ABSTRACT
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. Lonicera japonica 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 viability and weakened LDH activity, but also significantly up-regulated DDAHⅡ expression and activity, increased p-eNOS/eNOS ratio and NO content, and reduced ADMA content in HUVECs exposed to iron overload. Furthermore, Lut significantly attenuated intracellular/mitochondrial ROS generation, improved SOD, CAT, and GSH-Px activities, reduced MDA content, maintained MMP, inhibited 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 substrate), 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 effects.
Subject(s)
Humans , Endothelium, Vascular , Glucosides , Iron Overload , Luteolin , Reactive Oxygen SpeciesABSTRACT
Aim To investigate the damage of mitochondria in HUVECs cells by iron overload and the role of ADMA/eNOS/DDAHⅡ in it.Methods HUVECs cells were cultured and randomly divided into normal control (Ctrl) group, dextran iron (Iron) group and L-arginine (L-Arg) group.After 48 h, the survival rate of cells was detected by MTT assay;ADMA content and DDAHⅡactivity were measured by HPLC method;the expression of eNOS was determined by Western blot;LDH activity, MDA and NO content, and mitochondrial permeability transition pores(mPTP) openness were determined by colorimetric assay;ROS generation, mitochondrial membrane potential and apoptosis were determined by flow cytometry.Results After 48 h treatment with iron, the survival rate of HUVECs significantly decreased, while the activity of LDH in culture medium increased.The results showed that ADMA and MDA content significantly increased, NO content, DDAHⅡactivity, and the expression of eNOS markedly decreased, the generation of ROS was evidently elevated, mitochondrial membrane potential was lost apparently, mPTP openness was obvious, and the apoptosis of the HUVECs were worsened.However, as ADMA physiological antagonist, L-Arg significantly attenuated the above effects of iron.Conclusion Iron overload could damage mitochondrial function by eNOS and induce the apoptosis of HUVECs, in which ADMA/DDAHⅡ mechanism may also be engaged.