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Tanshinone IIA (TSA) is a widely used traditional Chinese medicine, which has been demonstrated to protect damaged liver cells and is currently administered in the treatment of liver fibrosis. Liver precursor cells, also termed oval cells, are key in the repair of liver tissues following injury. However, whether TSA improves the function of liver cells and protects the liver from injury by enhancing the growth and proliferation of hepatic oval cells remains to be elucidated. In the present study, low to moderate concentrations of TSA were observed to stimulate proliferation, did not induce apoptosis in WB-F344 rat hepatic oval cells and the increased expression levels of β-catenin. WB-F344 cells were treated with various concentrations of TSA (0-80 µg/ml) for 24, 48, 72 and 96 h. Cell proliferation was measured using a Cell Counting kit-8 (CCK-8) assay, a 5-ethynyl-2'-deoxyuridine assay and a carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. The CCK-8 assay demonstrated that treatment of WB-F344 cells with 20-40 µg/ml TSA for up to 72 h significantly increased proliferation. Similar results were observed in the subsequent EdU and CFSE assays. Furthermore, a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated that 20-40 µg/ml TSA treatment for up to 96 h did not induce apoptosis of the WB-F344 cells. Notably, the results of western blot, immunofluorescence and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that treatment of the WB-F344 cells with 20-40 µg/ml TSA for up to 72 h significantly increased the expression levels of β-catenin. These data indicated that TSA at concentrations between 20 and 40 µg/ml may induce WB-F344 cell proliferation by activating the canonical Wnt signaling pathway. The results of the present study suggest that TSA may be a useful natural agent to enhance repair and regeneration of the injured liver, and improve liver regeneration following orthotopic liver transplantation.

Elevated tissue inhibitor of metalloproteinase (TIMP)-1 expression contributes to excess production of extracellular matrix in liver fibrosis. However, there are few studies on sustained suppression of TIMP-1. We aimed to construct a recombinant adeno-associated virus (AAV) carrying small interfering RNAs (siRNAs) of TIMP-1 and investigate the long-term effects of RNA interference upon the TIMP-1 gene in rat hepatic stellate cells (HSCs). Five siRNA oligomers targeting rat TIMP-1 were designed and transfected into HSCs. A U6 promoter followed by the siRNA which had the strongest suppression effect was cloned into the AAV vector and packed into 293 cells to construct the recombinant AAV/siRNA-TIMP-1/neo. After infecting HSCs with this recombinant AAV, the transcription and expression levels of the TIMP-1 and matrix metalloproteinase-13 (MMP-13) genes were detected at 4 and 12 weeks. Three of the five designed siRNA oligomers had a suppressing effect on TIMP-1 expression in rat HSCs within 72 h. The transcription and expression levels of TIMP-1 were suppressed significantly (P<0.05) following recombinant AAV/siRNA1-TIMP-1/neo infection and lasted 12 weeks. TIMP-1 expression in rAAV/siRNA1-TIMP-1/neo-infected HSCs was suppressed by 60% after four weeks and 90% after twelve weeks when compared to the control recombinant AAV/neo and uninfected HSCs. Furthermore, the transcription and protein expression levels of MMP-13, the main substrate of TIMP-1, were elevated by approximately 40% at twelve weeks in rAAV/siRNA-TIMP-1/neo-infected HSCs. RNA interference exerts suppressive effect on the TIMP-1 gene in cultured HSCs for a longer time when a recombinant AAV is utilized as the gene delivery vector.

