Hydroxychloroquine-induced cytotoxicity in ARPE-19 cells involves PI3K/AKT-associated signaling: network toxicology and functional validation

Background Hydroxychloroquine (HCQ) is widely used for the long-term treatment of autoimmune and rheumatologic disorders, but retinal toxicity remains an important adverse effect. Although lysosomal and autophagic dysfunction have been implicated in HCQ-associated retinal injury, the signaling networks involved in the cellular response to HCQ remain incompletely understood. This study investigated the potential involvement of PI3K/AKT-associated signaling in HCQ-induced cytotoxicity. Methods Network toxicology and molecular docking were integrated to identify candidate HCQ-associated targets, followed by experimental validation in human retinal pigment epithelial ARPE-19 cells. Cell viability was evaluated by CCK-8 assay, and apoptosis was assessed by Annexin V/PI flow cytometry. The expression of predicted hub genes and proteins was examined by qRT-PCR and Western blotting. AKT phosphorylation was evaluated across HCQ concentration and time gradients. The AKT activator SC79 was further used in pharmacological rescue experiments to assess the functional involvement of AKT signaling in HCQ-induced apoptosis. Results Network analysis identified PIK3CA, PIK3R1, SRC, JAK2, and PTPN11 as candidate hub proteins associated with PI3K/AKT-related signaling. Molecular docking suggested potential interactions between HCQ and these proteins, with variable docking energies among targets. HCQ reduced ARPE-19 cell viability in a concentration-dependent manner, with a 24-h IC 50 of 59.51 μM, and increased apoptosis with increasing concentrations. qRT-PCR revealed concentration-dependent and gene-specific transcriptional responses rather than uniform suppression of all five hub genes, whereas Western blotting showed an overall reduction in the abundance of the candidate hub proteins after 24 h HCQ exposure. AKT phosphorylation exhibited dynamic regulation: short-term HCQ exposure increased p-AKT/AKT at higher concentrations, whereas an independent 30 μM time-course experiment showed an overall decline in AKT phosphorylation during prolonged exposure. Importantly, SC79 pretreatment enhanced p-AKT/AKT under HCQ exposure and partially attenuated HCQ-induced apoptosis. Conclusion These findings support the involvement of PI3K/AKT-associated signaling in HCQ-induced cytotoxicity in ARPE-19 cells. HCQ induces concentration- and time-dependent alterations in this signaling network, while pharmacological enhancement of AKT signaling partially reduces the apoptotic response. The results provide functional evidence that AKT signaling contributes to the cellular response to HCQ and support further investigation of PI3K/AKT-associated signaling in HCQ-related RPE injury.