摘要Objective Huaier,a traditional Chinese medicine(TCM)approved by the National Medical Products Administration(NMPA)of China for cancer therapy,demonstrates broad antitumor activity.However,its potential to overcome resistance to gefitinib,a first-generation epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI),in non-small cell lung cancer(NSCLC)and the underlying mechanisms remain unclear.This study aimed to determine whether Huaier aqueous extract enhances the efficacy of gefitinib against resistant NSCLC and to elucidate the molecular basis of this effect.Methods Cell proliferation was evaluated using the Cell Counting Kit-8 and colony formation assays.Apoptosis,reactive oxygen species(ROS),and lipid ROS were measured using flow cytometry,and mitochondrial morphology was examined using transmission electron microscopy.RNA sequencing and integrated bioinformatics analyses of GEO datasets were performed to identify ferroptosis-related genes,which were validated by qPCR and Western blotting.The in vivo efficacy was assessed using a PC-9GR xenograft model.Results Huaier aqueous extract significantly enhanced the sensitivity of gefitinib-resistant NSCLC cells to gefitinib in vitro,and suppressed tumor growth in vivo.Mechanistically,the combined treatment activated the ferroptosis pathway,accom-panied by the upregulation of acyl-CoA synthetase long-chain family member 4(ACSL4).Pharmacological inhibition of ferroptosis or ACSL4 partially attenuated the antitumor effect,confirming their key roles in mediating the synergistic activity of Huaier aqueous extract and gefitinib.Conclusions Huaier aqueous extract reversed gefitinib resistance in NSCLC cells by promoting ACSL4-dependent ferrop-tosis,thereby providing a promising therapeutic strategy for improving EGFR-TKI efficacy.