Quantitative proteomic analysis of the anti-ferroptosis effect of a proprietary hydroxy-α-sanshool extract in human keratinocytes using a tandem mass tag labeling approach.

Hydroxy-α-sanshool (HAS), a bioactive alkylamide from Sichuan peppercorns, is known for its antioxidant and neurosensory properties, yet the molecular mechanism underlying its skin-protective effects remains unclear. Herein, we report that HAS alleviates RSL-3-induced ferroptosis in human keratinocytes by restoring cell viability and reducing reactive oxygen species, lipid peroxidation, and iron accumulation. To elucidate the molecular mechanisms, a tandem mass tag (TMT)-based quantitative proteomic analysis was performed, identifying 5540 proteins with high labeling efficiency (99.1-99.3%). Comparative proteomic analysis revealed that HAS partially counteracted dysregulation of ferroptosis-associated proteins and modulated pathways involved in lipid metabolism, antioxidant defense, and RNA processing. Notably, peroxisome proliferator-activated receptor delta (PPARD) and nuclear factor erythroid 2-related factor 2 (NFE2L2) emerged as key upstream regulators responsible for restoring lipid metabolism and antioxidant defense, respectively, while network analysis suggested PPARD as a central node mediating ferroptosis resistance. These findings reveal a dual lipid-redox regulatory mechanism through which HAS counteracts ferroptotic stress in keratinocytes. Our findings support HAS as a sensation-active compound with promising skin-protection potential and shed light on the development of HAS-based active ingredients for enhanced skin health.