The Role Of SIRT6 In Inducing Macrophage Autophagy Flux And Promoting Cholesterol Efflux So As To Inhibit Macrophage Foam Cell Formation

IntroductionAtherosclerosis(AS)is the major complication and threat of type 2 diabetic patients,the treatment of AS is limited so intervention at the early stage of this disease is of great importance.Foam cell formation is the typical pathological change in the onset of AS,therefore identification of novel molecues that can reduce foam cell formation is the primary content of AS treatment.Studies revealed that autophagy mediated cholesterol efflux could effectively relieve foam cell formation,thus minishing artery plaque and leision area,so as to protect patients from AS progression SIRT6,a member of HD AC family,was reported to induce cell autophagy thro?gh negatively regulating IGF-AKT signaling pathway,but its involvement in macrophage and underlying mechanism was not revealed.Our previous study showed that SIRT6 was obviously down-regulated after ox-LDL treatment,and overexpressing SIRT6 could induce authophagy effectors and meanwhile,reduce foam cell formationObj ectiveThis study aims to observe the impact of SIRT6 on the regulation of macrophage autophagy and macrophage foam cell formation,and explore the possible mechanism underlying its protective role in AS progression.By identifying the possible target of SIRT6,it is likely to provide new insight into the clinical treatment of ASMethods1.100 nM PMA was added to the RPMI complete medium and culture cells for 72 h to induce THP1 differentiation into macrophages;50 ?g/ml ox-LDL was added into the RPMI complete medium and culture cells for 24 h to induce foam cell formation.2.Lentiveral mediated gene expression system was applied to establish stable cell lines expressing SIRT6 wildtype(LV-SIRT6),SIRT6 whose HDAC activity was absent(LV-H133Y)and SIRT6 knocked down(LV-shSIRT6)as well as their corresponding controls(LV-Vector and LV-shScram).The impact of SIRT6 on the regulation of macrophage autophagy flux and the formation of foam cells were carefully observed.3.Oil Red O staining was performed to examine foam cell formation in each cell line groups,and microplate reader was adopted to obtain data of the total cholesterol and cholesterol esters as well as cholesterol efflux in each cell line groups.4.Western blot was conducted to examine the protein levels of autophagy flux and lysosmal related protein as well as ABCG1;qRT-PCR was performed to test the mRNA levels of autophagy and lysosmal-biogenesis related genes and cholesterol efflux related genes in each cell groups;Lysotracker Red staining was used to observe the lysosomal biogenesis in each cell groups;mRFP-GFP-LC3 adenovirus was adopted to examine the autophagy flux among each cell groups;transmission electron microscope was used to observe the autophagic vacules biogenesis in each cell groups.5.Small interferin RNA against ATG5 was designed to block autophagy in THP1 cells;has-miR-33 and anti-miR-33 was transfected into THP1 cells to evaluate its role in regulating autophagy in macrophages.Results1.After the treatment of 50 ?g/mL ox-LDL for 24 h and foam cells was formed,the expression of SIRT6 and autophagy effectors were down regulated,meanwhile,while miR-33 expression was increased.2.Overexpression of wildtype SIRT6(LV-SIRT6)but not HDAC activity mutant SIRT6(LV-H133Y)led to decreased foam cell formation while knockdown SIRT6(LV-shSIRT6)resulted in aggravated macrophage foam cell formation.3.Overexpression of wildtype SIRT6 could largely induce autophagy effectors expression and autophagy flux as well as lysosomal biogenesis under ox-LDL condition,and knockdown SIRT6 had the opposite effect.Blocking autophagy pathway resulted in decreased expression of autophagy effectors in THP1 cells and increased macrophage foam cell formation4.Overexpression of wildtype SIRT6 could increase the levels of ABCA1 and ABCG1 and enhance macrophage cholesterol efflux but reduce miR-33 expression.Overexpression of has-miR-33 in THP1 cells could suppress the expression of autophagy effectors while overexpression of anti-miR-33 could induce the expression of autophagy effectors5.Overexpression of has-miR-33 in LV-SIRT6 cells could largely reverse the efficacy of SIRT6 in inducing macrophage autophagy flux and relieving foam cell formationConclusions1.Under pro-atherosclerotic condition(ox-LDL),the levels of SIRT6 expression and cell autophagy flux were inhibited in macrophages2.In macrophages under ox-LDL condition,SIRT6 relied on its HDAC activity to induce autophagy flux and lysosomal biogenesis so as to inhibit macrophage foam cell formation;and under normal condition,the impact of SIRT6 overexpression in macrophages was not dominant3.In macrophages under ox-LDL condition,SIRT6 relied on its HDAC activity to promote cell cholesterol efflux,thus decreasing the intracellular accumulation of lipids4.In macrophages under ox-LDL condition,SIRT6 at least partially through inhibiting the expression of miR-33 to induce autophage effectors expression and thus increasing cell autophagy flux,promoting cholesterol hydrolysis and decreasing cholesterol accumulation.