The Mechanism Of AIBP Inhibits High Glucose-induced Endothelial To Mesenchymal Transition And LncRNA NEAT1 Targeting Regulation

[Background and Objective]: Diabetic vascular complications are the most common chronic complication and main cause of death in diabetes mellitus(DM).The major vascular disease is mainly manifested as atherosclerosis(As)disease.In the development of diabetic vascular complications,hyperglycemia first stimulates endothelial cells,causing them to undergo endothelial-to-mesenchymal transition(EndMT),resulting in the original structural changes and loss of functions of endothelial cells.Promote the occurrence and development of As in large blood vessels.A large amount of evidence shows that exposure of human umbilical vein endothelial cells(HUVECs)to high glucose triggers mitochondrial damage,leading to mitochondrial division,excessive ROS production,membrane potential damage and decreased ATP production.Apolipoprotein A-I binding protein(AIBP)discovered in recent years is a binding protein of apolipoprotein A-I(apo A-I),which can slow down the formation of arterial plaque.However,the effect of AIBP on diabetic macrovascular disease is unclear.This research group and related studies have shown that AIBP protects macrophages by promoting mitochondrial autophagy,which suggests that AIBP may play a role in the process of mitochondrial function.Long non-coding RNA(lncRNA)is widely involved in all stages of life activities.It has been confirmed that nuclear enrichment abundant transcript 1(NEAT1)can promote epithelial-mesenchymal transition(EMT)and promote the formation of As.The results of the bio-information analysis in the early stage of this project show that there are potential binding sites between lncRNA NEAT1 and the AIBP gene promoter sequence.Therefore,this topic explored the impact of AIBP on high glucose-induced EndMT and mitochondrial changes,as well as the specific molecular mechanism of whether AIBP is regulated by NEAT1,and provides a theoretical basis and new targets for the prevention and treatment of diabetes vascular complications.[Methods]: Transfect endothelial cells with AIBP sh-RNA,use Western Blot(WB)and immunofluorescence to detect expression of AIBP,CD31,α-smooth muscle actin(α-SMA)in the cells.Transfection of AIBP overexpression lentiviral vector,WB and immunofluorescence detection of AIBP,CD31,α-SMA,vascular endothelial cadherin(VE-cadherin),vimentin in cells.The morphological changes of HUVECs were observed under a microscope,mitochondrial reactive oxygen species(ROS)and mitochondrial membrane potential(ΔMφ)levels were detected by flow cytometry,and ATP content was analyzed by chemiluminescence.The luciferase reporter gene experiment was used to verify the ability of NEAT1 to regulate AIBP.HUVECs were transfected with NEAT1 sh RNA,AIBP was silenced on this basis,and the levels of NEAT1,AIBP,CD31,and α-SMA were detected by real-time quantitative reverse transcription PCR(q RT-PCR).Flow cytometry was used to detect mitochondrial ROS,ΔMφ content,and chemiluminescence method to analyze ATP production.[Results]: WB and immunofluorescence results showed that CD31 expression decreased after AIBP knockdown,and α-SMA expression increased,suggesting that AIBP inhibits EndMT.After high glucose treatment,the WB and immunofluorescence results of HUVECs showed that the levels of AIBP,CD31,and VE-cadherin decreased,the levels ofα-SMA,vimentin increased,the mitochondrial ROS increased,the membrane potential damaged,and the ATP production decreased.Which proved that high glucose induced EndMT model was successfully constructed,with mitochondrial damage and AIBP levels decreased.Morphological observations showed that oval endothelial cells transformed into spindle-shaped mesenchymal cells after high glucose treatment.After HUVECs were transfected with AIBP lentivirus,compared with the high glucose group,CD31 levels increased,α-SMA levels decreased,mitochondrial ROS production decreased,ΔMφincreased,and ATP production increased,suggesting that AIBP reduces the occurrence of EndMT by reducing mitochondrial damage.Long Target software predictive analysis and luciferase reporter gene experiment results show that there are binding sites between NEAT1 and AIBP gene promoters.Under high glucose treatment,q RT-PCR showed that the expression of NEAT1 increased and the expression of AIBP decreased.Compared with the cells in the high-glucose group,the NEAT1 knockdown cells in the high-glucose treatment group showed increased expression of AIBP and inhibition of EndMT.[Conclusion]: 1.In a high glucose environment,AIBP of endothelial cells decreases,which damages mitochondrial function,which leads to increased mitochondrial ROS production and promotes EndMT.2.LncRNA NEAT1 down-regulates the expression of AIBP by inhibiting the activity of AIBP promoter,thereby promoting EndMT.