p25-CDK5-p53信号通路在甲醇致人神经母细胞瘤细胞凋亡中的作用
Role of p25-CDK5-p53 signaling pathway in methanol-induced apoptosis of human neuroblastoma cells
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摘要:
背景 职业接触甲醇后可引起中枢神经系统损伤。有研究表明，在应激或毒性刺激下，钙激活蛋白酶(calpain)使p35蛋白转化为p25蛋白，激活钙依赖性蛋白激酶5(CDK5)，p53蛋白积聚及磷酸化后出现神经细胞凋亡。甲醇是否可以激活calpain从而启动p25-CDK5-p53信号通路，通过经典线粒体途径使神经细胞凋亡，尚不明确。
目的 通过体外实验探究p25-CDK5-p53信号通路在甲醇致人神经母细胞瘤细胞(SK-N-SH细胞)凋亡中的作用。
方法 采用不同浓度甲醇(0、250、750、1 250 mmol/L，即对照、低、中、高浓度组)分别染毒SK-N-SH细胞24 h后，采用Annexin V-FITC/PI双染法检测细胞凋亡；实时荧光定量PCR技术检测calpain2、CDK5、p35、p25、p53 mRNA表达水平；Western blotting检测calpain2、CDK5、p25、p35、p53及p53磷酸化蛋白表达水平。
结果 对照组及甲醇低、中、高浓度组处理24 h后细胞凋亡率分别为(8.15±1.77)%、(12.06±0.71)%、(13.81±0.67)%、(19.88±2.40)%，组间差异有统计学意义(F=29.05，P < 0.01)。各浓度组calpain2、CDK5、p25、p53 mRNA相对表达水平差异均有统计学意义(F=10.02、61.48、28.91、21.12，P < 0.01)，各浓度组p35 mRNA相对表达水平差异无统计学意义(F=0.96，P>0.05)。与对照组相比，甲醇低、中浓度组calpain2、CDK5、p25 mRNA相对表达水平差异均无统计学意义(P>0.05)，甲醇中浓度组p53 mRNA相对表达水平升高(P < 0.01)，高浓度组calpain2、CDK5、p25、p53 mRNA相对表达水平升高(P < 0.01)。各浓度组calpain2、CDK5、p35、p25、p53磷酸化蛋白、p53蛋白相对表达水平差异具有统计学意义(F=32.29、17.60、35.02、35.92、93.72、11.00，P < 0.05)。与对照组相比，低、中、高浓度组calpain2、CDK5、p35、p25、p53磷酸化蛋白相对表达水平升高(P < 0.05)，中、高浓度组p53蛋白相对表达水平升高(P < 0.05)，低浓度组p53蛋白相对表达水平差异无统计学意义(P>0.05)。
结论 甲醇染毒后可使calpain2、p35、p25及CDK5蛋白表达水平增加，p53蛋白积聚及其磷酸化，和SK-N-SH神经细胞凋亡。
Abstract:Background Occupational exposure to methanol can cause damage to the central nervous system. Studies have shown that calpain can convert p35 into p25, activating calcium-dependent protein kinase 5 (CDK5) and leading to the accumulation and phosphorylation of p53 and the apoptosis of nerve cells. It is unclear whether methanol can activate calpain, initiate the p25-CDK5-p53 signaling pathway, and cause neuronal apoptosis through the classical mitochondrial pathway.
Objective This in vitro experiment is designed to investigate the role of p25-CDK5-p53 signaling pathway in the apoptosis of human neuroblastoma cells (SK-N-SH cells) induced by methanol.
Methods SK-N-SH cells were exposed to 0, 250, 750, and 1 250 mmol/L methanol (named control, low, medium, and high concentration groups, respectively) for 24 h. The apoptosis rate was detected with Annexin V-FITC/PI apoptosis kit. The relative mRNA expression levels of calpain2, CDK5, p35, p25, and p53 were measured by real-time fluorescence quantitative PCR.The protein expression levels of calpain2, CDK5, p25, p35, p53, and phosphorylated p53 were determined by Western blotting.
Results After 24 h of methanol exposure, the apoptosis rates of the control, low, medium, and high concentration groups were (8.15±1.77)%, (12.06±0.71)%, (13.81±0.67)%, and (19.88±2.40)%, respectively, and the difference among the four groups was statistically significant (F=29.05, P < 0.01). The difference of relative mRNA expression levels of calpain2, CDK5, p25, and p53 among the groups were statistically significant (F=10.02, 61.48, 28.91, and 21.12, respectively, P < 0.01), except the relative mRNA expression level of p35 (F=0.96, P>0.05). Compared with the control group, the relative mRNA expression levels of calpain2, CDK5, and p25 in the low and medium methanol concentration groups were not significantly different (P>0.05), the relative mRNA expression level of p53 in the medium methanol concentration group was increased (P < 0.01), and the relative mRNA expression levels of calpain2, CDK5, p25, and p53 in the high concentration group were increased (P < 0.01). The relative protein expression levels of calpain2, CDK5, p35, p25, phosphorylated p53, and p53 were significantly different among the designed groups (F=32.29, 17.60, 35.02, 35.92, 93.72, and 11.00, respectively, P < 0.05). Compared with the control group, the relative protein expression levels of calpain2, CDK5, p35, p25, and phosphorylated p53 were increased in the low, medium, and high concentration groups (P < 0.05), the relative protein expression levels of p53 were increased in the medium and high concentration groups (P < 0.05), and there was no significant difference in the relative protein expression level of p53 between the low concentration group and the control group (P>0.05).
Conclusion Methanol exposure may increase the expression levels of calpain2, p35, p25, and CDK5 proteins, induce p53 protein accumulation and phosphorylation, and result in the apoptosis of SK-N-SH cells.
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