摘要:
为探讨邻苯二甲酸二乙基己酯((DEHP)的细胞免疫毒性作用与机制,采用RT-PCR和ELISA方法,考察了0.05~1 μmol· L-1浓度范围内的DEHP对THP-1细胞白细胞介素-1β(IL-1β)及基质金属蛋白酶-8(MMP-8)基因和蛋白表达的影响;采用免疫印迹Western Blot方法检测DEHP对ERK1/2磷酸化水平的影响;以2',7'-二氯荧光素二乙酸酯(DCFH-DA)为荧光探针检测1~50 μmol· L-1DEHP对细胞内活性氧(ROS)产生的影响。结果显示,0.05和0.2 μmol· L-1 DEHP在6 h内显著诱导IL-1β和MMP-8基因表达(P <0.05或0.01);0.05~1 μmol· L-1 DEHP刺激细胞48 h,可诱导IL-1β蛋白表达,并表现出明显的剂量-效应关系,线性拟合的确定系数为0.937;0.05 μmol· L-1DEHP刺激细胞6或12 h,显著诱导MMP-8蛋白表达(P <0.05);0.2 μmol· L-1 DEHP在15~30 min内快速诱导ERK1/2磷酸化;1~50 μmol· L-1 DEHP浓度依赖性刺激细胞中ROS的产生;ERK/MAPK抑制剂PD98059显著抑制DEHP诱导的MMP-8分泌,但对IL-1β分泌未表现出抑制作用。研究表明,DEHP可能是经ERK/MAPK信号路径诱导MMP-8基因和蛋白的表达,经其他路径诱导IL-1β基因和蛋白的表达,诱导细胞内ROS的产生,从而激发炎症反应,进而损害免疫系统功能引发哮喘等炎症性疾病。此研究结果可为DEHP的暴露风险评估提供参考。
Abstract:
To investigate the cellular immunotoxicity of DEHP and its possible mechanism, RT-PCR and ELISA method were used to explore effect of DEHP on mRNA and protein expression of IL-1β and MMP-8 in THP-1 cells within the concentration range of 0.05 to 1 μmol·L-1. Western Blot method was applied to examine the effect of DEHP on the phosphorylation level of ERK1/2. The effect of 1-50 μmol·L-1 DEHP on the production of reactive oxygen species (ROS) was evaluated using 2', 7'-dichlorofluorescin diacetate (DCFH-DA) as a fluorescent probe. Results showed that DEHP (0.05 and 0.2 μmol·L-1) significantly induced mRNA expression of IL-1β and MMP-8 within 6 h (P < 0.05 or 0.01). DEHP (0.05-1 μmol·L-1) could induce the protein expression of IL-1β within 48 h stimulation, which displayed obvious dose-effect relationship with correlation coefficient of 0.937. MMP-8 protein expression was significantly enhanced by stimulation of 0.05 μmol·L-1 DEHP for 6 h or 12 h (P < 0.05). 0.2 μmol·L-1 DEHP rapidly induced the phosphorylation of ERK1/2 within 15-30 min. 1-50 μmol·L-1 DEHP dose-dependently stimulated the production of cellular ROS. ERK/MAPK inhibitor (PD98059) significantly suppressed DEHP-induced MMP-8 secretion, while it did not show inhibitory effect on IL-1β secretion. It was indicated that DEHP might induce the gene and protein expression of MMP-8 through ERK/MAPK signaling pathway, and induce the production of cellular ROS and the gene and protein expression of IL-1β through other pathways. The function of immune system was accordingly impaired and inflammatory disorders such as asthma were induced. This study could provide a reference for the risk assessment of DEHP exposure.
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