管花苷B对抗H_2O_2诱导的PC12细胞凋亡

Tubuloside B rescues the PC12 neuronal cells from H_2O_2-induced apoptosis

目的:观察肉苁蓉提取物管花苷B对H2O2诱导的PC12细胞损伤的影响。方法:用MTT法检测细胞存活率,以激光共聚焦显微镜荧光染色法检测细胞内活性氧的产生和线粒体膜电位的变化,DNA琼脂糖凝胶电泳和流式细胞仪检测细胞凋亡的发生,并用荧光酶标仪测定caspase-3的活性。结果:100μmol·L-1H2O2处理细胞24 h显著降低细胞的存活率;诱导细胞发生凋亡,凋亡率达48.0%;细胞内活性氧水平及caspase-3的活性显著升高;而线粒体膜电位却明显降低,红/绿荧光强度的比值由正常的5.97降低为0.41左右。而预先给予1、10或100 mg·L-1浓度的管花苷B处理细胞12 h,可显著提高细胞存活率;并可有效抑制DNA ladder的发生;流式细胞仪检测凋亡率分别降低到30.9%、18.3%和6.2%;激光共聚焦显微镜结果显示管花苷B可明显降低细胞内活性氧的水平;并可逐渐恢复线粒体的高能量状态;caspase-3的活性不断降低,并呈现了一定的剂量依赖性。结论:管花苷B能显著地抑制H2O2诱导的PC12细胞凋亡,其神经细胞保护作用可能与其降低细胞内活性氧水平,维持线粒体膜电位的高能状态和抑制caspase-3的活性有关。

AIM ： To investigate the neuroprotective effect of tubulaside B, one of the phenylethanoids isolated from the stems of Cistanehe salsa, on H2O2 - induced injury in PC12 cells. METHODS： PC12 cells were exposed to various doses of tubuloside B for 12 h, then treated with H2O2 at concentration of 100 μmoL/L for 24 h. The cell viability was observed with MTF assay. Reactive oxygen species and the mitochondrial membrane potential were measured with laser scanning confocal microscopy （ LSCM ）. The DNA content and percentage of apoptosis were assayed by DNA agarase gel electrophoresis and flow cytometry. The activation of caspase - 3 was detected with the caspase - 3 activity assay kit. RE- SULTS： Following treatment with H2O2 for 24 h, H2O2 induced a significant decrease in cell viability; DNA ladder was observed and apoptosis percentage was as high as 48.0%. Accumulation of intracellular ROS, increase in caspase - 3 activity and the decrease in mitochondrial membrane potential as indicated with the decrease of red/green ratios （ from 5.97 to 0.41 ） were detected. However, pretreatment with tubulaside B （ 1, 10 or 100 mg·L^-1 ） for 12 h exhibited cytoprotecrive effects in a dose - dependent manner. Tubuloside B obviously enhanced the cell viability, reduced formation of the DNA ladder, and significantly reduced the number of cells labeled with Annexin - V. The percentage of apoptasis/necrosis neurons was significantly decreased to 30.9% , 18. 3% and 6. 2% , respectively. LSCM showed that the tubulaside B attenuated the accumulation of ROS and the H2O2 - induced collapse of mitochondrial membrane potential in PC12 cells. The significant decrease in caspase - 3 activity was detected, compared to the H2 02 - treated cells at the same time point. CONCLUSION： Tubuloside B has the neuroprotective capacity to antagonize H2O2 -induced apoptasis and injury in PC12 cells, indicating it may be useful for treating some neurodegenerative diseases.