Effect of target-directed regulation of uncoupling protein-2 gene expression on ischemia-reperfusion injury of hepatocytes

Abstract

The effect of target-directed regulation of the uncoupling protein-2 (UCP-2) gene expression on the ischemia-reperfusion injury of hepatocytes under different conditions was investigated. The expression plasmid and RNAi plasmid targeting UCP-2 gene were constructed and transfected into normal hepatocytes and fatty liver cells, respectively. The expression of UCP-2 mRNA was detected by real time PCR. The cells were divided into normal cell group (NCG), group of normal cells transfected with empty vector (EVNCG), group of normal cells transfected with expression plasmid (EPNCG), fatty liver cell group (FCG) and group of fatty liver cells transfected with RNAi plasmid (RPFCG). The ischemia-reperfusion model in vitro was established. One, 6, 12 and 24 h after reperfusion, Annexin V/PI flow cytometry was used to measure cell necrosis rate, apoptosis rate and survival rate. Simultaneously, the intracellular ATP, ROS and MDA levels were determined. The results showed that 1, 6, 12 and 24 h after ischemia-reperfusion, the intracellular ROS, MDA and ATP levels and cell survival rate in EPNCG were significantly lower, and cell necrosis rate significantly higher than in NCG and EVNCG, but there was no significant difference in apoptosis rate among NCG, EVNCG and EPNCG (P>005). Six, 12 and 24 h after reperfusion there was no significant difference in ROS, MDA levels and apoptosis rate between FCG and RPFCG (P>0.05), but the ATP level and survival rate of cells in RPFCG were higher than in FCG (P<0.05). It was concluded that down-regulation of the UCP-2 gene expression in steatotic hepatocytes could alleviate the ischemia-reperfusion injury of liver cells.

Keywords

uncoupling protein 2 / ischemia-reperfusion / fatty liver cell / siRNA

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