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目的初步探讨橡胶防老剂N-异丙基-N’-苯基对苯二胺(4010NA)对细胞的毒性和致凋亡作用及对caspase-3、caspase-8蛋白表达的影响。方法以不同终浓度的4010NA(0、3、6、12、15和18μg/ml)处理正常人胚肺成纤维细胞(HELF)24 h后,应用MTT(噻唑蓝)比色实验、Hoechst 33258染色实验、western blotting法观察细胞增殖抑制率、细胞凋亡率和caspase-3、caspase-8的表达变化。结果 4010NA处理正常人胚肺成纤维细胞后,与阴性对照组比较,各剂量组的细胞生长抑制率和细胞凋亡/坏死率明显上升,并随着剂量的增加呈现剂量-反应关系,caspase-3、caspase-8的表达变化随剂量增加呈先增强后降低的趋势。结论橡胶防老剂4010NA对正常人胚肺成纤维细胞有细胞毒性,在低浓度时导致细胞程序性凋亡,其机制是启动细胞外凋亡信号通路;高浓度时则可直接导致HELF细胞的坏死。
Objective To investigate the effects of rubber antioxidant N-isopropyl-N’-phenyl-p-phenylenediamine (4010NA) on the cytotoxicity, apoptosis and the expression of caspase-3 and caspase-8 proteins. Methods Normal human embryonic lung fibroblasts (HELF) were treated with different concentrations of 4010NA (0, 3, 6, 12, 15 and 18μg / ml) for 24 h. MTT colorimetric assay, Hoechst 33258 staining The changes of cell proliferation inhibition rate, apoptosis rate and the expression of caspase-3, caspase-8 were observed by western blotting. Results 4010NA treated normal human embryo lung fibroblasts, compared with the negative control group, the cell growth inhibition rate and apoptosis / necrosis rate increased significantly with dose, the dose-response relationship, caspase- The expression of caspase-8 increased with the increase of dose and then decreased. CONCLUSION: The rubber antioxidant 4010NA is cytotoxic to normal human embryo lung fibroblasts and causes programmed cell apoptosis at low concentration. The mechanism is to activate the extracellular apoptotic signaling pathway. At high concentrations, it can directly induce the necrosis of HELF cells .