Targeted Blocking Of Ninj1 Protects Endothelial Function And Inhibits The Development Of Diabetic Peripheral Vascular Complications

Objective:To investigate the role of Ninj1 in the progression of diabetic peripheral vascular disease(DPVC)by elucidating the regulatory effect and mechanism of Ninj1 on the function of endothelial cells and macrophages under high glucose conditions,and to study the inhibitory effect of targeting Ninj1 on DPVC.Methods:?Muscle tissue and carotid atherosclerotic plaque of diabetic patients were obtained.The expression of Ninj1 protein,inflammatory factors,the density of blood vessels,the number of macrophages and co-localization of Ninj1 with endothelial cells and macrophages in diabetic and non-diabetic patients was analyzed by immunofluorescence,WB,and PCR.The clinical relevance of Ninj1 expression and diabetic DPVC were statistically analyzed.?Ninj1 expression in endothelial cells of diabetic mouse and in in vitro cultured endothelial cells and macrophages was detected through immunofluorescence,WB,and PCR.The correlation between Ninj1 expression and endothelial dysfunction or macrophage inflammation was analyzed.?Endothelial cell survival,apoptosis,tube formation ability and inflammation,as well as macrophage inflammation and transendothelial migration were analyzed by using Ninj1 neutralizing antibody.? Ninj1 neutralizing antibody was injected into the lower limb muscle to observe the effect of blocking Ninj1 on blood flow changes and vascular density in lower limbs of diabetic mice.Results:? The expression of IL-6,VCAM-1,TNF? or other inflammatory factors were significantly increased in diabetic patients and in peripheral blood mononuclear cells.The vascular density in lower extremity muscle of diabetic patients decreased with the increase of Ninjl expression.Co-localization was observed between Ninj1 and endothelial cells in diabetic muscle tissue.Significant macrophage infiltration with high level of Ninj1 expression was observed in atherosclerotic plaque in diabetic patients,and co-localization was observed between Ninj1 and macrophages cells;? Decreased vascular density in muscle tissue of diabetic mice is associated with an upregulation of Ninj1 expression;High glucose induced endothelial dysfunction and macrophage inflammation with high level of Ninj1 expression in vitro.Targeted blocking of Ninj1 can promote the survival of endothelial cells under high glucose by up-regulating the activity of eNOS and inhibit the apoptosis of endothelial cells by inhibiting the activation of caspase-3 and up-regulating the level of Bcl-2.In addition,targeted blocking of Ninj1 can inhibit macrophage inflammation and trans-endothelial migration.Further study found that targeted blocking Ninj 1 can inhibit ROS/p38 MAPK/NF-?B pathway-mediated inflammation and can activate the PI3K/Akt pathway.By using small molecule inhibitor,we found that targeted blocking of Ninj1 upregulated eNOS activity and BCL-2 expression through PI3K/Akt pathway.Targeted blocking of Ninj1 protects endothelial function under high glucose conditions by the above mechanisms.? Ninj1 neutralizing antibody effectively protected the blood flow and blood vessel density in lower extremities of diabetic mice.Conclusions:Ninj1 mediates endothelial dysfunction and inflammation induced by high glucose,whereas targeted blockade of Ninj1 may suppress inflammation and protect endothelial function under high glucose conditions.This study provides new ideas and theories for the prevention and treatment of DPVC progression and can be regarded as an potential effective complement to existing DPVC treatment options.