低渗非离子造影剂对肾小管上皮细胞凋亡的作用及其机制

Effect of low-osmolar non-ionic iodinated contrast medium on the apoptosis of renal tubniar epithelial cells and its mechanism

目的探讨低渗非离子型造影剂碘必乐诱导体外培养的人肾小管上皮细胞（HK-2细胞）凋亡的作用及机制。方法体外培养的HK-2细胞，分为阴性对照组、不同剂量（1．16、4．63、18．5、74、296gl/L）的碘必乐作用组（作用时间为6h）。通过流式细胞仪、Hoechst33258染色观察细胞凋亡的比例和形态学变化；Western印迹方法检测凋亡蛋白天冬氨酸半胱氨酸蛋白酶3（caspase-3）的表达水平，并选取作用最强的剂量（296gl／L）刺激2、4、6、12h，观察caspase-3表达变化。选取不同剂量碘必乐作用1h，与阴性对照组比较，观察细胞外信号调节激酶（ERK）和p38丝裂原活化蛋白激酶（MAPK）信号通路磷酸化水平的变化，并观察不同剂量p38MAPK抑制剂SB203580对碘必乐诱导的caspase-3和Bcl-2表达的影响。结果流式细胞仪检测结果示74、296gI／L碘必乐作用下细胞凋亡率较阴性对照显著升高（均P〈0．05）。Hoechst33258染色结果示296gI／L碘必乐可致明显的细胞凋亡形态学改变，凋亡细胞核呈致密浓染或核碎裂。Western印迹检测方法表明，碘必乐以剂量和时间依赖方式诱导细胞内caspase-3表达，以296gl/L作用12h表达最强；各剂量碘必乐作用下细胞内ERK1／2磷酸化水平与阴性对照组的差异均无统计学意义（均P〉0．05），而-定剂量的碘必乐处理组细胞内p38MAPK磷酸化水平较阴性对照组显著升高（均P〈0．05）。应用p38MAPK特异性抑制剂（30μmol／L）预处理2h可以阻断细胞内p38MAPK信号通路的磷酸化，抑制碘必乐诱导的细胞内caspase-3表达并部分上调Bcl-2表达，与碘必乐阳性对照组的差异均有统计学意义（P〈0．05）。结论低渗非离子型造影剂碘必乐以剂量和时问依赖方式诱导体外培养的HK-2细胞凋亡，其机制可能与上调caspase-3和下调抗凋亡蛋白Bcl-2有关，而p38MAPK信号通路的激活可能参与了该过程的调控。

Objective To evaluate the effect of low-osmolar non-ionic contrast medium （iopamidol） on apoptosis of human proximal tubular epithelial cells （HK-2） in vitro and to elucidate its possible mechanism. Methods Cultured HK-2 cells were divided into blank control group and iopamidol treated group （1.16, 4.63, 18.5, 74, 296 gI/L） and treated for 6 h. Then the HK-2 cells were treated with iopamidol（296 gI/L） for 0, 2, 4, 6, 12 h, respectively. The proportion and morphologic change of apoptotic cells were examined by flow eytometry and Hoechst 33258 staining. The protein expression of caspase-3 was determined by Western blotting. ERK phosphorylation （p-ERK1/2）/ERK1/2 and p-p38MAPK/p38MAPK were examined by Westernblotting 1 h after iopamidol treatment. The expression of caspase-3 and Bcl-2 was detected by Western blotting when HK-2 cells were pretreated with SB203580 （the inhibitor of p38MAPK）. Results The proportion of apoptotic cells treated with iopamidol of 74 gI/L and 296 gI/L increased significantly （all P〈0.05）. Iopamidol induced the expression of caspase-3 in HK-2 cells in dose- and time-dependent manner. The levels of p-ERK had no significant change in different iopamidol concentrations （all P〉0.05）. Iopamidol increased the level of p38MAPK significantly and the inhibitor of p38MAPK （30 μmol/L） significantly inhibited the production of caspase-3, but increased the level of Bcl-2 （P〈0.05）. Conclusion Iopamidol induces apoptosis of cultured HK-2 cells in dose- and time-dependent manner, which may be related to the activation of p38MAPK.