Study On Neuropathic Damage And Mechanism Of Nano Carbon Black Exposure

Background: Carbon black(CB)is a common ultrafine particle that is one of the main components of the smoke produced when the fuel is not completely burned.It is widely used in rubber,printing and coating industries.Because of its own structural characteristics,it has also widely used to study the biological toxicology of carbon-containing particulate matter in air pollution.With the commercialization of carbon black products and the severe condition of atmospheric pollution,the research on bio-toxicity of carbon black has attracted more attention.In recent years,studies have shown that exposure of carbon black can cause respiratory and cardiovascular diseases and is associated with the occurrence of various cancers.At present,studies have shown that CBs cause neuroinflammation,it also induced glutamate and ATP release in astrocytes.However,the effects of carbon black on nerve damage and its potential mechanism remain unclear.To evaluate the neurotoxicity of carbon black,the in vitro and in vivo experiments were conducted to investigate the neurotoxicity and mechanism of carbon black in the current work.Objective: A mouse model of chrolic long-term exposure to carbon black and PC-12 cell model of carbon black damage were established.MRI,transmission electron microscopy,metabolomics and molecular biology techniques were used to systematically explore the roles of carbon black exposure in neural damage.Methods: C57BL/6J male mice were randomly divided in 0,0.15 mg/ml,0.5 mg/ml,1.5 mg/ml groups(n=6).To make chronic animal models,mice in carbon black group were given nasal injection of 20 ?l carbon black for 3 months.The mice brains were detected by MRI imaging after the last injection.After the mice were sacrificed,electron microscope was performed in brain slices to characterize changes in olfactory bulb,striatum and hippocampus.Moreover,metabolomics was performed to characterize changes in metabolites in mice brains.CCK-8 cell counting kits were used to detect the PC-12 cells viability after different centrations of carbon black treatmentfor 24 h,meanwhile,the level of ATP in cells were observed by ATP detection kit,the total glutathione test kit and total SOD activity test kit were used to detect intracellular antioxidant enzyme viability.Mitochondrial division-associated protein Drp-1,oxidative stress protein NOX2,NOX4,4-HNE,ER stress-related protein GRP78,IRE1?,ATF6,PERK,CHOP,m TOR signaling pathway m TOR/p70s6 K,autophagy-related protein Lc3-?,Beclin1,ATG5,P62,insulin signaling pathway IRS1,AKT,GSK3?,GSK3?,neurodegenerative protein tau were detected by western blot with aim to investigate the nm carbon black induced neural damage.Results: The Carbon blacks were found in the olfactory bulb,striatum,and hippocampus of mice exposed to carbon black.The number of immature mitochondria in the mice exposed to carbon black was increased,and the mitochondrial cristas were damaged or even disappeared compared with the control group.In addition,the metabolomic analysis revealed that the metabolic profile of the low-dose(0.15 mg/ml)group was not significantly different from that of the control group,while the 0.5 mg/ml and 1.5 mg/ml groups had significant changes.Among them,sucrose,valine and phenylalanine were down-regulated,while the Lactic acid,glycine,aspartic acid,succinic acid and other substances were up-regulated.We found that the exposure of nano-carbon black mainly affected the biosynthesis of phenylalanine,tyrosine,tryptophan,valine,leucine and isoleucine,the metabolism of glycine,serine,threonine,alanine,aspartate and glutamate by pathway enrichment analysis.Exposure to carbon black obviously reduced cell viability and reduced ATP production.The m RNA levels of mitochondrial autophagy-related proteins PINK1,Parkin,DJ-1 and LRRK2 were abnormally expressed.Meanwhile,the m RNA and protein levels of Drpl were upregulated,which might exert an effect on the balance of mitochondrial dynamics.Moreover,exposure to carbon black led to the increase of ROS production and inhibition of antioxidant enzymes.These results were accompanyed by the upregulation of NOX2,NOX4,4-HNE protein levels in PC-12 cells.Exposure of carbon black resulted in increased expression of GRP78 and p-PERK,IRE1? and ATF6 proteins,resulting in upregulation of downstream CHOP protein.The carbon black also inhibited the m TOR/p70s6 K signaling pathway,generating increase of intracellular autophagy-related proteins Lc3-II,Beclin1,ATG5 and P62 levels,which may reflected the interfering with autophagic flow.Additionally,exposure to carbon black markedly increased the phosphorylation level of the IRS1 Ser307 site,a key molecule of the insulin pathway,and induced insulin signal disorders.Meanwhile,the phosphorylation of downstream AKT was inhibited, and the GSK3?,GSK3? proteins were obviously activated.In addition,up-regulation of tau protein phosphorylation sites(ser199,ser214,ser404)caused hyperphosphorylation of tau protein.Conclusions 1.Carbon black exposure cause neurotoxicity,it can enter the brain and widely distributed and cause mitochondrial damage and brain metabolism disorder in mice.2.Carbon black leads to impaired mitochondrial function in PC-12 cells,increasing mitochondrial division and causing oxidative stress and ER stress.3.Carbon black increases the level of autophagy in PC-12 cells.4.Carbon black disrupts insulin signaling pathway and induce hyperphosphorylation of tau protein in PC-12 cells.