| As the most important material basis for economic and social development in the21st century,nanomaterials have caused industrial innovation in the fields of chemical,medical,food and electronic technology,and penetrated into all aspects of our production and life.Among them,nano-silica?SiO2-NPs?has become one of the most used nanomaterials for the public.However,the characteristics of SiO2-NPs,such as small size effect,high stability and high reactivity,leads them extremely threaten to human life and health.In recent years,it has attracted close attention from governments and the public.Cardiovascular disease is the world disease with the highest mortality rate in the21st century.Epidemiological investigation shows that the exposure of fine particulate matter is closely related to cardiovascular disease.However,the exposure of nanoparticles in the population has become more and more common.Therefore,the study on the cardiovascular toxicity of nanomaterials and the mechanism analysis are conducive to providing scientific basis for their toxicity intervention and treatment.Part?:Vascular endothelial cells are the main cells that form the inner wall of blood vessels and play an important role in maintaining vascular homeostasis.Therefore,this chapter will take human umbilical vein endothelial cells?HUVECs?as an in vitro cell model to explore the molecular mechanism of SiO2-NPs induced vascular endotheliocyte damage.Three kinds of SiO2-NPs with different particle sizes were selected to act on HUVECs at different concentrations for different times.And then the toxic effects of SiO2-NPs on cells were detected using CCK-8.It was found that the smaller the particle size,the stronger the toxic effect of SiO2-NPs on HUVECs,which was time-dependent and dose-dependent.Compared with the control group,the LDH release in HUVECs cells in SiO2-NPs-treated group was significantly increased,with the up-regulation of intracellular ROS level,GPx enzyme activity in the cells was decreased,and the expressions of cytoinflammatory factors containing TNF-?,IL-1?,IL-6 and cell adhesion molecules containing ICAM-1 and VCAM-1 were significantly increased.These results suggested that SiO2-NPs caused oxidative stress and inflammatory injury in HUVECs.Transfer from nuclei to cytoplasm and the release of HMGB1,as well as acetylation level of HMGB1 were significantly increased with exposure to SiO2-NPs,which were detected through Immunofluorescence and immunoprecipitation respectively.Next,suppression of deacetylase Sirt1 by transiently transfecting Sirt1-siRNA to cells or pretreating cells with Sirt1 inhibitor Ex527,with subsequent exposure to SiO2-NPs,resulted in enhanced transfer from nuclei to cytoplasm and increased expression of inflammatory cytokines and adhesion molecules.While the reverse trend was observed when PCAF acetylase was interfered by transient transfection of PCAF-siRNA.These results suggest that the nucleo-cytoplasmic transfer and release of HMGB1 induced by SiO2-NPs need to be modified by acetylation of related kinases.To further validate the role of HMGB1,HMGB1-siRNA was transiently transfected to interfere with the expression of HMGB1.And inflammatory cytokines and adhesion molecules were found to be significantly down-regulated by SiO2-NPs at mRNA and protein levels.Next,after inhibition of caspase1 expression by Z-VAD-FMK?caspase family inhibitor?or AC-YYAD-CHO?caspase1 inhibitor?,it was found that the expression of HMGB1 was reduced,accompanied by the decreased expression of inflammatory cytokines and adhesion molecules.Likewise,the same effect was found after silencing NLRP3 by transiently transfecting NLRP3-siRNA to cells.These results indicate that NLRP3 inflammasome was involved in the regulation of transfer from nuclei to cytoplasm and release of HMGB1.pretreatment with the antioxidant N-acetylcysteine?NAC?or apocynin?APO,NADPH oxidase inhibitor?resulted in reduced expression of HMGB1 and proteins associated with NLRP3inflammasome.These results suggest that ROS play an important role in the regulation of NLRP3 in the nucleoplasmic transfer and release of HMGB1.Further exploration of the signaling pathway of HMGB1 revealed that SiO2-NPs simultaneously induced high expression of Toll-like receptors and RAGE receptors,and promoted the binding of these receptors to HMGB1,especially for TLR4.Silencing TLR4 and MyD88 by transient transfection of TLR4-siRNA and MyD88-siRNA reduced the expression of inflammatory cytokines and adhesion in mRNA and protein levels.At the same time,after exposure to SiO2-NPs,we also found the activation of NF-?B pathway.The results indicate that exposure of SiO2-NPs to HUVECs caused the generation of reactive oxygen species,which result in oxidative stress in the cells,activating NLRP3 inflammasome,promoting acetylation of HMGB1 and transfer from nuclei to cytoplasm and the subsequent release of it.Consequently,TLR4/MyD88 and NF-?B signaling was activated,causing inflammatory damage to cells.Part?:This section aims to explore the toxic effects of SiO2-NPs on vascular endothelium of mice exposed by respiratory means.We first observed the distribution and metabolism of nanomaterials in mice using fluorescent nano-silica?F-SiNPs?.Tracheal instillation of F-SiNPs for 24 hours,and then the isolated