| Background and objective:Diabetes-induced cardiovascular complications are important factors for the morbidity and mortality of diabetic patients,and vascular endothelial dysfunction are the early core pathological mechanisms of diabetic cardiovascular complications.In the complex mechanism of vascular endothelial injury in diabetes,the regulation of innate immune system on vascular endothelial cells has gradually attracted attention.Among them,the induction of innate immune system by "training immunity" is the innovative research direction of the mechanism of diabetic vascular complications.Monocyte is an important part of the innate immune system.However,the regulatory mechanisms of extracellular vesiceles(EVs)secreted by monocytes in vascular endothelial cells under the background of diabetes have not been further studied.Therefore,the aim of this study is to explore the new mechanism of high glucose-induced monocytes to regulate vascular endothelial cells via EVs pathway,in the hope of finding effective targets for protecting vascular endothelial cells in diabetes.Methods:In the first part of the experiment,EVs were isolated and purified from the cultured supernatant of monocytes(THP-1 cells)cultured in low glucose(5.5 mmol/L)and high glucose(33 mmol/L),which were further identified by transmission electron microscope,nanoparticle tracking analysis and western blotting(WB);The uptake of monocyte EVs by human umbilical vein endothelial cells(HUVECs)was tested by fluorescence labeling.The effects of monocyte EVs induced by high glucose on the proliferation,migration and oxidative stress level of HUVECs were tested,and the regulation of monocyte EVs on the signal pathway of nuclear factor E2 related factor 2(Nrf2)and nucleotide binding oligomerization domain like receptor protein 3(NLRP3)in HUVECs were examined.The second part of the experiment predicted and identified the small interfering RNA(microRNA,miRNA)that may be carried by high glucose-induced monocyte EVs which interfering with the Nrf2 expression in HUVECs through bioinformatics,RT qPCR and double luciferase reporter gene experiment.The regulatory effect of the selected miRNA on HUVECs cell proliferation,migration function,oxidative stress and Nrf2 and NLRP3 signal pathways were tested.Finally,a type 1 diabetic mouse model was developed and the protective effect of the miRNA on the aorta of diabetic mice was verified.Results:1.The results of transmission electron microscopy,nanoparticle tracking analysis and WB confirmed that THP-1 EVs could be stably isolated by the experimental protocols;PKH-67 labeled on THP-1 EVs could be stained to the surface of HUVECs co-cultured with THP-1 EVs;Both THP-1 EVs induced from low glucose and high glucose concentration could promote the proliferation of HUVECs,but there was no significant difference between groups;High glucose THP-1 EVs significantly inhibited the migration ability of HUVECs and increased the production of reactive oxygen species(ROS)in HUVECs,suggesting the elevation of oxidative stress level in HUVECs;WB results showed that high glucose THP-1 EVs inhibited the expression of Nrf2 signaling pathway in HUVECs and activated NLRP3 signaling pathway,suggesting that high glucose THP-1 EVs can promote endothelial cell inflammation.2.Through the verification of public miRNA database,RT-qPCR and double luciferase reporter gene,miR-142-5p was highly expressed in high glucose THP-1 EVs,and miR-1425p was significantly up-regulated in HUVECs after the co-culture with high glucose-induced THP-1 EVs;Regulating the expression level of miR-142-5p did not affect the proliferation of HUVECs;The up-regulation of miR-142-5p significantly inhibited the migration ability of HUVECs and increased the production of ROS,while down-regulation of miR-142-5p could alleviate the inhibition of high glucose on the migration ability of HUVECs and oxidative stress;The up-regulation of miR-142-5p inhibited Nrf2 signaling pathway and up regulated NLRP3 signaling pathway expression in HUVECs,while inhibition of miR-142-5p caused up regulation of Nrf2 signaling pathway and negative regulation of NLRP3 signaling pathway.3.The mice model of type 1 diabetes was successfully established,and miR-142-5p inhibitor(Antagomir)was injected via tail vein.After 14 weeks,aorta tissue was harvested:hematoxylin eosin staining showed no obvious morphological changes in blood vessels.Immunohistochemical results suggested that interleukin-1β in vascular wall of diabetic mice(Interleukin-1β,IL-1β)The expression level was significantly up-regulated in diabetic mice injected with miR-142-5p Antagomir group IL-1β is significantly reduced.Conclusion:1.Extracellular vesicles secreted by monocytes induced by high glucose can inhibit the migration ability of vascular endothelial cells and induce oxidative stress and inflammation of vascular endothelial cells;2.Extracellular vesicles secreted by monocytes induced by high glucose may shuttle miR142-5p to endothelial cells,which inhibiting the migration ability of vascular endothelial cells,induce oxidative stress and inflammation;3.The inhibition of miR-142-5p can effectively alleviate inflammation of blood vessel wall caused by diabetes.4.Inhibition of miR-142-5p is expected to become an innovative target for the prevention and treatment of diabetic angiopathy,with a prospect of clinical transformation. |
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