柴油废气颗粒物通过氧化损伤和炎症反应诱导血脑屏障损伤的体外实验研究

The adverse effects of diesel exhaust particulates on blood brain barrier through oxidative damage and inflammation in vitro

目的探讨柴油废气颗粒物(diesel exhaust particulates,DEP)是否通过氧化损伤和炎症反应导致血脑屏障损伤。方法用小鼠脑微血管内皮细胞bEnd.3细胞系构建血脑屏障体外模型,按照DEP染毒浓度不同将细胞分为对照组(0μg/mL)和实验组(DEP浓度为12.5、25、50、100μg/mL),染毒48 h后,检测谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活力,细胞内活性氧(reactive oxygen species,ROS)含量。酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测炎性细胞因子白细胞介素(interleukin,IL)-1β,IL-6,肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的分泌水平。Western blot检测紧密连接蛋白claudin-5和ZO-1的表达。结果bEnd.3细胞经不同浓度DEP染毒48 h后,100μg/mL浓度组与对照组相比,GSH-Px活力显著下降,差异具有统计学意义[(15.51±2.22)U/mg protein比(23.22±3.27)U/mg protein,q=4.741,P<0.05];活性氧含量升高,差异具有统计学意义;[(1.28±0.03)比(1.00±0.00),q=12.400,P<0.05];IL-1β分泌水平显著增加,25,50和100μg/mL浓度组与对照组相比差异具有统计学意义[(3.74±0.36)ng/L,(3.20±0.37)ng/L,(3.26±0.33)ng/L比(1.76±0.09)ng/L,q值分别为6.708、5.982、6.216,P值均<0.05];IL-6分泌水平显著增加,100μg/mL浓度组与对照组相比差异具有统计学意义[(57.79±4.20)ng/L比(20.31±1.12)ng/L,q=15.000,P<0.05];TNF-α分泌水平显著增加,50μg/mL和100μg/mL浓度组与对照组相比,差异具有统计学意义,[(20.88±4.77)ng/L,(24.41±6.53)ng/L比(7.54±2.81)ng/L,q值分别为4.715、5.962,P<0.05];claudin-5表达水平显著下降,12.5,25和100μg/mL浓度组与对照组相比,差异具有统计学意义[(0.87±0.16)、(0.92±0.17)、(0.37±0.12)比(1.24±0.11),q值分别为5.551、5.152、11.280,P<0.05];ZO-1表达水平显著下降,25、50和100μg/mL浓度组与对照组相比,差异具有统计学意义[(0.46±0.06)、(0.39±0.08)、(0.30±0.05)比(1.06±0.13),q值分别为6.724、7.572、8.470,P<0.05]。结论DEP可能通过氧化损伤和炎症反应,导致紧密连接蛋白表达降低进而造成血脑屏障损伤。

Objective To explore whether diesel exhaust particulates cause blood-brain barrier damage through oxidative damage and inflammation.Methods The blood-brain barrier(BBB)model in vitro was established by mouse brain microvascular endothelial cells(bEnd.3 cells).bEnd.3 cells were exposed to different concentrations of DEP(0,12.5,25,50,100μg/mL)for 48 h.The activity of glutathione peroxidase(GSH-Px)and the content of reactive oxygen species(ROS)were detected by special kits.The secretion of inflammatory cytokines(interleukin),IL-1β,IL-6,tumor necrosis factor-α(TNF-α)was detected by enzyme-linked immunosorbent assay(ELISA)kits.The tight junction protein,including claudin-5 and ZO-1 were measured by Western blot.Results After bEnd.3 cells were exposed to DEP for 48h,the activity of GSH-Px was decreased significantly in the 100μg/mL concentration group compared with the control group[(15.51±2.22)U/mg protein vs(23.22±3.27)U/mg protein,q=4.741,P<0.05]The content of ROS was increased significantly in the 100μg/mL concentration group compared with the control group[(1.28±0.03)vs(1.00±0.00),q=12.400,P<0.05].The secretory levels of IL-1βwere increased significantly in the 25,50 and 100μg/mL concentration groups compared with the control group[(3.74±0.36)ng/L,(3.20±0.37)ng/L,(3.26±0.33)ng/L vs(1.76±0.09)ng/L,q=6.708,5.982,6.216,all P values<0.05].The secretion level of IL-6 was increased significantly in the 100μg/mL concentration group compared with the control group[(57.79±4.20)ng/L vs(20.31±1.12)ng/L,q=15.000,P<0.05].The secretion level of TNF-αwas increased significantly in the 50 and 100μg/mL concentration groups compared with the control group[(20.88±4.77)ng/L,(24.4±6.53)ng/L vs(7.54±2.81)ng/L,q=4.715,5.962,both P values<0.05].The expression level of claudin-5 was decreased significantly in the 12.5,50 and 100μg/mL concentration groups compared with the control group[(0.87±0.16),(0.92±0.17),(0.37±0.12)vs(1.24±0.11),q=5.551,5.152,11.280,all P values<0.05].The expression of ZO-1 was decreased significantly in the 25,50 and 100μg/mL concentration groups compared with the control group[(0.46±0.06),(0.39±0.08),(0.30±0.05)vs(1.06±0.13),q=6.724,7.572,8.470,all P values<0.05].Conclusion DEP may cause blood brain barrier damage through oxidative damage and inflammation.