Yiqi Huoxue Decoction attenuates ischemia/hypoxia-induced oxidative stress injury in H9c2 cardiomyocytes

Yiqi Huoxue Decoction (YQHX) has been widely used for clinical treatment of ischemic heart disease. While oxidative stress plays a key role in the pathogenesis of ischemic heart disease, the function and molecular mechanism underlying antioxidative protective effects of YQHX on H9c2 cardiomyocytes against ischemia/hypoxia (I/H) have yet to be well clarified. H9c2 cells were subjected to 12 h of hypoxia with serum-free conditions and then treated with or without YQHX (100–400 μg/mL). Cell viability was examined using a CCK-8 assay. Maleic dialdehyde (MDA) and superoxide dismutase (SOD) activity were detected using commercial kits. Intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential were measured using fluorescence microscopy and confocal laser-scanning microscopy, respectively. Ultrastructural details of mitochondria in H9c2 cells were observed using transmission electron microscopy. The antioxidative protective pathway was assessed by measuring mRNA and protein expression of Nrf2 and HO-1, as well as AMPK activation. I/H injury gradually induced oxidative stress. Treatment with YQHX significantly increased cell viability and reversed I/H-induced oxidative stress, including reducing the production of oxidative stress products (ROS and MDA), increasing SOD levels, improving mitochondrial morphology, and increasing mitochondrial membrane potential. YQHX was also observed to increase I/H-induced expression of Nrf2 and HO-1, and the activation effects of YQHX were blocked by an AMPK inhibitor. In addition, HPLC analysis showed that YQHX contained two active antioxidative constituents (calycosin and ferulic acid). The results suggest that anti-oxidative effects exerted by YQHX in H9c2 cardiomyocytes may be linked to upregulation of the AMPK-mediated Nrf2/HO-1 pathway.