环孢霉素A对葡萄糖氧化酶诱导的HepG2线粒体功能损伤和细胞凋亡的保护作用

doi:10.3969/j.issn.1004-616x.2014.01.003

癌变·畸变·突变

环孢霉素A对葡萄糖氧化酶诱导的HepG2线粒体功能损伤和细胞凋亡的保护作用

于卫华，刘江正，王钊，刘颖，海春旭*

（第四军医大学军事预防医学系毒理学教研室，陕西西安 710032）

收稿日期:2013-10-10 修回日期:2013-11-28 出版日期:2014-01-30 发布日期:2014-01-30
通讯作者: 海春旭，E-mail：cx-hai@fmmu.edu.cn
作者简介:于卫华(1988- )，男，河南项城人，硕士，研究方向：氧化损伤。Tel：029-84774882-804；E-mail：18392181838@163.com

Protective effects of cyclosporine A on glucose oxidase- induced HepG2 cell apoptosis and mitochondria dysfunction

YU Wei-hua，LIU Jiang-zheng，WANG Zhao，LIU Ying，HAI Chun-xu*

(Department of Toxicology， School of Preventive Medicine， the Forth Military Medical University， Xi’an 710032， Shaanxi, China)

Received:2013-10-10 Revised:2013-11-28 Online:2014-01-30 Published:2014-01-30
Contact: HAI Chun-xu，E-mail：cx-hai@fmmu.edu.cn

摘要/Abstract

摘要：

目的： 探讨环孢霉素A(CsA)对葡萄糖氧化酶(GOX)诱导的肝细胞线粒体功能损伤和细胞凋亡的影响。方法：HepG2细胞分别给予不同浓度的GOX和CsA处理，MTT 法检测细胞活力，确定所要选用的剂量和时间点。以10 μmol/L的CsA 预处理 2 h，并给予25 U/L GOX 处理12 h作为CsA+GOX组，同时设立空白对照组、GOX阳性对照组、CsA阴性对照组。分别采用Annexin V/PI双染色结合流式细胞术检测细胞凋亡率；GENMED活体细胞线粒体膜通道孔荧光试剂盒检测线粒体膜通透性转换；JC-1染色激光共聚焦检测线粒体膜电位变化；ATP检测试剂盒检测细胞内ATP水平；液相氧电极法检测线粒体耗氧量。结果：MTT 结果显示GOX能诱导HepG2细胞活力下降，且呈现时间和剂量效应关系(P均<0.05)；与空白对照组比较，25 U/L GOX作用12 h可诱导HepG2细胞凋亡，同时伴有线粒体膜通道开放和膜电位下降，细胞内ATP水平和线粒体耗氧量下降(P均<0.05)，而10 μmol/L的CsA预处理2 h有一定的保护作用，可以有效阻断线粒体结构功能的损伤，降低凋亡率，CsA+ GOX组与GOX阳性对照组比较差异均具有统计学意义(P均<0.05)。结论：CsA对氧化应激诱导的肝细胞线粒体功能损伤和凋亡具有保护作用。

关键词: 葡萄糖氧化酶, 环孢霉素A, 急性肝损伤, 线粒体膜电位, 能量供应

Abstract:

OBJECTIVE: To explore the protective effects of cyclosporine A on glucose oxidase-induced liver cell apoptosis and mitochondria dysfunction. METHODS：Various concentrations of GOX and CsA were applied to HepG2 cells at different time points， and measured the cell viability using MTT assay， and the appropriate doses and time points selected. Pretreatment of HepG2 cells with 10 μmol/L CsA for 2 h， then 25 U/L GOX treatment for 12 h were used as experimental group，and blank control (RPMI-1640 for 14 h)， GOX group as positive control (pretreatment with RPMI-1640 for 2 h， treatment with 25 U/L GOX for 12 h)，CsA group as negative control (pretreatment with 10 μmol/L CsA for 2 h， cultured with RPMI-1640 for 12 h). AnnexinV- FITC apoptosis detection kit was applied to assess apoptosis by flow cytometry； mitochondrial membrane permeability transition was evaluated with the GENMED MPTP fluorescent kits. Mitochondrial membrane potential was observed by means of JC-1 staining and the fluorescence intensity was measured by LSCM. The level of ATP in HepG2 cells was measured by using ATP Assay Kit； and the oxygen consumption was calculated using liquid-phase oxygen measurement system. RESULTS：MTT assay indicated that GOX decreased cell viability in dose and time dependent manners， and 10 μmol/L CsA treatment for 2 h had some protective effect. 25 U/L GOX treatment for 12 h resulted in HepG2 cells apoptosis， associated with MPTP opening， the collapse of mitochondrial membrane potential， and the reductions of the ATP level and oxygen consumption. Pretreatment with CsA could prevent structural and functional injuries of the mitochondrial， decreasing apoptosis rate. CONCLUSION：CsA could protect HepG2 cells against mitochondrial dysfunction and apoptosis.

