铅、镉、汞、砷联合暴露SH-SY5Y细胞损伤和可能机制初探

A preliminary investigation of cell damage and possible mechanisms in SH-SY5Y cells exposed to a combination of lead,cadmium,mercury and arsenic

目的探讨铅(lead,Pb)、镉(cadmium,Cd)、汞(mercury,Hg)和砷(arsenic,As)联合暴露对SH-SY5Y细胞损伤和可能作用机制。方法构建SH-SY5Y和HMC3 Transwell共培养细胞模型,另以SH-SY5Y细胞单细胞培养作为组织阴性对照,以1、10、20和40倍的Pb、Cd、Hg和As模拟混合物暴露浓度(metal mixture,MM)处理细胞,其中40 MM分别为Pb 2.00μmol/L、Cd 0.01μmol/L、Hg 0.07μmol/L和As 0.06μmol/L,以含10%胎牛血清和1%青链霉素的DMEM/F12培养基为重金属联合暴露的阴性对照;CCK8检测细胞存活率,Real-time PCR检测APP和MT1G转录水平;以ELISA检测培养基上清液中TNF-α、IL-6和IFN-γ的含量。为评估重金属联合暴露后共培养细胞的氧化应激水平,采用流式细胞术检测SH-SY5Y/HMC3暴露于0、1、10、20和40 MM金属混合物后的ROS水平,以DMEM/F12完全培养基单细胞培养SH-SY5Y作为组织阴性对照。结果Pb、Cd、Hg和As联合暴露后,共培养模型和单培养模型中的SH-SY5Y细胞生长受到抑制(P<0.05)、MT1G mRNA表达增加、APP mRNA无明显变化,上清液中TNF-α含量剂量依赖性增加趋势,IL-6和IFN-γ呈下降趋势;与单一SH-SY5Y细胞损伤指标的测定结果相比较,共培养模型中SH-SY5Y细胞生长抑制程度较轻,APP mRNA相对较低,MT1G mRNA较高,TNF-α含量增加幅度较小,IL-6和IFN-γ下降趋势更明显。随暴露浓度的升高,ROS相对含量呈剂量依赖性增加[F(4,10)=320.4,P<0.05]。ROS相对含量与MT1G呈正相关(r=0.98,P=0.0015)。结论重金属联合暴露的神经炎症反应与ROS生成相关联,共培养的HMC3细胞可减轻共培养模型中SH-SY5Y细胞损伤的程度,MT1G可能在SH-SY5Y细胞损伤过程中提供保护作用。

Objective To investigate the damaging effect and potential underlying mechanism of combination exposure to lead(Pb),cadmium(Cd),mercury(Hg),and arsenic(As)on SH-SY5Y cells.Methods A co-culture cell model of SH-SY5Y and HMC3 was established using the Transwell system,alongside a single cell culture of SH-SY5Y cells serving as a tissue-negative control.The cells were exposed to concentrations of 1,10,20,and 40 times the simulated mixture of Pb,Cd,Hg,and As(metal mixture,MM),with the highest concentration of 40 MM comprising 2.00μmol/L Pb,0.01μmol/L Cd,0.07μmol/L Hgand 0.06μmol/L As.A negative control for the mixture exposure was established using DMEM/F12 medium containing 10%fetal bovine serum and 1%penicillin.Cell viability was evaluated using the CCK8 assay,the expression levels of APP and MT1G transcripts were analyzed by Real-time PCR,and the levels of TNF-α,IL-6 and IFN-γin the culture medium supernatant were measured by ELISA.To assess the level of oxidative stress in the cells after exposure,flow cytometry was used to detect the level of ROS after exposure of SH-SY5Y/HMC3 to 0,1,10,20,and 40 MM of metal mixtures,and single-cell culture of SH-SY5Y in DMEM/F12 complete medium was used as a tissue-negative control.Results After the combined exposure of Pb,Hg,Cd and As,both the co-culture and mono-culture models of SH-SY5Y cells exhibited inhibited growth(P<0.05),and increased expression of MT1G mRNA.No significant changes were observed in APP mRNA levels.The supernatant showed an increasing trend in TNF-αcontent and a dose-dependent decreasing trend in IL-6 and IFN-γ.Compared with the result of the single SH-SY5Y cell injury indicators,the co-culture model showed less growth inhibition of SH-SY5Y cells,relatively lower APP mRNA,higher MT1G mRNA,smaller increase in TNF-αcontent,and more obvious decreasing trend of IL-6 and IFN-γ.Relative ROS levels increased dose-dependently with increasing exposure concentrations(F(4,10)=320.4,P<0.05).Relative ROS levels were positively correlated with MT1G(r=0.98,P=0.0015).Conclusion The neuroinflammatory response to mixed exposure to heavy metals was associated with ROS generation,co-cultured HMC3 cells attenuated the extent of SH-SY5Y cell injury in the co-culture model,and MT1G may provide a protective role in the process of SH-SY5Y cell injury.