Study On The Induction Of Vascular Endothelial Cell Damage By Nanoplastics And Its Mechanism

Nanoplastics,as an emerging environmental pollutant,have attracted widespread global attention.Nanoplastics can penetrate biological barriers and accumulate in tissues such as the liver,lungs,kidneys,testes,and brain,posing potential health risks to organisms.Blood vessels are the key channel for nanoplastics to reach target organs.Current research has shown that plastic particles have been detected in human blood samples,so exploring the effects of nanoplastics on the cardiovascular system is of great significance.Nanoplastic particles formed by photodegradation,biodegradation,hydrolysis,and mechanical wear in the environment carry different functional groups,such as amino and carboxyl groups.Investigating the toxic effects of nanoplastics with different functional group modifications is of great reference significance for evaluating the biological effects of nanoplastics in the actual environment.Based on this,this study took polystyrene（PS）,a common environmental plastic,as representative,and investigated the toxicity and mechanism of action of PS,PS-NH₂ and PS-COOH nanoplastics on vascular endothelium.The main research content and conclusions are as follows:（1）Using mice as the research object,this study detected the body weight,blood routine indicators,vascular tissue slices,vascular ultrastructure,inflammatory factors,and coagulation factors of mice exposed to PS,PS-NH₂ and PS-COOH nanoplastics,the results showed that the blood vessels of mice exhibited varying degrees of inflammatory cell infiltration and ultrastructural changes.In addition,the levels of white blood cells and inflammatory factors in the blood of mice increased,and the expression of inflammatory and coagulation-related proteins in vascular tissues also increased.By comparison,it was found that PS-NH₂ nanoplastics had the most significant vascular inflammatory injury and coagulation dysfunction in mice compared with PS-COOH and PS nanoplastics.（2）Due to the fact that nanoplastics easily interact with proteins and other components in biological systems,this study further investigated the interaction between the three nanoplastics and a key coagulation protein,human fibrinogen（HF）.The effects of PS,PS-NH₂,and PS-COOH nanoplastics on the structure and coagulation function of HF were revealed through structural characterization and in vitro simulated fibrinogen polymerization tests.The results showed that all three types of nanoplastics can bind to HF,and PS-NH₂ has a stronger binding force with HF.Fluorescence spectroscopy experiments showed that the fluorescence quenching mechanism of nanoplastics on HF is static quenching.Thermodynamic parameter data indicated that the main driving force between nanoplastics and HF was hydrophobic interaction.The three types of nanoplastics can promote blood clotting and change the hydrophobic microenvironment of HF,with PS-NH₂ nanoplastics having the most significant effect.Based on the above research results,it is suggested that nanoplastics may have potential harm to the human blood system,and positively charged modified nanoplastics have the most significant toxic effects.（3）Based on the fact that nanoplastics can induce vascular inflammatory damage in mice,this study further investigated the effect and mechanism of nanoplastics on vascular endothelial cells using EA.hy 926 endothelial cells.Through cell proliferation assays,lactate dehydrogenase（LDH）level detection,reactive oxygen species（ROS）level detection,and cell migration assays,the toxic effects of PS,PS-NH₂,and PS-COOH nanoplastics on vascular endothelial cells were revealed.In addition,differentially expressed genes were screened by RNA-sequencing（RNA-seq）and signal pathway enrichment analysis was performed on differentially expressed genes.The results showed that the three types of nanoplastics inhibited vascular endothelial cell activity in a dose-dependent manner,and when the concentrations of PS,PS-NH₂,and PS-COOH nanoplastics were 50 μg/mL,25 μg/mL,and 50 μg/mL,respectively,cell activity was significantly inhibited.At the same time,this concentration of nanoplastics can induce cell membrane damage and ROS production,and has a significant inhibitory effect on cell migration.The results of RNA-seq showed that 147,4553,and 360 differentially expressed genes were screened in the PS vs Control,PS-NH₂ vs Control,and PS-COOH vs Control groups,respectively,with the PS-NH₂ vs Control group having the most differentially expressed genes,which may be related to the stronger cell toxicity induced by PS-NH₂ nanoplastics.KEGG enrichment analysis revealed that differentially expressed genes may induce vascular endothelial cell inflammatory damage through signaling pathways related to inflammation and immune response,including the Wnt signaling pathway and cytokine-cytokine receptor interaction pathway.（4）MicroRNA（miRNA）is an important genetic regulatory factor as a type of small RNA,which can participate in post-transcriptional regulation of gene expression by affecting the stability and translation of mRNA.Therefore,this study further screened differentially expressed miRNA in vascular endothelial cells exposed to nanoplastics through small RNA-seq,and analyzed the target object to be studied as miR-1908-5p and CEBPB in combination with mRNA-seq results.The targeting relationship between miR-1908-5p and CEBPB was verified through dual luciferase reporter gene assay.It was found that the 3’-UTR of CEBPB completely complemented the complementary region of hsa-miR-1908-5p.This study successfully constructed CEBPB WT（wild type）and CEBPB MUT（mutant type）recombinant vectors.When CEBPB WT was co-transfected,compared with the mimics NC group,the fluorescence enzyme activity of the hsa-miR-1908-5p mimics group was significantly reduced,while when CEBPB MUT was co-transfected,there was no significant difference in fluorescence enzyme activity between the mimics NC group and the hsa-miR-1908-5p mimics group.It was indicated that hsa-miR-1908-5p can complement the binding site of the 3’-UTR of the CEBPB gene,thereby significantly reducing the fluorescence enzyme activity.When the binding site of hsa-miR-1908-5p in the 3’-UTR is mutated,it cannot complement the binding site of the CEBPB gene’s 3’-UTR,and the fluorescence enzyme activity does not change significantly.According to the research results,it is speculated that the induction of vascular endothelial cell inflammatory damage by nanoplastics is related to the targeted inhibition of CEBPB by hsa-miR-1908-5p.This study preliminarily studied the harm and molecular mechanism of nanoplastics on the cardiovascular system,in order to provide a research basis to evaluate the potential risks of nanoplastics to human health.