Daphnetin protects oxidative stress-induced neuronal apoptosis via regulation of MAPK signaling and HSP70 expression.

Neurodegenerative disorders are characterized by progressive degeneration and loss of neurons in the brain. Oxidative stress is implicated in the pathogenesis of neurological disorders, although the pathological mechanism remains unelucidated. Daphnetin, an active ingredient extracted from Changbai daphne (Daphne Korean Nakai), exhibits various pharmacological effects, including anti-inflammatory, anti-oxidative and anti-tumor effects. However, the neuroprotective effects, as well as the specific mechanisms of daphnetin, remain unclear. Neuronal-like rat pheochromocytoma PC12 cells were pretreated with daphnetin for 2 h, then treated with or without H2O2 for various times. Cell morphology was detected using an inverted microscope, the apoptotic ratio was determined by Annexin V fluorescein isothiocyanate/propidium iodide assay, nuclear morphology was observed and photographed using a fluorescence microscope following 4',6-diamidino-2-phenylindole staining. The levels of pro-caspase 3, cleavage of poly ADP-ribose polymerase and caspase 3 were detected by western blotting. In addition, the activation of mitogen-activated protein kinase (MAPK) signal pathway and the expression of HSP70 were detected by western blotting. The present study demonstrated that daphnetin attenuated hydrogen peroxide (H2O2)-induced apoptosis in a concentration-dependent manner, reduced the cleavage of poly ADP ribose polymerase and caspase 3, and inhibited the phosphorylation of p38 MAPK and c-Jun N-terminal kinases (JNK) in H2O2-induced PC12 cells. In addition, daphnetin induced the expression of HSP70 in a dose- and time-dependent manner, and daphnetin-induced HSP70 expression was reduced by extracellular signal-regulated kinase (ERK) 1/2 inhibitor U0126 in PC12 cells. Therefore, the present results indicate that daphnetin protects PC12 cells against oxidative stress injury by regulating p38 MAPK and JNK signaling and increasing the expression of HSP70 via ERK signaling. This suggests that daphnetin may have the potential to treat certain neurodegenerative diseases. The present results not only provide insight into the potential use of daphnetin in H2O2-induced PC12 cell apoptosis, but also highlight the potential role of HSP70 in neuroprotection.