Iron overload injury is considered to be a part of blood stasis syndrome of arthralgia in traditional Chinese medicine.Its primary therapies include clearing heat and detoxification,activating blood circulation,and removing blood stasis.Lonicerajapon-ica flos (LJF) has long been known as an excellent antipyretic and antidote.Luteoloside (Lut) is one of the main components of LJF and exhibits antioxidant,anti-inflammatory,and cytoprotective properties.However,the protection of Lut against iron overload injury and its underlying mechanisms remain unclear.Therefore,HUVECs were exposed to 50 μmol·L-1 iron dextran for 48 h to establish an iron overload damage model and the effects of Lut were assessed.Our results showed that 20 μmol·L-1 Lut not only increased cell vi-ability and weakened LDH activity,but also significantly up-regulated DDAH Ⅱ expression and activity,increased p-eNOS/eNOS ra-tio and NO content,and reduced ADMA content in HUVECs exposed to iron overload.Furthermore,Lut significantly attenuated intra-cellular/mitochondrial ROS generation,improved SOD,CAT,and GSH-Px activities,reduced MDA content,maintained MMP,inhib-ited mPTP opening,prevented cyt c from mitochondria released into cytoplasm,reduced cleaved-caspase3 expression,and ultimately decreased cell apoptosis induced by iron overload.The effects of Lut were similar to those of L-arginine (an ADMA competitive sub-strate),cyclosporin A (a mPTP blocker agent),and edaravone (a free radical scavenger) as positive controls.However,addition of pAD/DDAH Ⅱ-shRNA adenovirus reversed the above beneficial effects of Lut.In conclusion,Lut can protect HUVECs against iron overload injury via the ROS/ADMA/DDAH Ⅱ/eNOS/NO pathway.The mitochondria are the target organelles of Lut\'s protective ef-fects.