李倩, 肖洪喜, 李睿, 唐黎明, 常秀丽, 周志俊. 百草枯促进小胶质细胞分泌IL-1β对神经干细胞增殖和神经元生成的影响[J]. 环境与职业医学, 2022, 39(9): 1045-1050. DOI: 10.11836/JEOM21605
引用本文: 李倩, 肖洪喜, 李睿, 唐黎明, 常秀丽, 周志俊. 百草枯促进小胶质细胞分泌IL-1β对神经干细胞增殖和神经元生成的影响[J]. 环境与职业医学, 2022, 39(9): 1045-1050. DOI: 10.11836/JEOM21605
LI Qian, XIAO Hongxi, LI Rui, TANG Liming, CHANG Xiuli, ZHOU Zhijun. Effects of paraquat on proliferation and neurogenesis of neural stem cells by promoting secretion of IL-1β by microglia[J]. Journal of Environmental and Occupational Medicine, 2022, 39(9): 1045-1050. DOI: 10.11836/JEOM21605
Citation: LI Qian, XIAO Hongxi, LI Rui, TANG Liming, CHANG Xiuli, ZHOU Zhijun. Effects of paraquat on proliferation and neurogenesis of neural stem cells by promoting secretion of IL-1β by microglia[J]. Journal of Environmental and Occupational Medicine, 2022, 39(9): 1045-1050. DOI: 10.11836/JEOM21605

百草枯促进小胶质细胞分泌IL-1β对神经干细胞增殖和神经元生成的影响

Effects of paraquat on proliferation and neurogenesis of neural stem cells by promoting secretion of IL-1β by microglia

  • 摘要: 背景

    百草枯(PQ)是广泛使用的除草剂,具有神经毒性。PQ介导的神经炎症对神经干细胞的影响研究仍然有限。

    目的

    探讨PQ通过小胶质细胞介导的神经炎症对神经干细胞增殖和神经元生成的影响。

    方法

    选用小胶质细胞系(BV2细胞)和原代神经干细胞作为研究对象。将BV2细胞置于PQ终浓度分别为0、1、3.3、10、33、100 μmol·L−1的完全培养基中培养6 h,检测其细胞存活率,选择对细胞活力没有影响的最高PQ浓度作为最终染毒浓度(33 μmol·L−1)。为了排除PQ对神经干细胞的直接影响,将BV2细胞置于PQ终浓度为33 μmol·L−1的完全培养基中培养6 h,再将培养基换成不含PQ的神经干细胞完全培养基继续培养24 h,用酶联免疫吸附法检测上清液中白细胞介素-1β(IL-1β)的浓度。此外,为了检测IL-1β对神经干细胞增殖和神经元生成的影响,从成年小鼠海马区分离提取的神经干细胞用上述BV2细胞上清液培养,分为4组:对照组上清+对照抗体组,对照组上清+IL-1β中和抗体组(10 ng·mL−1),PQ组上清+对照抗体组,PQ组上清+IL-1β中和抗体组(10 ng·mL−1),培养24 h后用流式细胞和免疫荧光法检测Ki67阳性的神经干细胞的比例,培养3~7 d后用流式细胞和免疫荧光法检测新生神经元的比例。

    结果

    与对照组相比,BV2细胞经过33 μmol·L−1 PQ处理后上清液中IL-1β含量上升(t=3.020,P<0.05)。用PQ处理过的BV2细胞上清液培养小鼠神经干细胞,与对照组相比,Ki67阳性的神经干细胞的比例下降(t=9.129,P<0.01),新生神经元的比例与对照组相比下降(t=4.638,P<0.01)。中和IL-1β后,共培养的神经干细胞中Ki67阳性的神经干细胞百分比高于未中和组(t=22.05,P<0.01),新生神经元的比例也高于未中和组(t=11.09,P<0.01)。免疫荧光技术检测同样显示,中和33 μmol·L−1 PQ处理的BV2细胞上清液中的IL-1β后,Ki67阳性的神经干细胞数量高于未中和组,神经元的数量也高于未中和组。

    结论

    PQ促进小胶质细胞分泌IL-1β,导致神经干细胞增殖下降,神经元生成减少。研究结果提示小胶质细胞介导的神经炎症参与PQ导致的神经干细胞损伤。

     

    Abstract: Background

    Paraquat (PQ) is a widely used herbicide that exerts neurotoxicity. The effects of PQ on neural stem cells (NSCs) through microglia mediated neuroinflammation remain limitedly studied.

    Objective

    To investigate the effects of PQ on the proliferation and neurogenesis of NSCs through neuroinflammation mediated by microglia.

    Methods

    Microglial cell lines (BV2 cells) and primary NSCs were used. BV2 cells were exposed to 0, 1, 3.3, 10, 33, and 100 μmol·L−1 of PQ for 6 h followed by viability assessment. The highest PQ concentration that had no effect on cell viability was selected as the final exposure concentration (33 μmol·L−1). To exclude the direct effect of PQ on NSCs, after the BV2 cells were cultured in complete medium containing 33 μmol·L−1 PQ for 6 h, the BV2 culture medium was replaced by NSCs complete medium without PQ for 24 h. The concentration of interleukin-1β (IL-1β) in supernatant was detected by enzyme-linked immune sorbent assay. Besides, in order to detect the effects of IL-1β on NSCs proliferation and neurogenesis, NSCs isolated from hippocampus of adult mice were cultured in the supernatant obtained above and divided into four groups: control supernatant + control antibody, control supernatant + IL-1β neutralizing antibody (10 ng·mL−1), PQ supernatant + control antibody, PQ supernatant + IL-1β neutralizing antibody (10 ng·mL−1). Proportion of Ki67-positive NSCs was detected by flow cytometry (FCS) and immunofluorescence after 24 h culture, and neurogenesis was detected by FCS and immunofluorescence after 3-7 d of culture.

    Results

    The IL-1β concentration in the supernatant of BV2 cells was significantly increased after the 33 μmol·L−1 PQ exposure compared with the control group (t=3.020, P<0.05). After the NSCs were cultured with the supernatant of PQ-treated BV2 cells, the proportion of Ki67-positive NSCs (t=9.129, P<0.01) and the proportion of newborn neurons (t=4.638, P<0.01) were significantly decreased compared to the control group. After neutralizing IL-1β, the proportion of Ki67-positive NSCs (t=22.05, P<0.01) and the proportion of newborn neurons (t=11.09, P<0.01) were significantly higher than those in the un-neutralized group. The results of immunofluorescence detection also showed that after neutralizing IL-1β secreted by 33 μmol·L−1 PQ-treated BV2 cells, the number of Ki67-positive NSCs and the number of newborn neurons were significantly higher than those in the un-neutralized group.

    Conclusion

    The secretion of IL-1β by microglia is increased after PQ treatment, resulting in a decrease in the proliferation and neurogenesis of NSCs. These results suggest that neuroinflammation is involved in NSCs damage caused by PQ.

     

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