To clarify the molecular mechanism of maduramicin induced ap
To clarify the molecular mechanism of maduramicin-induced apoptosis in chicken myocardial cells, the mRNA levels of bcl-2, bax, and cytochrome C, as well as the mRNA levels and activities of caspase-3/8/9 were determined. The mRNA expressions and activities of initiator casapase-9 and executioner caspase-3 all increased in a concentration-dependent manner. Coincidentally, cytochrome C mRNA expression increased upon the activation of the caspase cascade, suggesting that the intermediate signaling molecule cytochrome C might have been released from the mitochondria, which in turn activated caspase-9 and subsequently, caspase-3. These data demonstrate that this intrinsic pathway was related to maduramicin-induced apoptosis. Simultaneously, an increase in the activity and mRNA expression of caspase-8 was observed in chicken myocardial egfr inhibitors exposed to maduramicin, thereby suggesting that an extrinsic pathway was involved in maduramicin induced- apoptosis. These results are in accordance with previous in vitro and in vivo reports (Lv and Bao, 2009; Ren et al., 2015; Zang et al., 2015). The mRNA expression of the anti-apoptotic factor bcl-2 was upregulated by maduramicin, and the ratio of bax/bcl-2 increased slightly (data not shown). However, the role of bcl-2 and bax in regulating maduramicin-induced apoptosis requires further investigation. Calcium ions play an important role in the regulation of cell apoptosis (Pinton et al., 2008). Several ionophores can alter the concentration gradients of ions, thereby resulting in an imbalance in cellular ions and calcium overload (Calo et al., 2002). Boehmerle and Endres (2011) demonstrated that salinomycin causes an increase of cytoplasmic Ca2+ levels in murine dorsal root ganglia neurons (DRGNs), which in turn triggers caspase- dependent apoptosis. We thus hypothesized that an overload of intracellular Ca2+ in chicken myocardial cells may be related to maduramicin-induced apoptosis. The results of the present study support our hypothesis, as the application of maduramicin induced an increase in intracellular Ca2+ levels in all treatment groups. Nevertheless, the source of intracellular Ca2+ levels remains unclear. Thus, we performed flow cytometry using Ca2+-free medium and thapsigargin, which indicated that the observed cytoplasmic Ca2+ levels was due to an influx from the extracellular medium and its release from the endoplasmic reticulum. However, the specific relationship between increased cytoplasmic Ca2+ levels and cell apoptosis requires further investigation. To our knowledge, various biological processes including apoptosis are associated with crosstalks between ROS and Ca2+. The interactions between ROS and calcium signaling can be considered as bidirectional, wherein ROS can regulate cellular calcium signaling, whereas calcium signaling is essential for ROS production (Gordeeva et al., 2003). At the same time, intracellular ROS increased and GSH decreased when chicken myocardial cells were incubated with maduramicin (0.5 μg/mL and 5 μg/mL). These findings prompted us to investigate the contribution of oxidative stress in the maduramicin-induced damage observed in myocardial cells. Thus, the antioxidant NAC was utilized to inhibit ROS, and then we evaluated the effect of maduramicin on cytotoxicity and cell apoptosis in chicken myocardial cells. The results demonstrated that NAC did not impart any visible effects on cell apoptosis and viability at all concentrations. These findings suggest that maduramicin caused generation of ROS but oxidant stress was not directly relevant to maduramicin-induced apoptosis and cytotoxicity. Furthermore, excess ROS production, if not detoxified, may induce mitochondrial damage and depolarization. Loss of MMP is one of the initial symptoms of mitochondria-mediated apoptosis, and a dysfunction in the mitochondria generally results in biochemical changes (Kominsky et al., 2002; Xiong et al., 2014). In the current study, flow cytometry and fluorescence microscopy demonstrated that maduramicin causes mitochondrial depolarization in chicken myocardial cells. This depolarization induces permeabilization of the mitochondrial membrane, thereby resulting in the release of apoptosis-related proteins and molecules to the cytoplasm, which consequently triggers apoptosis. In summary, we conclude that an intrinsic mitochondrial apoptosis pathway and extrinsic pathway participate in maduramicin- induced apoptotic cell death (Fig. 11).