Suppression of oxidative stress-induced hepatocyte injury by calpain antisense

J Surg Res. 2003 May 1;111(1):23-7. doi: 10.1016/s0022-4804(03)00047-7.

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-mu 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-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calpain / genetics*
  • Calpain / physiology
  • Cell Survival / drug effects
  • Fluorescein-5-isothiocyanate
  • Fluorescent Dyes
  • Gene Expression
  • Hepatocytes / drug effects
  • Hepatocytes / enzymology*
  • Hepatocytes / ultrastructure*
  • Humans
  • L-Lactate Dehydrogenase / metabolism
  • Liver Neoplasms
  • Oligonucleotides, Antisense / genetics*
  • Oxidative Stress*
  • Transfection
  • Tumor Cells, Cultured
  • tert-Butylhydroperoxide / pharmacology*

Substances

  • Fluorescent Dyes
  • Oligonucleotides, Antisense
  • tert-Butylhydroperoxide
  • L-Lactate Dehydrogenase
  • Calpain
  • Fluorescein-5-isothiocyanate