Key words: glucose oxidase, cyclosporine A, acute liver injury, mitochondrial membrane potential, energy supply

于卫华，刘江正，王钊，刘颖，海春旭. 环孢霉素A对葡萄糖氧化酶诱导的HepG2线粒体功能损伤和细胞凋亡的保护作用[J]. 癌变·畸变·突变, doi: 10.3969/j.issn.1004-616x.2014.01.003.

YU Wei-hua，LIU Jiang-zheng，WANG Zhao，LIU Ying，HAI Chun-xu. Protective effects of cyclosporine A on glucose oxidase- induced HepG2 cell apoptosis and mitochondria dysfunction[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2014.01.003.

参考文献

[1] Talukder MA，Zweier JL，Periasamy M. Targeting calcium transport in ischaemic heart disease[J]. Cardiovasc Res， 2009， 84(3)：345-352.

[2] Nobili V，Pastore A，Gaeta LM，et al. Glutathione metabolism and antioxidant enzymes in patients affected by nonalcoholic steatohepatitis[J]. Clin Chim Acta，2005，355(1)：105-111.

[3] Madsen KG，Olsen J，Skonberg C，et al. Development and evaluation of an electrochemical method for studying reactive phase-I metabolites：correlation to in vitro drug metabolism[J]. Chem Res Toxicol，2007，20(5)：821-831.

[4] Szydlowska K，Tymianski M. Calcium，isehemia and excitotoxicity [J]. Cell Calcium，2010，47(2)：122-129.

[5] Rasheva Vl，Domingos PM. Cellular responses to endoplasmic reticulum stress and apoptosis[J]. Apoptosis， 2009， 14(8)： 996-1007.

[6]吴伟，徐蔚. 线粒体通透性转换孔结构和功能的研究进展[J]. 医学信息：上旬刊，2011，24(3)：1753-1754.

[7] Takeyama N，Miki S，Hirakawa A，et al. Role of mitochondrial permeability transition and cytochrome C release in hydrogen peroxide induced apoptosis[J]. Exp Celt Res， 2002， 274(1)： 16-24.

[8] Bernardi P. Mitochondrial transport of cations：channels， exchan- gers，and permeability transition[J]. Physiol Rev， 1999，79 (4)：1127-1155.

[9] Emerit J，Edeas M，Bricaire F. Neurodegenerative diseases and oxidative stress[J]. Biomed Pharmacother， 2004， 58(1)： 39-46.

[10] Kinnally KW，Antonsson B. A tale of two mitochondrial channels MAC and PTP in apoptosis[J]. Apoptosis，2007，12 (5)： 857-868.

[11] Perl A，Nagy G，Gergely P，et al. Apoptosis and mitochondrial dysfunction in lymphocytes of patients with systemic lupus erythematosus[J]. Methods Mol Med， 2004， 102(1)：87-114.

环孢霉素A对葡萄糖氧化酶诱导的HepG2线粒体功能损伤和细胞凋亡的保护作用

Protective effects of cyclosporine A on glucose oxidase- induced HepG2 cell apoptosis and mitochondria dysfunction

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 5

编辑推荐

Metrics

本文评价

[1]	孔德钦, 刘江正, 于卫华, 张涛, 龙子, 王欣, 张晓迪, 柏桦, 海春旭. 百草枯诱导AML12细胞凋亡过程中线粒体活性氧的作用[J]. 癌变·畸变·突变, 2016, 28(1): 1-7.
[2]	金磊, 王帅, 王欣, 海春旭, 李文丽. 高脂诱导BRL-3A细胞胰岛素抵抗模型的建立[J]. 癌变·畸变·突变, 2016, 28(1): 46-50,55.
[3]	张伟;王玲;徐培渝;肖恒怡. 姜黄素联合低浓度长春新碱抑制肝癌细胞系生长的实验研究[J]. , 2012, 24(4): 270-274,.
[4]	周小玲;孙平楠;康祥锦;刘冬玲;谢庆东. 乙肝病毒表面蛋白对人精子活力及线粒体膜电位的影响[J]. , 2010, 22(2): 100-103.
[5]	邹家杰;张　婷;汤树生;陈　倩;靳　溪;陈开跑;肖希龙. 喹乙醇诱导HepG2细胞线粒体的氧化损伤[J]. , 2009, 21(5): 372-376.