A combination therapy could prove more effective than any one therapy alone through the regulation of multiple targets.Thus,the studies reported were designed to test the hypothesis that the combination of gastrodin and isorhynchophylline inhibits PC12 cells from MPP⁺-induced apoptosis via the mitochondrial pathway.To test this hypothesis,PC12 cells were treated with gastrodin or/and isorhynchophylline for 1 h prior to the challenge with MPP+for additional 24 h in the presence or absence of PD98059,DPI,or/and LiCl.Then,we measured DNA fragmentation,caspase-3/7 activity,cell proliferation,mitochondrial membrane potential,supernatant cytochrome c concentration,total intracellular NAD and NADH concentration,and Akt phosphorylation by ELISA,MTS,flow cytometry,colorimetric assays,and Western Blot analysis.Results:The combination of gastrodin and isorhynchophylline inhibited PC12 cells from MPP⁺-induced apoptosis.Accompanying this,the combination of gastrodin and isorhynchophylline decreased caspase-3/7 activity,enhanced cells proliferation,increased JC-1 aggregates/JC-1 monomers ratio,decreased supernatant cytochrome c concentration,and elevated NAD⁺ and NAD⁺/NADH ratio in MPP⁺-challenged PC12 cells.PD98059,LY294002,or LiCl can partially reverse these changes mediated by the combination of gastrodin and isorhynchophylline.The combination of gastrodin and isorhynchophylline increased phosphorylation of Akt.Our results support our hypothesis and suggest attenuation of MPP+-induced apoptosis in PC12 cells by the combination of gastrodin and isorhynchophylline may relate to Akt phosphorylation.