ACE2基因过表达减轻血管紧张素Ⅱ诱导的神经细胞氧化应激

Over-expression of ACE2 gene alleviates Ang Ⅱ-induced oxidative stress in neural cells

目的:探讨过表达血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)基因对血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)诱导的小鼠神经母细胞瘤Neuro-2A细胞氧化应激和NADPH氧化酶(NADPH oxidase, NOX)表达的影响。方法:构建ACE2重组慢病毒载体,以感染复数(multiplicity of infection,MOI)=10转染Neuro-2A细胞72 h,测定ACE2基因转染效率和ACE2蛋白表达,并检测神经细胞标志物以鉴定Neuro-2A细胞。Neuro-2A细胞分为7组:空白对照(control)组、慢病毒载体(eGFP)组、重组ACE2(ACE2-eGFP)组、AngⅡ刺激(Ang Ⅱ)组、 Ang Ⅱ+慢病毒载体(Ang Ⅱ-eGFP)组、Ang Ⅱ+ACE2(Ang Ⅱ-ACE2-eGFP)组和Ang Ⅱ+重组ACE2+A779(Ang Ⅱ-ACE2-eGFP-A779)组。采用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)测定Ang(1-7)水平,DHE染色法检测活性氧簇(reactive oxygen species,ROS)水平,Western blot检测MAS受体及NOX亚基(NOX2、NOX4、p47^phox和p67^phox)蛋白表达。结果:AngⅡ显著增加Neuro-2A细胞ROS水平(P<0.01),上调NOX2、NOX4、p47^phox和p67^phox蛋白表达(P<0.01),但下调MAS蛋白表达(P<0.01)。ACE2过表达抑制AngⅡ诱导的ROS增加(P<0.05),下调NOX2、NOX4、p47^phox和p67^phox蛋白表达(P<0.01),还可增加神经细胞Ang(1-7)含量(P<0.01)和MAS受体表达(P<0.01)。MAS受体拮抗剂A779能阻断ACE2下调NOX表达的作用(P<0.05)。结论:ACE2过表达通过MAS受体对抗AngⅡ诱导的神经细胞氧化应激。

AIM: To investigate the effect of over-expression of angiotensin-converting enzyme 2(ACE2) gene on angiotensin II(Ang Ⅱ)-induced oxidative stress and NADPH oxidase(NOX) expression in mouse neuroblastoma Neuro-2 A cells. METHODS: The recombinant lentivirus encoding ACE2 gene was constructed and transfected into the Neuro-2 A cells at a multiplicity of infection(MOI) of 10 for 72 h. The transfection efficiency of ACE2 gene and protein expression of ACE2 were detected, and the Neuro-2 A cells were identified by detection of a neural cell marker. The Neuro-2 A cells were divided into 7 groups: control group, eGFP group, ACE2-eGFP group, Ang Ⅱ treatment group, Ang Ⅱ-eGFP group, Ang Ⅱ-ACE2-eGFP group and Ang Ⅱ-ACE2-eGFP-A779 group. The Ang(1-7) level was determined by ELISA. The level of reactive oxygen species(ROS) in the cells was measured with a method of DHE staining. The protein expression of MAS receptor and NOX subunits(NOX2, NOX4, p47^phox and p67^phox) was detected by Western blot. RESULTS: Ang Ⅱ signi-ficantly increased ROS levels(P<0.01) and up-regulated the protein expression of NOX2, NOX4, p47^phox and p67^phox(P<0.01), but down-regulated MAS protein expression(P<0.01). Over-expression of ACE2 inhibited Ang Ⅱ-induced increase in ROS, down-regulated the protein expression of NOX2, NOX4, p47^phox and p67^phox,and still increased the Ang(1-7) level(P<0.01) and MAS receptor expression(P<0.01). An antagonist of the MAS receptor, A779, blocked the down-regulating effect of ACE2 on NOX expression(P<0.05). CONCLUSION: ACE2 over-expression antagonizes Ang Ⅱ-induced oxidative stress via MAS receptor in the neural cells.