Suppression of oxidative stress-induced hepatocyte injury by calpain antisense.
BACKGROUND
Calpain, a cytosolic Ca(2+)-dependent proteinase, plays a pivotal role in cell injury. In this study, we investigated the effect of calpain-mu antisense oligonucleotide on oxidative stress induced-hepatocyte injury.


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-mu 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.
Taniguchi
Kazunori
K
Department of Surgery and Clinical Oncology, Graduate School of Medicine Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Umeshita
Koji
K
Sakon
Masato
M
Miyoshi
Hideyuki
H
Tokunaga
Masaru
M
Ariyoshi
Hideo
H
Yamano
Tomoki
T
Kaneda
Yasufumi
Y
Monden
Morito
M
D002154:Calpain / Q000235:genetics* / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D015870:Gene Expression; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008113:Liver Neoplasms; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D018384:Oxidative Stress; D014162:Transfection; D014407:Tumor Cells, Cultured; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
D005456:Fluorescent Dyes; D016376:Oligonucleotides, Antisense; D020122:tert-Butylhydroperoxide; D007770:L-Lactate Dehydrogenase; D002154:Calpain; D016650:Fluorescein-5-isothiocyanate