Manganese overexposure can induce neurotoxicity, lead to manganism and result in clinical manifestations similar to those of parkinsonism. However, the underlying molecular mechanism is still unclear. This study demonstrated that MnCl2 induces mitophagy and leads to neurotoxicity by promoting BNIP3-mediated reactive oxygen species (ROS) generation. Human neuroblastoma SH-SY5Y cells were used throughout our experiments. Cell viability was detected by cell proliferation/toxicity test kits. Mitochondrial membrane potential was measured by flow cytometry. ROS generation was detected using a microplate reader. Protein levels were evaluated by Western blot. Transmission electron microscopy was used to evaluate mitochondrial morphology. Co-immunoprecipitation was used to verify the interaction between BNIP3 and LC3. MnCl2 led to loss of mitochondrial membrane potential and apoptosis of SH-SY5Y cells by enhancing expression of BNIP3 and conversion of LC3-I to LC3-II. Moreover, MnCl2 reduced expression of the mitochondrial marker protein TOMM20 and promoted interaction between BNIP3 and LC3. The results also indicated that a decrease in BNIP3 expression reduced the mitochondrial membrane potential loss, attenuated apoptosis and reduced mitochondrial autophagosome formation in SH-SY5Y cells after MnCl2 treatment. Finally, we found that manganese-induced ROS generation could be reversed by the antioxidant N-acetyl cysteine (NAC) or silencing BNIP3 expression. BNIP3 mediates MnCl2-induced mitophagy and neurotoxicity in dopaminergic SH-SY5Y cells through ROS. Thus, BNIP3 contributes to manganese-induced neurotoxicity by functioning as a mitophagy receptor protein.

中文翻译：

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氯化锰 (II) 通过在 SH-SY5Y 细胞中通过 BNIP3 介导的氧化应激促进线粒体自噬导致多巴胺能神经毒性

锰过度暴露可引起神经毒性，导致锰中毒并导致与帕金森症相似的临床表现。然而，潜在的分子机制仍不清楚。该研究表明，MnCl2 通过促进 BNIP3 介导的活性氧 (ROS) 的产生，诱导线粒体自噬并导致神经毒性。在我们的整个实验中都使用了人神经母细胞瘤 SH-SY5Y 细胞。通过细胞增殖/毒性测试试剂盒检测细胞活力。通过流式细胞术测量线粒体膜电位。使用酶标仪检测 ROS 的产生。通过蛋白质印迹评估蛋白质水平。透射电子显微镜用于评估线粒体形态。共免疫沉淀用于验证 BNIP3 和 LC3 之间的相互作用。MnCl2通过增强BNIP3的表达和LC3-I向LC3-II的转化导致SH-SY5Y细胞线粒体膜电位的丧失和凋亡。此外，MnCl2 降低线粒体标记蛋白 TOMM20 的表达并促进 BNIP3 和 LC3 之间的相互作用。结果还表明，在MnCl2处理后，BNIP3表达的降低减少了SH-SY5Y细胞中线粒体膜电位的损失，减弱了细胞凋亡并减少了线粒体自噬体的形成。最后，我们发现锰诱导的 ROS 生成可以通过抗氧化剂 N-乙酰半胱氨酸 (NAC) 或沉默 BNIP3 表达来逆转。BNIP3 通过 ROS 在多巴胺能 SH-SY5Y 细胞中介导 MnCl2 诱导的线粒体自噬和神经毒性。因此，BNIP3 通过作为线粒体自噬受体蛋白发挥作用，导致锰诱导的神经毒性。