丙烯酰胺通过内质网应激诱导运动神经元VSC4.1细胞凋亡的研究
Acrylamide induced apoptosis in VSC4.1 cells through endoplasmic reticulum stress
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摘要:
目的 探讨丙烯酰胺对运动神经元VSC4.1细胞的毒性作用及可能机制。
方法 分别用质量浓度（以下简称"浓度"）为20、60、100 g/L丙烯酰胺（低、中、高暴露组）处理VSC4.1细胞48 h（未暴露丙烯酰胺的细胞作对照组），检测细胞的活力、凋亡情况、内质网应激凋亡途径关键因子C/EBP同源蛋白（CHOP）、天冬氨酸特异性半胱氨酸蛋白酶12（Caspase 12）和未折叠蛋白反应相关基因蛋白（GRP78、ATF6、IRE1）的表达水平，并观察内质网应激抑制剂（4-苯基丁酸）和抗氧化剂（牛磺酸）对丙烯酰胺致细胞毒性的影响。
结果 暴露组的细胞活力随着丙烯酰胺暴露浓度的升高而下降，分别为（88.5±6.2）%、（59.3±4.1）%和（37.6±3.4）%（χ趋势2=13.04，P < 0.05）与对照组（90.7±8.4）%相比，差异均有统计学意义（P < 0.05）。Hochest33258染色后，暴露组细胞核固缩浓染，发出较强蓝色荧光，呈凋亡特征性形态。暴露组的细胞凋亡率随丙烯酰胺暴露浓度的升高而增加，分别为（9.8±0.9）%、（27.4±2.3）%和（53.1±4.8）%（χ趋势2=6.98，P < 0.05）。除低暴露组的Caspase 12外，其余暴露组的CHOP和Caspase 12表达水平与对照组的差异均有统计学意义（P < 0.05）；暴露组GRP78、ATF6、IRE1的表达水平均随丙烯酰胺浓度的增加而升高（P < 0.05）。在中暴露组中加入低、中、高浓度（20、60、100 mg/L）的4-苯基丁酸，细胞活力分别是对照组（不加4-苯基丁酸的中暴露组）的（125±9）%、（154±12）%、（222±20）%，差异均有统计学意义（P < 0.05）。在中暴露组中加入低、中、高浓度（20、40、60 mmol/L）的牛磺酸，细胞凋亡率是对照组（不加牛磺酸的中暴露组）的（75±8）%、（60±6）%、（35±4）%，差异均有统计学意义（P < 0.05）；GRP78、ATF6、IRE1的表达也随牛磺酸浓度递增而降低，差异均有统计学意义（P < 0.05）。
结论 丙烯酰胺对VSC4.1细胞具有明显的毒性作用，可诱导细胞凋亡，其机制可能与内质网应激有关。
Abstract:Objective To explore the toxicity of acrylamide on motor neuron VSC4.1 cells and relevant mechanism.
Methods Acrylamide (20, 60, 100 g/L) was added into the culture medium of VSC4.1 cells, and the cells without acrylamide exposure were treated as controls. After 48 h, cell viability, staining, and apoptosis were evaluated. C/EBP-homologous proteins (CHOP), cysteine-containing aspartate-specific protease 12 (Caspase 12) and the unfolded protein response related gene (GRP78, ATF6, IRE1) were also examined. Meanwhile, the effects of endoplasmic reticulum stress inhibitor, 4-phenylbutyric acid (4-PBA), and antioxidant, taurine, on the VSC4.1 cells intoxicated with acrylamide were observed.
Results The cell viability was decreased with higher acrylamide concentrations, and the survival rates of the 20, 60, and 100 g/L acrylamide groups were (88.5±6.2)%, (59.3±4.1)%, and (37.6±3.4)%, respectively (χtrend2=13.04, P < 0.05), compared with the control group (P < 0.05). Nucleus pycnosis, strong blue fluorescence, and characteristic morphology of cell apoptosis were observed using Hochest 33258 staining after acrylamide exposure. The apoptosis rates of the 20, 60, and 100g/L acrylamide groups were (9.8±0.9)%, (27.4±2.3)%, and (53.1±4.8)%, respectively, compared with the control group (χtrend2=6.98, P < 0.05). The protein expression levels of CHOP and Caspase 12 were decreased markedly in each treated group compared with the control group (P < 0.05) except the Caspase 12 of the 20 g/L acrylamide group. After 20, 60, and 100 mg/L 4-PBA were added into the VSC4.1 cells intoxicated with 60 g/L acrylamide, the cell viabilities were (125±9)%, (154±12)%, and (222±20)%, respectively, of the cells without 4-PBA treatment (P < 0.05). After 20, 60, and 100mg/L taurine were added into the VSC4.1 cells intoxicated with 60 g/L acrylamide, the cell apoptosis rates were (75±8)%, (60±6)%, (35±4)%, respectively, of the cells without taurine treatment (P < 0.05). The expression levels of 3 CHOP (GRP78, ATF6, IRE1) were also decreased with higher levels of taurine (P < 0.05).
Conclusion Acrylamide could induce apoptosis in VSC4.1 cells and the relevant mechanism may be associated with endoplasmic reticulum stress.
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