蛋白激酶C-δ抑制剂Rottlerin抑制造影剂诱导的肾小管上皮细胞凋亡  

Protein Kinase C-Delta Inhibitor Rottlerin Prevents Renal Tubular Epithelial Cells from Contrast-Media-Induced Apoptosis

作　　者：李晓宁[1] 肖劲松[2] 喻明霞[3] 胡雪峰 水华[1] 吴小燕[1] 

机构地区：[1]武汉大学中南医院肾内科,湖北武汉430071 [2]武汉大学中南医院神经内科,湖北武汉430071 [3]武汉大学中南医院检验科,湖北武汉430071 [4]湖北省鄂州市梁子湖区人民医院内科,湖北鄂州436060

出　　处：《武汉大学学报（医学版）》2011年第6期789-792,I0002,共5页Medical Journal of Wuhan University

摘　　要：目的:研究蛋白激酶C-δ(PKC-δ)抑制剂Rottlerin在造影剂诱导的肾小管上皮细胞凋亡中的作用及其机制。方法:体外培养的大鼠肾小管细胞(NRK-52E)分别与不同碘浓度(25,50,100,150gI/L)的碘帕醇(非离子型造影剂)共孵育2h;或者以100gI/L碘帕醇先后处理NRK-52E细胞0.5,1,2,4h,相同渗透浓度(420mOsm/kg)的甘露醇处理4h作为对照;部分细胞用2μmol/L Rottlerin与100gI/L碘帕醇共同处理2h。Hoechst 33342染色检测凋亡细胞核形态并计算凋亡细胞百分比;并以DEVD-AFC为底物,用荧光酶标仪测量Caspase-3酶活性;用DCFH-DA荧光探针检测细胞内活性氧产物(ROS)水平;用Western blot检测PKC-δ蛋白表达和磷酸化。结果:碘帕醇与NRK-52E细胞共孵育后,细胞内PKC-δ蛋白表达和Tyr-311位点磷酸化增加,细胞内ROS产生增加,Caspase酶活性上升。碘帕醇呈浓度和时间依赖性诱导NRK-52E细胞凋亡。Rottlerin可明显抑制以上变化。结论:PKC-δ抑制剂Rottlerin通过抑制PKC-δ活化和细胞内氧化应激抑制碘帕醇诱导的NRK-52E细胞凋亡,为造影剂肾病的预防和治疗提供了新靶点和新药物。Objective： To investigate the role of protein kinase C-delta（PKC-δ） inhibitor Rottlerin in regulating renal tubular cells apoptosis and the underlying mechanisms.Methods： Cultured renal tubular epithelial cells（NRK-52E cells） were treated with different concentrations of non-ionic contrast media,iopamidol（25,50,100,150 gI/L）,for 0.5,1,2,and 4 h respectively;or treated with 100 gI/L iopamidol with or without the presence of 2 μmol/L Rottlerin for 2 h;420 mOsm/kg mannitol was set as osmotic control.After iopamidol treatment,NRK-52E cells were stained with Hoechst 33342 for apoptosis percentage estimation.Caspase-3 activity was measured by using DEVD-AFC as substrate.Intracellular reactive oxygen species（ROS） was measured with DCFH-DA probe and flow cytometry.PKC-δ expression and phosphorylation were detected with Western blot.Results： Iopamidol induced PKC-δ expression and Tyr-311 site phosphorylation,ROS production,caspase activation and apoptosis.Iopamidol-induced NRK-52E cells apoptosis was both in dose-and time-dependent manners.Rottlerin inhibited all of those stress responses and apoptosis.Conclusion： Rottlerin inhibits contrast media-induced NRK-52E cells apoptosis via inhibiting PKC activation and intracellular oxidative stress.It may provide a new target and new drug in preventing and treating contrast-induced acute kidney injury.

关 键 词：造影剂 肾小管 细胞凋亡 蛋白激酶C-δ ROTTLERIN 

分 类 号：R692.9[医药卫生—泌尿科学] R981.1[医药卫生—外科学]