Impact of lead oxide nanoparticle exposure on the polarization of microglia cells in mouse hippocampus

Abstract

Objective To investigate the effect of exposure to lead oxide nanoparticles (PbO NPs) on the polarization of microglia in mouse hippocampus.

Methods i) Specific pathogen-free male C57 mice were randomly divided into control group, low-, medium- and high-dose groups, with 10 mice in each group. Mice in these three dose groups were intraperitoneally injected with PbO NPs suspension at doses of 5, 10 and 20 mg/kg per day, respectively, and mice in the control group were intraperitoneally injected with the same volume of 0.9% sodium chloride solution, five days per week for four weeks. ii) BV-2 cells were treated with PbO NPs at doses of 0.0, 2.5, 5.0 and 10.0 mg/L for 24 hours. iii) BV-2 cells were randomly divided into control group, PbO NPs group and triggering receptor expressed on myeloid cells 2 (TREM2) high expression + PbO NPs group. The cells in the control group received no treatment. The cells in PbO NPs group were exposed to 10.0 mg/L PbO NPs suspension for 24 hours. Cells in TREM2 high expression + PbO NPs group were transfected with Trem2 high expression plasmid, and then exposed to 10.0 mg/L PbO NPs suspension for 24 hours. iv) The mRNA expression of M1 markers [nitric oxide synthase ( iNos), cyclooxygenase 2 ( Cox2), chemokine receptor 7 ( Ccr7)], M2 markers [arginin-1 (Arg-1), transforming growth factor-β ( Tgf-β), chemokine receptor 2 ( Ccr2)] and Trem2 of microglia was detected by real-time fluorescent quantitative polymerase chain reaction. The protein expression of iNOS, ARG-1 and TREM2 was detected by Western blotting.

Results i) During the experiment, there was no significant difference in body weight of mice among these four groups ( P>0.05). The relative expression of Cox2 and Ccr7 mRNA in the hippocampus of the mice increased in the low-dose group and the iNos, Cox2 and Ccr7 mRNA increased in the medium- and high-dose groups, compared with the control group (all P<0.05). The relative mRNA expression of Tgf-β in the hippocampus of the mice of low-dose group and Arg-1, Tgf-β and Ccr2 in the medium- and high-dose groups was decreased compared with the control group (all P<0.05). The mRNA relative expression of iNos, Cox2 and Ccr7 was increased (all P<0.05), while the mRNA relative expression of Arg-1, Tgf-β and Ccr2 was decreased (all P<0.05) in the hippocampus of the mice of high-dose group compared with the low-dose group. The relative expression of Trem2 mRNA and TREM2 protein in the hippocampus of mice of the medium- and high-dose groups was lower than those in the control group (all P<0.05). The relative expression of Trem2 mRNA and TREM2 protein in the hippocampus of mice of the high dose group was lower than those in the low- and the medium-dose groups (all P<0.05). With the increase of PbO NPs exposure dose, the relative expression of iNOS protein in hippocampus tissues of mice increased ( P<0.01), and the relative expression of ARG-1 protein decreased ( P<0.01). ii) With the increase of PbO NPs exposure dose, the relative expression of iNOS protein increased ( P< 01), and the relative expression of ARG-1 protein decreased ( P<0.01) in BV-2 cells. The relative expression of iNOS protein in BV-2 cells of PbO NPs group and TREM2 high expression + PbO NPs group was increased (all P<0.05), and the relative expression of ARG-1 protein decreased (all P<0.05) compared with the control group. The relative expression of iNOS protein decreased ( P<0.05), and the relative expression of ARG-1 protein increased ( P<0.05) in BV-2 cells of TREM2 high expression + PbO NPs group compared with the PbO NPs group.

Conclusion Exposure to PbO NPs could increase the M1 polarization and decrease the M2 polarization of microglia, with a dose-effect relationship. The M1 polarization of microglia decreased and M2 polarization increased after overexpression of Trem2 gene. The regulation of microglia polarization by TREM2 may be involved in the neurotoxic effects of PbO NPs.

