ABSTRACT
Background Lead exposure induces microglial cell death, of which the mechanism is unclear. Ferroptosis is a new death form and its role in microglia death has not been reported. Objective To investigate the role of ferroptosis in microglia following lead exposure in order to provide a theoretical basis for the mechanism of lead neurotoxicity. Methods Microglial cell line BV-2 cells were co-cultured with 0, 10, 20 and 40 μmol·L−1 lead acetate for 24 h. The 40 μmol·L−1 lead acetate group with iron chelator (DFO) was named the 40+DFO group. Changes in BV-2 cell morphology after lead exposure were observed under an inverted microscope; tissue iron kit and glutathione kit were used to detect intracellular iron and glutathione (GSH) respectively; flow cytometry was applied to detect lipid reactive oxygen species (lipid ROS) immunofluorescence intensity. Western blotting and qPCR were adopted to detect the expressions of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), transferrin receptor 1 (TFR-1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1) protein and mRNA. Results Compared with the control group, the number of BV-2 cells decreased with increasing doses of lead and the cells showed a large, round amoeboid shape. The intracellular levels of iron of BV-2 cells were (1.08±0.04), (1.29±0.03), and (1.72±0.10) mg·g−1 (calculated by protein, thereafter) in the 10, 20, and 40 μmol·L−1 lead acetate groups, respectively, significantly higher than that in the control group (P<0.05), and the intracellular level of iron in the 40+DFO group, (1.34±0.10) mg·g−1, was lower than that in the 40 μmol·L−1 lead acetate group, (1.72±0.03) mg·g−1 (P<0.05). Compared with the control group, the TFR-1 and DMT1 protein and mRNA expressions were increased in BV-2 cells in the 10, 20, 40 μmol·L−1 lead acetate groups (P<0.05), especially in the 40 μmol·L−1 lead acetate group; the FPN1 protein expression did not change significantly, but the FPN1 mRNA expressions in BV-2 cells in the 10, 20, 40 μmol·L−1 lead acetate groups were significantly decreased (P<0.05). Compared with the control group, the intracellular GSH level decreased and the lipid ROS content increased in all three lead acetate groups; compared with the 40 μmol·L−1 lead acetate group, the GSH level increased by 12.30% and the lipid ROS content decreased by 13.00% in the 40+DFO group (P<0.05). The expressions of GPX4 protein were reduced to 50.00%, 35.00%, and 17.00% of that of the control group in the 10, 20, and 40 μmol·L−1 lead acetate groups respectively, while the expressions of GPX4 mRNA were also significantly reduced; the expressions of SLC7A11 protein and mRNA in the 20 and 40 μmol·L−1 lead acetate groups were lower than that in the control group, with the most significant decrease in the 40 μmol·L−1 lead acetate group (P<0.05). Conclusion Lead exposure could induce ferroptosis in BV-2 cells, in which iron transport imbalance and oxidative damage might be involved.
ABSTRACT
Objective:To investigate the effect of nano lead oxide (nano-PbO) exposure on learning and memory as well as spatial exploration ability in the mice, and the role of leukocyte infiltration of brain tissue in neurobehavioral damage caused by nano-PbO exposure.Methods:A total of 60 male SPF grade Kunming mice were divided into control group, low-dose nano-PbO group, medium-dose nano-PbO group and high-dose nano-PbO group according to body mass matching method, with 15 mice in each group.Mice in low, medium and high dose groups of nano-PbO were intraperitoneally injected with 5 mg·kg -1, 10 mg·kg -1, 20 mg·kg -1 nano-PbO, respectively. And mice in the control group were intraperitoneally injected with the same volume of 0.9% normal saline.The frequency of intervention was once a day for 28 days.Morris water maze test and open field test were used to detect the ability of learning and memory and spatial exploration of mice. Western blot was used to detect the protein expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) in hippocampus of mice, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) in mouse microvessels and lymphocyte function-associated antigen-1 (LAF-1) in mouse blood leukocyte. The proportion of leukocytes in mouse brain was detected by flow cytometry. All statistical analyses were performed by SPSS 20.0. Morris water maze data were analyzed by repeated measures ANOVA, the other data among multiple groups were compared by one-way ANOVA, and Tukey's test was used for further pairwise comparison.Pearson correlation analysis was performed to evaluate the correlation between neurobehavioral indexes and the proportion of white blood cells, TNF-α and IL-1β in brain tissue. Results:Morris water maze results showed that the escape latency of the four groups of mice had a significant interaction between group and time( F=3.21, P<0.05). The escape latencies of mice in middle and high dose groups of nano-PbO were higher than that in the control group (both P<0.05), and the numbers of crossing the platform of the two groups were lower than that in the control group (both P<0.05). The results of open field test showed that there was a statistically significant difference in the residence time of the mice in the four groups ( F=119.10, P<0.01). The total standing times of mice in the middle group and high dose group of nano-PbO were lower than that in the control group (both P<0.01). The results of Western blot showed that the levels of TNF-α and IL-1β in hippocampus tissue of mice were significant differences among the four groups ( F=7.21, 9.89, both P<0.05). The levels of TNF-α and IL-1β in the hippocampus of mice in the high-dose nano-PbO group were higher than those in the control group (TNF-α: (0.35±0.10), (1.03±0.30), P<0.05; IL-1β: (0.32±0.10), (0.50±0.15), P<0.05). The results of flow cytometry analysis showed that the proportions of leukocytes in the brain tissue of mice in the low, medium and high dose groups of nano-PbO were (9.99±1.09)%, (13.03±0.94)% and (16.51±3.89)%, respectively. Among them, the proportions of leukocytes in the middle and high dose groups of nano-PbO were significantly higher than that in the control group((8.13±1.29)%) (both P<0.05). The results of correlation analysis showed that the proportion of leukocytes, levels of TNF-α, IL-1β protein of hippocampus in the medium, high dose groups of nano-PbO were negatively correlated with the behavioral indexes ( r=-0.815, -0.744, -0.578, all P<0.01; r=-0.771, -0.836, -0.704, all P<0.05; r=-0.823, -0.876, -0.695, all P<0.05). The results of Western blot showed that the levels of ICAM-1 and VCAM-1 in cerebral microvessels of mice in the four groups were significantly different ( F=5.51, 16.19, both P<0.05). The levels of ICAM-1 and VCAM-1 in the middle and high dose groups of nano-PbO were higher than those in the control group(ICAM-1: (1.07±0.16), (1.21±0.35), (0.59±0.19), all P<0.05; VCAM-1: (0.68±0.12), (1.92±0.23), (0.23±0.05), both P<0.05). In addition, there was a significant difference in the level of LFA-1 protein in blood leukocytes of mice in the four groups ( F=41.80, P<0.05). The levels of LFA-1 in the middle and high dose groups of nano-PbO were higher than that in the control group((0.33±0.06), (0.89±0.23), (0.05±0.01), both P<0.05). Conclusion:The nano-PbO exposure can lead to cognitive impairment and increased inflammatory factors in the hippocampus of mice, which may be related to the increase of ICAM-1 and VCAM-1 in vascular endothelial cells, which promotes leukocyte infiltration into brain tissue.