Nanoplastic Particles Promote Atherosclerosis By Inducing Long Chain Lipoacylcarnitine Accumulation And MARCO Expression

BackgroundPlastic pollution has become a global environmental problem.The risk of human exposure to microplastics is everywhere.Therefore,the study of cardiovascular toxicity of nanometer plastic particles is carried out,and the toxicity and molecular mechanism of different particle size nanoparticles are explained,and the basis of scientific basis for the exposure limit and intervention of nanometer plastic particles in the environment is provided.ObjectiveTo investigate the effect of nanoplastic particle exposure on the progression of atherosclerotic plaques in ApoE knockout mice.Methods1.Distribution of nanoplastic particles in the aorta of ApoE knockout miceUsing green fluorescent polystyrene nanoplastics with a particle size of 50 nm(PS-NPs),gavage ApoE gene knockout mice(0,2.5,25,and 250 mg/kg),blood fluorescence intensity was detected after 24 h to detect fluorescent particles in aortic sections.2.Effects of nanoplastic particles on atherosclerosis(AS)in ApoE knockout mice.ApoE gene knockout male mice were subjected to 19w subchronic oral exposure experiments(0,2.5,25 and 250 mg/kg)using polystyrene nanoplastics(PS-NPs)with a particle size of 50 nm.The weight and blood pressure of each group of animals were monitored,and the left carotid artery vascular elasticity index of mice was measured by small animal Doppler color ultrasound technology the day before the end of the experiment.After the test,the aortic tissue of each group of mice was pathologically examined to observe the plaque area and pathological components.3.Exploration of the mechanism of nanoplastic particles promoting atherosclerosis in ApoE knockout mice.(1)Lipidomics assays were performed on plasma,and mechanisms related to lipid metabolism promotion were explored through orthogonal partial least squares discriminant analysis(OPLS-DA),differential lipid classes and differential lipid molecules analysis.(2)Transcriptomic detection was performed on aortic tissue,differentially expressed genes were screened according to aortic transcriptomic data,and changes at the aortic gene level were explored through differential gene pathway enrichment analysis.4.Verification of atherosclerotic mechanism of nanoplastic particles promoting ApoE knockout mice.(1)The mRNA and protein expression levels of genes related to differential lipid molecule metabolism were detected by qRT-PCR and Western blot methods,and the function of differential lipid molecules was explored in mouse macrophages.(2)The mRNA and protein expression levels of differential genes were verified by qRT-PCR and Western blot methods,and the mechanism of MARCO-related toxicity was verified by knockdown and overexpression of differential genes.Results1.Distribution of PS-NPs in blood and aorta in ApoE knockout miceAfter 24 hours of oral contamination of fluorescent PS-NPs,the concentration of fluorescent PS-NPs in plasma increased with the increase of dose,and fluorescent PS-NPs could be observed in aortic tissue sections in the 250 mg/kg dose group.2.PS-NPs promote atherosclerosis in ApoE knockout mice.In the PS-NPs infected group,the systolic and diastolic blood pressure increased,the left carotid vascular wall thickened,and the longitudinal strain rate of blood vessels decreased.Pathological staining showed that the area of atherosclerotic plaques in the intima of the aorta increased with the increase of infection dose,and the pathological characteristics of plaques in the 250 mg/kg dose group showed more characteristics of advanced atherosclerosis.These results suggest that PS can reduce vascular elasticity and accelerate the development of atherosclerosis.3.PS-NPs activate the phagocytic function of macrophages in aortic and promote macrophage lipid accumulation by upregulating the expression of MARCO.Analysis of aortic Transcriptome data showed that the aortic humoral immunity of mice in the 250 mg/kg group was activated,macrophage recognition and phagocytic pathways were activated,and the expression of macrophage surface scavenger receptor MARCO was significantly upregulated.Experiments in vitro showed that PS-NPs could upregulate the expression of macrophage MARCO and increase the accumulation of lipids in macrophages.Overexpression of MARCO increases macrophage lipid phagocytosis while MARCO knockout reduces macrophage lipid phagocytosis,moreover,MARCO knockout reverses PS-NPs-enhanced lipid uptake.The results showed that PS-NPs activated the phagocytic function of aortic macrophages in aortic and promoted macrophage lipid accumulation by upregulating the expression of MARCO.4.PS-NPs interferes with liver lipid metabolism,leading to increased Long Chain Lipoacylcarnitine(LCAC)content in plasma.Plasma LCAC concentration increased in PS-NPs groups,and PS-NPs may increase plasma LCAC concentration by decreasing liver acylcarnitase transferase 2 expression.5.LCAC also promotes lipid phagocytosis by up-regulating MARCO expression in macrophages.48 h after LCAC exposure,MARCO mRNA and protein levels in macrophages increased.After co-exposure of LCAC and oxidized low-density lipoprotein,lipid phagocytosis of macrophages increased.Salvage function experiments showed that MARCO knockdown could significantly reduce the increase of macrophage lipid phagocytosis induced by LCAC.6.PS-NPs acts synergically with LCAC on macrophages,up-regulating MARCO expression and promoting lipid phagocytosis in macrophages.Orthogonal experimental data of two factors and three levels showed that the cholesterol level in macrophages was higher after combined exposure of PS-NPs and LCAC than that after single exposure.Analysis of variance showed that the combined effect of the two was statistically significant.Therefore,PS-NPs and LCAC had a synergic effect and could jointly up-regulate MARCO expression and lipid phagocytosis in macrophages.Conclusion(1)PS-NPs can enter the blood,aorta and liver tissue of ApoE-/-mice.(2)PS-NPs promotes atherosclerosis in ApoE-/-mice.(3)PS-NPs activated the phagocytic function of aortic macrophages and promoted lipid accumulation in macrophages by up-regulating MARCO expression.(4)PS-NPs interferes with liver lipid metabolism,leading to increased plasma Long Chain Lipoacylcarnitine(LCAC)content,and promotes macrophages to phagocytosis of lipids.(5)LCAC also promotes lipid phagocytosis by up-regulating MARCO expression in macrophages.(6)PS-NPs acts synergically with LCAC on macrophages,up-regulating MARCO expression and promoting lipid phagocytosis in macrophages.