Objective: To investigate the effects of herbal compound 861 (Cpd861) on hepatocarcinogenesis induced by diethylntrosamine and 2-acetylaminofluorene (DEN-AAF) in female Sprague Dawley rats., Methods: Liver preneoplastic foci were induced using the DEN-AAF method in female Sprague Dawley rats, which were then treated with Cpd861. For quantitative assessment of liver preneoplastic foci, the placental form of glutathione-S-transferase (GST-P) positive foci were measured using immunohistochemical staining and image analysis. GST-P protein expression was measured by Western blotting, mRNA expression was assessed by Northern blotting., Results: Treatment using DEN-AAF caused a significant decrease in body weight and increase in liver weight compared to the control group. Oral Cpd861 administration essentially prevented DEN-AAF-induced body weight loss and liver weight increase. When 2-AAF was followed by treatment with Cpd861, there was a decrease in the number of large foci as compared to 2-AAF alone. However, there were still considerable numbers of small mixed clear/vacuolated cell foci, some of which were positive for GST-P. Significant increase in GST-P protein and mRNA expression were observed in the DEN-AAF group, while treatment with Cpd861 inhibited the increase. The effect of Cpd861 on hepatocarcinogenesis occurred in a concentration-dependent manner., Conclusion: Chinese herbal compound Cpd861 prevents hepatocarcinogenesis in DEN-AAF-induced liver preneoplastic lesions in rats.

Objective: To evaluate the long-term efficacy and safety of lamivudine therapy for the treatment of chronic hepatitis B and the clinical influence of emergence of tyrosine methionine aspartic acid (YMDD) motif mutation of hepatitis B virus (HBV)., Methods: This multicenter, double-blind, randomized, placebo controlled trial began in 1996. A total of 429 patients with HBsAg, HBeAg and HBV CNA positives were enrolled. They were randomized to receive either lamivudine 100 mg daily (n = 322) or placebo (n = 107) on 3 : 1 ratio for the first 12 weeks. Thereafter all patients were offered open label lamivudine treatment and assessed every 4 weeks for a total of 104 weeks., Results: After 1 year treatment 72.7% patients (285/392) had a sustained serum HBV DNA response. HBV DNA continued to be substantially suppressed at the second year, except in patients with the emergence of YMDD mutation whose mean HBV DNA levels increased to 86 Meq/ml (bDNA assay) but were much more lower than that of pre-treatment baseline level. lamivudine therapy resulted in increased HBeAg loss and HBeAg/anti-HBe seroconversion, which were correlated with both baseline alanine transaminase (ALT) levels and also with duration of lamivudine treatment. HBeAg loss was achieved in 26.8% of patients with ALT > 1-fold upper limit of normal at 2 yeas and in 35.6% and 55.6% of patients with ALT > 2-fold upper limit of normal and ALT > 5-fold upper limit of normal, respectively. For HBeAg seroconversion, these figures were 17.4%, 22.2%, and 33.3% respectively. By the end of 2 years, ALT levels were remained in normal ranges in 50.3% whose ALT were abnormal before treatment, and in 83% whose ALT were mormal before treatment. YMDD mutation were developed in 49.7% of the patients. Their serum HBV DNA levels were slightly increased to bDNA median level 86 Meq/ml and 15% of the patients they were ALT exceeded baseline levels. Four patients clinically flared-up and recovered after stop treatment. The adverse drug reactions (ADRs) of lamivudine were mild to moderate, only two patients were reported as drug related severe ADR., Conclusion: Sustained HBV replication and clinical improvement could be obtained by the long-term lamivudine therapy with good tolerance and safety.

Objective: To explore the effect of herbal compound 861 (Cpd 861) on MMP-2 expression and its enzymatic activities, and the antifibrotic mechanism of this herbal compound., Methods: Forty five female rats were randomly divided into normal control (sham operation) group, common bile duct ligation (BDL) group and Cpd 861 therapeutic group. In the last group, daily gastric feeding of Cpd 861 (9 g/kg.bw) started on day 7 after BDL operation. At 49 days, all animals were sacrificed and mRNA expression of MMP-2 in liver tissue was evaluated by semi-quantitive RT-PCR. In addition, enzymatic activities of MMP-2 were analyzed by zymography., Results: In comparison with model group, MMP-2 mRNA levels in Cpd 861 therapeutic group were significantly decreased. MMP-2 enzymatic activities were not detectable in normal group, slightly elevated in model group, higher in Cpd 861 therapeutic group., Conclusions: Cpd 861 decreases the mRNA level of MMP-2, but transiently increases the enzymatic activities of MMP-2. The latter effect of Cpd 861 may be mediated by decreasing TIMPs, the inhibitors of MMPs, during resolution stage of fibrosis. This is probably one of the mechanisms whereby herbal Cpd 861 exerted its antifibrotic action in this kind of experimental liver fibrosis.

