Liver-specific overexpress Glutathione Reductase to prevent Thioacetamide Induce Oxidative stress in Zebrafish

• Main Authors: Jhih-Cyun Pan, 潘智群
• Other Authors: Chuian-Fu Ken,PH.D.
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/74199643484622755186

碩士 === 國立彰化師範大學 === 生物技術研究所 === 101 === Thioacetamide (TAA) is one of the hepatotoxin that can cause liver fibrosis, cirrhosis and hepatocarcinoma, but the mechanism was still unclear. previous study, we have shown TAA can induce hydrogen peroxide, but not superoxide production. The oxidative stress markers include lipid peroxidation and protein carbonylation were higher in TAA treated group than control. In this study, histopathological alterations include liver cell diffuse and fatty infiltration were showed after TAA treated for 2-3 weeks. We next generate transgenic fish with liver-specific glutathione reductase (GR) overexpression by using Tol2 transposon system. The GR mRNA is increase about two-fold, GR protein is increase about 1.5-fold, and GR activity is increase about 2.5-5 fold. When exposed to 4mM H2O2 and 32mM TAA for 96hr, the survival rate was increased and the abnormal embryos were decreased in GRF1 TG-fish than WT-fish. Further , the TAA induced Caspase-3 activation leading cell to apoptosis is also reduced in GRF1 TG-fish embryos by acridine orange staining. The GRF1 TG-fish larvaes were used to test the inhibition of TAA induced oxidative stress. The hydrogen peroxide and oxidative stress markers of GRF1 TG-fish were significantly lower than WT-fish after TAA treatment. The TAA induced cell apoptosis is also reduced in GRF1 TG-fish larvae by acridine orange staining. Our results showed TAA induced H2O2 that damage to living cells or embryos, and overexpress GR can prevent TAA induced oxidative damages.