缺血性脑卒中是一种脑血管病,这与DNA损伤有关。许多研究表明,Ku70是DNA损伤的关键调节因子。这里,我们旨在探讨Ku70在缺血性脑卒中中的作用及其潜在的分子机制。在我们的研究中,采用氧糖剥夺/复氧法(OGD/R)诱导神经干细胞(NSCs)构建缺血性脑卒中细胞模型。CCK8测定,Brdu/GFP染色,流式细胞术和TUNEL染色检查细胞增殖,细胞周期和凋亡,分别。通过实时定量PCR和蛋白质印迹分析检测相对mRNA和蛋白质水平。分别。免疫荧光染色检测Ku70阳性细胞。使用彗星测定法来确定DNA损伤。进行动物实验以评估移植NSC和Ku70过表达的NSC对神经功能缺损的影响。梗死体积,大脑中动脉闭塞(MCAO)模型的脑水肿和血脑屏障(BBB)完整性。我们的数据发现,在OGD/R后,NSCs中Ku70的表达降低。Ku70的过表达减少了OGD/R诱导的NSCs的DNA损伤和凋亡。Ku70的击倒促进ATM/p53的活性。此外,KU60019(ATM特异性抑制剂)逆转了Ku70沉默对OGD/R诱导的神经干细胞DNA损伤和凋亡的促进作用。在动物实验中,NSC过表达Ku70的移植增强了细胞存活,改善电机功能,梗死体积减少,减轻MCAO小鼠模型的脑水肿和BBB功能障碍。总之,Ku70过表达通过调节ATM/p53通路抑制OGD/R诱导的神经干细胞DNA损伤和凋亡,NSC过表达Ku70的移植在MCAO小鼠模型中发挥神经保护作用。
Cerebral ischemic stroke is a cerebrovascular disease, which is related to DNA damage. Many researches have shown that
Ku70 is a key regulator for DNA damage. Here, we aimed to explore Ku70 roles in cerebral ischemic stroke and its potential molecular mechanism. In our study, neural stem cells (NSCs) were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) for constructing cerebral ischemic stroke cell model. CCK8 assay, Brdu/GFP staining, flow cytometry and TUNEL staining were performed to examine cell proliferation, cell cycle and apoptosis, respectively. Relative mRNA and protein levels were detected by quantitative real-time PCR and western blot analysis, respectively.
Ku70 positive cells were examined by immunofluorescence staining. Comet assay was employed to determine DNA damage. Animal experiments were performed to assess the effect of transplanting NSCs and Ku70-overexpressed NSCs on neurological deficits, infarct volume, brain edema and blood‒brain barrier (BBB) integrity in middle cerebral artery occlusion (MCAO) model. Our data found that
Ku70 expression was decreased in NSCs after OGD/R. Overexpression of
Ku70 reduced DNA damage and apoptosis of OGD/R-induced NSCs. Knockdown of
Ku70 promoted the activity of ATM/p53. Moreover, KU60019 (ATM-specific inhibitor) reversed the promoting effects of Ku70 silencing on DNA damage and apoptosis in OGD/R-induced NSCs. In animal experiments, transplantation of NSCs-overexpressed Ku70 enhanced cell survival, improved motor function, reduced infarct volume, relieved brain edema and alleviated BBB dysfunction in MCAO mice models. In conclusion, Ku70 overexpression repressed the DNA damage and apoptosis in OGD/R-induced NSCs by regulating ATM/p53 pathway, and transplantation of NSCs-overexpressed Ku70 played neuroprotective effects in MCAO mice models.