Abstract
Aminolevulinic acid synthase 1(ALAS 1) is the rate-limiting enzyme for heme synthesis, which is the essential source of intracellular oxidative stress under specific conditions. This study aimed to identify the role of ALAS1 in the oxidative injury of cardiomyocyte induced by doxorubicin. H9c2 cardiac myocytes were treated with a 5-μM concentration of doxorubicin for 24 h. ALAS1 small interfering RNA (siRNA) or rALAS1 plasmid was transfected into H9c2 cardiac myocytes to establish ALAS1 knockdown or over-expressing H9c2 cells. Apoptotic cells were detected using flow cytometric analysis. ALAS1 expression was detected by real-time PCR and western blot assay. The levels of heme were detected by ELISA. MitoSOX Red was introduced for detection of mitochondrial superoxide in H9c2 cells and was validated with confocal fluorescence microscopy. Doxorubicin increase the expression of ALAS1, and heme promotes the oxidative stress and apoptosis in H9c2 cardiac myocytes. Over-expression of ALAS1 can produce similar apoptotic effect on H9c2 cells with doxorubicin, although at a relatively low level. Inhibition of ALAS1 by ALAS1 siRNA decreased heme and mitochondrial superoxide levels, results in inhibition of doxorubicin-induced apoptosis. ALAS1 plays a pivotal role in the doxorubicin-induced oxidative stress and apoptosis in H9c2 cardiac myocytes.
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