PINK1介导缺氧损伤时星形胶质细胞对神经元的保护作用

The Protection Effects of Astrocytes on Ischemic Neuron Mediated by PINK1

目的探究星形胶质细胞内PTEN诱导假定激酶1(PINK1)缺失对缺血时神经保护作用的影响及其作用机制。方法离体培养原代星形胶质细胞,使用小干扰RNA(si RNA)沉默PINK1表达,氧糖剥夺(OGD)建立细胞缺氧模型,分为4组:PINK1沉默组(si RNA+转染剂)、空质粒组(空质粒+转染剂)、转染剂组(只加转染剂)和对照组(星形胶质细胞),各组均与神经元共培养;另设立神经元单独培养组。免疫荧光染色观察神经元凋亡情况。定量PCR及ELISA检测星形胶质细胞促红细胞生成素(EPO)及血管内皮生长因子(VEGF)表达量;Western blot检测星形胶质细胞内缺血诱导因子(HIF)及核因子κB(NF-κB)通路相关蛋白水平。结果 OGD损伤后神经元凋亡率较高,与星形胶质细胞共培养后神经元凋亡率显著降低(P<0.05)。PINK1基因沉默后共培养神经元凋亡增加,星形胶质细胞EPO及VEGF分泌量减少、胞内EPO及VEGF转录水平降低(P<0.05);HIF-1、HIF-2与NF-κB通路活化水平均显著降低(P<0.05)。结论星形胶质细胞对OGD损伤神经元有保护作用,其作用通过EPO及VEGF实现;PINK1基因沉默后星形胶质细胞对缺血神经元保护作用减弱,可能与NF-κB通路活化水平降低、HIF激活受损进而下调EPO和VEGF表达量有关。

Aim To explore the ischemic neuroprotection effect of astrocyte PINK1 and its underlying mechanisms. Methods Small interfere RNA was used for the silencing of PINK1 in astrocytes, astrocytes were randomly divided into 4 groups： a PINK1 knockdown（si RNA with transfection agent） group, a empty vehicle（empty plasmid with transfection agent） group, a transfection agent（transfection agent only） group and a control group, then co-culturing them with neurons. Oxygen-glucose deprivation（OGD） was usedto build an ischemic cell model. After that, neuron aptosis rate after ischemic was assessed by Annexin V-PI dyeing. EPO and VEGF secreted by astrocytes were measured by ELISA. Quantitative PCR was used to assess the expression levels of EPO and VEGF in astrocytes. Western blotting was adopted to detected hypoxia induced factor（HIF） and phosphorylation levels of NF-κB pathway in astrocytes. Results The neuron aptosis rate after OGD signifi cantly decreased while co-cultured with astrocytes. The survival rate of co-cultured neuron decreased when co-culture astrocyte PINK1 was silenced. The m RNA and excretion levels of EPO and VEGF signifi cantly decreased in PINK1 silenced astrocytes, as well as HIF levels and phosphorylation levels of NF-κB pathway. Conclusion Astrocytes can protect co-culture neurons from ischemic damage, this protection effect was mediated by EPO and VEGF. The loss of PINK1 in astrocyte will attenuate HIF induction, thus reduce the excretion of EPO and VEGF, ameliorate the neuroprotection effect. The signal conduction between PINK1 and HIF may be mediated by NF-κB pathway.