Confirmation of enhanced expression of heme oxygenase-1 gene induced by ultrasound and its mechanism: Analysis by cDNA microarray system, real-time quantitative PCR, and Western blotting

Purpose. The present study was undertaken to reconfirm heme oxygenase-1 (HO-1) induction by ultrasound, and elucidate the mechanism by which this occurs. Methods. After exposure of human lymphoma U937 cells to 1 MHz continuous ultrasound (US), gene profiling by using cDNA microarray analysis, cell viability by using the trypan blue dye exclusion test, mRNA expression by using real-time quantitative polymerase chain reaction, and protein expression by using Western blotting were examined. As an indicator of cavitation, hydroxyl radical formation was studied by using electron paramagnetic resonance-spin trapping. Results. The cDNA microarray analysis reconfirmed HO-1 induction in human lymphoma U937 cells after exposure to US, and further identified one upregulated and two downregulated genes. When U937 cells were exposed to US for 1min, HO-1 induction, as examined by real-time quantitative polymerase chain reaction and Western blotting, was observed at intensities higher than the cavitational threshold. When a potent antioxidant, N-acetyl-l-cysteine, was added to the culture medium before or after sonication, the induction was attenuated, indicating that reactive oxygen species are involved in HO-1 induction. A decrease in mitochondrial membrane potential and generation of superoxide anion radicals were also observed in the cells exposed to US. Conclusion. We used a cDNA microarray system to confirm upregulation of the HO-1 gene and to discover new genes that respond to ultrasonic cavitation. Increased intracellular oxidative stress secondary to the sonomechanical effects arising from ultrasonic cavitation is suggested to be the mechanism of enhancement of HO-1 expression.