抗霉素诱导肾小管上皮细胞铁死亡的实验研究  

Experimental study on ferroptosis of renal tubular epithelial cells induced by antimycin A

作　　者：陈芳[1] 胡韬韬[1] 陈丹[1] 陈菁[1] 黄晓丽[1] 梁鸿卿[1] 熊飞[1] 邹荣[1] CHEN Fang;HU Tao-tao;CHEN Dan;CHEN Jing;HUANG Xiao-li;L;XIONG Fei;ZOU Rong(Wuhan Integrated TCM ＆ Western Medicine Hospital,Wuhan 430022,China)

机构地区：[1]武汉市中西医结合医院,武汉430022

出　　处：《内科急危重症杂志》2018年第4期320-323,共4页Journal of Critical Care In Internal Medicine

基　　金：国家自然科学基金81600530;武汉市卫生和计划生育委员会科研项目(WX16C36)

摘　　要：目的:观察抗霉素诱导肾小管上皮细胞发生铁死亡(ferroptosis)的现象,初步探讨铁死亡参与急性肾损伤的病理生理机制。方法:以1、2. 5、5、10μmol/L抗霉素A(antimycin A)干预人近曲小管上皮细胞(HK-2) 2、4、8、16、24h。MTT法检测细胞存活率,乳酸脱氢酶检测细胞活性,透射电镜检测细胞线粒体形态,Western blot检测凋亡关键蛋白(caspase3)活化,流式细胞术检测细胞内活性氧(ROS)。结果:HK-2细胞存活率随着抗霉素作用剂量和时间的增加而下降(P <0. 01)。2. 5μmol/L抗霉素A干预HK-2细胞2h后,细胞上清液乳酸脱氢酶(LDH)释放明显增加(P <0. 01),电镜观察发现铁死亡特征性改变,细胞线粒体膜密度增加,线粒体嵴减少,细胞内ROS含量明显增加(P <0. 05)。与抗霉素模型组相比,铁螯合剂去铁胺(deferoxamine)、铁死亡特异性拮抗剂Ferrostatin-1预处理HK-2细胞能明显减少抗霉素A损伤后LDH的释放、线粒体损伤及ROS的产生(P <0. 05),且该过程不伴有caspase3活化。结论:在缺氧诱导肾小管上皮细胞损伤过程中,铁死亡可能是肾小管上皮细胞较早出现的损伤方式;抑制细胞铁死亡的发生可以减轻缺氧对肾小管上皮细胞的损害。Objective： To investigate the occurrence and molecular mechanisms of antimycin A-induced ferroptosis in renal tubular epithelial cells（ HK-2）. Methods： MTT assay was used to detect cell viability rate after treatment with 1,2. 5,5. 0,and 10. 0 μmol/L antimycin A. The leakage of lactate dehydrogenase（ LDH） was measured by LDH kit. The mitochondrion injury was observed by the electron microscopy. The activity of caspase-3 was measured by Western blotting. The LDH C11-BODIPY fluorescent probe was used to test the production of lipid ROS by flow cytometry. Results： MTT assay showed that antimycin A markedly increased the death of HK-2 cells in a certain period of time and concentration. The leakage of LDH distinctly increased after treatment with antimycin A in HK-2 cells. After treatment with antimycin A for 2 h,the characteristic mitochondrion injuries such as small mitochondria with condensed mitochondrial membrane densities and reduction of mitochondria crista were observed. The production of ROS detected by C11-BODIPY fluorescence probe was also increased（ P〈0. 05）. The pre-treatment with deferoxamine or ferrostatin-1 prevented the LDH leakage,mitochondria injuries and ROS increase without activation of caspase-3（ P〈0. 05）. Conclusion： Ferroptosis participated in the hypoxia injuries in renal tubular epithelial cells induced by antimycin A,which may be therapeutically targeted by ferroptosis inhibition.

关 键 词：铁死亡 急性肾损伤 抗霉素A 去铁胺 

分 类 号：R692.6[医药卫生—泌尿科学]