氯化多氟烷基醚磺酸经氧化应激损伤神经干细胞的增殖与分化

韩雅昕; 冯龙飞; 周志俊; 常秀丽

doi:10.11836/JEOM25077

氯化多氟烷基醚磺酸经氧化应激损伤神经干细胞的增殖与分化

Chlorinated perfluoroalkyl ether sulfonate impairs proliferation and differentiation of neural stem cells via oxidative stress

摘要

摘要:
背景氯化多氟烷基醚磺酸（Cl-PFAES，商品名F-53B）是一种新兴全氟/多氟烷基化合物（PFAS），现有研究表明可对人体造成多器官毒性并可穿过血脑屏障，但其对神经干细胞的毒性效应及机制尚不明确。
目的探讨F-53B通过氧化应激对神经干细胞增殖和分化的影响，并初步探讨其毒性作用与线粒体功能损伤及自噬相关基因（PINK1/Parkin）表达变化的潜在关联。
方法以新生C57BL/6小鼠原代神经干细胞为模型，分别暴露于0、33、100 μmol·L⁻¹的F-53B 24 h，采用细胞计数试剂盒（CCK-8）检测细胞活力，5-乙炔基-2' 脱氧尿苷（EdU）实验评估增殖能力，免疫荧光染色观察分化表型，二氢乙锭（DHE）和MitoSOX™ Red探针检测细胞内及线粒体活性氧（ROS）水平，MitoTracker Green观察线粒体形态，三磷酸腺苷（ATP）试剂盒检测能量代谢，实时定量聚合酶链式反应（qPCR）测定PINK1和Parkin基因表达。
结果 F-53B 100 μmol·L⁻¹染毒组显著降低细胞活力，为对照组的93.6%（P<0.01），EdU⁺细胞比例减少（P<0.01）提示增殖抑制。染毒组神经干细胞分化结果表明神经元生成数量减少、轴突缩短，同时星形胶质细胞数量增加。F-53B 100 μmol·L⁻¹暴露导致细胞内ROS升高至对照组的122%（P<0.01），线粒体ROS（MitoROS）升至135%（P<0.001），线粒体呈现碎片化形态，ATP水平下降至62.4%（P<0.001），PINK1和Parkin mRNA表达显著下调（P<0.05）。
结论 F-53B可能通过引起氧化应激，进而破坏线粒体形态和功能，并抑制Pink1/Parkin介导的线粒体自噬通路，最终导致神经干细胞增殖抑制与分化异常。本研究为F-53B的神经毒性解释提供了新的机制。

Abstract:
Background Chlorinated perfluoroalkyl ether sulfonate Cl-PFAES, trade name F-53B, a novel per- and polyfluoroalkyl substance (PFAS), has been shown to induce multi-organ toxicity in humans and cross the blood-brain barrier. However, its toxic effects and underlying mechanisms on neural stem cells (NSCs) remain unclear.
Objective To investigate the impact of F-53B on NSCs proliferation and differentiation through oxidative stress and explore its potential molecular mechanisms in associations with mitochondrial function damage and the expression of autophagy-related gene (PINK1/Parkin).
Methods Primary NSCs isolated from neonatal C57BL/6 mice were used as a model and exposed to F-53B at concentrations of 0, 33, or 100 μmol·L⁻¹ for 24 h. Cell viability was assessed using the cell counting kit-8 (CCK-8) assay, while proliferation was evaluated by the 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay. Immunofluorescence staining was performed to observe differentiation phenotypes. Intracellular and mitochondrial reactive oxygen species (ROS) levels were quantified using dihydroethidium (DHE) and MitoSOX probes, respectively. Mitochondrial morphology was observed using MitoTracker Green. ATP level was measured with a commercial kit. Additionally, real-time quantitative polymerase chain reaction (qPCR) was conducted to quantify the expression of PINK1 and Parkin genes.
Results Exposure to 100 μmol·L⁻¹ F-53B significantly reduced cell viability to 93.6% of the control group (P<0.01), and decreased the proportion of EdU⁺ cells (P<0.01), indicating proliferation inhibition. The differentiation analysis showed a reduction in neuronal generation, axonal shortening, and an increase in astrocytes. The 100 μmol·L⁻¹ F-53B exposure elevated intracellular ROS to 122% (P<0.01) and mitochondrial ROS (MitoROS) to 135% (P<0.001) of the control levels, leading to mitochondrial fragmentation. The ATP levels after the F-53B exposure decreased to 62.4% relative to the control group (P<0.001). Furthermore, the mRNA expression levels of PINK1 and Par after the F-53B exposure were notably reduced (P<0.05).
Conclusion F-53B may induce oxidative stress, thereby disrupting mitochondrial morphology and function while inhibiting the PINK1/Parkin-mediated mitophagy pathway, ultimately leading to impaired neural stem cell proliferation and abnormal differentiation. This study provides new insights into the neurotoxicity mechanisms of F-53B.

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