目的

探讨纳米金棒 （AuNRs）对A549 细胞的生物毒性效应及其可能机制。

方法

将正常培养A549 细胞分别给予0、0.5、1、2、4 μg/mL AuNRs 处理6、12、24 h 后进行检测。使用CCK-8 实验、LDH 实验分别观察细胞活力与细胞膜损伤；使用透射电镜观察AuNRs 在细胞内的分布和细胞形态；使用Western blot 检测自噬相关蛋白ATG16、ATG4、Beclin1、LC3- Ⅱ、P62 的表达；使用激光扫描共聚焦显微镜检测ROS；使用试剂盒检测GSH/GSSG、T-AOC。数据方差齐性检验后进行单因素方差分析；组间两两比较采用Student-Newman-Keuls 检验。

结果

CCK-8 实验结果表明，与0 μg/mL 相比，4 μg/mL AuNRs 处理A549 细胞6 h 后细胞活力[（80.05 ± 3.45）%比 （100 ± 2.47%）］降低 （P ＜ 0.01）；2、4 μg/mL AuNRs 处理A549 细胞12 h 后细胞活力[（71.36 ± 3.87）%、（39.47 ± 4.16）%比 （100 ± 3.12）%］降低 （P ＜ 0.01）；1、2、4 μg/ mL AuNRs 处理A549 细胞24 h 后细胞活力[（91.83 ± 1.98）%、（56.53 ± 3.57）%、（28.65 ±3.09）%比 （100 ± 2.34）%］降低 （P ＜ 0.01）。LDH 实验表明，细胞LDH 渗漏增加且具有统计学意义 （P ＜ 0.01）。4 μg/mL AuNRs 处理6 h 后，透射电镜观察发现AuNRs 能够进入A549 细胞并以单个颗粒或聚集体形式存于细胞质和部分膜囊泡中。Western blot 结果表明，分别用0、0.5、1、2、4 μg/mL AuNRs 处理A549 细胞6 h 后，ATG16、ATG4、Beclin1 和LC3-Ⅱ蛋白表达增加，而自噬底物蛋白P62 表达降低 （P ＜ 0.01）。给予100 μmol/L MnTBAP 或10 mmol/L NAC 处理，AuNRs 引起细胞ROS 和LC3 表达增加能够被逆转，同时T-AOC [（92.37 ± 8.49）%比 （68.46 ±6.38）%，（110.49 ± 7.39）%比 （51.26 ±7.39）%］、GSH/GSSG[（92.56 ± 5.52）%比 （62.48 ±5.52）%，（104.49 ± 4.84）%比 （57.39 ± 4.84）%］水平升高 （P 均 ＜ 0.05）。

结论

AuNRs 能够进入A549 细胞引发细胞毒性效应，且细胞毒性效应呈剂量、时间依赖性增加，其机制可能与AuNRs 导致A549 细胞抗氧化应激能力降低进而引起细胞自噬有关。

Objective

To investigate the biological toxic effect and mechanisms of gold nanorods （AuNRs） on A549 cells.

Methods

Normal cultured A549 cells were treated with different concentrations of AuNRs for 6， 12 or 24 hours for subsequent detection. CCK-8 test and LDH test were used to observe cell viability and cell membrane damage. The distribution and morphology of AuNRs in cells were observed by transmission electron microscope. Western blot was used to detect the expression of autophagy related proteins ATG16， ATG4， Beclin1， LC3-Ⅱ and P62. ROS was detected by laser scanning confocal microscope. The levels of GSH/GSSG and T-AOC were detected by kits. One way ANOVA was performed after the homogeneity test of data variance and Student- Newman-Keuls test was used for pairwise comparison between different groups.

Results

CCK-8 results showed that compared with the 0 μg/mL， the cell viability of the 4 μg/ mL AuNRs [（80.05 ± 3.45）% vs （100 ± 2.47）%］ was decreased at 6 h after treated with AuNRs （P ＜0.01）. The cell viability of 2 μg/mL and 4 μg/mL AuNRs [（71.36 ± 3.87）% and （39.47 ± 4.16）%vs （100 ± 3.12）%］ were decreased （P ＜ 0.01） at 12 h after treated with AuNRs； The cell viability of 1 μg/mL， 2 μg/mL and 4 μg/mL AuNRs [（91.83 ± 1.98）%， （56.53 ± 3.57）% and （28.65 ± 3.09）%vs （100 ± 2.34）%］ were decreased （P ＜ 0.01） at 24 h after treated with AuNRs. LDH test showed that the leakage of LDH was increased significantly （P ＜ 0.01）. Transmission electron microscopy showed that AuNRs could enter A549 cells and exist in the cytoplasm and some membrane vesicles in the form of single particles or aggregates 6 h after treatment with 4 μg/mL AuNRs. Western blot showed that after treated with different concentrations of AuNRs （0， 0.5， 1， 2， 4 μg/mL）for 6 h， the expression levels of ATG16， ATG4， Beclin1 and LC3- Ⅱ proteins were significantly increased， while the expression level of autophagy substrate protein P62 was significantly decreased in A549 cells（P ＜ 0.01）. After treated with 100 μmol/L MnTBAP or 10 mmol/L NAC， the increased expression of ROS and LC3 induced by AuNRs could be reversed， and the content of T-AOC [（92.37 ± 8.49）%vs （68.46 ± 6.38）%， （110.49 ± 7.39）% vs （51.26 ± 7.39）%］ and GSH/GSSG [（92.56 ± 5.52）%vs （62.48 ± 5.52）% ， （104.49 ± 4.84）% vs （57.39 ± 4.84）%］ were increased （all P ＜ 0.05）.

Conclusion

AuNRs can enter A549 cells and induce cytotoxicity effect， which in a dose-dependent and time-dependent manner. The mechanism may be related to the reduction of antioxidative stress ability of A549 cells caused by AuNRs.