Rifampicin inhibits apoptosis in rotenone-induced differentiated PC12 cells by ameliorating mitochondrial oxidative stress
Rifampicin inhibits apoptosis in rotenone-induced differentiated PC12 cells by ameliorating mitochondrial oxidative stress
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DOI：
10.3969/j.issn.1673-5374.2010.04.002
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发表时间：
2010-02-01
影响因子：
6.1
通讯作者：
Tao, Enxiang
中科院分区：
文献类型：
--
作者：
Chen, Shiwen;Sun, Yuanlin;Tao, Enxiang
背景：已有研究表明利福平具有神经保护作用，但其确切机制尚不清楚。目的：探讨利福平预处理对鱼藤酮诱导分化的PC 12细胞线粒体氧化应激的保护作用。在中山大学附属第二医院神经内科进行了一项重复测量、基于细胞的研究，材料：PC 12细胞为中山大学中山医学院生理实验室捐赠。方法：在含10%胎牛血清的DMEM/F12培养基中，加入100 ng/mL 7S神经生长因子，培养9d，诱导PC 12细胞分化。根据不同的处理条件将细胞分为6组：对照组，用正常培养基培养，利福平组，用300 μ mol/L鱼藤酮处理26小时，鱼藤酮组，用2.5 μ mol/L鱼藤酮处理24小时，利福平预处理组，分别用100、200、300 μ mol/L利福平预处理2 h，再用2.5 μ mol/L鱼藤酮预处理24 h。采用荧光显微镜和流式细胞术，分别测定线粒体膜电位，使用罗丹明123染色。使用2 '，7'-二氯荧光素-二乙酸盐染色通过流式细胞术分析细胞内活性氧物质的形成，并使用酶标仪测量细胞内还原型谷胱甘肽。通过3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴化物测定法测定细胞活力。结果：鱼藤酮诱导分化后的PC 12细胞凋亡增加，线粒体膜电位降低，活性氧形成，还原型谷胱甘肽减少（P < 0.01）。结论：利福平预处理分化后的PC 12细胞，通过改善线粒体功能和氧化应激，阻断鱼藤酮诱导的细胞凋亡。
BACKGROUND: Previous studies have shown that rifampicin exhibits neuroprotective effects, but the precise mechanisms remain unclear. Rifampicin is thought to exert the neuroprotective effect as a hydroxyl free radical scavenger.OBJECTIVE: To investigate the protective effects of rifampicin pretreatment on rotenone-induced mitochondrial oxidative stress in differentiated PC12 cells.DESIGN, TIME AND SETTING: A repeated measure, cell-based study was performed at the Department of Neurology, Second Affiliated Hospital, Sun Yat-sen University, China between December 2007 and November 2008.MATERIALS: PC12 cells were a kind gift from the Physiology Laboratory of Zhongshan Medical School, Sun Yat-sen University, China. Rotenone and rifampicin were purchased from Sigma, USA.METHODS: PC12 cells were differentiated by culturing with 100 ng/mL 7S nerve growth factor for 9 days in Dulbecco's modified Eagle's medium/Nutrient Mix F12 (DMEM/F12) supplemented with 10% fetal bovine serum. The cells were assigned to six groups according to various treatment conditions: control, cultured with normal media; rifampicin group, treated with 300 mu mol/L rotenone for 26 hours; rotenone group, treated with 2.5 mu mol/L rotenone for 24 hours; rifampicin pretreatment groups, pretreated with 100, 200, and 300 mu mol/L rifampicin for 2 hours, respectively, followed by 2.5 mu mol/L rotenone for 24 hours.MAIN OUTCOME MEASURES: Mitochondrial membrane potential was measured by fluorescence microscopy and flow cytometry, respectively, using rhodamine 123 staining. Intracellular reactive oxygen species formation was analyzed by flow cytometry using 2', 7'-dichlorofluorescin-diacetate staining, and intracellular reduced glutathione was measured with a microplate reader. Cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Cell apoptosis was detected by Hoechst 33342 staining and flow cytometry.RESULTS: Increased apoptosis in rotenone-induced, differentiated, PC12 cells was accompanied by the loss of mitochondrial transmembrane potential, the formation of reactive oxygen species, and reduced glutathione depletion (P < 0.01). Rotenone-induced mitochondrial dysfunction was blocked in a dose-dependent manner by rifampicin (P < 0.05 or P < 0.01).CONCLUSION: Pretreatment of differentiated PC12 cells with rifampicin blocked rotenone-induced apoptosis by ameliorating mitochondrial dysfunction and oxidative stress.