Study On The Mechanism Of Antioxidants And Phase ? Enzymes In Oxidative Damage Induced By Diesel Engine Particulate In Mice

ObjectiveDue to the advantages of energy efficiency,engine durability and powerperformance,diesel engine are widely used in various industries.Diesel engine particles(DEP),a major component of diesel engine exhaust(DEE),are a complex mixture of nano-dimension elemental carbon cores surrounded by polycyclic aromatic hydrocarbons(PAHs)and metals.Exposure to DEP have been associated with several adverse respiratory health events,including pulmonary fibrosis,chronic alveolitis,and pulmonary edema.Nrf2 is a transcription factor that is vital in protecting oxidative damage,especially in the lungs,which directly interface with pollutants and oxidant stressors from the external environment.Nrf2 regulate multiple genes,such as antioxidant enzymes and phase ? enzymes.However,none of the previous studies has focused on the relationship between DEP and Nrf2 regulated enzyme system.To provide a scientific basis for elucidating the mechanism of oxidative damage in DEP and to investigate the role of Nrf2 and Nrf2 regulated enzyme system in the lungs in DEP exposure.We collected DEP from the vent of a workshop in a diesel engine manufacturing factory.We measured particle size distribution,PAHs and metals of DEP.Based on this,DEP suspension was used to construct intratracheal instillation mice model.The lung surface-associated protein A(Surfactant Protein A,SPA)and Club cell protein(Club Cell secretory 16)were measured to detect lung injury.The ROS level and MDA level were measured to detect the oxidative damage of the lung.Further,we mainly measured Nrf2 and Nrf2-related antioxidant enzymes(Superoxide dismutase,SOD;Catalase,CAT;Glutathione peroxidase,GSH-Px)and phase ? enzymes(Heme oxygenase-1,HO-1;NAD(P):quinone oxidoreductase 1,NQO1;Glutamate cysteine ligase catalytic subunit,GCLC;Glutamate cysteine ligase modifier subunit,GCLM)in C57BL6/J and Nrf2-/-mice.Methods and Results1.The analysis of DEP components:The particle size distribution of DEP revealed that 84.3%of DEP had a size below 100nm.The result revealed that totaled 15 PAHs are absorbed on DEP,carcinogenic and non-carcinogenic PAHs accounted for 38.3%and 61.7%of the total PAHs,respectively.Regarding metal contents,we identified 32 metals in the DEP.Transitional metals accounted for 15.6%of the total metals.2.Oxidative damage induced by DEP:After single exposure to DEP,the pathological changes in the lungs showed a time-dependent pattern.At 6 h and 12 h,the pathological changes included a disordered alveolar wall,thickened pulmonary septal and inflammatory cell infiltration.SPA and CC16 levels in lung showed a transient increase.The ROS level in lung showed a trend of increased first and then decreased.in addition,the MDA increased after 12 h.3.Antioxidants and phase ? enzyme system in oxidative damage induced by DEP1)Time-course effects:108 male C57BL/6J mice were randomly divided into 12 groups(n=9 in each group),including 6 experimental groups(administered 100?g DEP)and 6 control groups(administered PBS+0.05%Tween-80).The mice sacrificed after 30 min,6h,12h,24h,48h,and 72h.The result revealed that,activity of CAT and GSH-Px were decreased.All the other enzymes showed a trend of increased first and then decreased,at either mRNA expression level or protein level.2)Dose effect relationship:100 male C57BL/6J mice were randomly divided into 4 groups(n=25 in each group).The mice were IT instilled 3 times a week(total,12 times)with 12.5 ?g,50 ?g,and 100 ?g DEP.The result showed that,the mRNA expression of Nrf2 decreases after exposure to 50 ?g and 100 ?g DEP exposure.The activity of SOD and GSH-Px were decreased after 100 ?g DEP exposure.The expression of HO-1,NQO1,GCLC and GCLM were decreased after 50 ?g or 100?g DEP exposure at mRNA expression level or protein level.3)Confirm the role of Nrf2 in DEP exposure:12 male Nrf2-/-and 12 male Nrf2+/+mice were IT instilled with 50 ?g DEP thrice a week(total,6 times).After DEP exposure,the Nrf2-/-mice exhibited more severe histopathological lung damage than that of Nrf2+/+mice.The result revealed that,activity levels of SOD,CAT,and GSH-Px in the Nrf2-/-mice exposed to 50 ?g DEP were lower than those of both the Nrf2-/-control group and Nrf2+/+50 ?g group.After Nrf2-/-mice exposure to DEP exposure,the expression of HO-1,NQO1,and GCLC were decreased at both mRNA level and protein level.Exposure to DEP in the Nrf2-/-group significantly decreased the mRNA expression of Gclm compared with the Nrf2-/-control group.Conclusion1.The particle size of DEP is mostly ultra-fine particles.The PAHs adsorbed on DEP are mainly non-carcinogenic PAHs,and the DEP adsorbed transition metals,which can cause oxidative damage.The DEP used in the present study are typical DEP particles,which can use to evaluate the damage induced by DEP.2.After repeated DEP exposure,Nrf2 and Nrf2 regulated enzymes decreased after exposure to medium and high doses of DEP.With the increase in the DEP exposure dose,the cell damage in the lungs occurred extracellularly first,and then intracellularly(outside in),as revealed by the mRNA expression of Sod isoforms.3.Single DEP exposure resulted in lung tissue injury.Nrf2 and Nrf2-regulated antioxidant and phase ? enzymes showed time-dependent changes.The decrease of CAT and GSH-Px indicate the DEP exposure damaged the lung defense.4.After exposure to DEP,Nrf2-/-mice showed more severe lung damage than that of Nrf2+/+mice.When DEP exposure,DEP suppress the enzymes directly,additionally,not by the regulation of Nrf2.