• 关键词：纳米氧化锌颗粒  大鼠气管上皮细胞  细胞凋亡  氧化应激  离子通道

• 基金项目：浙江省自然科学基金（No.LY15B070014）；浙江省新苗人才计划资助项目（No.2017R423082）；杭州市"131"人才计划项目；浙江省"151"人才计划项目；杭州师范大学卓越人才计划

• 陆香君
• 生命与环境科学学院, 杭州师范大学, 杭州 310016

• 唐娟
• 生命与环境科学学院, 杭州师范大学, 杭州 310016

• 蔡恩祺
• 生命与环境科学学院, 杭州师范大学, 杭州 310016

• 朱维琴
• 生命与环境科学学院, 杭州师范大学, 杭州 310016

• 丁颖
• 生命与环境科学学院, 杭州师范大学, 杭州 310016

• 王娇娇
• 生命与环境科学学院, 杭州师范大学, 杭州 310016

• 吴应珠
• 生命与环境科学学院, 杭州师范大学, 杭州 310016

• 牛丽丽
• 生命与环境科学学院, 杭州师范大学, 杭州 310016

• 张杭君
• 1. 生命与环境科学学院, 杭州师范大学, 杭州 310016;2. 杭州师范大学生态系统保护与恢复杭州市重点实验室, 杭州师范大学, 杭州 310036

• 摘要：纳米氧化锌具有广泛的工业用途，其生态安全性受到广泛关注，针对纳米氧化锌诱导的呼吸道细胞毒性及其作用机理研究尚不广泛.本研究分别采用不同浓度和粒径（30 nm和90 nm）的氧化锌颗粒物处理大鼠气管上皮细胞（rat tracheal epithelial cells，RTE cells），暴露时间为12 h，通过检测细胞内锌元素含量，细胞增殖抑制率，细胞凋亡率，凋亡相关caspsae 3基因与蛋白相对表达量，细胞内金属硫蛋白活性，ROS和MDA含量、细胞内Ca²⁺-ATP酶和Na⁺/K⁺-ATP酶活性来分析纳米氧化锌诱导细胞毒效应机理.在90 nm纳米氧化锌高浓度暴露时，其细胞内锌元素浓度为0.845 μg·L^-1，约为低浓度暴露组的4.7倍，是30 nm低浓度暴露组的9倍；纳米颗粒物诱导的细胞增殖和凋亡毒效应具有剂量和尺寸依赖效应；30 nm处理组的pro-caspase 3和cleaved-caspase 3蛋白表达量均高于90 nm暴露组；暴露浓度为10 mg·L^-1的90 nm处理组的金属硫蛋白增加量为0.533 μg·L^-1，增幅达到46%；不同粒径氧化锌颗粒物处理后，细胞内ROS和MDA含量显著上升，且30 nm处理组结果均高于90 nm处理组；纳米氧化锌颗粒物暴露诱导细胞Ca²⁺-ATP酶和Na⁺/K⁺-ATP酶活性显著下降，30 nm氧化锌颗粒物暴露组，其Na⁺/K⁺-ATP酶活性分别是对照组的1.8倍和3.5倍.纳米氧化锌颗粒物进入RTE细胞，通过干扰锌在细胞内代谢，诱导细胞内ROS和MDA水平升高，产生氧化应激，进而诱导细胞凋亡是导致纳米氧化锌产生细胞毒性的主要原因之一.纳米氧化锌会导致细胞内Ca²⁺-ATPase和Na⁺/K⁺-ATPase活性下降，离子通道失调，破坏细胞内离子平衡，进一步造成细胞凋亡.

• Abstract：Zinc oxide nanomaterials (ZnO NPs) have a wide range of industrial applications with extensive attention for their biosafety. However, little was known about the respiratory cytotoxicity and the mechanism induced by ZnO NPs. The objective of the study is to investigate the effects of ZnO NPs on rat tracheal epithelial (RTE) cells. RTE cells were treated with ZnO NPs of different concentrations and diameters (30 nm and 90 nm) for 12 h. Cytotoxicity effects and mechanism were detected by indexes of intracellular zinc content, cell proliferation inhibition percentage, apoptotic cell percentage, metallothionein activity, ROS and MDA content, intracellular Ca²⁺-ATPase and Na⁺/K⁺-ATPase activities and relative expressions of apoptosis related gene and protein of caspsae 3. The results showed that the intracellular zinc concentration was 0.845 μg·L^-1 under the exposure of 90 nm ZnO NPs with a high concentration, which was about 4.7-fold of the low concentration exposure group and 9-fold of the 30 nm low concentration exposure group. The increment of metallothionein in the 90 nm treatment group was 0.533 μg·L^-1. The increase of the content of ROS and MDA in the treated group was significantly higher than that in the control group. The activities of Ca²⁺ -ATPase and Na⁺/K⁺ -ATPase were significantly decreased, and the Na⁺/K⁺ -ATPase activity treated by 30 nm ZnO NPs was 1.8-fold and 3.5-fold higher than that in the control group, respectively. The effect of nanoparticle-induced cell proliferation and apoptosis was dose-dependent and size-dependent. The expression levels of pro-caspase 3 and cleaved-caspase 3 in the 30 nm treatment group were higher than those in the 90 nm exposure group in same exposure dose. These results suggested that ZnO NPs disrupted the metabolism process of zinc and increased the intracellular ROS and MDA content, which led to oxidative stress and further promoted apoptosis. In addition, ZnO NPs can decreased intracellular Ca²⁺ -ATPase and Na⁺/K⁺-ATPase activity, disorder Iron channel, destruct of intracellular ion balance, and further cause apoptosis.

• Key words：ZnO NPs  rat tracheal epithelial cells  apoptosis  oxidative stress  iron channel

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