软脂酸通过上调还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶表达促进血管内皮细胞凋亡

摘要

目的 探讨软脂酸(PA)诱导的血管内皮细胞凋亡中还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的作用.方法 人脐静脉内皮细胞(HUVEC)贴壁培养后分为对照组；PA(200、400、800 μmol/L)浓度组(分别为PA200组、PA400组、PA800组)；NADPH氧化酶抑制剂apocynin(50、100、200 mol/L)干预组(分别为apo50+PA400组、apo100+PA400组、apo200+PA400组).采用流式细胞仪检测细胞凋亡率；提取细胞蛋白,采用Western blot技术检测NADPH氧化酶亚基p47phox蛋白表达的水平.倒置共聚焦荧光显微镜检测活性氧的产生.结果 PA呈浓度依赖性诱导HUVEC凋亡；与对照组比较,PA400、PA800组HUVEC凋亡率明显升高,P47phox蛋白表达明显增强(P＜0.05),PA400组活性氧产生明显升高(P＜0.05)；与PA400组比较,apo100+PA400组、apo200+ PA400组HUVEC凋亡率明显降低,p47phox蛋白表达明显减弱,活性氧产生明显降低(P＜0.05).结论PA呈浓度依赖性诱导HUVEC凋亡,apocynin能部分抑制PA的上述作用,其机制与下调NADPH氧化酶亚基p47phox蛋白表达及活性氧产生有关.%Objective To study the effect of reduced form of NADPH oxidase on palmitic acid (PA)-induced apoptosis of vascular endothelial cells. Methods Human umbilical vein endothelial cells(HUVEC) after adherent culture were divided into control group,PA group(which was further divided into PA200 group,PA400 group and PA800 group),NADPH oxidase inhibitor(apoc-ynin) intervention group (which was further divided into apo50 + PA400 group, apolOO + PA400 group and apo200 +PA400 group). Apoptosis of vascular endothelial cells was detected by flow cytometry. Expression level of NADPH p47phox protein was measured by Western blot. Active oxygen generation was assayed by inverted focal fluorescence microscopy. Results PA induced apoptosis of HUVEC in a concentration-dependent manner. The apoptosis rate of HUVEC, expression level of p47phox protein,and active oxygen generation were significantly higher in PA400 group and PA800 group than in control group and significantly lower in apol00 + PA400 group and apo200 + PA400 group than in PA400 group(P<0. 05). Conclusion PA induces apoptosis of HUVEC in a concentration-dependent manner. Apocynin can partly inhibit the effects of PA by down-regulating the expression of NADPH p47phox protein, which may be related with the generation of active oxygen.