Zhenbao pill attenuates hydrogen peroxide–induced apoptosis by inhibiting autophagy in human umbilical vein endothelial cells

The Zhenbao pill (ZBP) is composed of 29 traditional Chinese medicines and has been proven to exhibit a valid therapeutic effect in nervous system diseases, such as stroke and hemiplegia sequelae. Whether ZBP has a protective effect on vascular endothelial cells remains unknown. In this study, we established hydrogen peroxide (H2O2)-induced oxidative injury in human umbilical vein endothelial cells (HUVECs) as an in vitro model to investigate the pharmacological effects of ZBP. Following the intragastric administration of ZBP (0.25, 0.5, and 1 g/kg for seven days) in rats, drug-containing serum was obtained and cultivated with HUVECs before H2O2 treatment. The viability of HUVECs in the presence of H2O2 was measured by Cell Counting Kit-8 assay, lactate dehydrogenase assay, and flow cytometry. Furthermore, we estimated the effects of ZBP on the production of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Autophagic puncta were detected using a fluorescence microscope. Western blotting and real-time polymerase chain reaction were used to detect the expression levels of several genes associated with apoptosis and autophagy. Drug-containing serum separated from rats at 1 h after intragastric administration of ZBP (0.5 g/kg) significantly offered a protective effect to HUVECs and reduced cell apoptosis rates. Meanwhile, ZBP-containing serum also repressed ROS production induced by H2O2 exposure and maintained MMP. Further investigation revealed that ZBP-containing serum effectively reduced the accumulation of autophagic puncta. ZBP-mediated inhibition on cell autophagy was found to contribute to ameliorating cell apoptosis. Western blotting also confirmed that ZBP maintained AKT and mTOR phosphorylation and antagonized the imbalance of BCL2/BAX, thereby protecting cells from apoptosis. Taken together, our data indicate that ZBP inhibits ROS production, mitochondrial damage, cell autophagy, and cell apoptosis. ZBP can offer protection to vascular endothelial cells against oxidative injury through the antagonism of apoptosis and autophagy. Thus, this study enhances the understanding of the therapeutic effects and mechanisms of ZBP in the process of recovery from myocardial and cerebral ischemic stroke.