The Role And Underlying Mechanism Of PAOA-flavone Promoting HNF1b SUMOylation In Macrophage Lipid Accumulation And Aortic Atherosclerosis

Objective（s）（1）To probe the expression of hepatocyte nuclear factor 1beta（HNF1b）and macrophage sortilin-mediated lipid metabolism regulated by Polygonatum odoratum flavone（PAOA-flavone）.（2）To explore the role of PAOA-flavone promoting SUMO-activating enzymes E1（SAE1）-catalyzed SUMOylation of the HNF1b protein in the atheroprotective efficacy.Method（s）Human myeloid leukemia mononuclear cells（THP-1）-derived macrophages were treated with 50μg/ml ox-LDL or in combination with 1000μg/ml PAOA-flavone,the expression levels of SAE1,HNF1b and sortilin were determined by qRT-PCR and WB.Bioinformatics was used to predict the SUMOylation sites of the HNF1b protein,and a co-inmunoprecipitation（Co-IP）assay was used to confirm small ubiquitin-related modifier 1（SUMO1）modification of the HNF1b protein.Intracellular lipid droplets（LDs）were stained with Oil red O,and cellular lipid contents were assessed by the colorimetry in ox-LDL-treated THP-1 macrophages.Under the intervention of HNF1b and SAE1 in high-fat diet（HFD）-fed apolipoprotein E knockout(ApoE^-/-)mice,the plasma lipid profile was assessed using the enzymatic oxidation method,the formation of atherosclerosis（AS）plaque in aortic trunk and its three branches was observed by stereomicroscope,histological staining and immunofluorescence staining were conducted to analyze AS lesions and the expression of SAE1,HNF1b and sortilin in the cryosections of aortic root.Result（s）PAOA-flavone elevated HNF1b expression and inhibited intracellular sortilin levels and cholesterol accumulation in THP-1 lipid-laden macrophages;the peptide sequences of human and mouse HNF1b proteins had multiple sites for SUMOylation,as calculated by the SUMOplot^TMAnalysis Program and JASSA website,further experiments confirmed that PAOA-flavone facilitated the SUMOylation of the HNF1b protein.What’s more,an enzyme of SUMOylation,SAE1,was discovered to intimately correlate with HNF1b and SUMO1 by GENEMANIA and ASSISTANT for Clinical Bioinformatics website,PAOA-flavone incubation was verified to increase the levels of SAE1 mRNA and protein and SAE1 overexpression led to HNF1b upregulation and sortilin suppression and to repress lipid accumulation.Small interfering RNA（siRNA）of SAE1 was observed to antagonize PAOA-flavone-induced upregulation of SAE1 and HNF1b.Based on this,when co-transfected the lentiviruses of the SAE1 siRNA and HNF1b oe into THP-1macrophages incubated with PAOA-flavone,HNF1b oe negated the antagonistic role of SAE1 siRNA in PAOA-flavone-induced upregulation of SAE1 and HNF1b and suppression of sortilin expression,lipid accumulation and LD formation.The recombinant adeno-associated viruses（AAVs）of HNF1b and SAE1 short hairpin RNA（shRNA）were used to infect Apo E^-/-mice fed a HFD supplemented with PAOA-flavone,HNF1b oe counteracted the antagonistic effect of SAE1 shRNA on PAOA-flavone-prevented aortic walls against lipid deposition and atherogenesis in vivo.Conclusion（s）PAOA-flavone administration promoted SAE1-catalyzed SUMOylation of HNF1b protein to reduce macrophage sortilin expression,cellular lipid content,and the atherosclerotic lesions in aortic wall.