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目的用多巴胺诱导的SH-SY5Y细胞凋亡模型,研究从小蜡树皮提取的单体化合物liriodendrin的神经保护作用。方法应用MTT法检测liriodendrin对细胞存活率的影响。应用Annexin V-FITC与PI双染流式法检测liriodendrin对多巴胺诱导的细胞凋亡的影响。应用荧光染料DCFH-DA及Rhodamine 123对细胞内活性氧簇(ROS)及线粒体膜电位(ΔΨm)进行了检测。此外,应用RT-PCR方法,对细胞内P53基因的转录水平进行了检测。结果在经过10–8,10–7,10–6,10–5及10–4mol·L–1浓度liriodendrin处理后,细胞存活率与多巴胺处理组相比有显著性提高。流式细胞术结果显示liriodendrin具有显著的抗细胞凋亡作用。同时liriodendrin可明显改善多巴胺引起的ΔΨm下降,并可以逆转多巴胺诱导的ROS水平升高。此外,liriodendrin还可以降低多巴胺引起的P53基因转录水平的升高。结论Liriodendrin对多巴胺诱导的细胞损伤有明显保护作用。推测主要机制可能与其调节细胞氧化应激反应及细胞凋亡的信号转导通路有关,提示该化合物可以作为治疗退行性神经疾病如帕金森氏病等的候选化合物。
Objective To study the neuroprotective effect of liriodendrin, a monomeric compound extracted from bark of small wax, using a dopamine-induced model of apoptosis in SH-SY5Y cells. Methods The effect of liriodendrin on cell viability was detected by MTT assay. The effect of liriodendrin on dopamine-induced apoptosis was examined by Annexin V-FITC and PI double staining. Fluorescence dyes DCFH-DA and Rhodamine 123 were used to detect reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) in the cells. In addition, the transcriptional level of P53 gene in cells was detected by RT-PCR. Results After treatment with liriodendrin at concentrations of 10–8, 10–7, 10–6, 10–5, and 10–4 mol·L–1, the cell viability was significantly increased compared to the dopamine-treated group. Flow cytometry results showed that liriodendrin has a significant anti-apoptotic effect. At the same time, liriodendrin can significantly improve dopamine-induced decline in ΔΨm, and can reverse dopamine-induced increase in ROS levels. In addition, liriodendrin can also reduce dopamine-induced elevation of P53 gene transcription. Conclusion Liriodendrin has obvious protective effect on dopamine-induced cell injury. It is speculated that the main mechanism may be related to signal transduction pathways that regulate cell oxidative stress response and apoptosis, suggesting that the compound may be used as a candidate compound for treating degenerative neurological diseases such as Parkinson’s disease.