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ABSTRACT: Diethyldithiocarbamate (DDC) could block collagen synthesis in hepatic stellate cells through the inhibition of reactive oxygen species derived from hepatocyte cytochrome P450 2E1 (CYP2E1). However, the effect of DDC on matrix metalloproteinase-1 (MMP-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human hepatic stellate cells (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were upregulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H2O2 treatment abrogated DDC-induced MMP-1 upregulation and collagen I decrease, while catalase treatment slightly upregulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 upregulation. ERK1/2, Akt and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 upregulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 upregulation through the stimulation of ERK1/2. Our data indicate that DDC significantly upregulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H2O2 inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis.
Bioscience Reports 04/2013; · 2.38 Impact Factor
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Min Cong, Tianhui Liu,
Ping Wang,
Xu Fan,
Aiting Yang,
Yanfeng Bai,
Zhen Peng,
Peng Wu,
Xiaofei Tong,
Jing Chen,
Hai Li,
Rui Cong,
Shuzhen Tang,
Baoen Wang,
Jidong Jia,
Hong You
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ABSTRACT: Elevated tissue inhibitor of metalloproteinase 1 (TIMP-1) expression contributes to excess production of extracellular matrix in liver fibrosis. Herein, we constructed a recombinant adeno-associated virus (rAAV) carrying siRNA of the TIMP-1 gene (rAAV/siRNA-TIMP-1) and investigated its effects on liver fibrosis in rats. Two models of rat liver fibrosis, the carbon tetrachloride and bile duct ligation models, were treated with rAAV/siRNA-TIMP-1. In the carbon tetrachloride model, rAAV/siRNA-TIMP-1 administration attenuated fibrosis severity, as determined by histologic analysis of hepatic collagen accumulation, hydroxyproline content, and concentrations of types I and III collagen in livers and sera. Levels of mRNA and active matrix metalloproteinase (MMP) 13 were elevated, whereas levels of mRNA and active MMP-2 were decreased. Moreover, a marked decrease was noted in the expression of α-smooth muscle actin, a biomarker of activated hepatic stellate cells (HSCs), and transforming growth factor-β1, critical for the development of liver fibrosis. Similarly, rAAV/siRNA-TIMP-1 treatment significantly alleviated bile duct ligation-induced liver fibrosis. Furthermore, this treatment dramatically suppressed TIMP-1 expression in HSCs from both model rats. These data indicate that the administration of rAAV/siRNA-TIMP-1 attenuated liver fibrosis by directly elevating the function of MMP-13 and diminishing activated HSCs. It also resulted in indirect decreased expression of type I collagen, MMP-2, and transforming growth factor-β1. In conclusion, rAAV/siRNA-TIMP-1 may be an effective antifibrotic gene therapy agent.
American Journal Of Pathology 03/2013; · 4.89 Impact Factor
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ABSTRACT: The activation of hepatic stellate cells (HSCs) is closely associated with liver fibrosis in chronic hepatitis B virus (HBV) infection. However, the molecular mechanisms leading to HSC activation remain unclear. It has been reported that the platelet-derived growth factor-B (PDGF-B)/PDGF receptor-β (PDGFR-β) signaling pathway is involved in this process. Thus, we investigated whether HBV and its protein contribute to HSC proliferation by the PDGF-B/PDGFR-β signaling pathway. HBV particles were purified from the supernatant of HepG2.2.15 cells by ultracentrifugation and the cell lines carrying HBV preS, e, c or x genes were obtained. After incubation with HBV particles or co-cultured with the cell lines expressed in the viral protein, the proliferation of LX-2 cells, an HSC cell line, were detected by flow cyto-metry and real-time PCR and the expression of molecules related to the PDGF-B/PDGFR-β signaling pathway were further measured. Our results indicated that HBV particles, c and x proteins promoted LX-2 proliferation and increased the mRNA levels of PDGF-B, PDGFR-β, collagen-I and α-smooth muscle actin (α-SMA), as well as the phosphorylation of PDGFR-β; however, the expression protein levels of PDGF-B and PDGFR-β remained unchanged. In conclusion, HBV particles and HBV c and x proteins promote HSC proliferation and fibrogenesis in vitro and the PDGF-B/PDGFR-β signaling pathway is important in this process.
