Basic—Liver, Pancreas, and Biliary TractHepatitis C Virus Regulates Transforming Growth Factor β1 Production Through the Generation of Reactive Oxygen Species in a Nuclear Factor κB–Dependent Manner
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Cell Cultures
Huh7.5.1 cells (human hepatocellular carcinoma)17 and HCV JFH1-infected Huh7.5.1 cells16 were grown in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (FBS) medium. The JFH1-infected cells used in this study were analyzed between day 6 and 20 after infection.
ROS Measurements and Cell Viability Assay
ROS levels were measured as previously described.7, 8, 18 Briefly, Huh7.5.1 cells or JFH1 cells were seeded in 100 μL of 10% FBS Dulbecco's Modified Eagle's Medium in 96-well clear-bottom white assay plate
HCV Increases TGF-β1 mRNA and ROS Production in JFH1 Cells
We first examined whether HCV infection has an effect on ROS production in JFH1 cells. We found a >2-fold increase of ROS production in JFH1 cells compared with Huh7.5.1 cells (Figure 1A;P = .001). To visualize ROS activity in Huh7.5.1 cells and JFH1 cells, fluorescent images of the cells were obtained. We confirmed that live cells infected with JFH1 HCV contained higher levels of ROS than uninfected Huh7.5.1 cells (Figure 1B). We found that HCV infection enhances TGF-β1 expression in a
Discussion
HCV infection can lead to liver fibrosis, cirrhosis, and hepatocellular carcinoma through multiple mechanisms. HCV-induced TGF-β1 production appears to play a key role in the development of hepatic fibrosis. TGF-β1 is the most potent known profibrogenic factor. However, the molecular mechanism by which HCV induces TGF-β1 is not well characterized. It has been hypothesized that oxidative stress could contribute to the development of liver fibrosis by inducing the overproduction of TGF-β1 and
Acknowledgments
The authors thank the following investigators and institutes for supplying the reagents: Dr Ralf Bartenschlager, University Heidelberg, Germany; and Dr Takaji Wakita, Second Department of Virology, National Institute of Infectious Diseases, Tokyo, Japan (infectious HCV virus JFH1 DNA construct); and Dr Frank Chisari, Scripps Institute, CA (Hu7.5.1 cells).
W.L. and W.-L.T. contributed equally to this work.
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Conflicts of interest The authors disclose no conflicts.
Funding This work was supported in part by NIH grants R01 AI069939 and U19 AI082630 (R.T.C.).