Superoxide Dismutase Gene Expression in the Endotoxin-Treated Rat Lung

Yoo, Chul Gyu; Suh, Gee Young; Kim, Young Whan; Han, Sung Koo; Shim, Young Soo; Kim, Keun Youl; Han, Yong Chol

Abstract

BACKGROUND
It is well known that oxygen free radicals (OFR) play a vital role in the various type of acute lung injury. Among various antioxidant defense mechanisms, the superoxide dismutases (SOD) are thought to be the first line of antioxidant defense by catalyzing the dismutation of two superoxide radicals to yield hydrogen peroxide and oxygen. Eukaryotic cells contain two types of intracellular SOD : cytosolic, dimeric copper/zinc- containing enzyme (CuZnSOD) and mitochondrial, tetrameric manganese-containing enzyme (MnSOD). The purpose of this study is to evaluate the time-dependent gene expression of MnSOD and CuZnSOD in the endotoxin-treated rats, and to compare with the manifestations of LPS-induced acute lung injury in rats.
METHODS
Total RNA from rat lung was isolated using single step phenol extraction 0, 1, 2, 4, 6, 12, 18, 24 hours after E. coli endotoxin injection (n=3, respectively). RNA was separated by formaldehyde-containing 1.2% agarose gels elctrophoresis, transblotted, baked, prehybridized, and hybridized with 32P-labeled cDNA probes for rat MnSOD and CuZnSOD, which were kindly donated by Dr. Ho (Duke University, Durham, NC, USA). The probes were labeled by nick translation. Blots were washed and autoradiography were quantitated using laser densitometry. Equivalent amounts of total RNA/gel were assessed by monitoring 285 and 185 rRNA.
RESULTS
Endotoxin caused a rise in steady-state MnSOD mRNA levels by 4h with peak mRNA accumulation by 6h. Continued MnSOD mRNA expression was observed at 12h. CuZnSOD mRNA expression was observed from 1h to 24h with peak levels by 18h.
CONCLUSION
These results suggest that SOD palys an important defensive role in the endotoxin-induced acute lung injury in rats.