International Journal of Molecular Medicine 10/2012; · 1.98 Impact Factor
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Xiaoning Wu,
Yu Wang,
Yan Cui,
Qixuan Bai,
Xingyu Ze, Tianhui Liu,
Min Cong,
Ping Wang,
Xinmin Li,
Gang Yin,
Xiaojuan Ou,
Hong You,
Jidong Jia
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ABSTRACT: Aim: To investigate direct effects of hepatitis B virus (HBV) on collagen type I in vitro. Methods: Collagen type I were measured after LX-2 cell cultured with purified or serum HBV for 12, 24 and 48 h. Furthermore, evidence of HBV infection to LX-2 were detected, and different inhibitors were used to identify pathways regulating collagen I expression. Results: The 3 × 10(5) IU/mL purified/serum HBV increased collagen type I mRNA expression by 2.2-/3.2- and 1.3-/1.5-fold at 24 and 48 h, respectively. Collagen type I protein in the supernatant of purified/serum HBV group also increased compared to the control group (408.0 ± 8.0/384.4 ± 6.8 vs 262.7 ± 15.7 ng/mL, P < 0.05). However, the 3 × 10(7) IU/mL purified/serum HBV increased collagen type I expression similar to that of 3 × 10(5) IU/mL, while 3 × 10(3) IU/mL group showed no effect. Human HBV immunoglobulin alleviated HBV-induced collagen I expression, but no evidence of HBV infection was found. Neutralization of transforming growth factor beta, tumor necrosis factor alpha, platelet-derived growth factor, extracellular signal-regulated kinase and TGF-β receptor had no obvious inhibitory effects; only inhibition of p38 mitogen-activated protein kinase decreased collagen type I mRNA expression by 0.5-/0.4- and 0.4-/0.3-fold at 24 and 48 h, respectively. It reduced collagen type I protein in the purified/serum HBV group for 48 h (252.1 ± 14.1/251.7 ± 18.8 vs 403.9 ± 4.9/385.0 ± 4.2 ng/mL, P < 0.05). Conclusion: HBV directly promotes collagen type I expression of LX-2 cells without infection in vitro.
Hepatology Research 03/2012; 42(9):911-21. · 2.20 Impact Factor
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ABSTRACT: Fibrosis is a hallmark histologic event of chronic liver diseases and is characterized by the excessive accumulation and reorganization of the extracellular matrix (ECM). The gold standard for assessment of fibrosis is liver biopsy. As this procedure has various limitations, including risk of patient injury and sampling error, a non-invasive serum marker for liver fibrosis is desirable. The increasing understanding of the pathogenesis of hepatic fibrosis has suggested several markers which could be useful indicators of hepatic fibrogenesis and fibrosis. These markers include serum markers of liver function, ECM synthesis, fibrolytic processes, ECM degradation and fibrogenesis related cytokines. Recently, neo-epitopes, which are post-translational modifications of proteins, have been successfully used in bone and cartilage diseases which are characterized by extensive ECM remodeling. Increasing numbers of studies are being undertaken to identify neo-epitopes generated during liver fibrosis, and which ultimately might be useful for diagnosing and monitoring fibrogenesis. To date, the metalloproteinases generated fragment of collagen I, III, IV and VI have been proven to be elevated in two rat models of fibrosis. This review summarizes the recent efforts that have been made to identify potentially reliable non-invasive serum markers. We used the recently proposed BIPED (Burden of disease, Investigative, Prognostic, Efficacy and Diagnostic) system to characterize potential serum markers and neo-epitope markers that have been identified to date.
Biomarker insights 01/2012; 7:105-17.
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ABSTRACT: Serum alanine aminotransferase (ALT) increase is a well-known phenomenon during interferon treatment for chronic hepatitis B. However, little is known about these increases during nucleoside/nucleotide treatment and the effects on long-term clinical outcomes.
A total of 170 treatment-naive hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients were treated with a nucleoside/nucleotide analogue for at least 2 years and followed up for 1 more year post-treatment. Clinical characteristics were detected and analysed at baseline and at every 3-month interval.
Two patterns of ALT increase, virus- and host-induced, were detected. Virus-induced increases were characterized by a rapid increase in serum ALT and HBV DNA typically after 2 years of treatment, and were more common than host-induced ALT increases (15.9% versus 6.5%; P<0.05) with a median ALT increase of 5.7-fold the upper limit of normal (ULN). Host-induced ALT increases were characterized by moderately increased ALT (median 2.5-fold ULN) with a slow decrease in HBV DNA that occurred mainly in the first year of treatment (63.6%). Most importantly, host-induced increases were associated with favourable long-term treatment outcomes in HBV DNA undetectable rate (82% versus 0%), HBeAg seroconversion (82% versus 7%) and histological improvement. Moreover, interferon-γ-expressing T-helper cells were increased in patients with host-induced ALT increases.
Two patterns of ALT increases may occur during nucleoside/nucleotide analogue treatment. Host induced ALT increases, accompanied by decreased HBV DNA, lead to better long-term clinical outcomes.
Antiviral therapy 01/2011; 16(3):299-307. · 3.16 Impact Factor
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ABSTRACT: 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.
