氯化镉对大鼠肾上皮细胞丝裂原活化蛋白激酶表达及其磷酸化的影响

Effect of cadmium chloride on the expression and phosphorylation of mitogen-activated protein kinase in normal rat kidney epithelial cells

目的 探讨氯化镉染毒大鼠正常肾上皮（normal rat kidney epithelial,NRK）细胞对丝裂原活化蛋白激酶（MAPK）表达及其磷酸化水平的影响.方法 应用不同剂量（0、1、5、10、20、40 μmol/L）氯化镉染毒NRK细胞24 h和同一剂量氯化镉（10 μmol/L）于不同时间（0、0.5、1.0、2.0、4.0、8.0 h）染毒NRK肾细胞.采用蛋白免疫印迹法检测氯化镉染毒后NRK细胞中MAPK表达水平,并用磷酸化抗体检测MAPK磷酸化水平.结果 不同氯化镉剂量和不同染毒时间染毒NRK细胞,发现MAPK表达无明显改变,但MAPK磷酸化蛋白表达量较对照组升高.氯化镉浓度在10 μmol/L时p-ERK1/2表达明显,表达量为1. 00±0.06;20 μmol/L和40 μmol/L剂量组表达量分别为2.58±0.11、2.76±0.23,比10 μmol/L组高1.58倍和1.76倍,差异有统计学意义（F=827.70,P＜0.01）;氯化镉浓度为20 μmol/L（2.47±0.20）和40 μmol/L（3.73±0.25）时p-p38MAPK表达量比对照组（1.00±0.02）高1.47倍和2.73倍,差异有统计学意义（F=280.06,P＜0.01）.p-ERK1/2和p-p38MAPK表达量与氯化镉浓度存在剂量-效应关系（p-ERK1/2相关系数为r=0.919,t=4.69,P=0.009;p-p38MAPK相关系数为r=0.945,t=5.79,P=0.004）.MAPK磷酸化蛋白表达水平也与染毒时间相关,p-ERK1/2在染毒1 h（1.26±0.11）时表达明显,染毒4 h（1.51±0.07）和8 h（3.53±0.23）时表达量是对照组（1.00±0.02）的1.5倍和3.5倍,差异有统计学意义（F=427.82,P＜0.001）;p-p38MAPK在染毒1 h（1.31±0.07）时表达也明显增加,染毒4 h（3.53±0.32）和8 h（4.41±0.38）时表达水平是对照组（1.00±0.03）的3.5倍和4.4倍,差异有统计学意义（F=280.06,P＜0.001）.结论 氯化镉染毒NRK细胞可引起MAPK蛋白磷酸化水平明显升高,可能与MAPK的激活有关.

Objective To explore the effect of cadmium chloride on the expression and phosphorylation of mitogen-activated protein kinase （MAPK） in normal rat kidney epithelial （NRK）cells. Methods The NRK cells were incubated with cadmium chloride either at respective dose （0,1,5,10,20,40 μmol/L） for 24 h or at same dose （10 μmol/L） for respective time （0,0. 5,1.0,2.0,4.0,8.0 h）. Western blotting was applied to test the expression of MAPK in NRK cells （ERK1/2,p38,JNK）;and phosphor-specific antibody to detect the phosphorylated MAPK （p-ERK1/2, p-p38, p-JNK）. Results There was no significant difference in the MAPK expression among the groups either treated with different doses or for different time; however,the level of phosphorylated MAPK was comparatively higher than it in control group. There was an obvious expression of p-ERK1/2 at 1.00 ±0. 06 in the group incubated with 10 μmol/L CdCl2; and the expression in the 20 μmol/L and 40 μmol/L CdCl2 group was 2. 58 ± 0. 11,2. 76 ±0. 23 respectively,which was 1.58 and 1.76 times more than it in 10 μmol/L CdCl2 group. The differences were statistically significant （F= 827.70, P〈 0. 01）. The respective expression of p-p38MAPK in the 20 μmol/L （2. 47 ±0. 20） and 40 μmol/L CdCl2 group （3.73 ±0. 25）was 1.47 and 2. 73 times more than it in control group （1.00 ± 0. 02）, and the differences were also statistically significant （F = 280. 86, P 〈0. 01）. There was a dose-effect relationship of the concentration of cadmium in the expression of p-ERK1/2（r = 0. 919, t = 4. 69, P = 0. 009） and p-p38MAPK （r = 0. 945, t = 5.79, P = 0. 004）. Additionally,phosphorylated MAPK expressed in a time-dependent manner. The expression of p-ERK1/2 was obvious in the group incubated for 1 h （1. 26 ±0. 11） ,and the respective expression in the 4 h group （1.51 ±0. 07）and 8 h group （3.53 ± 0. 23） was 1.5 and 3.5 times of it in the control group （1.00 ± 0. 02）. The differences were statistically significant （F = 427. 82, P 〈 0. 001）. The expression of p-p38MAPK increased significantly in 1 h group （1.31 ±0. 07）; while the respective expression in 4 h group （3.53 ±0. 32） and 8 h group （4. 41 ±0. 38） was 3.5 and 4. 4 times of it in control group （1.00 ±0. 03）. The differences were also statistically significant （F =280. 06,P〈0. 001）. Conclusion Cadmium chloride could significantly enhance the phosphorylation of MAPK in NRK cells; and it is probably associated with the activation of MAPK.