Li Xin, Chan Lijun, Zhang Huajun, Zhang Hongmei, Niu Qiao
Center of Disease Control of Taiyuan Iron Steel Company, Taiyuan 030003, China.
E-mail:
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2014 Mar;32(3):202-6.
To study the effect of arsenic on neuronal cell apoptosis and the mRNA and protein expression of calpain 1, calpain 2, and cyclin-dependent kinases 5 (cdk5)/p25 and to provide a scientific basis for the research on neurotoxic mechanism of arsenic trioxide (As2O3).
Primary cultured rat neurons were divided into untreated control group, dimethyl sulfoxide (DMSO) solvent control group, and 1, 5, and 10 µmol/L As2O3 treated groups. Eight hours after being treated with As2O3, cell apoptosis rate was determined by flow cytometry, the mRNA expression of calpain 1, calpain 2, cdk5, and p35 was measured by real-time fluorescence quantitative PCR, and the protein expression of calpain 1, calpain 2, cdk5, p35, and p25 was measured by Western blot.
Compared with those in the untreated control group and DMSO solvent control group, the cell apoptosis rates in the 5 and 10 µmol/L As2O3 treated groups were significantly increased (P < 0.05). The mRNA expression levels of calpain 1 were 6.36±3.26, 7.11±5.13, and 7.47±2.59 in the 1, 5, and 10 µmol/L As2O3 treated groups, respectively, and the mRNA expression levels of cdk5 were 1.27±0.19, 1.54±0.04, and 1.79±0.21 in the 1, 5, and 10 µmol/L As2O3 treated groups, respectively, which were significantly higher than those in the untreated group (0.72±0.15 and 1.77±0.87) and those in the DMSO solvent control group (0.96±1.23 and 1.18±0.09) (P < 0.05). The mRNA expression levels of p35 in the 1 and 5 µmol/L As2O3 treated groups were 2.17±0.59 and 2.51±0.51, respectively, which were significantly higher than that in the untreated control group (1.26±0.37) (P < 0.05). The protein expression levels of calpain 1 were 0.37±0.10, 0.42±0.13, and 0.51±0.18 in the 1, 5, and 10 µmol/L As2O3 treated groups, respectively, which were significantly higher than those in the untreated control group (0.11±0.08) and DMSO solvent control group (0.13±0.07) (P < 0.05). In the 5 and 10 µmol/L As2O3 treated groups, the protein expression levels of cdk5 were 0.34±0.12 and 0.37±0.21, while the protein expression levels of p25 were 0.31±0.23 and 0.55±0.16, all of which were significantly higher than those in the untreated control group and DMSO solvent control group (P < 0.05). The protein expression levels of p35 were reduced in the 5 µmol/L As2O3 treated group (0.31±0.23) and 10 µmol/L As2O3 treated group (0.26±0.16), as compared with those in the untreated control group and DMSO solvent control group (P < 0.05). The mRNA and protein expression of calpain 2 showed no significant differences between all groups (P > 0.05).
The calpain 1-cdk5/p25 pathway may be involved in the process of As2O3-induced neuronal cell apoptosis.
研究砷对神经元细胞凋亡及钙蛋白酶1、钙蛋白酶2、细胞周期蛋白依赖性激酶5（cdk5）/p25的mRNA和蛋白表达的影响，为三氧化二砷（As2O3）神经毒性机制的研究提供科学依据。
将原代培养的大鼠神经元分为未处理对照组、二甲基亚砜（DMSO）溶剂对照组以及1、5和10 μmol/L As2O3处理组。用As2O3处理8小时后，通过流式细胞术测定细胞凋亡率，用实时荧光定量PCR检测钙蛋白酶1、钙蛋白酶2、cdk5和p35的mRNA表达，用蛋白质免疫印迹法检测钙蛋白酶1、钙蛋白酶2、cdk5、p35和p25的蛋白表达。
与未处理对照组和DMSO溶剂对照组相比，5和10 μmol/L As2O3处理组的细胞凋亡率显著升高（P < 0.05）。1、5和10 μmol/L As2O3处理组中钙蛋白酶1的mRNA表达水平分别为6.36±3.26、7.11±5.13和7.47±2.59，cdk5的mRNA表达水平分别为1.27±0.19、1.54±0.04和1.79±0.21，均显著高于未处理组（0.72±0.15和1.77±0.87）以及DMSO溶剂对照组（0.96±1.23和1.18±0.09）（P < 0.05）。1和5 μmol/L As2O3处理组中p35的mRNA表达水平分别为2.17±0.59和2.51±0.51，显著高于未处理对照组（1.26±0.37）（P < 0.05）。1、5和10 μmol/L As2O3处理组中钙蛋白酶1的蛋白表达水平分别为0.37±0.10、0.42±0.13和0.51±0.18，显著高于未处理对照组（0.11±0.08）和DMSO溶剂对照组（0.13±0.07）（P < 0.05）。在5和10 μmol/L As2O3处理组中，cdk5的蛋白表达水平分别为0.34±0.12和0.37±0.21，p25的蛋白表达水平分别为0.31±0.23和0.55±0.16，均显著高于未处理对照组和DMSO溶剂对照组（P < 0.05）。与未处理对照组和DMSO溶剂对照组相比，5 μmol/L As2O3处理组（0.31±0.23）和10 μmol/L As2O3处理组（0.26±0.16）中p35的蛋白表达水平降低（P < 0.05）。各组间钙蛋白酶2的mRNA和蛋白表达无显著差异（P > 0.05）。
钙蛋白酶1-cdk5/p25通路可能参与As2O3诱导的神经元细胞凋亡过程。
