Study On Mechanism Of Iron Oxide Nanoparticles Exacerbating Atherosclerosis And Plaque Instability In ApoE^-/- Mice

The aggravation of air pollution exposes people to high concentrations of air particulate pollutants for a long time.Air particulate matter,especially fine particulate matter,is considered to be the“initiator”of increasing the risk of cardiovascular diseases and thus affecting human health.Since airborne particulate matter is a complex mixture composed of many components,it is important to have a thorough understanding of the health effects of a single substance in particulate matter,so that we can fully understand the toxicity of particulate matter and take effective control measures.Magnetite nanoparticles（Fe₃O₄ NPs）are the main components of fine particulate matter.Their large surface area,low density and prolonged suspension in the air increase the risk of human inhalation.The fate of Fe₃O₄ NPs in the body after respiratory exposure,their effects on cardiovascular disease,their correlation with the occurrence of cardiovascular diseases,and their potential mechanism remain to be investigated.Part Ⅰ:In order to investigate the effects of Fe₃O₄ NPs on the development of cardiovascular diseases,a respiratory exposure model of mice was established by nasal drip.Male ApoE^-/-mice were given Fe₃O₄ NPs by nasal drip weekly and continued for three months.ApoE^-/-mice were examined after 3 months to evaluate the effect of Fe₃O₄ NPs on the development of cardiovascular disease.Body weight change is a direct visual parameter of pathology and is often used to assess mouse growth and health status.Body weight of mice was recorded before weekly respiratory exposure to Fe₃O₄ NPs,and the growth curve of ApoE^-/-mouse was plotted after administration.The results showed that respiratory exposure to Fe₃O₄ NPs did not affect the health status of ApoE^-/-mice,and the body weight of ApoE^-/-mice showed an obvious upward trend.The blood from heart,liver,spleen,lung,kidney,brain was collected to further detect the biological distribution of Fe₃O₄ NPs in ApoE^-/-mice.The results showed that Fe₃O₄NPs was redistributed through the air-blood barrier and accumulated mainly in the liver.Fe₃O₄ NPs down-regulated the mRNA expression level of cytoplasmic protein（ZO-1）and affected the structural integrity of the air-blood barrier in mice during metastasis.In addition,respiratory exposure to Fe₃O₄ NPs resulted in significant lung lesions in ApoE^-/-mice,with thickened pulmonary septa,alveolar congestion,and a large number of inflammatory cell infiltration.Not only that,ApoE^-/-mice lung inflammation reaction and oxidative damage,the mRNA expression levels of classical inflammatory cytokines such as tumor necrosis factor a,Interleukin 6 and Interleukin-1βwere significantly up-regulated,and the lung oxidation and antioxidant were unbalanced in ApoE^-/-mice.The translocation of Fe₃O₄ NPs led to iron deposition in the liver of ApoE^-/-mice and enhanced iron metabolism in the liver of ApoE^-/-mice,which was manifested as the up-regulated mRNA expression level of Hepcidin（Hamp）and Bone morphoprotein 6and recombinant SMAD family member 5.Fe₃O₄ NPs exposure affected the overall iron level of ApoE^-/-mice,Plasma iron and total iron binding capacity and transferrin saturation had increased.Increased concentration of Malondialdehyde（MDA）and decreased concentration of Catalase（CAT）in the plasma of ApoE^-/-mice suggested that exposure to Fe₃O₄ NPs caused systemic oxidative stress in ApoE^-/-mice.After exposure to Fe₃O₄ NPs,hepatic cell vacuolation increased and hepatic cholesterol including total cholesterol,low density lipoprotein cholesterol,and free cholesterol and triglyceride levels were increased;mRNA expression levels of genes related to cholesterol and triglyceride synthesis included fatty acid synthetase,acetyl coenzyme A desaturase,scd1,lipin1,sterol regulatory element binding protein 1c and Elovl fatty acid synthetase6 