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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。
Objective To investigate the effect of PTEN-induced hypoxia-inducible kinase 1 (PINK1) deficiency on neuroprotective effect during ischemia and its mechanism. Methods Primary astrocytes were cultured in vitro. PINK1 was silenced by si RNA. Oxygen deprivation (OGD) was used to establish cell hypoxia model and divided into four groups: PINK1 silencing group (si RNA + transfection agent ), Empty plasmid group (empty plasmid + transfection agent), transfection agent group (transfected with transfection agent only) and control group (astrocyte), all groups were co-cultured with neurons; another set up neurons alone Cultivation group. Immunofluorescence staining to observe neuronal apoptosis. The expression of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in astrocytes were detected by quantitative PCR and ELISA. The expression of astrocyte inducible factor 1 (HIF) and nuclear factor kappa B -κB) pathway-related protein levels. Results The apoptotic rate of neurons after OGD injury was high, and the apoptosis rate of neurons after co-cultured with astrocytes was significantly decreased (P <0.05). PINK1 gene silencing co-cultured neurons apoptosis increased, astrocyte EPO and VEGF secretion decreased, intracellular EPO and VEGF transcription decreased (P <0.05); HIF-1, HIF-2 and NF-κB pathway Activation levels were significantly lower (P <0.05). Conclusion Astrocytes can protect OGD-injured neurons through EPO and VEGF. Astrocytes can attenuate the protection of ischemic neurons after PINK1 gene silencing, which may be related to the decrease of activation of NF-κB pathway , Impaired HIF activation and down-regulated the expression of EPO and VEGF.