转录因子Nrf2在缺氧性肺血管重构中的作用机制

The action mechanism of transcription factor Nrf2 in hypoxic pulmonary vascular remodeling

目的探究转录因子Nrf2在缺氧性肺血管重构中的作用机制的研究。方法C57BL/6J小鼠(周龄10周左右)和Nrf2基因敲除(Nrf2-/-)的小鼠用于本次实验,所有小鼠具有相同的遗传背景。将实验小鼠分为WT小鼠对照组(野生型小鼠不做手术处理);WT小鼠损伤组(小鼠进行肠缺血/再灌注手术处理,进行肺损伤模型构建);Nrf2-/-小鼠损伤组(Nrf2-/-小鼠进行肠缺血/再灌注手术处理);人肺微血管内皮细胞的氧葡萄糖剥夺和复氧人PMVEC从科学细胞研究实验室购买。细胞分为Nrf2 siRNA转染组(Nrf2抑制表达);Sirt1过表达组(用Sirt1慢病毒载体转染PMVEC)。免疫荧光和免疫组化检测Nrf2的表达;通过蛋白质印迹分析实验小鼠的HIF-1α、VEGF和CD31的蛋白表达;组织学及胶原定量检测小鼠肺损伤;细胞计数试剂盒检测细胞的增殖;通过PCR实时分析细胞CD31、NOX4和Nrf2的mRNA表达。结果WT小鼠损伤组较WT小鼠对照组Nrf2的荧光染色增加(P<0.05)。WT小鼠损伤组较Nrf2-/-小鼠损伤组HIF-1α、VEGF和CD31的蛋白表达升高(P<0.05)。WT小鼠损伤组较Nrf2-/-小鼠损伤组白细胞浸润量降低(P<0.05),Nrf2-/-小鼠损伤组较WT小鼠损伤组肺损伤评分升高(P<0.05),WT小鼠损伤组较Nrf2-/-小鼠损伤组胶原蛋白沉淀降低(P<0.05)。第12小时Sirt1过表达组较Nrf2 siRNA转染组细胞增殖升高(P<0.05),第24小时Nrf2 siRNA转染组较Sirt1过表达组细胞增殖降低(P<0.05)。Sirt1过表达组较Nrf2 siRNA转染组CD31、NOX4和Nrf2的mRNA表达升高(P<0.05)。结论Sirt1可通过刺激Nrf2表达,通过NOX4介导的基因调控维持缺氧性血管生成方面起重要作用。

Objective To investigate the action mechanism of transcription factor Nrf2 in hypoxic pulmonary vascular remodeling.Methods The C57BL/6J mice(10 weeks of age)and Nrf2 knockout(Nrf2-/-)mice enrolled in the study.All the mice had the same genetic background.The experimental mice were divided into WT mouse control group(wild-type mice were not treated);WT mouse injury group(the mice were subjected to intestinal ischemia/reperfusion surgery,and a lung injury model was constructed);Nrf2-/-Mouse injury group(Nrf2-/-mice undergoing intestinal ischemia/reperfusion surgery).Moreover human lung microvascular endothelial cells deprived of oxygen glucose and deoxygenated human PMVEC were divided into Nrf2 siRNA transfection group(Nrf2 expression inhibition);Sirt1 overexpression group(PMVEC transfected with Sirt1 lentivirus vector).Immunofluorescence and immunohistochemistry were used to detect the expression levels of Nrf2,and the expression levels of HIF-1α,VEGF and CD31 protein were detected by Western Blot,and the lung injury of mice was detected by histological and collagen quantitative assay,cell proliferation was measured by cell count kit and the expression levels of CD31,NOX4 and Nrf2 mRNA were detected by Realtime PCR.Results As compared with that in WT mouse control group,the fluorescence staining of Nrf2 was significantly increased in WT mouse injury group(P<0.05).The expressions levels of HIF-1α,VEGF and CD31 protein in WT mouse injury group were significantly higher than those in Nrf2-/-mouse injury group(P<0.05).As compared with that in Nrf2-/-mouse injury group,the amount of leukocyte infiltration was significantly decreased in WT mouse injury group(P<0.05),and the lung injury scores in Nrf2-/-mouse injury group were significantly higher than those in WT mouse injury group(P<0.05).Moreover the collagen precipitation in WT mouse injury group was significantly lower than that in Nrf2-/-mouse injury group(P<0.05).In addition the cell proliferation in Sirt1 overexpression group at 12 hours was significantly higher than that in Nrf2 siRNA transfection group(P<0.05),which in Nrf2 siRNA transfection group at 24 hours was significantly lower than in Sirt1 overexpression group(P<0.05).The expression levels of CD31,NOX4 and Nrf2 mRNA in Sirt1 overexpression group were significantly higher than those in Nrf2 siRNA transfection group(P<0.05).Conclusion Sirt1 can play an important role in maintaining hypoxic angiogenesis by stimulating Nrf2 expression and NOX4-mediated gene regulation.