increased,suggesting that Fe₃O₄ NPs promoted lipid synthesis in ApoE^-/-mice liver.Hepatic lipid deposition induced hepatic inflammation and oxidative stress,hepatic fibrosis further promoted the occurrence of Nonalcoholic fatty liver disease（NALFD）in ApoE^-/-mice,and abnormal lipid levels（TC,TG,LDL-C,FC）in mice.Increased lipid plaque formation in the aorta.Fe₃O₄NPs accelerated the progression of aortic root plaque in ApoE^-/-mice,and increased the plaque formation area,plaque lipid content and collagen content.In addition,Fe₃O₄ NPs promoted the formation of unstable plaques,increased the number of platelets and platelet-activated areas in plaques,and the formation of necrotic cores in plaques.Unstable plaques develop from the infiltration of M1 macrophages and the decrease in the number of M2 macrophages.Exposure to Fe₃O₄ NPs significantly up-regulated the mRNA expression levels of M1macrophage-related markers（IL-6,IL-1β,TNF-αand MCP-1）in the aorta of ApoE^-/-mice,and down-regulated M2 macrophage-related markers in the aorta of ApoE^-/-mice,including the mRNA expression levels of Arginine1 and Interleukin-12.Immunofluorescence results showed that respiratory exposure to Fe₃O₄ NPs increased the number of M1-type macrophages in ApoE^-/-plaques,further leading to increased plaque instability.Part Ⅱ:The effects of Fe₃O₄ NPs on phenotypic transformation of macrophages was further explored at the cellular level.J774A.1 macrophages were exposed at concentration gradients（0,50,100,200,400μg/m L）in reference to the administration concentration of Fe₃O₄ NPs.Lactate dehydrogenase release and increased cell apoptosis were the main factors affecting the cell viability of J774A.1 macrophages.Previous studies have shown that oxidative stress caused by iron nanoparticles is one of the important mechanisms affecting cell viability.The results showed that Fe₃O₄ NPs increased the content of lipid peroxide in J774A.1 macrophages in a concentration-dependent manner,and correspondingly decreased the level of intracellular antioxidant molecule Glutathione.The imbalance of intracellular oxidation and antioxidant resulted in oxidative stress in J774A.1 macrophages.Mitochondria are the main producers of oxidative stress and Reactive oxgen spicies（ROS）.Fe₃O₄NPs-induced oxidative stress in J774A.1 macrophages increases the intracellular ROS level.In addition,Fe₃O₄ NPs affected mitochondrial ATP production capacity,decreased mitochondrial membrane potential,and further affected mitochondrial function.J774A.1 Macrophages take up Fe₃O₄NPs in a time-dependent manner,increasing intracellular iron levels and reaching the maximum uptake at 12 h.Concentration gradient exposure of Fe₃O₄ NPs affected intracellular iron homeostasis in macrophages J774A.1,and up-regulated the mRNA expression levels of transferrin receptor related to iron uptake,Ferritin-H/L and Hepcidin.Changes in intracellular ROS and iron homeostasis may affect phenotypic transformation of macrophages.The specific phenotype of macrophage polarization after exposure to Fe₃O₄ NPs was determined by detecting macrophage phenotypic markers.The results show that Fe₃O₄NPs exposure promoted the polarization of macrophages towards M1 macrophages,and the mRNA expression levels of M1 macrophage-related markers include Tumor necrosis factor-α（TNF-α）,Interleukin-6,Interleukin-1β,Inducible nitric oxide synthase,CD11c and Chemokine ligand 3（CCL3）were significantly up-regulated.Inhibition of macrophages’polarization into M2-type macrophages and the mRNA expression levels of related markers of M2-type macrophages include suppressor of cytokine singaling 3,Arginine 1,Interleukin-12,interleukin-10,mannose receptor CD206,and hypoxia-induced mitosis factor（Fizz1）were significantly down-regulated.These results indicated that Fe₃O₄ NPs promoted the polarization of macrophages into M1-type macrophages,which may be related to the increased ROS production in macrophages and the changed intracellular iron homeostasis.