溶酶体膜通透化在铀诱导肾近端小管上皮细胞死亡中的作用及机制  

Mechanism of lysosomal membrane permeabilization in uranyl acetate-induced death of renal proximal tubule epithelial cells

作　　者：钟登琴 李强 张旭霞[1] 王梦梦 王睿云 陈红红[1] Zhong Dengqin;Li Qiang;Zhang Xuxia;Wang Mengmeng;Wang Ruiyun;Chen Honghong(Institute of Radiation Medicine,Shanghai Medical College,Fudan University,Shanghai 200032,China)

机构地区：[1]复旦大学上海医学院放射医学研究所,上海200032

出　　处：《中华放射医学与防护杂志》2022年第3期161-167,共7页Chinese Journal of Radiological Medicine and Protection

基　　金：国家自然科学基金面上项目(81972971)。

摘　　要：目的探究铀(Uranium,U)暴露诱导人肾近端小管上皮HK-2细胞溶酶体膜通透化(LMP)致细胞死亡的作用及机制。方法以100、300、600μmol/L铀染毒HK-2细胞24 h,分别采用DCFH-DA荧光探针法和MitoSOX荧光探针法检测不同浓度铀染毒的HK-2细胞内氧自由基(reactive oxygen species,ROS)和线粒体超氧化物的生成。将HK-2细胞分为:空白对照组、单纯N-乙酰半胱氨酸(NAC)或CA-074 Me组、单纯铀染毒组和铀联合NAC或CA-074 Me组,采用双色免疫荧光法检测HK-2细胞内半乳凝素-1(Galectin-1)与溶酶体相关膜蛋白-1(LAMP-1)共定位情况以检测溶酶体膜通透化(lysosomal membrane permeability,LMP)程度,或检测Cathepsin B与LAMP-1非共定位情况以反映溶酶体内Cathepsin B的释放情况,钙黄绿素(Calcein-AM)-碘化丙啶(PI)双染色法检测HK-2细胞死亡情况,单色免疫荧光法检测HK-2细胞内Cleaved-caspase-3表达以检测细胞凋亡情况。结果100、300、600μmol/L铀染毒HK-2细胞24 h诱导细胞内ROS或线粒体超氧化物生成与0μmol/L铀对照组比较明显增加,分别为1.1~2.5倍或4.0~28倍(t_(ROS)=17.98、11.84、11.75,P<0.05;t线粒体超氧化物=6.14、16.02、13.06,P<0.05),且随铀浓度增加而显著增加(t_(ROS)=10.10、10.37、5.59,P<0.05;t线粒体超氧化物=21.50、15.16、5.93,P<0.05)。与空白对照组相比,600μmol/L铀染毒24 h使HK-2细胞中Galectin-1和LAMP-1共定位率及Cathepsin B和LAMP-1非共定位率显著增加,分别为5.4~6.7倍或1.5~2.1倍(t_(Galectin-1)=15.85、12.70,P<0.05;t_(Cathepsin) B=5.95、6.69,P<0.05),而给予NAC处理则能抑制其增加(t_(Galectin-1)=4.74,P<0.05;t_(Cathepsin) B=4.51,P<0.05);而且,600μmol/L铀染毒24 h使HK-2细胞死亡率及Cleaved-caspase-3表达水平较空白对照组显著增加,分别为28~47倍或2.4~6.0倍(tPI=30.40、10.34,P<0.05;t_(Cleaved-caspase-3)=18.49、9.52,P<0.05),而给予CA-074 Me处理则能显著降低铀暴露HK-2细胞的死亡率及Cleaved-caspase-3表达水平(tPI=6.76,P<0.05;t_(CObjective To explore the mechanism of lysosomal membrane permeabilization(LMP)inuranyl acetate-induced death of human kidney proximal tubular epithelial HK-2 cells.Methods HK-2 cells were exposed to uranyl acetate at concentrations of 100,300 and 600μmol/L for 24 h,then in tracellular reactive oxygen species(ROS)and mitochondrial superoxide were measured by DCFH-DA and MitoSOX probe,respectively.HK-2 cells were divided into four groups:blank control group,NAC or CA-074 Me group,uranyl acetate exposure group and uranyl acetate exposure plus NAC or CA-074 Me group.Two-color immune of luorescence staining was used to detect the co-localization of galectin-1 and lysosomal associated membrane protein-1(LAMP-1)to measure the extent of LMP,and to detect the non-co-localization of cathepsin B and LAMP-1 to reflect the release of cathepsin B in lysosomes.Calcein-AM/PI double staining method was used to detect cell death.One-color immune of luorescence staining of cleaved-caspase-3 expression was used to detect apoptosis.Results Intracellular ROS and mitochondrial superoxide levels were significantly increased in HK-2 cells after exposure with 100,300 and 600μmol/L uranyl acetate for 24 h,about 1.1-2.5 times or 4.0-28 times,respectively(t_(ROS)=17.98,11.84,11.75,P<0.05;tmitochondrial superoxide=6.14,16.02,13.06,P<0.05),and they also increased with uranyl acetate concentrations(t_(ROS)=10.10,10.37,5.59,P<0.05;tmitochondrial superoxide=21.50,15.16,5.93,P<0.05).The percentage of co-localization of galectin-1 and LAMP-1 and the percentage of non-co-localization of cathepsin B and LAMP-1 were markedly increased in HK-2 cells after exposure with 600μmol/L uranyl acetate for 24 h,5.4-6.7 times or 1.5-2.1 times,respectively(t_(Galectin-1)=15.85,12.70,P<0.05;t_(Cathepsin) B=5.95,6.69,P<0.05),but these increases were inhibited by NAC(t_(Galectin-1)=4.74,P<0.05;t_(Cathepsin) B=4.51,P<0.05).Moreover,the cell death rate and the cleaved-caspase-3 expression level were also significantly increased in HK-2 cells after exposure with 600

关 键 词：铀 溶酶体膜通透化 氧自由基 溶酶体依赖性细胞死亡 HK-2细胞 

分 类 号：X503[环境科学与工程—环境工程] R114[医药卫生—卫生毒理学]