心脑舒通胶囊对氧糖剥夺/复氧损伤原代神经元的保护作用

Protective effect of Xinnao Shutong Capsule on primary neurons injured by oxygen-glucose deprivation/reoxygenation

关键词:  心脑舒通胶囊  氧糖剥夺/复氧  神经元  突触  微管相关蛋白2

Key words:  Xinnao Shutong Capsule  oxygen-glucose deprivation/reoxygenation  neuron  synapse  MAP-2

摘要:

[目的] 探讨心脑舒通胶囊对氧糖剥夺/复氧（OGD/R）损伤的原代神经元细胞活力及微管相关蛋白2（MAP-2）表达的影响。[方法] 取新生24 h内SD大鼠的大脑皮质进行神经元原代培养，并通过免疫荧光法进行MAP-2目的蛋白鉴定；建立神经元OGD/R损伤的体外模型，CCK-8检测不同浓度的心脑舒通胶囊对体外正常培养和OGD/R损伤后神经元细胞活力的影响；选取最佳浓度的心脑舒通胶囊进行MAP-2的荧光表达强度实验，通过倒置荧光显微镜进行拍照处理，并通过软件对神经元突触长度进行测量及统计处理，Western blot法检测MAP-2的蛋白表达情况。[结果] 神经元原代培养成功，且MAP-2染色阳性。CCK-8结果显示心脑舒通胶囊在10、25、50 μg/mL浓度对正常培养神经元细胞有细胞毒作用（P<0.05），因此选取低于10 μg/mL浓度的心脑舒通胶囊进行后续实验。而在OGD/R损伤条件下，与模型组比较，心脑舒通胶囊在3.125、6.25 μg/mL浓度能显著增加OGD/R损伤神经元的细胞活力（P<0.05）；免疫荧光实验结果显示，与对照组比较，模型组MAP-2荧光表达强度及神经元突触长度明显降低（P<0.05）；与模型组比较，心脑舒通胶囊在3.125 μg/mL浓度能显著增加MAP-2的荧光表达强度及神经元突触长度（P<0.05）。Western blot实验结果显示，与对照组比较，模型组MAP-2蛋白表达显著降低（P<0.05），与模型组比较，心脑舒通胶囊能显著增加MAP-2的蛋白表达（P<0.05）。[结论] 心脑舒通胶囊能促进OGD/R损伤神经元活力，其作用机制与促进细胞骨架蛋白MAP-2表达及突触稳定性有关。

Abstract:

[Objective] To investigate the effects of Xinnao Shutong (XNST) Capsules on the viability of primary neurons and the expression of microtubule-associated protein-2 (MAP-2) in oxygen-glucose deprivation/reoxygenation cells. [Methods] Newborn SD rats were used to obtain cerebral cortical neurons for primary culture and MAP-2 immunofluorescence identification. Oxygen-glucose deprivation/reoxygenation (OGD/R) injury model was established and CCK-8 was used to detect different concentrations of XNST Capsules on the cell vitality. Immunofluorescence method was used to observe the fluorescence intensity of MAP-2,and the neuronal synaptic length was measured by Image J software. Western blot method to detect MAP-2 protein expression. [Results] The primary culture of neurons was successful,and MAP-2 staining was positive. CCK-8 results showed that XNST Capsules had cytotoxic effects on normal cultured neurons at concentrations of 10,25,and 50 μg/mL (P<0.05). Compared with the OGD/R group,XNST Capsules at 3.125 and 6.25 μg/mL concentration can significantly increase the cell viability (P<0.05). Compared with the control group,the MAP-2 fluorescence intensity and neuron synaptic length in the OGD/R group were significantly reduced (P<0.05). Compared with the OGD/R group,3.125 μg/mL XNST Capsules could significantly increase MAP-2 fluorescence intensity expression and neuronal synaptic length (P<0.05). Western blot results showed that compared with the control group,the MAP-2 protein expression of the model group was significantly reduced (P<0.05). Compared with the model group,XNST Capsules can significantly increase the expression of MAP-2 protein (P<0.05). [Conclusion] XNST Capsules can promote the neuron viability induced by OGD/R,and its mechanism is related to the promotion of cytoskeletal protein MAP-2 expression and synaptic stability.