血管内皮生长因子对氧糖剥离再灌注诱导的大鼠原代神经元损伤的保护作用及机制

The effect and mechanism of vascular endothelial growth factor on hypoxia-induced injury of primary neurons of rat

目的探讨血管内皮生长因子(VEGF)对氧糖剥离再灌注(OGD/R)诱导的大鼠原代神经元损伤的保护作用及机制。方法培养SD大鼠原代神经元,并将细胞随机分为对照组、OGD/R组以及VEGF 25、50、100 ng/mL组,采用OGD/R诱导细胞损伤。采用CCK8检测细胞活性,采用Hoechst染色检测细胞凋亡,酶联免疫法检测各组细胞培养液乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)水平;采用免疫印迹法检测各组pro-Caspase-3、Bax、Bcl-2、cleaved Caspase-3、核因子相关因子2(Nrf2)、醌氧化还原酶(NQO1)、血红素氧合酶-1(HO-1)、磷酸化核因子κB抑制因子α(p-IκBα)及核因子-κB(NF-κB)P65的表达水平;利用siRNA沉默Nrf2,采用RT-PCR检测Nrf2和NF-κB P65的表达。结果与对照组比较,OGD/R组细胞活性、SOD水平、GSH-Px水平以及Nrf2、NQO1和HO-1蛋白表达水平均明显降低(P<0.05或P<0.01),细胞凋亡率、Bax/Bcl-2比值、cleaved-Caspase-3/pro-Caspase-3、LDH、MDA表达水平以及p-IκBα和NF-κB p65表达水平明显升高(P<0.05或P<0.01);与OGD/R组比较,VEGF 25、50、100 ng/mL组细胞活性以及SOD和GSH-Px表达水平明显升高,而细胞凋亡率、Bax/Bcl-2比值、pro-Caspase-3/cleaved Caspase-3比值以及LDH和MDA表达水平显著降低,Nrf2、NQO1和HO-1蛋白表达显著上调,p-IκBα和NF-κB p65表达明显下调(均P<0.05);此外,si-Nrf2能明显减弱VEGF(100 ng/mL)对Nrf2和NF-κB p65表达的调控作用,减弱VEGF(100 ng/mL)对细胞活性的促进作用及细胞凋亡的抑制作用(P<0.05或P<0.01)。结论VEGF能通过调控Nrf2/HO-1和NF-κB通路减轻OGD/R诱导大鼠原代神经元损伤。

Objective To investigate the effect and mechanism of vascular endothelial growth factor(VEGF)on hypoxia-induced injury of primary neurons of rat.Methods The primary neurons of SD rat were cultured,the cells were divided into control,oxygen glucose deprivation/reperfusion(OGD/R),VEGF(25 ng/mL),VEGF(50 ng/mL)and VEGF(100 ng/mL)groups.All the cells were injured by OGD/R.Cell viability was measured by CCK8 assay.Cell apoptosis was determined by Hoechst staining.The concentrations of lactate dehydrogenase(LDH),superoxidate dismutase(SOD),glutathion peroxidase(GSH-Px)and malondialdehyde(MDA)were measured by kits.Western blot was performed to measure protein levels,including pro-Caspase-3,Bax,Bcl-2,cleaved Caspase-3,Nrf2,NOQ1,HO1,p-IκBα,NF-κB,and P65.Nrf2 was silenced by siRNA,the expressions of Nrf2,NF-κB,and P65 were measured by RT-PCR and the above experiments were repeated.Results In the OGD/R group,the cell viability,the concentrations of SOD and GSH-Px,the protein levels of Nrf2,NQO1,and HO-1 were decreased significantly(P<0.05 or P<0.01);the cell apoptosis rate,the ratio of Bax/Bcl-2 and cleaved-Caspase-3/pro-Caspase-3,the levels of LDH and MDA,the protein levels of p-IκBα,NF-κB,and p65 were increased compared with the control group(P<0.05 or P<0.01).In the VEGF groups,the cell viability,the concentrations of SOD and GSH-Px,the protein levels of Nrf2,NQO1,and HO-1 were significantly increased;the cell apoptosis rate,the ratio of Bax/Bcl-2 and cleaved-Caspase-3/pro-Caspase-3,the concentrations of LDH and MDA,the protein levels of p-IκBα,NF-κB,and p65 were decreased compared with the OGD/R group(all P<0.05).In addition,si-Nrf2 eliminated the effects of VEGF(100 ng/mL)on Nrf2,NF-κB,and p65,and attenuated the effects of VEGF(100 ng/mL)on cell viability and cell apoptosis(P<0.05 or P<0.01).Conclusions VEGF alleviated the OGD/R-induced neuron injury of rats via regulating Nrf2/HO-1/NF-κB signaling pathway.