Ginsenoside Rf Suppresses Oxidative Stress–Induced Insomnia by Modulating Mitophagy Through the AMPK/mTOR Signaling Pathway

Neuro‐oxidative damage is a key determinant in the pathogenesis and progression of sleep–wake disturbances, whereas mitophagy serves a crucial role in maintaining cellular homeostasis and mitigating oxidative stress (OS)–induced impairments. Ginsenoside Rf (G‐Rf), a bioactive compound derived from ginseng ( Panax ginseng C. A. Mey), has been recognized for its regulatory effects on mitophagy. However, its potential therapeutic efficacy in OS‐associated insomnia remains largely unexplored. This study aimed to elucidate the neuroprotective effects and mechanistic pathways of G‐Rf in OS‐induced insomnia via a combined approach of network pharmacology and experimental validation. G‐Rf’s effects on sleep–wake regulation, locomotor activity, and cognitive function were evaluated using the DAM2 behavioral monitoring system in an OS‐induced Drosophila model. Potential targets were identified via TCMSP, SwissTargetPrediction, OMIM, and GeneCards databases, followed by GO and KEGG enrichment analyses. Experimental validation was conducted in OS‐exposed Drosophila and glutamate‐induced PC12 cells. ROS levels and apoptosis were quantified through flow cytometry, while Western blot analysis examined apoptosis‐related proteins, AMPK/mTOR, and mitophagy markers. G‐Rf effectively restored sleep–wake homeostasis in OS‐induced Drosophila, improving locomotor activity as well as learning and memory functions. It also enhanced glutathione synthesis and elevated catalase activity in brain tissue. Network pharmacology analysis identified the AMPK/mTOR signaling pathway as a key regulatory mechanism, which was further validated in Drosophila brain tissue. In PC12 cells, G‐Rf reduced lactate dehydrogenase release, reduced ROS accumulation, and suppressed apoptosis. Moreover, G‐Rf preserved mitochondrial membrane potential and regulated apoptosis‐associated proteins by upregulating Bcl‐2 while downregulating Bax and Caspase‐3 expression. Furthermore, G‐Rf stimulated AMPK signaling while suppressing mTOR activity, thereby promoting mitophagy, as evidenced by elevated Beclin‐1 expression, enhanced LC3‐I to LC3‐II conversion, and reduced p62 levels. This study is the first to identify that G‐Rf alleviates OS‐induced neuronal apoptosis and insomnia via AMPK/mTOR‐mediated mitophagy, establishing a previously uncharacterized therapeutic axis associating mitochondrial autophagy with neuroprotection and sleep regulation.