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The role of antioxidant enzymes on hepatic stellate cell activation and viability

By F.W. Haijer


Introduction: Liver fibrosis and cirrhosis are serious diseases listed in the top 10 causes of death in the Netherlands. Currently, there is no satisfactionary therapy available. Elucidating the mechanisms of fibrogenesis is essential for development of an effective anti-fibrotic therapy . Liver fibrosis is characterized by an increased production of extracellular matrix (ECM) by hepatic stellate cells (HSCs). During fibrogenesis HSCs transdifferentiate from a quiescent to an active myofibroblast-like phenotype that produces excessive amounts of ECM. This ECM is partly composed of collagen type 1. Other genes induced in the activated HSCs are transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA). A major research question is the mechanism of HSC activation. It has been postulated that superoxide anions promote HSC activation. The electron transport chain in mitochondria is a major source of superoxide anions. Manganese superoxide dismutase (MnSOD) is active as an antioxidant enzyme in mitochondria. CuZnSOD is mainly responsible for dismutation of superoxide anions in the cytosol. Catalase is an important antioxidant enzyme in detoxification of hydrogen peroxide that is produced by SODs. Previous results from our group showed that MnSOD expression is reduced during stellate cell activation whereas no effect is observed on CuZnSOD expression. Catalase is induced during the first part of activation, but is similar to the quiescent level when HSCs are activated. Aim: of this study is to determine the role played by anti-oxidant enzymes in HSC activation. Methods: Experiments are performed in a human HSC cell line (LX-2). MnSOD and CuZnSOD and catalase were downregulated using RNAi. Protein was quantified using Western Blot. Morphology was observed by microscopy and mRNA expression was visualized using rt-PCR. Results: Our data showed that MnSOD was effectively downregulated at mRNA and protein level. Downregulation resulted in a morphological change of LX-2 cells to a stretched morphology. Rt-PCR analysis showed that activation markers (α-SMA, TGF-β and collagen type 1) were induced at mRNA level. Downregulation of CuZnSOD at mRNA level did not show differences in the expression of activation markers. Downregulation of catalase slightly reduces expression of activation markers, while MnSOD is slightly induced. Conclusion: Our results suggest that downregulation of MnSOD is linked to the activation of LX-2 cells. This effect seems to be specifically located in mitochondria as downregulation of CuZnSOD does not have effects on LX-2 cell activation. A lower MnSOD level after downregulation of MnSOD might be involved in reduction of activation markers. Taken together, the reduced expression of MnSOD during HSC activation may be the driving force of HSC activation.

Year: 2010
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