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_(Cleaved-caspase-3)=13.47,P<0.05)。结论铀暴露诱导HK-2细胞内ROS爆发导致LMP,致使溶酶体内Cathepsin B泄露至胞质中进而触发溶酶体依赖性细胞死亡和线粒体途径的细胞凋亡。
Objective 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 600μmol/L uranyl acetate for 24 h,about 28-47 times or 2.4-6.0 times,respectively(tPI=30.40,10.34,P<0.05;t_(Cleaved-caspase-3)=18.49,9.52,P<0.05),and these increases were obviously diminished by CA-074 Me(tPI=6.76,P<0.05;t_(Cleaved-caspase-3)=13.47,P<0.05).Conclusions Exposure to uranyl acetate induces a burst of intracellular ROSthat leads to LMP and consequently causes leakage of cathepsin B from lysosomes to cytoplasm,in turn triggering the lysosomal-dependent cell death and mitochondrial-regulated apoptosis of HK-2 cells.
作者
钟登琴
李强
张旭霞
王梦梦
王睿云
陈红红
Zhong Dengqin;Li Qiang;Zhang Xuxia;Wang Mengmeng;Wang Ruiyun;Chen Honghong(Institute of Radiation Medicine,Shanghai Medical College,Fudan University,Shanghai 200032,China)
出处
《中华放射医学与防护杂志》
CAS
CSCD
北大核心
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)。
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
Uranium
Lysosomal membrane permeability
Reactive oxygen species
Lysosomal-dependent cell death
HK-2 cells
作者简介
通信作者:陈红红,Email:hhchen@shmu.edu.cn。
