长春西汀对帕金森病细胞模型保护作用的研究

Study on Protective Effect of Vinpocetine on Parkinson Disease Cell Model

关键词:  长春西汀  帕金森模型  6-羟基多巴胺

Key words:  vinpocetine  Parkinson disease model  6-hydroxydopamine

摘要:

目的 研究长春西汀对帕金森病细胞模型的保护作用和机制。方法 以100 μmol·L^-1 6-羟基多巴胺损伤SH-SY5Y细胞构建帕金森病模型，分为模型组和给药组，给药组在模型组的基础上分别加入不同浓度的长春西汀孵育24 h，另设空白组仅加入维生素C平行操作。以CCK-8法检测细胞存活率摸索长春西汀的有效浓度，流式细胞法检测细胞的凋亡率，Hoechst 33258染色观察细胞的凋亡状况，比色法检测过氧化氢酶（catalase，CAT）、超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）的含量，ELISA法检测白细胞介素1β（interleukin-1β，IL-1β）、白细胞介素6（interleukin-6，IL-6）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）等炎症因子的含量。Western blotting检测N-甲基-D-天冬氨酸受体1（N-methyl-D-aspartate receptor 1，NMDAR1），α-突触核蛋白，cleaved caspase-9的表达量。结果 与模型组相比，给药组细胞随着长春西汀浓度的增加，存活率逐渐上升而凋亡率随之下降且可观察到的凋亡小体数量逐渐减少，细胞中CAT、SOD含量随药物浓度增加而递增，MDA生成量则相应降低，IL-1β、IL-6、TNF-α的含量随药物浓度增加而递减（P<0.05或P<0.01）。Western blotting结果表明，与模型组相比，给药组细胞的NMDAR1、cleaved caspase-9的含量均降低而α-突触核蛋白的表达量显著增加（P<0.05或P<0.01）。结论 长春西汀可通过下调NMDAR1和上调α-突触核蛋白的表达，抑制caspase-9的激活，降低氧化应激和炎症因子的生成，对帕金森病模型的神经细胞产生保护作用。

Abstract:

OBJECTIVE To study the protective effect and mechanism of vinpocetine on Parkinson disease cell model. METHODS The Parkinson disease model was established with SH-SY5Y cells injured by 100 μmol·L^-1 6-hydroxydopamine, they were divided into model group and administration groups. Vinpocetine of different concentrations were added to the administration groups and incubated for 24 h on the basis of model group, while vitamin C was added to the blank group under parallel operation. The cell survival rate was detected by CCK-8 kit to define the suitable concentration of vinpocetine. The apoptosis rate and apoptotic morphology were detected by flow cytometry and Hoechst 33258 staining. The content of catalase(CAT), superoxide dismutase(SOD) and malondialdehyde(MDA) were measured by colorimetry. The content of interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α) were measured by ELISA. Western blotting was used to measure the expression of N-methyl-D-aspartate receptor 1(NMDAR1), α-synuclein and cleaved caspase-9. RESULTS Compared with the model group, with the increase of vinpocetine concentration, the survival rate of cells in the administration groups increased, while the apoptosis rate decreased along with number of apoptotic bodies decreased gradually; the content of CAT and SOD increased while the production of MDA decreased regularly due to the increase of vinpocetine concentration; the content of IL-1β, IL-6 and TNF-α decreased regularly when the vinpocetine concentration enhanced(P<0.05 or P<0.01). The results of Western blotting showed that, compared with model group, the content of NMDAR1 and cleaved caspase-9 in the administration groups was lower while the expression of α-synuclein was increased(P<0.05 or P<0.01). CONCLUSION Vinpocetine can protect the nerve cells of Parkinson disease model by downregulating NMDAR1, upregulating α-synuclein, inhibiting the activation of caspase-9, mitigating the injury of oxidative stress and the production of inflammatory factors.