葫芦素B破坏PC-12神经元样细胞微丝骨架的研究

Research of cucurbitacin B-induced disruption of microfilament cytoskeleton in PC-12 neuron-like cells

目的探讨葫芦素B对神经细胞actin细胞骨架(微丝)的影响。方法实验主要分为3组:NGF+1 nmol·L^(-1) CuB,NGF+10 nmol·L^(-1) CuB及NGF+1 000 nmol·L^(-1) CuB。以神经生长因子诱导的PC-12神经样细胞为模型,经不同浓度的葫芦素B处理后,用免疫荧光显微术观察细胞神经突的收缩以及actin和cofilin的亚细胞分布。结果 PC-12经神经生长因子诱导分化为具有多个树突的神经样细胞,葫芦素B处理可引起神经突快速收缩,其收缩的速率与葫芦素B剂量和处理时间有关。1~3 min, NGF+1 nmol·L^(-1) CuB,NGF+10 nmol·L^(-1) CuB及NGF+1 000 nmol·L^(-1)神经突收缩的平均速率分别为(5.82±2.27),(18.25±3.38),(29.24±9.94)μm·min^(-1);3~5 min,神经突收缩的平均速率明显较慢,依次为(3.70±2.12),(12.32±5.64),(18.31±2.37)μm·min^(-1)。免疫荧光分析显示,收缩后的神经突中可观察到actin聚集和cofilin-actin棒状结构形成。结论葫芦素B能破坏神经突中的微丝结构,提示葫芦素B可能具有神经毒性。

Objective To explore the effects of cucurbitacin B on the actin cytoskeleton in neural cells. Methods The experiments were divided into three groups: NGF + 1 nmol·L^(-1) CuB, NGF + 10 nmol·L^(-1) CuB and NGF + 1 000 nmol·L^(-1) CuB. The nerve growth factor-induced PC-12 neuron-like cells were treated with different doses of cucurbitacin B. And then the shrinkage of actin cytoskeleton and the subcellular distribution of cofilin-actin rods by cucurbitacin B treatment were observed by microscopy or revealed by immunofluorescence microscopy. Results The nerve growth factor induced PC-12 to differentiate into neuron-like cells with dendrites. Cucurbitacin B treatment made the dendrites shrinked, and the shrinkage speed was dose-and time-dependent. The average rate of neurite contraction win 1-3 min of NGF + 1 nmol·L^(-1) CuB, NGF + 10 nmol·L^(-1) CuB and NGF + 1 000 nmol·L^(-1) CuB groups were(5.82±2.27),(18.25±3.38) and(29.24±9.94) μm·min^(-1), the rate of neurite contraction was significantly slower in the late stage of cucurbitin B treatment(3-5 min), which were(3.70±2.12),(12.32±5.64),(18.31±2.37) μm·min^(-1), respectively.Immunofluorescence microscopy revealed that actin aggregation and cofilin-actin rods were observed in shrinked dendrites of cucurbnitacin B-treated cells. Conclusion Cucurbitacin B disrupted the microfilament cytoskeleton in neural cells,implicating that this agent might have potential neurotoxicity.