Abstract

摘要： 目的 探讨纳米氧化铅(PbO NPs)暴露对小鼠海马组织小胶质细胞极化的影响。 方法 (1)将无特定病原体级雄 性C57小鼠随机分为对照组、低剂量组、中剂量组和髙剂量组, 每组10只。后3组小鼠每天分别腹腔注射剂量为5、10和 20 mg/kg的PbO NPs混悬液, 对照组小鼠予腹腔注射等体积0.9%氯化钠溶液, 每周5 d, 共4周。(2)采用剂量为0.0、2.5、5.0和10.0 mg/L的PbO NPs混悬液处理BV-2细胞24 h。(3)⑶将BV-2细胞随机分为对照组、PbO NPs组和髓样细胞触发受 体2(TREM2)髙表达+PbO NPs组。其中, 对照组细胞不做任何处理;PbO NPs组细胞予质量浓度为10.0 mg/L的PbO NPs 混悬液染毒24 h;TREM2髙表达+PbO NPs组细胞在转染 Trem2髙表达质粒的基础上, 予质量浓度为10.0 mg/L的PbO NPs 混悬液染毒24h。(4)小胶质细胞M1型标志物[一氧化氮合成酶( iNos)、环氧化酶2( Cox2)、趋化因子受体7( Ccr7)]、M2型标志物[精氨酸-1( Arg-i)、转化生长因子-β( Tgf-β)、趋化因子受体2( Ccr2)]和髓样细胞触发受体2( Trem2)的 mRNA表达均采用实时荧光定量聚合酶链反应法检测; iNOS、ARG-1和TREM2蛋白表达均采用蛋白质印迹法检测。 结果 (1)实验期间, 4组小鼠体质量比较, 差异无统计学意义( P>0.05)。与对照组比较, 低剂量组小鼠海马组织中 Cox2、 Ccr7和中剂量组、髙剂量组细胞 iNos、 Cox2、 Ccr7的mRNA相对表达水平均升髙( P 值均<0.05), 低剂量组小鼠海马组 织中 Tgf-β和中剂量组、髙剂量组细胞 Arg-1、 Tgf-β、 Ccr2的mRNA相对表达水平均下降( P 值均<0.05)。与低剂量组比较, 髙剂量组小鼠海马组织中 iNos、 Cox2和 Ccr7的mRNA相对表达水平均升髙( P值均<0.05), Arg-1、 Tgf-β和 Ccr2的mRNA 相对表达水平均下降( P 值均<0.05)。中和髙剂量组小鼠海马组织中 Trem2 mRNA和TREM2蛋白的相对表达水平均低 于对照组( P 值均<0.05), 髙剂量组小鼠海马组织中上述2个指标分别低于低剂量组和中剂量组( P 值均<0.05)。随 着PbO NPs染毒剂量的增加, 小鼠海马组织中iNOS蛋白相对表达水平升髙( P<0.01), ARG-1蛋白相对表达水平下降 ( P<0.01)。(2)随着PbO NPs染毒剂量的增加, BV-2细胞中iNOS蛋白相对表达水平升髙( P<0.01), ARG-1蛋白相对表达 水平下降( P<0.01)。与对照组比较, PbO NPs组和TREM2髙表达+PbO NPs组BV-2细胞中iNOS蛋白相对表达水平均升 髙( P 值均<0.05), ARG-1蛋白相对表达水平均下降( P 值均<0.05);与PbO NPs组比较, TREM2髙表达+PbO NPs组BV-2 细胞中iNOS蛋白相对表达水平下降( P<0.05), ARG-1蛋白相对表达水平升髙( P<0.05)。 结论 PbO NPs暴露可导致小 胶质细胞M1型极化增加, M2型极化减少, 呈一定程度的剂量-效应关系; Trem2基因髙表达后, 小胶质细胞M1型极化下 降, M2型极化升髙。TREM2调控小胶质细胞极化可能参与了 PbO NPs的神经毒性作用。