鱼藤酮诱导PC12细胞凋亡及线粒体膜电位变化

Rotenone induces apoptosis of PC12 cells and alteration in mitochondrial membrane potential

目的探讨鱼藤酮对大鼠PC12细胞的毒性作用及可能机制。方法将培养的PC12细胞分为正常对照组、鱼藤酮50、100、500、1 000、2 000 nmol·L-1组,分别用MTT比色法检测细胞存活率,Hoechst 33258染色观察细胞核形态及凋亡比例,免疫组织化学法检测α-突触核蛋白(ɑ-synuclein)的表达,JC-1染色检测线粒体膜电位变化。结果 MTT检测发现,与正常对照组相比,鱼藤酮50、100、500、1 000、2 000nmol·L-1组细胞存活率下降(P<0.05),分别为(83.0±9.3)%、(79.5±11.8)%、(41.0±7.0)%、(26.4±5)%、(10.2±3.5)%。Hoechst 33258染色发现,鱼藤酮组细胞核固缩、裂解;与正常组比较,鱼藤酮组细胞凋亡率分别增加了6%,21%,64%,80%,89%,差异具有统计学意义(P<0.05)。免疫组化检测ɑ-synuclein结果显示,鱼藤酮组细胞出现ɑ-synuclein聚集,部分形成球状包涵体。鱼藤酮50、100、500、1 000、2 000 nmol·L-1组线粒体膜电位的红/绿荧光强度比值分别为1.21±0.13、0.85±0.1、0.32±0.18、0.25±0.09、0.18±0.04,明显低于正常对照组1.92±0.15,差异具有统计学意义(n=3,P<0.01)。结论鱼藤酮对PC12细胞具有毒性作用,可诱导细胞凋亡和ɑ-synuclein聚集,其机制可能与降低线粒体膜电位有关。

Abstract：Aim To investigate the toxicity of rotenone on rat PC12 cells and its potential mechanisms. Meth-ods The cultivated rat PC12 cells were divided into control and rotenone-treated groups at various concen-trations （50, 100, 500, 1 000, 2 000 nmol . L-l）. The cell viability was assessed by MTT assay. Hoechst 33258 was employed to observe nucleus morphological changes and apoptotic rate. The expression of u-synu-clein protein was detected by immunocytochemistry. The mitochondrial membrane potential （MMP） was measured by JC-1 staining. Results As assayed by MTT, the cell viability with different concentrations of rotenone（50, 100, 500, 1 000, 2 000 nmol . L-t） was significantly decreased, reaching 83.0% , 79.5%, 41.0.0%, 26.4% and 10.2%, respective-ly, compared with the control group （P 〈 0.05）. The pyknosis and fragmentation of nucleus were observed in rotenone exposure well by Hochest 33258 staining. The apoptotic rate was significantly increased in rotenone-treated cells by 6% ,21% ,64% ,80% and 89% , re-spectively, as compared with control group （P 〈 0. 05 ）. The a-synuclein accumulated evidently in rote-none treated groups, partially formed u-synuclein-posi- tive cytoplasmic inclusions, which were detected by immunocytochemistry technic. The MMP showed by red/green fluorescence ratio in rotenone treated groups（50, 100, 500, 1 000, 2 000 nmol ~ L-＇ ）was significantly reduced to 1.21 _＋ 0. 13, 0. 85 _＋ 0. 1, 0.32_＋0. 18, 0.25 -＋0.09, and 0. 18 -＋ 0. 04,respec-tively, compared with 1.92 _＋ 0. 15 of control cells as detected by JC-1 staining （P 〈 0. 01 ）. Conclusions Rotenone induces toxicity in PCI2 ceils by promoting apoptosis and a-synuclein aggregation. It is suggested that decreased MMP levels might be involved in the mechanism.