Objective: To explore the clinical and histopathological features of primary biliary cirrhosis (PBC)., Methods: The clinical, laboratory, as well as histological features of 27 cases of PBC were retrospectively analyzed., Results: The male to female ratio was 1:8 (3:24), aged from 22 to 69 years. The main clinical manifestations included: fatigue (62.9%, 17/27), jaundice (59.2%, 16/27) and pruritus (29.6%, 8/27), with all of patients having markedly elevated serum alkaline phosphatase and gamma-glutamyl transpeptidase and 95.8% (23/27) of the patients being positive for anti-mitochondrial antibody (AMA). The main histopathological changes were: necroinflammation of interlobular bile ducts (100%, 27/27), lymphocyte aggregation or lymphocyte follicles (15%, 4/27), granuloma (26%, 7/27), decreased number of interlobular bile ducts and smaller bile duct proliferation (55%, 15/27), feathery degeneration of hepatocytes (59%, 16/27); bilirubinostasis in hepatocytes and/or canaliculi (52%, 14/27); fibrosis and distortion of lobules (26%, 7/27), pseudolobular formation (11%, 3/27)., Conclusions: The main clinical features of PBS are fatigue and pruritus, markedly elevated phosphatase and gamma-glutamyl transpeptidase, and positive AMA with or without jaundice. Its histopathological hallmarks are (1)necroinflammmation and ductopenia involved mainly in interlobular bile ducts; (2)lymphocyte aggregation, granuloma formation and bile ductular proliferation in the portal area; and (3)feathery degeneration of hepatocytes.

Objective: To observe the in vitro effect of compound 861 (Cpd 861) on tissue inhibitor of metalloprotenase 1 (TIMP1) mRNA levels of HSC-T6 cell., Methods: HSC-T6 cells were exposed in different concentrations of Cpd 861 (0.25~1.0 mg/ml) for 48 hours. The TIMP1 level was measured by the quantitative reverse-transcription polymerase chain reaction (RT-PCR)., Results: The TIMP1 mRNA levels of HSC-T6 cells at different concentrations of Cpd 861 were lower (2.50 0.71, 0.50 0.01, 0.11 0.03) than those of the normal control (3.78 0.67, P<0.05 or P<0.01)., Conclusions: The antifibrotic mechanism of Cpd 861 is partly due to its downregulation on TIMP1 mRNA levels of HSC-T6 cells.

Objective: To study the clinical features of patients with primary biliary cirrhosis (PBC) in order to improve the doctors' awareness of the disease., Methods: General status, clinical manifestations and laboratory findings of 40 patients with PBC were reviewed.Thirty-seven patients were females (37/40), and the mean age at diagnosis was 50.5 +/- 7.8 years. The time interval from initial symptoms or preliminary diagnosis to final diagnosis was 24.0 +/- 23.6 months., Results: The most frequently reported symptoms were fatigue (67.5%, 27/40), jaundice (60%, 24/40) and pruritus (32.5%, 17/40). Eight patients (20%) had associated auto-immune diseases (Sjogren's syndrome and/or rheumatoid a(c)arthritis). Serum alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (gamma-GT) levels were markedly elevated (520.3 +/- 382.3 IU/L and 648.6 +/- 529.1 IU/L, respectively) in all patients, while alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were mildly elevated (82.6 +/- 54.5 IU/L and 100.7 +/- 47.2 IU/L, respectively). Twenty-four patients (60%) had a total bilirubin level >/= 34.2 micromol/L. Thirty-five patients (87.5%) had elevated serum immunoglobin M,and 97.5% of patients (39/40 ) were anti-mitochondrial antibody (AMA)/AMA-M2 positive., Conclusion: Elevated serum ALP and gamma-GT levels, together with a positive AMA/AMA-M2, can help the diagnosis of PBC. Liver biopsy is useful to confirm the diagnosis and to differentiate histopathological stages.

