肾小球内皮细胞Nrf2激活在ANCA相关性血管炎中的作用及其机制

Roles of glomerular endothelial cell Nrf2 activation in ANCA associated vasculitis and its mechanism

目的探讨核转录因子E2相关因子2(Nrf2)在抗中性粒细胞胞浆抗体(anti-neutrophil cytoplasmic antibody,ANCA相关性血管炎(ANCA-associated vasculitis,AAV)中的作用及调控机制。方法采用AAV患者和健康志愿者血清刺激人条件永生化肾小球内皮细胞(conditionally immortalized human glomerular endothelial cells,ciGEnCs)。分别设置空白对照组,健康对照组,AAV血清组,AAV血清组+抗氧化剂Tempol(3 mmol/L预处理)。通过DCFH-DA荧光探针检测细胞内活性氧簇(ROS)的生成;免疫荧光法检测Nrf2在细胞中的定位;免疫印记(Western blot)检测Nrf2、HO-1、NQO1蛋白表达。结果AAV患者血清可以促进ciGEnCs的ROS增加(相比较空白对照组与健康对照组,P值均为0.0004),抗氧化剂Tempol可抑制AAV患者血清诱导的ROS生成(P=0.0180);抗氧化剂Tempol可激活抗氧化反应因子Nrf2并向核内转位,并且Nrf2蛋白表达明显增高(P=0.0143);抗氧化剂Tempol可上调Nrf2及下游HO-1、NQO1蛋白表达(P值分别为0.0298、0.0006、0.0082)。结论抗氧化治疗可能缓解AAV血管内皮损伤,有望成为延缓AAV进展的治疗策略。

This study was performed to investigate the roles and regulatory mechanism of nuclear factor erythroid 2 related factor 2(Nrf2)in antineutrophil cytoplasmic antibody(ANCA)associated vasculitis(AAV).The serum of AAV patients and healthy volunteers were collected to stimulate conditionally immortalized human glomerular endothelial cells(ciGEnCs)and divided into healthy control group,AAV serum group,and AAV serum group+Tempol(3 mmol/L)pretreatment group,with untreated cells as blank control group.The intracellular reactive oxygen species(ROS)was detected by DCFH-DA fluorescent probe;the localization of Nrf2 in cells was detected by immunofluorescence assay;and the expressions of Nrf2,HO-1 and NQO1 were detected by Western blotting.Data showed that serum of AAV could promote the increase of ROS in ciGEnCs,as compared with the blank control and healthy control groups.Antioxidant tempol could inhibit the ROS production induced by AAV serum,activate Nrf2 and transfer it into the nucleus,and up-regulate the expression of Nrf2,downstream HO-1 and NQO1 proteins.In conclusion,antioxidant therapy may alleviate the vascular endothelial injury of AAV,thus becoming a treatment strategy to delay the progression of AAV.