The Function And Mechanism Of O-GLcNAcylation Of SIRT3 Involved In Vascular Endothelial Glycocalyx Degradation Induced By Oscillating Shera Stress Combined With Inflammation

Background:The intact vascular function is essential for the normal operation of the human blood circulatory system.As a protective barrier between the vascular wall and blood,the endothelial glycocalyx(EGC)is a key factor to maintain the normalization of vascular function,endothelial dysfunction caused by glycocalyx degradation is the initiating factor in the progress of cardiovascular disease.However,how endothelial glycocalyx maintains its homeostasis in the complex vascular pathophysiological microenvironment remains unclear.With the development of blood biomechanics,researchers have discovered that there exits different modes of fluid shear stess in different parts of arteries and blood vessels,and it contributes to diverse regulatory mechanisms for endothelial glycocalyx and endothelial functions.Some studies have reported that oscillating shear stress(OSS)could induce disorders of the hexosamine biosynthetic pathway of endothelial cells.However,as the end product of the hexosamine biosynthetic pathway,the uridine diphosphate-N-acetylglucosamine(UDP-GlcNAc)can be transferred to functional protein for corresponding O-N-Acetyl glucosamine transferase(OGT)mediated protein O-linked?-N-acetylglucosamine(O-GlcNAc)post-translational modification,hence regulating the activity and structual stability of downstream target proteins.SIRT3,an NAD⁺-dependent mitochondrial deacetylase,which plays a dominant role in regulating the level of protein acetylation,is responsible for various pathophysiological processes of blood vessels.The enzyme activity and protein stability of SIRT3 are regulated by protein post-translational modification.Actively exploring the role of SIRT3 glycosylation modification in endothelial glycocalyx may provide a new perspective for the elucidation of glycocalyx damage mechanism and the prevention and treatment of glycocalyx-related vascular diseases.Objective:Based on the disturbance of the hexosamine pathway under OSS and the important role of SIRT3 in maintaining the vascular function,this study aims to investigate the function and molecular mechanisms of O-GlcNAcylation SIRT3 involved in endothelial glycocalyx injury.Methods:1 Establish and verify experimental model(1)Animal model was established by partial ligation of the mouse carotid artery combined with lipopolysaccharide(LPS) stimulation;a digital color ultrasonic diagnostic instrument was used to detect changes in carotid blood flow velocity and direction;immunofluorescence staining was adopted to detect HS and glycocalyx components in vascular endothelial cells glycoprotein changes;transmission electron microscopy(TEM) was employed to detect glycocalyx shedding;Western blot was used to analyze endothelial glycocalyx components including heparan sulfate(HS),syndecan 1(SDC1)and inflammation factors vascular cell adhesion molecule 1(VCAM1),intercellular adhesion molecule 1(ICAM1)changes.(2)Cell model was applied by culturing HUVEC at low oscillatory shear stress in the presence of LPS stimulation; immunofluorescence staining was used to detect cell endothelial characteristics;the ordinary optical microscopes was applied to observe the morphology of endothelial cells and the cell growth direction after different model of shear stress; immunofluorescence staining was utilized to detect sugar glycoprotein expression in the glycocalyx;Western blot was used to analyze the expression levels of HS and SDC1 in the EGCcomponents.2 Western blot was used to detect changes in LKB1-AMPK-p47^phox pathway and glycocalyx degrading enzyme Hyal2.3 Role of SIRT3 and O-GlcNAc glycosylation in EGC damage(1)Western blot was used to detect the changes of SIRT3 and OGT protein expression in mouse carotid artery tissue and aortic tissue, the total protein acetylation level and total O-GlcNAc level in the carotid artery tissue also were detected;Serum glucose levels in mice treated with LPS were detected by a biochemical analyzer;(2)Under the cell model,the expression of SIRT3 protein was analyzed by Western blot;meanwhile,after the intervention of the OGA inhibitor PUGNAc,WGA staining was used to detect whether the EGC expression is restored;(3)After interfering the endothelial cells with overexpressed SIRT3 and OGT plasmid,WGA staining was applied to observe the changes in the glycocalyx of the cells.4 Prediction and verification of O-glycosylation site of SIRT3(1)After using siRNA to interfere with the expression of OGT and SIRT3,respectively,the interaction between SIRT3 and OGT was analyzed by co-immunoprecipitation;(2)Sequence alignment analysis of the SIRT3 conservative sequence regions of 8 different species,and then use the Glycosylation Predictor system was used to analyze the potential glycosylation sites of human SIRT3;(3)293T cells were treated with OGT overexpression plasmid