Necrostatin-1对阿尔茨海默病细胞模型的保护作用

Neuroprotective effects of necrostatin-1 on the cellular model of Alzheimer's disease

目的:研究程序性坏死在阿尔茨海默病(AD)中的作用及Necrostatin-1(Nec-1)对其保护机制。方法:采用25μmol/L Aβ_(25-35)作用于原代培养的小鼠皮层神经元36 h,复制AD细胞模型,分别加入不同浓度的Nec-1进行干预,采用MTT法评价细胞的存活率,试剂盒检测LDH的漏出量,用活细胞碘化丙啶(PI)染色观察细胞坏死的情况,用Western Blot检测细胞的RIPK1、RIPK3和磷酸化MLKL蛋白的表达情况。结果:与对照组相比,模型组细胞存活率明显降低(P<0.05),上清中LDH含量明显升高,PI阳性细胞数量明显增多,有聚集成团现象,细胞RIPK1、RIPK3和MLKL蛋白表达量水平均明显升高(P<0.01)。Nec-1处理后对Aβ细胞毒性作用有明显的减轻,RIPK1、RIPK3和磷酸化MLKL蛋白表达量较模型组明显下降(P<0.05,P<0.01)。结论:Aβ_(25-35)模拟的AD细胞模型中发生了程序性细胞坏死,Nec-1可能通过对抗程序性细胞坏死发挥神经保护作用。

Objective： To investigate the role of programmed necrosis and the protective mechanism of necrostatin-1（ Nec-1） in Alzheimer＇s disease（ AD）. Methods： AD cellular model was established,Aβ（25-35） was used to treat the primary cultured cortical neurons from fetal mice at the final concentration of 25 μmol/L for 36 hours. Cells were treated with different concentrations of Nec-1. MTT assay was used to observe the cell vitality; the kit was adapted to measure LDH leakage quantity; living cells propidium iodide（ PI） staining was used to observe the cell necrosis. Western Blot was used to detect the expressions of RIPK1,RIPK3 and phosphorylated MLKL（ p MLKL） protein. Results： The cell vitality was decreased,the LDH content in supernatant was significantly increased（ P〈0. 05） and the numbers of PI positive cells were obviously increased and the expressions of RIPK1,RIPK3 and p MLKL were significantly higher in AD model compared with the control group（ P〈0. 01）. Moreover,the neurons in AD model often gather to form a glomeration. The toxicity effectives of Aβ（25-35） was significantly reduced after the Nec-1 treatment,the expressions of RIPK1,RIPK3 and p MLKL were significantly lower than that in the model group（ P〈0. 05,P〈0. 01）. Conclusion： Necroptosis occurred in AD cell model induced by Aβ（25-35）. Nec-1 plays a neuroprotective role in the pathological processby fightingagainst programmed cell necrosis.