Supplementary Material for: Effect of Cryptotanshinone on Measures of Rat Cardiomyocyte Oxidative Stress and Gene Activation Associated with Apoptosis

Background: Oxidative stress is a key factor that results in cardiomyocyte apoptosis and cardiovascular diseases. Cryptotanshinone (CTS), one of the major bioactive constitutes extracted from the root of the plant Salvia miltiorrhizaBunge, has been widely studied for various disease treatments. However, the roles of CTS on cardiomyocytes remain unclear. Methods: In the present study, neonatal rat cardiomyocytes were pretreated with CTS for 4 h before being exposed to H2O2. Cell viability for the cells with or without pretreatment with CTS before exposure to H2O2 was evaluated by the MTT assay. Production of lactate dehydrogenase (LDH), nitric oxide (NO), prostaglandin E2 (PGE2), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxides (GSH-Px) was quantified by corresponding detection kits. The mRNA levels of Bcl-2 antiapoptotic and Bax-like proapoptotic genes were quantified with RT-PCR. Production of reactive oxygen species (ROS) was qualified and quantified with a dichlorofluorescein diacetate cellular ROS detection assay kit. The extracellular signal-related kinase (ERK) phosphorylation and nuclear factor κB (NF-κB) activation were measured by Western blot. Results: Our results revealed that the CTS pretreatment could enhance cell viability and promote Bcl-2 antiapoptotic gene expression. Additionally, CTS could abolish the H2O2-induced production of NO, LDH, and PGE2. Consistent with these findings, CTS could inhibit ROS and MDA production and promote SOD, CAT, and GSH-Px activities. Mechanistically, CTS may achieve these processes by inhibiting ERK and NF-κB signal pathways. Conclusion: CTS protects cardiomyocytes against the H2O2-induced cellular injuries through ERK and NF-κB inactivation and ROS scavenging. Therefore, CTS is a promising reagent against ROS-induced cardiomyopathy.