Inactivation of the AMPKα/CncC/GPX4 axis mediates rotenone-induced ferroptosis in the silkworm (Bombyx mori) model

Ferroptosis is a regulated, iron-dependent form of cell death characterized by excessive lipid peroxidation and redox imbalance. Although extensively studied in mammals due to its involvement in neurodegeneration, cancer, and metabolic diseases, its occurrence and regulatory mechanisms in insects-particularly non-model organisms-remain poorly understood. The silkworm, Bombyx mori, offers a promising invertebrate model owing to its physiological similarity to vertebrates and its established utility in toxicological research. Here, rotenone, a mitochondrial complex I inhibitor and known environmental toxin, was used to induce neurotoxicity in both B. mori larvae and silkworm-derived BmN cells. Transcriptome profiling revealed widespread alterations in gene expression, with significant enrichment in pathways related to mitochondrial dysfunction, oxidative stress, and ferroptosis-related biological processes. Notably, key components of the AMPKα/CncC/GPX4 signaling axis, which regulates antioxidant defense and cellular metabolism, were disrupted. Functional assays further confirmed the hallmark features of ferroptosis, including reactive oxygen species accumulation, glutathione depletion, mitochondrial membrane potential loss, lipid peroxidation, and iron dyshomeostasis. These effects were significantly attenuated by treatment with Ferrostatin-1, a selective ferroptosis inhibitor. Collectively, these findings provide the first comprehensive evidence that ferroptosis occurs in insects and is a key mechanism underlying rotenone-induced toxicity in B. mori. Furthermore, this study establishes the silkworm as a valuable invertebrate model for ferroptosis research and offers new insights into the evolutionarily conserved mechanisms of oxidative cell death.