Abstract

Objective Condyloma acuminatum (CA), a common sexually transmitted disease caused by human papillomavirus (HPV) infection, is closely associated with the abnormal proliferation and differentiation of host keratinocytes. Brusatol (BRU), a quassinoid compound derived from the medicinal plant Brucea javanica, has gained attention for its ability to regulate multiple biological processes through the phosphoinositide 3-kinase/Protein Kinase B (PI3K/AKT) signaling pathway. It may also reduce Nuclear factor erythroid 2-related factor 2 (Nrf2) expression to induce ferroptosis in cancer cells. However, its regulatory role in ferroptosis of HPV-infected keratinocytes remains unreported. Therefore, this study was designed to examine the role of BRU in inducing ferroptosis in CA keratinocytes via the PI3K/AKT/Nrf2 pathway, with the aim of identifying a promising therapeutic strategy for CA. Methods To simulate the pathological process of CA, a pEGFP-N1-HPV6 recombinant plasmid vector was transfected into HaCaT human epidermal keratinocyte cells. The cytotoxicity of BRU was determined by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell membrane integrity was assessed via the Lactate Dehydrogenase release assay. Lipid peroxidation accumulation was detected using C11-BODIPY581/591 fluorescent staining. The oxidative stress level was evaluated by measuring the glutathione/oxidized glutathione (GSH/GSSG) ratio. Cellular iron content was measured to assess iron homeostasis. Nrf2 nuclear translocation was examined by immunofluorescence. Cell proportions were analyzed by flow cytometry. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome enrichment analyses were performed to identify signaling pathways involved in BRU-induced ferroptosis. The protein expression levels of Glutathione Peroxidase 4 (GPX4), Solute Carrier Family 7 Member 11 (SLC7A11), Nrf2, phosphorylated-PI3K (p-PI3K)/total-PI3K, and p-AKT/total-AKT were detected by Western blot. Th