毛蕊花苷对MPP^+诱导的SHSY5Y细胞凋亡的保护作用

Verbascoside rescues the SHSY5Y neuronal cells from MPP^+-induced apoptosis

目的观察肉苁蓉提取物毛蕊花苷对MPP+诱导的SHSY5Y细胞损伤的影响。方法用MTT法检测细胞存活率,以流式细胞仪检测细胞内活性氧的产生和线粒体膜电位的变化,以及细胞凋亡的发生,并用荧光酶标仪测定caspase-3的活性,蛋白印迹测定Bcl-2的表达水平。结果200μmol·L-1MPP+处理细胞24h降低细胞的存活率;诱导细胞发生凋亡,凋亡率达38.9%;细胞内活性氧水平及caspase-3的活性升高;而线粒体膜电位却明显降低。而预先给予0.1、1或者10mg·L-1浓度的毛蕊花苷处理细胞12h,可提高细胞存活率;流式细胞仪检测凋亡率分别降低到29.5%,15.3%和8.6%,而且细胞内活性氧的水平明显降低,并可逐渐恢复线粒体的高能量状态;caspase-3的活性不断降低,Bcl-2的表达水平增高,并呈现一定的剂量依赖性。结论毛蕊花苷能抑制MPP+诱导的SHSY5Y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 verbascoside,one of the phenylethanoids isolated from the stems of Cistanche salsa, on MPP^＋-induced injury in SHSY5Y cells. Methods SHSY5Y cells were exposed to various doses of verbascoside for 12 h,and then treated with 200 μmol · L^-1 MPP^＋ for 24 h. The cell viability was observed with MTT assay; reactive oxygen species, the mitochondrial membrane potential and the percentage of apoptosis were measured by flowcytometer; the activation of easpase-3 was measured with the easpase-3 activity assay kit;the expression of Bel-2 was measured with Western blot. Resuits Following treatment with MPP^＋ for 24 h,MPP＆ ＋ induced a significant decrease of cell viability;apoptosis percentage were 38.9% ;accumulation of intracellular ROS,increase of caspase-3 activity and the decreases in mitochondrial membrane potential were detected. However, pretreatment with verbascoside （0. 1,1 or 10 mg · L^-1） for 12 h exhibited eytoprotective effects in a dose-dependent manner. Verbascoside obviously enhanced cell viability, and significantly reduced the number of cells labeled with Annexin-V. The percentage of apoptosis neurons was significantly decreased to 29.5% , 15.3% and 8.6% respectively. Floweytometer showed the verbascoside attenuated the aceumulation of ROS and the MPP^ ＋-induced collapse of mitochondrial membrane potential in SHSY5Y cells. And significant decreases were detected in caspase-3 activity compared with the MPP^＋ -treated cells at the same time point. Moreover, pretreatment with verbascoside promoted the expression of Bcl-2. Conclusions verbascoside had the neuroprotective capacity to antagonize MPP^＋-induced apoptosis in SHSY5Y cells, andmight be useful in treating Parkinson disease.