heart,liver,spleen,lung and kidney of the mice were taken.By fluorescence imaging experiments,it was found that F-SiNPs could enter the blood circulatory system through the air-blood barrier,then enriched by the liver and excreted through the kidneys.After 24 hours of tracheal instillation of SiO2-NPs,the lungs of the mice were subjected to transmission electron microscopy.The results showed that the exposure of SiO2-NPs caused damage to the structure of the air-blood barrier in mice,which made the nanomaterials easily enter the blood circulation system.To investigate the toxic effects of SiO2-NPs on the vascular endothelim,the mice were randomly divided into four groups.And nasal instillation was executed to expose different doses of SiO2-NPs suspension to mice,with equal and the control mice were given the same amount of physiological saline.After 15 days of continuous administration,the blood,lung and aortic valve of the mouse were taken.Quantitative analysis of silicon in blood by ICP-OES revealed that intranasal instillation of SiO2-NPs resulted in an increase of silicon in the blood of mice,and as the dose of SiO2-NPs increased,silicon in the blood is up-regulated.Subsequent Western blot analysis showed that the exposure of SiO2-NPs increased the expression of inflammatory factors TNF-?,IL-1?and adhesion molecules sICAM-1 and sVCAM-1 in the serum of mice,and with the exposure of increased SiO2-NPs,the inflammatory factors were up-regulated.These inflammatory and adhesion factors are hallmark proteins when vascular endothelial cells are damaged.Further analysis revealed that the exposure of SiO2-NPs induced an increase in the release of lactate dehydrogenase in the serum and disrupted the body's redox balance,resulting in a decrease in GSH content and a decrease in GPx activity.HE staining and SR staining of the lungs of mice showed that the exposure of SiO2-NPs caused obvious inflammatory damage and fibrosis in the lungs,but HE staining and oil red O staining of blood vessels did not show obvious pathological tissue damage in the blood vessels of mice.Studies have shown that vascular injury requires a long-term process,which indicates that slight oxidative stress damage and up-regulation of inflammatory factors after short-term acute exposure to SiO2-NPs do not destroy the integrity of vascular cells and cause severe vascular histopathological damage.In order to investigate whether SiO2-NPs-induced vascular damage is caused by silica particles or silicon,we exposed HUVECs to SiO2-NPs and silicates with the same silicon content,and by CCK-8 and LDH experiments.It was found that SiO2-NPs induced HUVECs to release a large amount of LDH and inhibited cell proliferation,and with the increase of SiO2-NPs exposure,the cytotoxicity became more obvious;whether it was high-dose or low-dose silicate exposure,none of the dose caused cytotoxic damage in HUVECs.In this chapter,experiments have shown that exposure of SiO2-NPs by nasal instillation will cause slight oxidative stress damage and inflammatory damage in the vascular endothelium of mice in a short period of time,but it will not cause serious pathological tissue damage of blood vessels,and this vascular endothelium damage is caused by silica particles rather than by silicon ions.Part?:This section aims to study the complex behavior of SiO2-NPs after contact with body fluids in mice.Since the extremely high surface energy and reactivity of SiO2-NPs,once they enter the body,they will adsorb some proteins,lipids and some other substances in the body fluid,thereby forming biomolecular-nanomaterial complex.Moreover,the biomolecules that make up the biological crown are dynamically exchanged.In the above experiments,we demonstrate that the SiO2-NPs exposed by respiratory means can destroy the structure of the air-blood barrier and directly enter the blood circulation system.Based on the above conclusions,this chapter will simulate the complex behavior of SiO2-NPs in contact with body fluids in vitro.In vitro,we incubated SiO2-NPs with pulmonary surfactant?PS?or mouse serum?MS?,and the results of malvern particle size analyzer analysis,BCA protein quantification and coomase bright blue staining showed that SiO2-NPs can adsorbed some protein and form a protein corona on the nanomaterial.By proteomic analysis and western blotting experiments,we found that the transfer of SiO2-NPs from PS to MS resulted in the exchange of the coronary layer and specific enrichment of apolipoprotein A-I?Apo A-I?.Through circular dichroism experiments,it was found that the adsorption of SiO2-NPs changes the structure of Apo A-I.After three months of exposure to SiO2-NPs by nasal drip,Elisa results showed that Apo A-I content in blood was significantly reduced,and LDH content in blood was significantly increased.In summary,the experiments in this chapter demonstrate that SiO2-NPs exposed through the nasal instillation can react with pulmonary surfactant to form PS-SiO2-NPs.After crossing the air-blood barrier,the protein corona exchange occurs in contact with blood,lead to enrichment of Apo A-I and damage to the structure of Apo A-I,and then excreted by the body,resulting in a decrease in Apo A-I content in the blood,thereby promoting vascular endothelial damage. |
PDF Full Text Request