or OGA inhibitor PUGNAc,then we constructed SIRT3 point mutation plasmid,and verified SIRT3 potential glycosylation sites by immunoprecipitation;(4)SIRT3 enzyme activity detection kit was employed to quantitativ-ely analyze the influence of SIRT3 glycosylation sites on its enzyme activity.5 Effect of O-GlcNAc SIRT3 on endothelial glycocalyx and its function(1)After transfection of endothelial cells with SIRT3 point mutation plasmid,immunofluorescence was applied to analyse the glycocalyx HS and glycoprotein expression;Western blot was used to analyse the glycocalyx components including HS,SDC1, and glycocalyx degrading enzyme Hyal2 expression,as well as LKB1/AMPK/p47^phox Changes in signal pathways;(2)CCK-F kit was used to detect the adhesion ability of monocyte THP-1 to endothelial cells after endothelial cells transfection with SIRT3 point mutation plasmid.Results:1 Establish and verify experimental model(1)After partial ligation of the mouse carotid artery,digital color ultrasound diagnosis showed that the carotid blood flow rate was decreased,and the flow direction was bidirectional;after partial ligation of the mouse carotid artery combined with LPS treatment, the vascular endothelial glycocalyx shed off significantly,and the glycocalyx components including HS,glycoprotein,SDC1 expression were down-regulated,and the expression of inflammatory factors VCAM1 and ICAM1were increased;(2)Both white light microscopy and immunofluorescence staining showed that the endothelial cells used in the experiment had endothelial cell characteristics,and the growth direction of HUVEC was chaotic after OSS treatment;after the cells were treated with OSS combined with LPS,the expressions of glycoprotein,HS and SDC1 were reduced.2 Both animal model and cell model showed that in the shaking shear stress combined with LPS treatment group,the phosphorylation level of LKB1 and AMPK was decreased,and the expression of p47^phox and Hyal2 was increased.3 Role of SIRT3 and O-GlcNAc in glycocalyx damage(1)After mice were treated with high concentration of LPS as a single factor,the serum glucose level was decreased;(2)After partial carotid artery ligation and LPS treatment in mice,the protein SIRT3,OGT and O-GlcNAc levels in carotid artery tissue protein showed a decreasing trend,and the total acetylation level was increased;SIRT3 in the aortic arch and OGT protein expression was also down-regulated;(3)After endothelial cells were treated with oscillating shear stress combined with LPS,the protein expression of SIRT3 and OGT was decreased;and after the intervention of OGA inhibitor,the damaged glycocalyx would be restored;(4)After the intervention of SIRT3 and OGT siRNA,the expression of glycocalyx on the surface of endothelial cells was reduced; after treatment with SIRT3 and OGT overexpression plasmids,the expression of glycocalyx could be restored.4 Prediction and verification of O-GlcNAc site of SIRT3(1)Co-immunoprecipitation showed that SIRT3 interacted with OGT,and after interfering with the expression of SIRT3 and OGT,the interaction was weakened;(2)Sequence alignment analysis showed that there were two conserved sequence regions in SIRT3 from different species,and there existed 6 potential glycosylation sites(150,152,159,320,321,329)in the human SIRT3 sequence,but analysis of the protein structure of SIRT3 revealed that only positions 159 and 329 were involved in protein synthesis;(3)The transfection results of SIRT3 point mutations showed that the mutation of serine at position 159 did not significantly change the interaction between SIRT3 and OGT,as well as the O-GlcNAc modification and lysine acetylation modification of SIRT3,while the mutation of serine at position 329 weakened the interaction between SIRT3 and OGT,which significantly reduced the O-GlcNAc modification of SIRT3,and significantly increased the lysine acetylation modification;(4)Analysis of enzyme activity results showed that the mutation of erine 329 of SIRT3 significantly reduced the enzyme activity of IRT3 when OGT was overexpressed.5 Effect of O-GlcNAc SIRT3 on endothelial glycocayx and unction(1)After endothelial cells were transfected with SIRT3 point utation plasmid,the expressions of endothelial glycocalyx omponents HS,glycoprotein,and SDC1 were down-regulated, he expression of degrading enzyme Hyal2 was increased,and the hosphorylation levels of LKB1 and AMPK were decreased;(2)After endothelial cells were transfected with SIRT3 point utation plasmid,the adhesion of monocyte THP-1to endothelial ells was increased.Conclusion:1 The OSS combined with LPS stimulation could cause EGC amage;2 SIRT3 and O-glycosylation were definitely involved in the rocess of EGC damage;3 SIRT3 at serine 329 could occur OGT mediated the O-glycosylation,and O-glycosylation of SIRT3 would increase its nzyme activity;4 SIRT3 O-glycosylation site mutation could accelerate EGC hedding through the LKB1-AMPK-p47^phox axis,and enhanced he adhesion of monocytes to endothelial cells.