Study On The Mechanism Of Macrophage Energy Metabolism Pathway Remodeling Induced Atherosclerosis By PFOS Targeting FTH

Objective:Cardiovascular diseases related to Atherosclerosis（AS）are the main cause of death globally,and the study of the pathogenesis factors of AS is currently one of the important public health issues.High-fat diet is recognized AS an important risk factor for the development of AS.According to the study,the proportion of fat intake is increasing year by year,which poses a great threat to the health of Chinese residents.However,data on the impact of exposure to environmental risk factors on the development of AS in such highly susceptible populations are lacking.Perfluorooctane sulfonate（PFOS）which can be detected in global human blood samples is a widely distributed perfluorochemical.Evidence from epidemiological studies shows that PFOS exposure is an important risk factor for the occurrence and development of AS.However,the direct link between PFOS exposure and AS and its underlying mechanism remains unclear.Macrophages are the main cells in the innate immune response to AS,and changes in the relative proportions of their phenotypes affect the progression of AS.Studies have shown that macrophage phenotypes are regulated by intracellular metabolic states.Therefore,this study focused on the phenotypic remodeling process of energy metabolism regulation in macrophages under the highly susceptible model,and revealed the key cellular events and molecular mechanisms of PFOS exposure causing the development of AS.Methods:In this study,experimental exposure models of mice and cells were established,and the specific methods involved were as follows:1.Animal model:Wild-type C57BL/6J mice were selected to construct a high-fat diet（HFD）combined with the PFOS gavage exposure model.After 8 weeks of exposure,the aorta of each group of mice was taken.AS plaque area,aortic wall thickness,and endothelial injury were detected by oil red O staining,H&E,EVG,and other histopathological staining methods.Flow cytometry,PCR,and immunofluorescence were used to analyze the proportion and number of subtypes of macrophages and the expression of key enzymes in the energy metabolism of macrophages.2.Cell model:set up the oxidized low-density lipoprotein（ox-LDL）,stimulating source of mice bone marrow macrophage（BMDM）research model.The phenotype and function of macrophages were determined by Western Blot,oil red O staining,and immunofluorescence.3.In the co-treatment experiment of glycolysis inhibitor 2-DG and PFOS under the condition of ox-LDL,Western blot,and q PCR were used to explore the regulatory effect of PFOS exposure-induced energy metabolism remodeling on promoting phenotypic remodeling of macrophages.4.Using the methods of Western Blot,molecular docking,flow cytometry,and immunofluorescence,we focused on the key regulatory role of PFOS on the expression of ferritin heavy chain（FTH）and Fe²⁺level.By the co-treatment experiment of Fe²⁺chelating agent CQ,to comprehensively elucidate the role of PFOS expose-induced changes in Fe²⁺level in regulating and controlling the remodeling of energy metabolism in macrophages.Results:1.Compared with the high-fat diet control group,the area of atherosclerotic plaque in the aorta of mice exposed to PFOS for 8 weeks increased significantly;The thickness of the aortic wall increases with inflammatory infiltration and endothelial damage;In addition,the proportion of macrophages in the aorta of PFOS exposed mice increased,and macrophage foam increased.It is suggested that PFOS exposure can promote the development of atherosclerosis in mice under the condition of high-fat diet.2.The results of mouse aortic flow cytometry showed that under high-fat diet conditions,the number and proportion of classic activated macrophages（CAM,pro-inflammatory phenotype）in the mouse aorta increased after 8 weeks of PFOS exposure,while alternating activated macrophages（AAM,anti-inflammatory phenotype）decreased.The transcriptional levels of CAM and AAM markers in the mouse aorta were measured,which were consistent with the above results;The results of cell experiments showed that after exposure to ox-LDL,the expression level of macrophage CAM markers increased,the transcription level of macrophage CAM markers increased,and the transcription level of AAM markers decreased after PFOS exposure.Moreover,macrophage adhesion ability and ox-LDL uptake ability were enhanced.PFOS can induce macrophage phenotype remodeling,promote the generation of classic activated macrophages（CAM）,and inhibit the generation of alternating activated macrophages（AAM）;And PFOS affects the normal physiological function of macrophages and aggravates atherosclerosis.3.After exposure to ox-LDL,PFOS exposure resulted in a decrease in total ATP content and expression of mitochondrial respiratory chain complex proteins in macrophages;The glucose uptake rate,the content of lactic acid and pyruvate increased,and the expression of hexokinase（Hk）increased;The transcription levels of lactate dehydrogenase A（Ldha）,glucose transporter 1（Glut1）,and pyruvate kinase（Pkm）genes are upregulated.The above results suggest that PFOS exposure promotes the energy metabolism of macrophages from oxidative phosphorylation to glycolysis remodeling,leading to the reduction of overall energy production.4.The results of the co-treatment experiment of glycolysis inhibitor 2-DG and PFOS showed that compared with the exposure group co treated with ox-LDL and PFOS,the co-treatment of 2-DG and PFOS resulted in a decrease in the expression of i NOS protein in macrophages and the transcription level of CAM marker decreased,while the transcription levels of AAM markers Fizz1 and Ym-1 increased.The results suggest that inhibiting glycolysis levels can alleviate the energy metabolism remodeling of macrophages induced by PFOS,and further alleviate the phenotype remodeling of macrophages induced by PFOS.This indicates that the energy metabolism remodeling induced by PFOS exposure plays an important regulatory role in promoting macrophage phenotype remodeling.5.PFOS exposure increases the expression of FTH protein in BMDM cells.Molecular docking results show that PFOS and FTH bind at Glu64,Glu61,Glu140,His65 and other iron storage functional sites,and the molecular docking binding energy is-7.1kcal/mol,indicating that PFOS can stably bind to FTH and affect its iron storage function.Further detection of intracellular Fe²⁺content revealed that PFOS exposure to BMDM cells significantly increased intracellular Fe²⁺content.After co-treatment with Fe²⁺chelating agent CQ and PFOS,the energy metabolism remodeling of macrophages caused by PFOS was alleviated.The increase in Fe²⁺induced by PFOS exposure plays an important regulatory role in promoting macrophage glycolysis.Conclusions:It was the first time to explore the risk regulation,key cellular events,and molecular mechanism of PFOS exposure on the occurrence and development of atherosclerosis under the condition of high fat.It was found that PFOS affected its iron-storage function by targeting FTH,which led to the increase of Fe²⁺level in macrophages,promoted the up-regulation of macrophages’glycolysis level,and induced macrophages to differentiate into pro-inflammatory phenotypes.Then it can further aggravate the occurrence and development of atherosclerosis.This study reflects the risk of PFOS exposure in highly susceptible populations and the characteristics of key events,providing a new perspective and key data for the study of the mechanism of atherosclerosis induced by PFOS exposure.