四硫代钼酸铵作为新型硫化氢供体的鉴定及其对皮肤细胞的保护作用  

Identification of ATTM as a novel H_2 S donor and investigation of its protective effect on Ha Ca T skin cells

作　　者：孟富慧 陈利[1] 徐适[2] 鲜明[2] 张辉[1] 李建华[1] 董颀[1] 杨春涛[1] 

机构地区：[1]广州医科大学生理教研室,广东广州511436 [2]华盛顿州立大学化学教研室,美国华盛顿州普尔曼市99164

出　　处：《中国病理生理杂志》2015年第12期2271-2276,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81200606);广东省科技计划(No.2012B031800313);广东省自然科学基金资助项目(No.2015A030313458);广州市属高校科技计划(No.1201410467);广州市科技计划(No.201510010201)

摘　　要：目的：本研究旨在检测金属络合物四硫代钼酸铵（ATTM）的硫化氢（H2S）释放能力并进一步观察其减轻氧化应激诱导的皮肤细胞损伤的作用。方法：反应瓶法检测ATTM在细胞培养基中释放的H2S,二氯二氢荧光素二乙酸酯或罗丹明123染色结合荧光显微镜照相术分别检测细胞内活性氧簇（ROS）的含量和线粒体膜电位（ΔΨm）,用试剂盒检测细胞存活率和乳酸脱氢酶（LDH）的释放。结果：与H2S供体GYY4137类似,ATTM在细胞培养基中可剂量依赖性地释放H2S。在25~400μmol/L的浓度范围内,ATTM处理对人皮肤细胞（Ha Ca T细胞）的存活率无明显影响（P〉0.05）。紫外线照射或外源性给予ROS供体（过氧化氢,H2O2）处理均可增加Ha Ca T细胞内ROS的含量。400μmol/L H2O2处理可明显降低Ha Ca T细胞的存活率（P〈0.01）,而在H2O2处理前给予ATTM预处理,细胞存活率明显提高,其中在100和200μmol/L浓度时ATTM具有最好的细胞保护作用（P〈0.01）。400μmol/L H2O2处理还可损害细胞的ΔΨm和细胞膜并使LDH释放增加（P〈0.01）。而在细胞遭受损伤前给予200μmol/L ATTM预处理可明显改善ΔΨm的水平（P〈0.05）并抑制LDH从细胞内释放（P〈0.01）。结论：ATTM具有释放H2S的能力并可保护人皮肤细胞对抗氧化应激诱导的损伤效应。AIM： To investigate the ability of a metal complex ammonium tetrathiomolybdate（ ATTM） to release H2 S and its cytoprotective effect on an oxidative injury model. METHODS： Released H2 S was absorbed in a reaction flask from ATTM dissolved in the cell medium. Staining with dichlorodihydrofluorescein diacetate or rhodamine 123 followed by photofluorography was conducted for the observation of reactive oxygen species（ ROS） and mitochondrial membrane potential（ ΔΨm） levels,respectively. Cell viability and release of lactate dehydrogenase（ LDH） from the cells were measured with commercial kits. RESULTS： Similar to another H2 S donor GYY4137,ATTM had an ability to release H2 S in the cell medium in a dose-dependent manner. Treatment of human skin Ha Ca T cells with ATTM at concentrations of 25~ 400 μmol / L didn＇t significantly alter cell viability. Exposure of the cells to ultraviolet rays or a ROS donor H2O2 increased the intracellular ROS levels. Treatment with 400 μmol / L H2O2 significantly reduced the viability of Ha Ca T cells（ P〈0. 01）. However,before the treatment with H2O2,pretreatment with ATTM at 100 and 200 μmol/L markedly prevented the H2O2-induced cell injury（ P〈0. 01）. In addition,the treatment with H2O2 triggered ΔΨmloss（ P〈0. 01） and LDH release from the cells（ P〈0. 01）. Prior to suffering from H2O2 injury,the preconditioning with 200 μmol / L ATTM significantly improved ΔΨmlevels（ P〈0. 05） and attenuated LDH release from the cells（ P〈0. 01）. CONCLUSION：ATTM is capable of releasing H2 S and protecting human skin cells against oxidative injury.

关 键 词：HACAT细胞 硫化氢 四硫代钼酸铵 氧化应激 

分 类 号：R962[医药卫生—药理学] R75[医药卫生—药学]