOS is elevated in chronic hepatitis B patients and correlated well with the severity of the disease, which indicated that inducible nitric oxide synthase may have a critical role in the pathogenesis of chronic viral hepatitis B.

Objective: To investigate the factors related to the outcome of patients with chronic severe hepatitis B and effectiveness of antivirus therapy., Methods: The effects of the factors including age, prothrombin activity (PTA), serum HBeAg, Anti-HBe, HBV-DNA load, with or without complication, antivirus therapy and so on, on outcome of 330 patients with chronic severe hepatitis B were analyzed in this retrospective study., Results: The mortality of patients with chronic severe hepatitis B was significantly higher among patients at higher age, with lower PTA, and with more complications. The mortality of patients with HBV-DNA more than 1x10(5) copies/ml (52.3 percent) was higher than that of patients whose HBV-DNA was less than 1x10(5) copies/ml (32.9 percent). There was no correlation between serum HBeAg or anti-HBe and the mortality. The mortality of patients with HBV-DNA higher than 1x10(5) copies/ml (30.38 percent) who were treated with lamivudine in 2005 was lower than that of patients whose HBV-DNA was less than 1x10(5) copies/ml (54.64 percent) who were not treated with any antiviral therapy in 2001., Conclusion: The higher serum virus load is the key factors of the mortality in addition to the other factors such as older age, lower PTA, more complication in the patients with chronic severe hepatitis B. The usage of antivirus therapy may be associated with lower mortality.

Objective: To investigate nitric oxide (NO) and nitric oxide synthase (NOS) in patients with chronic hepatitis B., Methods: Nitric oxide and nitric oxide synthase, including inducible NOS (iNOS) and constitutive NOS (cNOS), were measured in patients and control groups, then were statistically analyzed., Results: NO and iNOS were significantly higher in patients with hepatitis B than in the controls (P < 0.05). NO and iNOS were significantly higher in patients with increased ALT than in the controls and in patients with normal ALT (P < 0.05). NO was significantly higher in patients with normal ALT than in the controls (P < 0.05). cNOS were not significant different among these groups. NO and iNOS significantly correlated with ALT in patients with hepatitis B (r=0.367 and r=0.474). No significant relationship was found among NO, NOS and HBV DNA. Among different genotype groups, NO and NOS had no significant difference., Conclusion: NO and NOS were higher in patents with chronic hepatitis B. In patients with increased ALT, NO's damage was severe. In patients with normal ALT, there was no significant damage caused by NO. NO should be detected in patients with hepatitis B in addition to HBV markers.

Background: To study the expression of endothelial nitric oxide synthase (eNOS) and its mRNA in liver tissues of patients with chronic hepatitis B (CHB) and the relationship between its expression and pathological changes of hepatic tissues and the clinical significance., Methods: 48 cases of specimens of liver tissues with CHB were observed and analyzed using immunohistochemical method and in situ nucleic acid hybridization., Results: eNOS was expressed in hepatocytes, sinusiod endothelial cells and vessel endothelial cells, being predominantly expressed in hepatocytes. The number and distribution of eNOS mRNA positive cells were similiar to those of its protein. Increase in expression of eNOS was correlated with degrees of inflammatory activity and fibrosis (r=0.6855 [P<0.05]; r=0.4828 [P<0.05]; respectively), and with decreasing PA and ALB (F=3.4101 [P<0.05]; F=9.1714 [P<0.0]). Changes of expression of TGF-alpha-R were parallel to those of eNOS., Conclusions: eNOS may be involved in pathological lesion of CHB by affecting the expression of TGF-R in liver tissues with CHB.

To study the expression of FasL mRNA in liver tissues of CHB, we investigated FasL mRNA by in situ hybridization in liver tissues from patients with chronic hepatitis B. FasL oligonucleotide probes were synthesized and labeled with DIG-Oligonucleotide 3'-End Labeling Kit. FasL mRNA was found in cytoplasm of liver-infiltrating mononuclear cells, hepatocytes and some sinus cells. FasL mRNA-positive hepatocytes were present mainly at the edges of 'piecemeal necrosis(PN)' and 'bridging necrosis (BN)'. These results demonstrated that human hepatocytes themselves can express FasL in chronic hepatitis B. We deem that FasL-positive hepatocytes might express cytotoxic activity and eliminate Fas-positive hepatocytes by both 'fratricide' and 'suicide'. These results also emphasize that apoptosis caused by CTL through Fas-mediated pathway may play an important role in CHB.

