MitoNEET-induced Ferroptosis Contributes To Diabetic Vascular Injury

Objectives: Diabetes is one of the top ten causes of death worldwide,and cardiovascular complications are the most serious complications of diabetes.Long-term hyperglycemia and high free fatty acids can produce a large number of reactive oxygen species in endothelial cells through a variety of metabolic pathways,and induce oxidative stress response and dysfunction,which is an important link of diabetic vascular injury.Ferroptosis is a newly discovered iron-dependent cell death induced by lipid peroxidation.The iron-sulfur cluster protein mitoNEET can mediate mitochondrial iron transport under oxidative state.Whether it mediates mitochondrial ferroptosis to cause diabetic vascular injury is an important scientific question to be addressed in this study.Methods: In this study,the blood glucose,blood lipid,glucose tolerance,endothelium-dependent relaxation function,HE staining,ROS staining and mitochondrial electron microscopy were compared between diabetic mice and control mice to clarify the characteristics of vascular injury in diabetes.Furthermore,db/db mice were given ferroptosis inhibitors Ferrostatin-1(Fer-1),mitochondrial antioxidants mitoTEMPO,iron chelators Desferrioxamine(DFO)and other drugs to explore the effect of interfering with mitochondrial ferroptosis on vascular injury in db/db mice.Finally,the down-regulation of GPX4 expression,ROS production and mitochondrial function by mitoNEET in diabetic endothelial cells were elucidated,and the role and mechanism of mitoNEET in diabetic vascular injury through mitochondrial ferroptosis were revealed.Results: The structure and function of blood vessels were impaired in diabetic mice.The levels of blood glucose(P < 0.01),blood lipid(P < 0.01),glucose tolerance(P <0.01)and endothelium-dependent vasodilation(P < 0.01)were increased in diabetic mice.The level of serum endothelin-1(ET-1)was increased(P < 0.01).The morphological results showed that the structure of the descending aorta was disordered,the production of reactive oxygen species was increased(P < 0.01),and the structure of mitochondria was damaged.The addition of ferroptosis inhibitor Ferrostatin-1(Fer-1),mitochondrial antioxidant mitoTEMPO,iron chelator Desferrioxamine(DFO)and other reagents to interfere with mitochondrial oxidation and antioxidation effectively alleviated the vascular damage caused by diabetes,especially after the addition of ferroptosis inhibitor Fer-1,although there was no significant change in blood glucose in the intervention group.However,injection glucose tolerance(P < 0.05)and endothelium-dependent vasodilation function(P < 0.05)were improved.The level of serum endothelin-1(ET-1)was decreased(P < 0.05).The morphological results showed that after adding Fer-1,mitoTEMPO and DFO,the tissue structure of the descending aorta of mice was more orderly,the degree of fibrosis was alleviated,the production of reactive oxygen species was reduced(P < 0.01),and the damage of mitochondrial structure was recovered to some extent.mitoNEET,as a key molecule of iron transport on the outer mitochondrial membrane,induced ferroptosis in endothelial cells by overexpression of gene(P < 0.01)and protein(P < 0.001)in the diabetic endothelial cell model constructed by high glucose and fat,and the gene expression of GPX4,the key factor of ferroptosis,decreased(P <0.01).The generation of reactive oxygen species was increased(P < 0.001),and the mitochondrial membrane potential was decreased.Treatment with ferroptosis inhibitors,mitochondrial antioxidants,iron chelators,and other drugs significantly decreased the gene expression(P < 0.05)and protein expression(P < 0.05)of mitoNEET,increased the gene expression of GPX4(P < 0.05),inhibited the ferroptosis of endothelial cells,and reduced the deposition of reactive oxygen species(P < 0.001).The mitochondrial membrane potential was increased(P < 0.05),and mitochondrial function was improved.Conclusion: The vascular structure and function are impaired in diabetic mice,and ferroptosis is one of the causes of this damage.Ferroptosis inhibitors can alleviate vascular damage in diabetes by inhibiting ferroptosis.mitoNEET,as a key molecule of mitochondrial iron transport,mediates ferroptosis under high glucose and lipid environment,thereby accelerating the structural changes and functional damage of blood vessels.