谷精草提取物对6-OHDA所致PC12细胞及斑马鱼神经损伤模型的保护作用

Effect of Eriocaulon buergerianum Extract on Protecting PC12 cells and Zebrafish from 6-hydroxydopamine-induced Neuronal Cell Damage

目的探讨谷精草乙醇提取物(Eriocaulon buergerianum extract,EBE)对6-羟基多巴胺(6-hydroxy-dopamine,6-OHDA)在PC12细胞和斑马鱼上引起的神经损伤的保护作用及其机制。方法采用4-甲基偶氮唑蓝比色法(MTT法)和乳酸脱氢酶释放法(LDH法)检测PC12细胞的活力;Hoechst 33324荧光染色观察细胞核形态的改变分析细胞凋亡;DAF-FM diacetate荧光染色检测细胞内一氧化氮(NO)的含量;采用免疫印迹法检测诱导型一氧化氮合酶(iNOS蛋白)的表达水平。免疫染色方法观察斑马鱼脑部多巴胺神经元的变化。结果 EBE能够剂量依赖性的提高6-OHDA损伤的PC12细胞的存活率,减少6-OHDA引起的PC12细胞凋亡。免疫染色的结果表明EBE可以抑制6-OHDA在斑马鱼上所引起的多巴胺神经元的减少。作用机理研究证明EBE能够降低6-OHDA刺激的PC12细胞内NO含量增加。免疫印记杂交的结果显示EBE能够降低iNOS的表达。结论 EBE对6-OHDA诱导的PC12细胞损伤具有保护作用,能减少6-OHDA引起的细胞凋亡,并且可以抑制6-OHDA在斑马鱼上引起的多巴胺神经元减少,其作用机制可能与降低NO的产生和iNOS的表达量有关。

Objective To investigate the protective effect of Eriocaulon buergerianum ethanol extract （EBE）against 6- hydroxydopamine （6-OHDA）-induced damage in PC12 cells and zebrafish, and to explore the underlying mechanism. Methods The viability and cellular damage of PC12 cells were detected by methyl thiazolyl tetrazolium （MTD assay and lactate dehydrogenase （LDH）assay, respectively. Nuclear morphological changes in apoptotic cells were e- valuated by DNA staining with Hoechst 33342 dye. lntracellular nitric oxide（NO）was quantified by DAF-FM diacetate fluorescence staining. The expression of inducible nitric oxide syuthase（iNOS）was determined by western blotting. Immunostaining method was used to observe changes of dopaminergic neurons in zebrafish brain. Results EBE increased the viability of 6-OHDA-induced PC12 cells damage in a dose-dependent manner, and reduced 6-OHDA- induced PC12 cells apoptosis. In addition, EBE inhibited 6-OHDA-induced dopaminergic neuron loss in zebrafish. Results of therapeutic mechanism showed that EBE suppressed the increase of both NO production and iNOS expres- sion induced by 6-OHDA in PC12 cells. Conclusions EBE exerts neuroprotective effects by inhibiting the 6-OHDA- induced apoptosis in PC12 cells. Moreover, EBE exhibits obviously neuroprotective activity against 6-OHDA stimulated dopaminergic （DA）neuron loss in zebrafish. The underlying mechanism maybe relation to suppressing NO production and down - regulating iNOS expression.