Protective Effects Of Nobiletin On Iron Overload Induced Damages In HUVECs By Regulation Of ROS/ADMA/DDAH?/eNOS/NO Pathway

Objective: To investigate the effect of nobiletin(Nob)against HUVECs suffered iron overload injury and its relationship with ROS/ADMA/DDAHII/eNOS/NO pathway.Method: The experiments were randomly divided into 7 groups:(1)Control group;(2)DTF group;(3)20 ?M Nob + DTF group;(4)20 ?M Nob + pAD /DDAHII-shRNA + DTF group;(5)1 mM L-Arg + DTF group;(6)1 ?M CsA + DTF group;(7)100 ?M Eda + DTF group.MTS was used to detect cell viability,lactate dehydrogenase(LDH)activity was measured by kit,and the extent of mitochondrial permeability transition pore(mPTP)was detected by microplate reader;flow cytometry was used to measure intracellular reactive oxygen species(ROS)and mitochondrial membrane potential;Apoptosis was detected by TUNEL or Annexin V-FITC/PI double staining.The expression of DDAHII,eNOS,p-eNOS and Cleaved Caspase-3 protein were assayed by Western blotting.The ADMA content and DDAHII activity were detected by HPLC.Results: 1.Nobiletin can improve the survival rate of iron overload endothelial cells and reduce the activity of lactate dehydrogenase LDH.2.20?M Nob significantly reduced ROS production,increased DDAHII and p-eNOS protein expression and NO content,reduced ADMA content,inhibited mPTP opening,stabilized cell membrane potential,finally reduced apoptosis.While infected pAD/DDAHII-shRNA adenovirus down-regulated DDAHII expression,the above effects were abolished or attenuated.Conclusion: Nobiletin protects iron overload HUVECs by inhibiting ROS production,up-regulating DDAHII and eNOS expression,thereby reducing ADMA content and increasing NO content to reduce apoptosis.