纳米二氧化硅致Nrf-2基因缺陷人永生化表皮细胞氧化损伤的研究
Oxidative damage of Nrf-2 gene silenced human immortalized epidermal cells induced by silica nanoparticles
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摘要:
目的 探讨纳米二氧化硅(nano-SiO2)致核因子E2相关因子2(Nrf-2)基因缺陷型人永生化表皮细胞的氧化应激损伤效应。
方法 采用RNA干扰技术构建人永生化表皮细胞(HaCaT)Nrf-2基因缺陷细胞模型，以终质量浓度(后称浓度)为2.5、5.0、10.0 mg/L的nano-SiO2(15 nm粒径)分别染毒HaCaT细胞和Nrf-2基因缺陷细胞24 h，检测细胞内活性氧(ROS)、8-羟化脱氧鸟苷(8-oHdG)、丙二醛(MDA)、总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-Px)等氧化应激指标，并对细胞总蛋白和核蛋白中Nrf-2蛋白的表达水平进行分析。
结果 5.0、10.0 mg/L nano-SiO2可引起HaCaT细胞的活力明显降低(P＜0.05)，而2.5 mg/L nano-SiO2开始即可引起Nrf-2基因缺陷细胞活力呈剂量依赖性降低(r=-0.914，P < 0.05)，Nrf-2基因缺陷细胞的活力均明显低于同浓度组的HaCaT细胞(P＜0.05)。各浓度组细胞内ROS、8-oHdG和MDA水平均呈剂量依赖性增高，而T-SOD和GSH-Px的含量则呈剂量依赖性降低；与同浓度组HaCaT细胞相比，Nrf-2基因缺陷细胞中相关指标的变异程度更大，差异有统计学意义(P＜0.05)。正常HaCaT细胞中，低剂量组总Nrf-2蛋白和低、中剂量组核Nrf-2蛋白的表达水平增高(P＜0.05)；而随着nano-SiO2暴露浓度的进一步增高，两种蛋白的表达水平又呈降低的趋势。与HaCaT细胞的阴性对照比较，Nrf-2基因缺陷细胞中总Nrf-2蛋白表达水平下降，核Nrf-2蛋白表达水平未见差异。
结论 5.0 mg/L nano-SiO2可体外诱导HaCaT细胞发生氧化应激性损伤，Nrf-2基因缺陷可通过改变细胞的抗氧化防御能力，增加HaCaT细胞对nano-SiO2的敏感性。
Abstract:Objective To investigate the oxidative damage of nuclear factor E2-related factor 2 (Nrf-2) gene silenced human immortalized epidermal cells induced by silica nanoparticles (nano-SiO2).
Methods RNA interference technology was used to construct a Nrf-2 gene silenced cell model of human immortalized epidermal cells (HaCaT) (HaCaT-SiRNA-Nrf2). Then the HaCaT and HaCaT-SiRNA-Nrf2 cells were exposed to different final concentrations (2.5, 5.0, and 10.0 mg/L) of nano-SiO2 (15 nm) for 24 h. Cellular reactive oxygen species (ROS), 8-hydroxy-2-deoxyguanosine (8-oHdG), malondialdehyde (MDA), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) were detected for each group. Western blot was used to detect total protein and nuclear protein expression levels of Nrf-2.
Results The cell viability of HaCaT cells decreased significantly in the 5.0 and 10.0 mg/L nano-SiO2 groups (P < 0.05), but 2.5, 5.0, and 10.0 mg/L nano-SiO2 decreased the viability of HaCaT-SiRNA-Nrf2 cells in a dose-dependent manner (r=-0.914, P < 0.05), and the viabilities of HaCaT-SiRNA-Nrf2 cells were significantly lower in all dose groups compare with those of HaCaT cells (P < 0.05). The cellular ROS, 8-oHdG, and MDA levels significantly increased (P < 0.05), while the T-SOD and GSH-Px levels decreased (P < 0.05) in a dose-independent manner for both HaCaT and HaCaT-SiRNA-Nrf2 cells. The variances of related indicators for HaCaTSiRNA-Nrf2 cells were statistically higher than the variances for HaCaT cells in the same concentration group (P < 0.05). The expression levels of total Nrf-2 protein in low-dose group and the expression levels of nuclear Nrf-2 protein in low- & medium-dose groups were significantly increased in normal HaCaT cells (P < 0.05), then decreased with the increase of exposure concentrations. Total Nrf-2 protein expression levels in HaCaT-SiRNA-Nrf2 cells also showed a similar trend described above, but there was no significant difference in nuclear Nrf-2 protein expression levels compare with the HaCaT control group (P > 0.05).
Conclusion Nano-SiO2 could induce oxidative damage in HaCaT cells at 5.0 mg/L. Nrf-2 gene silencing would increase the sensitivity of HaCaT cells exposed to nano-SiO2 by changing antioxidant defense capability.
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