Objective: To study the clinical features of primary biliary cirrhosis (PBC) in order to facilitate cognition of the disease., Methods: General status, clinical manifestations and laboratory findings of 45 patients with PBC were reviewed., Results: Among the 45 patients, 42 were females and the mean age at diagnosis was (50.8 +/- 8.1) years. The mean time interval between the first visit to physicians to the time of correct diagnosis was about 2 years. The most frequent symptoms were fatigue (66.7%, 30/45), then jaundice (55.6%, 25/45) and pruritus (40%, 18/45). Nine patients (20%) were asymptomatic. Nine patients (20%) were associated with other auto-immune diseases (Sjogren's syndrome and/or rheumatoid arthritis). Serum alkaline phosphatase (ALP) and glutamyl transpeptidase (gamma-GT) levels were markedly elevated in all the patients [(498.5 +/- 369.8) IU/L and (595.2 +/- 518.4) IU/L, respectively], whereas ALT and AST levels were mildly to moderately elevated [(83.9 +/- 58.8) IU/L and (100.8 +/- 48.8) IU/L, respectively]. 25 patients (55.6%) had a total bilirubin level >/= 34.2 micromol/L. 40 patients (88.9%) had elevated serum IgM and 43 patients (95.6%) were anti-mitochondrial antibody (AMA)/AMA-M2 positive., Conclusion: In China, PBC is probably not so rare as it was thought before. It is mostly found in middle-aged women. The most frequent symptom is fatigue and some patients are asymptomatic at early stage. Elevated serum ALP and gamma-GT levels together with positive AMA/AMA-M2 can help to diagnose PBC. Liver biopsy is useful to confirm the diagnosis and differentiate histopathological stages.

Objective: Protein induced by vitamin K absence or antagonist II (PIVKA II), also called des-gamma carboxy prothrombin (DCP), is a sensitive marker for the diagnosis of hepatocellular carcinoma (HCC), in Japan and the United States since the sensitive kits were available (1998). PIVKA II is not used in clinical diagnosis in China so far. The aim of this study was to assess the diagnostic value of PIVKA II in Chinese patients with HCC., Methods: Serum PIVKA II and alpha-fetoprotein (AFP) levels were determined in 60 patients with HCC and 30 patients with cirrhosis not carrying HCC., Results: The mean serum concentration of PIVKA II in HCC patients (784.3 +/- 1364.1 mean +/- s) was higher than that in cirrhosis patients (16.1 +/- 31.7); this difference was highly significant (P < 0.0001). When the cutoff level of 40 mAU/ml was used as the level of discriminating HCC from cirrhosis, 51.7% of patients (31/60) with HCC had PIVKA II values above this level (sensitivity). Only 4 patients with cirrhosis had such high PIVKA II levels. Thus, the specificity of this test was 86.7% (26/30). Total accuracy was 62.2% [(31 + 26)/(60 + 30)]. Seven of 19 small HCCs (36.84%) had PIVKA II values above the cutoff level. Concentrations of AFP above 20 ng/ml were observed in 34 of 60 patients with HCC (56.7%) and in 11 patients with cirrhosis (36.7%). Eleven of 26 patients with HCC (46.2%) without increased AFP had concentrations of PIVKA II greater than 40 mAU/ml. No significant correlation was found between serum levels of AFP and PIVKA II that were measured in 60 HCC patients (rs = 0.101, P = 0.247). Combining the information from PIVKA II and AFP showed an increase of approximately 21.6% over AFP and 26.7% over PIVKA II alone. For small HCC patients, combining the information from PIVKA II and AFP showed an increase of approximately 15.8% over AFP alone and 21.1% over PIVKA II alone., Conclusion: PIVKA II is a useful early diagnostic marker for HCC and may be more sensitive when combined with AFP in Chinese patients.