The Mechanisms Of Atmospheric Fine Particles Penetrating Vascular Barrier To Promoting Vascular Calcification

Air pollution has become one of the major threats to human health,and a huge health challenge in China.Fine particles(PM_2.5,an aerodynamic diameter less than 2.5 ?m)have a wide range of sources,including vehicle emissions,coal burning and construction dust.The urban rapid development and industrial structure changes show a significant impact on the emission of PM_2.5.In addition,PM_2.5 contains different components and a variety of toxic substances,which pose a great threat to human health.In recent years,there has been a significant change in the disease spectrum of Chinese population,and cardiovascular disease has become the primary disease threatening the health of Chinese.The increasing concentration of PM_2.5 in the environment also contributes to the rate of incidence and death of cardiovascular diseases.Vascular endothelial cells have barrier function in the inner wall of vessels,and the injury of vascular endothelial cells plays an important role in the occurrence of cardiovascular diseases.Vascular calcification is the pathological basis of cardiovascular disease,and the degree of vascular calcification is positively correlated with the severity of cardiovascular disease.This study intends to evaluate the risk of PM_2.5 and different components from Taiyuan,Shanxi Province on human health,explore the path that PM_2.5 penetrates the vascular barrier and impacts on organelles,reveal the effect of PM_2.5 exposure on vascular calcification and molecular mechanisms.The main research contents are described as below:?1?Health risk assessment of PM_2.5 and its different components.Through collection and detection of PM_2.5 in Taiyuan city,it indicated that PM_2.5 is irregularly arranged,with particle sizes mainly between 300-350 nm,and has a negatively charge in different media.The metal enrichment factors analysis showed that the enrichment factors of heavy metals Cr,Cd,As,Hg and Mn in PM_2.5 are significantly larger than 100,indicating that this group of elements is mainly affected by human activities.The accumulation index indicated that Cd,Hg,Cr and As accumulate in large quantities in PM_2.5,which are mainly caused by the large amount of coal burning and automobile exhaust emissions of Taiyuan.Furthermore,carcinogenic analysis of heavy metals showed that Cr has significant carcinogenic effects on human.The accumulation of non-carcinogenic heavy metal Hg is a serious threat to human health.Moreover,heavy metals in PM_2.5 also make ecological environment risks,with Cd and Hg contributing 90%.In addition,the PAHs such as CHR,Bb F,Ba P and Bb A in PM_2.5 have high carcinogenicity to human.?2?The manners of PM_2.5 entering HUVEC cells and its effects on organelle structure and function.The CCK8 experiment was used to analyze the effect of PM_2.5 exposure on survival rate of HUVEC cells.The results showed when the PM_2.5 exposure concentration was 10 ?g/cm2 and the time of duration was 6 hours,there was no significant cytotoxicity.SDS-PAGE Gel results showed that PM_2.5 exposure can penetrate cell membranes and enter cells,and it increased significantly with the change of time and concentration.Furthermore,our results suggested that PM_2.5 enters the cells through a complex manner,including phagocytosis,macropinocytosis,clathrin and caveolin-1 mediated endocytosis.Next,we separated and detected the manner of different components of PM_2.5 into the cells.The results showed that the metal components entered the cell mainly through macropinocytosis,the organic components entered the cell mainly through clathrin-mediated endocytosis,and the water-soluble components through caveolin-1 mediated endocytosis.In addition,PM_2.5 and its metal components exposure significantly strengthened the depolarization of mitochondrial and induced mitochondrial division.At the same time,PM_2.5 exposure reduced the number of lysosomes and induced the changes of lysosomal membrane structure.?3?The effects and mechanisms of PM_2.5 exposure on vascular calcification.Human vascular smooth muscle cells?HASMC?were treated ?-GP for 3 days to form the calcifying vascular cells in vitro.The results of Von Kossa staining showed that PM_2.5 exposure promoted deposition of hydroxyl phosphate,decreased OPG expression,and increased of Osteocalcin and RANKL expressions in calcifying vascular cells.PM_2.5-induced the expression of ULK1 to activated autophagy,leading to vascular calcification in calcifying vascular cells.After pretreatment with autophagy inhibitors,autophagy and vascular calcification caused by PM_2.5 exposure were significantly inhibited.Moreover,PM_2.5 exposure also promoted vascular calcification by activated the expression of inflammatory factors and the production of ROS.The 3'-UTR region of ULK1 was used to predicted the target mi RNAs.The results implied that mi R-26a-5p could directly target ULK1.Western blot and q RT-PCR results showed that transfection with mi R-26a-5p mimic and inhibitor significantly activated/inhibited autophagy and vascular calcification in calcifying vascular cells.In addition,6 weeks-old Apo E-/-mice were fed a high-sugar and high-fat diet and exposed to winter PM_2.5,the results showed that PM_2.5 exposure promoted vascular calcification in the artery of Apo E-/-mice.Autophagy and vascular calcification induced by PM_2.5 exposure in the artery of Apo E-/-mice were significantly inhibited after 3-MA pretreatment.?4?Antagonistic effect of Rutin on promoting vascular calcification caused by PM_2.5 exposure.By using Von Kossa staining to detected the intervention effect of Rutin on promoted vascular calcification by PM_2.5 exposure.The results showed that Rutin significantly inhibited the effects of PM_2.5 on vascular calcification,and the expressions of calcification related proteins OPG,Osteocalcin and RANKL were meaningfully reversed.Moreover,Rutin inhibited OPG/RANKL signaling pathway and ROS generation,which were up-regulated by PM_2.5 exposure.These results implied that Rutin antagonized the promoting of PM_2.5 on vascular calcification.Taken together,this study uncovers that PM_2.5 and its different components have serious hazards to the environment and human health.We found PM_2.5 entered cells with different manners,which further led to the damage of structure and function of mitochondria and lysosomes.PM_2.5 exposure promoted vascular calcification via activated mi R-26a-5p/ULK1-mediated autophagy,OPG/RANKL signal pathway and inflammatory factors.Moreover,our data showed that Rutin treatment reversed the toxicity produced by PM_2.5.Collectively,the study clarified the mechanisms of PM_2.5 penetrating blood vessels to cause vascular calcification,providing a theoretical basis for the prevention and control of cardiovascular diseases caused by PM_2.5.