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Skeletal myoblast (SKM) transplantation is a promising approach to regenerate tissue and improve the function of the injured heart. However, the number of survival cells transplanted to host myocardium is quite poor due to high rate of apoptosis; diazoxide (DZ) is a highly selective mito-KATP channel opener that may reduce cell apoptosis by relieving reactive oxygen species (ROS) damage. The aim of this study is to explore the protective effects of DZ on L6 SKM damage induced by hydrogen peroxide (H(2)O(2) ) in vitro. Different dose and time of H(2)O(2) and DZ treatment were performed and only 24 hr of 1.00 mmol/L H(2) O(2) treatment and 200 μmol/L DZ pretreatment for 30 min were used for further experiment. L6 SKMs were cultured and divided into control group (no treatment), H(2)O(2) group (24 hr of 1.00 mmol/L H(2) O(2) treatment) and DZ + H(2)O(2) group (pretreated with 200 μmol/L DZ for 30 min before 24 hr of 1.00 mmol/L H(2) O(2) treatment). Compared with control group, H(2)O(2) treatment caused cell damage, increased lactate dehydrogenase release, cell apoptosis, and bax gene expression, while reduced cell proliferation and decreased bcl-2 expression. DZ pretreatment may protect cells from damage induced by H(2)O(2) and reduce cell apoptosis by increasing bcl-2 and decreasing bax expression. DZ pretreatment may also promote cell proliferation measured by both PCNA expression and flow cytometry method. These results suggest that DZ may protect L6 SKMs from damage induced by H(2)O(2) by maintaining integrity of cell membrane, reducing apoptosis and increasing proliferation in vitro., (Copyright © 2012 Wiley Periodicals, Inc.)