To study the relationship between abnormal expression of oncogene protein and HBV replication, we detected the expression of protein products C-erbB-2 P185, ras P21 and tumorsuppressor gene product P53 and HBeAg in the serum of 64 patients with chronic HBV infection by using immunohistochemical technique and ELISA. The results were as follows. The positive detection rates of serum HBeAg in C-erbB-2 P185 and ras P21 oncogenes positive groups were 89.4% and 84.6% respectively, but only 20% and 48% in C-erbB-2 P185 and ras P21 negative groups. It suggested that there is a close correlation between the over-expression of C-erbB-2 P185, ras P21 and replication of HBV.

In order to study the significance of expression of Bcl-1 and Bax proteins in the liver tissues, 35 cases of chronic viral hepatitis (CH) were immunohistochemically studied. The results showed that Bcl-2 and Bax were mainly colocated in cytoplasm of the mononuclear cells(MN) in the areas of focal necrosis, bridging necrosis and piecemeal necrosis (PN). Some hepatocytes surrounding these areas expressed Bcl-2 and Bax or expressed Bax only. Pathological examination found that these hepatocytes Suffered from hydropic or acidophilic degeneration. In the severe viral hepatitis, survival hepatocytes also expressed Bcl-2 and Bax. It is suggested that the expression of Bcl-2 and Bax in the liver tissues correlated with the apoptosis of hepatocytes in CH and influenced the progress of severe viral hepatitis.

33 specimens of liver biopsy from patients with different types of viral hepatitis were detected for transforming growth factor-beta 3(TGF-beta 3) by immunohistochemistry. The results showed that TGF-beta 3 mainly distributed in the sinusal walls cells in sinus portal, areas fibrous, septum, hepatocytes in pseudolobes and necrotic areas of severe hepatitis. The expressing intensity of TGF-beta 3, was stronger in the cirrhosis, chronic severe hepatitis than that in the chronic and acute hepatitis. As the expression of TGF-beta 3 increased, the level of serum bilirubin ascended and plasmozyme activity was prolonged. It may be concluded that the expression of TGF-beta 3 is correlated with severe hepatitis and the progress of the liver cirrhosis.

The expression of platelet-derived growth factor(PDGF)-BB and PDGF receptor-beta(PDGFR-beta) in liver tissues from viral hepatitis was detected by immunohistochemistry. The results showed that the expression of PDGF-BB and PDGFR-beta in the liver tissues correlated closely with the degree of liver fibrosis. The levels of their expression in the liver cirrhosis and fibrosis stage S3-4 of chronic viral hepatitis(CH) were much higher than those in acute viral hepatitis and fibrosis stage S0-2 of CH. Meanwhile, the expression of PDGF and its receptor was associated with the expression of desmin-positive cells pro-collagen III peptide in liver tissues and tissue inhibitor of metalloprotenase-1 in serum. It is suggested that PDGF-BB and its receptor-beta may promote the progress of liver fibrosis by means of the activation, proliferation and differentiation of satellite cells.

To evaluate the role of apoptosis on liver injury of chronic hepatitis B, we studied the expression of Fas ligand and HBsAg immunohistochemically. Sixty-eight liver biopsy samples from patients with type B chronic liver disease were immunostained for Fas ligand and thirty-two of which were immunostained for HBsAg.35/68(51.47%) cases showed FasL immunoreactive in liver-infiltrating mononuclear cells, and more expression of FasL were found in cytoplasm of hepatocytes. Both were closely linked to the degree of inflammation in liver(P = 0.008 and P = 0.011, respectively). But the expression of FasL had no close relationship with that of HBsAg in liver tissue (P > 0.05). These findings suggest that apoptosis caused by CTL through Fas-mediated pathway plays an important role in liver cell injury by hepatitis B virus infection. At the same time, FasL-positive hepatocytes may express cytotoxic activity and eliminate themselves by both fratricide and suicide; 'Bystander effect' can be used to account for the difference expression between FasL and HBsAg.