松果菊苷对TNFα诱导的SH-SY5Y细胞凋亡的保护作用

Echinacoside rescues the SHSY5Y neuronal cells from TNFα- induced apoptosis

目的 探讨肉苁蓉提取物松果菊苷对TNFα诱导的SH SY5Y细胞凋亡的保护作用。方法 用MTT法检测细胞存活率,DNA琼脂糖凝胶电泳和流式细胞仪检测细胞凋亡的发生,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,并用荧光酶标仪测定caspase 3的活性。结果 100μg·L-1 TNFα处理细胞 36h显著降低细胞的存活率;诱导细胞发生凋亡,凋亡率达37%;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,红 /绿荧光强度的比值由正常的 5 97降低为 0 35左右。而预先给予 1, 10或者 100mg·L-1浓度的松果菊苷处理细胞 2h,可提高细胞存活率;并可有效抑制DNAladder的发生;流式细胞仪检测凋亡率分别降低到25 9%, 18 3% 和 8 2%;激光共聚焦显微镜结果显示松果菊苷可明显抑制细胞内活性氧产生;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 松果菊苷能抑制TNFα诱导的SH SY5Y细胞凋亡,其神经细胞保护作用可能与降低细胞内活性氧水平,抑制caspase 3的活性和维持线粒体膜电位的高能状态有关。

Aim To investigate the neuroprotective e ffect of echinacoside, one of the phenylethanoids isolated from Cistanche salsa, on tumor necrosis factor-alpha (TNFα)-induced apoptosis in SH-SY5Y neuronal cells. Methods Cell viability was analyzed using MTT assay. Apoptotic cells were detected using DNA fragmentation and flow cytometric analys is. The level of intracellular reactive oxygen species(ROS) and the potential of mitochondrial membrane were determined by laser scanning confocal microscopy (L SCM). The activity of caspase-3 was measured with microplate spectrofluorometer . Results Treatment of cultured SH-SY5Y cells with 100 μg· L -1 TNFα for 36 h induced a significant decrease of cell viability, and t he percentage of apoptosis increased to 37%. The level of intracellular ROS and activity of caspase-3 enhanced, and the red/green ratios of mitochondrial membr ane decreased to 0.35 from 5.97. However, simultaneous treatment with echinac oside (1, 10, or 100 mg·L -1) exhibited cytoprotective effects, reduced fo rmation of the DNA ladder, prevented the accumulation of ROS and caspase-3, reconverted the potential of mitochondrial membrane, and the percentage of apoptosis/necrosis neurons was significantly decreased to 25.9%, 18.3% and 8.2% in a dose-dependent manner . Conclusion These results demonstrate that echinacoside has the neuroprotective capacity to antagonize TNFα-induced apoptosi s in SH-SY5Y cells. The antiapoptotic action of echinacoside may be partially d ependent on an anti-oxidative stress effects, inhibition of caspase-3 activity , and maintain of mitochondria function.