Ninjinyoeito and Kamikihito Preserve Mitochondrial Function by Reducing Oxidative Stress in a Rotenone‐Induced PC12 Cell Model

ABSTRACT Aim Oxidative stress and mitochondrial dysfunction are central pathological features of neurodegenerative diseases. Ninjinyoeito (NYT) and Kamikihito (KKT), traditional Kampo medicines, show cognitive benefits in Alzheimer's disease; however, their effects on mitochondrial function and the underlying mechanisms remain unclear. This study investigated the effects of NYT and KKT on mitochondrial function in a rotenone (ROT)‐induced cellular model of neurodegeneration. Methods PC12 cells were pretreated with NYT or KKT for 3 h before ROT exposure. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels were measured. Activation of nuclear factor erythroid 2‐related factor 2 (Nrf2) was assessed using an ARE reporter assay. mRNA and protein expression of antioxidant‐related factors, including heme oxygenase‐1 (HO‐1) and Nrf2, were analyzed using RT‐PCR and Western blotting. HO‐1 protein expression was also examined after treatment with the 19 constituent herbs of NYT and KKT. Results ROT‐induced concentration‐dependent cytotoxicity, mitochondrial depolarization, and ROS overproduction. Both NYT and KKT significantly improved cell viability, preserved MMP, and reduced ROS accumulation. They significantly activated ARE reporter activity and increased expression of HO‐1 , GCLC , and GCLM , but not NQO1 . They also elevated HO‐1 and Nrf2 protein levels. Of the 19 herbs tested, 18—including Glycyrrhizae Radix and Gardeniae Fructus —upregulated HO‐1. Conclusion NYT and KKT protect against mitochondrial dysfunction and oxidative stress by activating cellular antioxidant defenses, particularly through induction of HO‐1 and glutathione synthesis components via Nrf2 signaling. These effects may reflect cooperative contributions from multiple herbs, suggesting potential for therapeutic applications in conditions involving oxidative damage and mitochondrial dysfunction.