International Journal of Molecular Medicine 11/2009; 24(5):685-92. · 1.98 Impact Factor
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Hong You,
Hong Ma, Tianhui Liu,
Min Cong,
Ping Wang,
Xiaojuan Ou,
Xiaoming Wang,
Jiangbo Ren,
Hongyi Li,
Baoen Wang,
Jidong Jia
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ABSTRACT: Pretreatment alanine transaminase (ALT) elevation may be used as a predictor for higher hepatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B patients. However, the role of dynamic changes of on-treatment ALT for seroconversion is unknown. A total of 170 naïve HBeAg-positive chronic hepatitis B patients were treated with a nucleoside/nucleotide analogues (NA), either lamivudine, adefovir, entecavir, or telbivudine, for at least 2 years and followed up for 1 more year. Clinical characteristics were detected and analysed at baseline and at 3-month intervals. On-treatment ALT predicted HBeAg seroconversion more accurately than baseline ALT. Among the patients with on-treatment ALT </=1 x UNL, 1-</=2 x UNL, 2-</=5 x UNL and >5 x UNL, HBeAg seroconversion was 11.4, 5.4, 24.4 and 65.0% (odds ratio = 1.0, 0.4, 2.5 and 14.4, respectively), respectively. Moreover, two models/types of seroconversion were observed. Type I was characterized by rapidly decreased ALT and HBV DNA during the first 3-month interval, but with high HBeAg reversion rate (50%) after consolidation treatment. Type II was a slow decreased DNA procedure accompanied by significant elevated ALT with less reversion (23%). Receiver operating characteristic curve analysis showed a 1.9-fold increased ALT ratio (present visit ALT: previous visit ALT) accompanied by at least a 0.8 log decreased HBV DNA may be used to classify these two seroconversion types. We conclude that on-treatment elevated ALT levels is a better predictor for seroconversion after NAs treatment, and HBV DNA profiles may help to identify different models of seroconversion.
Journal of Viral Hepatitis 07/2009; 16(12):876-82. · 4.09 Impact Factor
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ABSTRACT: Rep78, the rep gene product of adeno-associated virus (AAV), has been shown to inhibit the replication of several DNA viruses. This study investigated the effects of Rep78 on replication of Hepatitis B virus (HBV) and possible mechanisms of inhibition. We have shown that HBV DNA replication and secretion of HBsAg and HBeAg in HepG2 2.2.15 cells were inhibited by Rep78. We have also demonstrated, using in vitro transcription and luciferase assay, that Rep78 binds to the HBV core promoter (HBV CP) and inhibits HBV CP activity. Furthermore, after Rep78 and HBV core protein expression plasmids were co-transfected into HepG2 cells, the expression of HBV core protein was inhibited significantly. These results suggest that Rep78 can inhibit the replication of HBV, correlating strongly with suppression of HBV CP activity.
Biochemical and Biophysical Research Communications 06/2009; 385(1):106-11. · 2.48 Impact Factor
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ABSTRACT: Hepatic oval cells, progenitor cells in the liver, can differentiate into hepatocytes and bile duct cells both in vitro and in vivo. Although hepatic stellate cells are another important cell component in the liver, less attention has been focused on the relationship between hepatic oval cells and hepatic stellate cells.
Hepatic oval cells were isolated from rats fed a choline-deficient diet supplemented with 0.1% ethionine for 6 weeks and characterized by electron microscopy, flow cytometry, reverse transcription polymerase chain reaction, Western blot and bi-direction differentiation. After treatment with transforming growth factor-beta1 (TGF-beta1), changes in cell viability, morphology, extracellular matrix (ECM) expression and immune phenotype were analysed in these cultured and adherent hepatic oval cells.
The primary cultured hepatic oval cells were positive for the oval cell-specific markers OV-6, BD-1/BD-2 and M2PK as well as the hepatocyte markers albumin and alpha-foetoprotein. These hepatic oval cells differentiated bipotentially into hepatocytes or bile duct-like cells under appropriate conditions. It is noteworthy that these bipotential hepatic oval cells expressed ECM genes stably, including collagens, matrix metalloproteinases and tissue inhibitor of mellatoproteinase. Furthermore, except for growth inhibition and morphological changes in the hepatic oval cells after exposure to TGF-beta1, there was an increased expression of ECM genes, the onset expression of snail and loss expression of E-cadherin. During this process, TGF-beta1 treatment induced an upregulation of marker genes for hepatic stellate cells in hepatic oval cells, such as desmin and GFAP.
Except for the expression of ECM, the cultured hepatic oval cells could induce an increased expression of hepatic stellate cell markers by TGF-beta1 through an epithelial-mesenchymal transition process, which might indicate the contribution of hepatic oval cells to liver fibrosis.
Liver international: official journal of the International Association for the Study of the Liver 05/2009; 29(4):575-84. · 3.82 Impact Factor
