Geniposide ameliorates ferroptosis through the ROS/p38 MAPK/NCOA4/GPX4-mediated ferritinophagy in a co-culture system of HMC3 and SH-SY5Y cells

Ferritinophagy plays a crucial role in ferroptosis, regulating cell death in response to reactive oxygen species (ROS) and iron metabolism. Geniposide (GEN) is an iridoid glycoside that has been reported to exhibit antioxidant and anti-inflammatory effects. Whether GEN can modulate hydrogen peroxide (H₂O₂)-induced inflammation and ferroptosis in human neuronal SH-SY5Y cells remains elusive. In the present study, we utilize the transwell co-culture system, which contains the human microglial cell line HMC3 (upper chamber) and the neuroblastoma cell line SH-SY5Y (lower chamber), to investigate the mechanisms of action of GEN and its neuroprotective effects. GEN inhibited ROS generation, decreased the malondialdehyde (MDA) level, attenuated p38 MAPK phosphorylation, nuclear receptor coactivator 4 (NCOA4), and SQSTM1 (p62) protein expression following H₂O₂ exposure. Meanwhile, GEN increases the intracellular GSH level and glutathione peroxidase 4 (GPX4) protein expression, protecting cells from oxidative damage. Therefore, GEN could modulate NCOA4-mediated ferritinophagic flux and alleviate ferroptosis. In addition, overexpression of NCOA4 significantly induced ferroptosis in both HMC3 and SH-SY5Y cells, but GEN mitigated these effects. NCOA4-mediated ferritinophagy collectively contributes to the process by which hydrogen peroxide induces oxidative stress and leads to cell ferroptosis. In conclusion, GEN alleviates hydrogen peroxide-induced inflammation and ferroptosis through the ROS/p38 MAPK/NCOA4/Gpx4-mediated ferritinophagy signaling pathway.