Suppression of oxidative stress-induced hepatocyte injury by Calpain antisense

Abstract

Background. Calpain, a cytosolic Ca 2+-dependent proteinase, plays a pivotal role in cell injury. In this study, we investigated the effect of calpain-μ antisense oligonucleotide on oxidative stress induced-hepatocyte injury. Materials and methods. Hemagglutinating virus of Japan liposome complex with one of three types of antisense oligonucleotide (AS-1, AS-2, AS-3) or scramble oligonucleotide was added to the culture medium of HuH7 cells and incubated for 6 days. The expression of calpain-μ protein was examined by Western blotting. After the addition of tert-butyl hydroperoxide, bleb formation was examined by phase contrast microscopy, and cell viability was assessed by the release of lactate dehydrogenase. Results. Incubation of HuH7 cells with AS-2 resulted in a decrease in the amount of calpain on day 4 and a further decrease to almost undetectable levels on day 6, whereas scramble oligonucleotide had no effect. Bleb formation was observed 120 min after the addition of tert-butyl hydroperoxide in scramble oligonucleotide-treated cells as in untreated cells. In contrast, it was rarely observed in AS-2-treated cells. Lactate dehydrogenase release was significantly suppressed in AS-2-treated cells, compared with that in scramble oligonucleotide treated-cells. Conclusions. Our findings suggest that calpain activation is involved in the pathogenesis of oxidative stress injury and that transfection of calpain antisense may potentially protect against ischemia/reperfusion liver injury.