环状RNA-ZNF292对缺氧缺血性脑损伤细胞氧化及凋亡的影响

Effects of circular RNA-ZNF292 on cell oxidation and apoptosis in hypoxic-ischemic brain injury

目的建立原代神经元缺氧缺血性脑损伤细胞模型,探讨环状RNA-ZNF292(cZNF292)对脑缺血损伤后神经元氧化应激及凋亡的影响。方法选取胎龄为18 d的胎鼠培养原代神经元,采用4、10 mmol/L Na_(2)S_(2)O_(4)和无糖培养基处理细胞1、2、3、4 h后复氧培养0、5、15、30 h,建立氧糖剥夺/复氧细胞模型。通过细胞骨架蛋白和β3-微管蛋白免疫荧光染色观察原代神经元的形态变化,FITC标记鬼笔环肽染色观察不同浓度Na_(2)S_(2)O_(4)对原代神经元存活率的影响。采用ELISA检测培养上清液中活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)和乳酸脱氢酶(LDH)的表达水平。采用蛋白质印迹法检测细胞中caspase 3及细胞周期蛋白G1(CCNG1)的表达水平。结果成功建立缺氧缺血性脑损伤细胞模型和cZNF292敲减细胞模型,并发现缺氧培养4 h时复氧、10 mmol/L的Na_(2)S_(2)O_(4)对原代神经元的生长抑制率最高,且在复氧培养15 h时细胞中cZNF292表达水平最高,因此选择缺氧培养4 h、复氧培养15 h、10 mmol/L Na_(2)S_(2)O_(4)为最佳实验条件。氧糖剥夺使细胞培养基中ROS和MDA水平上升,SOD和LDH水平下降(P均<0.05);而敲减cZNF292后,细胞培养基中ROS和MDA水平下降,SOD和LDH水平上升(P均<0.05)。敲减cZNF292可使caspase 3和CCNG1表达下降(P均<0.05)。结论脑缺血可以诱导原代神经元cZNF292表达增加,而敲减cZNF292可以缓解原代神经元在缺血缺氧环境下发生的氧化损伤、抑制细胞凋亡及增殖。

Objective To establish a cell model of primary neuronal hypoxic-ischemic brain injury and explore the effects of circular RNA-ZNF292(cZNF292)on neuronal oxidative stress and apoptosis after hypoxic-ischemic brain injury.Methods The primary neurons were cultured in fetal rats on the 18^(th) day of pregnancy.The cells were treated with 4,10 mmol/L Na_(2)S_(2)O_(4)and glucose-free medium for 1,2,3 and 4 h,and reoxygenated for 0,5,15 and 30 h to establish the oxygen and glucose deprivation/reoxygenation cell model.The morphological changes of primary neurons were observed by immunofluorescence staining of cytoskeletal proteins andβ3-tubulin,the effects of different concentrations of Na_(2)S_(2)O_(4)on the survival rate of primary neurons were observed by fluorescein isothiocyanate(FITC)-phalloidin staining.The expression levels of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD)and lactate dehydrogenase(LDH)in the culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of caspase 3 and cyclin G1(CCNG1)were detected by Western blotting.Results The hypoxic-ischemic brain injury cell model and cZNF292-knockdown model were successfully established and the highest inhibition rate was observed in 10 mmol/L Na_(2)S_(2)O_(4)and reoxygenation 4 h after deoxygenation.The expression level of cZNF292 was the highest in cells after reoxygenation culture for 15 h.Therefore,hypoxia culture for 4 h,reoxygenation culture for 15 h and 10 mmol/L Na_(2)S_(2)O_(4)were selected as the best experimental conditions.Oxygen and glucose deprivation increased the levels of ROS and MDA,and decreased the levels of SOD and LDH in cell culture medium(all P<0.05);after knockdown of cZNF292,the levels of ROS and MDA were decreased,and the levels of SOD and LDH were increased(all P<0.05).Knockdown of cZNF292 decreased the expression of caspase 3 and CCNG1(both P<0.05).Conclusion Cerebral ischemia can increase expression of cZNF292 in primary neurons,while knockdown of cZNF292 can alleviate the oxidative damage,inhibit cell apoptosis and proliferation of primary neurons in the environment of ischemia and hypoxia.