纳米二氧化硅颗粒对血管内皮细胞的毒性及其氧化损伤作用

摘要

目的：探讨纳米二氧化硅（SiO2）颗粒的血管内皮细胞毒性，阐明其作用机制。方法：选用粒径约60 nm的纳米 SiO2颗粒，以体外培养的人脐静脉内皮细胞（HUVECs）为模型，分为对照组和纳米 SiO2颗粒暴露组（浓度分别为12.5、25.0、50.0和100.0 mg·L-1），采用 MTT法测定细胞活力；乳酸脱氢酶（LDH）释放法检测细胞膜的完整性；流式细胞术（FCM）检测细胞内活性氧（ROS）水平；实时荧光定量 PCR法检测细胞内核因子E2相关因子2（Nrf2）、血红素加氧酶1（HO-1）、超氧化物歧化酶2（SOD2）和γ-谷氨酰半胱氨酸合成酶催化亚单位（GCLC）mRNA表达水平。结果：MTT检测，与对照组比较，纳米 SiO2颗粒暴露组细胞活力降低，呈现明显的剂量依赖效应；当作用时间为12 h 时，仅100.0 mg· L-1纳米 SiO2颗粒暴露组细胞活力显著降低（P＜0.05）；当作用时间延长至24 h，25.0～100.0 mg· L-1纳米 SiO2颗粒暴露组细胞活力明显降低（P＜0.05）；同一浓度作用下，随着作用时间的延长，细胞活力也呈现下降趋势，呈时间效应关系。LDH 和 FCM检测，与对照组比较，除12.5 mg·L-1组外，其余纳米SiO2颗粒暴露组细胞培养液中LDH活力和细胞内ROS水平均明显升高（P＜0.05），且随着暴露剂量的增加而逐渐升高。实时荧光定量 PCR 法检测，与对照组比较，100.0 mg·L-1纳米 SiO2颗粒暴露组，细胞内 Nrf2、HO-1、SOD2和 GCLC mRNA表达水平均显著升高（P＜0.05）。结论：纳米 SiO2颗粒具有降低细胞活力、破坏细胞膜完整性、诱导 ROS生成和转录调控氧化还原因子等血管内皮细胞毒性，氧化损伤是纳米 SiO2颗粒发挥血管内皮细胞毒性的作用机制之一。%Objective To investigate the cytotoxicity of silica nanoparticles on vascular endothelial cells, and to clarify its action mechanism.Methods The 60 nm silica nanoparticle was selected and the invitro cultured human umbilical vein endothelial cells (HUVECs)were used as cell model.The HUVECs were divided into control and silica nanoparticle exposure groups with concentrations of 12.5,25.0,and 100.00 mg·L-1 .MTT assay was used for the determination of cell viability,lactate dehydrogenase (LDH)release assay for membrane integrity,flow cytometry (FCM)for intracellular reactive oxygen species (ROS)content,and real-time PCR assay for intracellular NF-E2-related factor 2 (Nrf2 ), heme oxygenase-1 (HO-1 ), superoxide dismutase 2 (SOD2 ) and glutamate-cysteine ligase catalytic subunit (GCLC)mRNA levels.Results The MTT results showed that the cell viabilities in each silica nnaoparticle exposure group were decreased compared with control group in a dose-dependent manner. Upon the silica nanoparticle exposure for 12 h,the cell viability was declined significantly only in 100 mg·L-1 exposure group compared with control group (P<0.05).When exposured for 24 h,the cell viabilities in 25.0, 50.0,and 100.0 mg·L-1 exposure groups were declined significantly compared with control group (P<0.05). Under the exposure to silica nanoparticle with the same dose, the cell viabilities were decreased along with the elongation of exposure time.LDH assay and FCM showed that except for that in 12.5 mg·L-1 exposure group, both the LDH activities in media and intracellular ROS levels in other exposure groups were increased compared with control group (P<0.05 ). The results of real-time fluorescence PCR showed that the mRNA levels of Nrf2, HO-1,SOD2 and GCLC in 100 mg·L-1 silica nanoparticle exposure group were increased significantly compared with control group (P<0.05).Conclusion Silica nanoparticles have toxicity to vascular endothelial cells,which includes reducing cell viability,membrane integrity destruction,induction of ROS generation,and tranSCriptional regulation of redox-related factors. Oxidative damage is one of the mechanisms of vascular endothelial toxicity mediated by silica nanoparticles.