作者：徐家云，刘章锁,马沙，娄丹，王宜娟，卢丹萍
【摘要】 目的探讨AngⅡ对腹膜间皮细胞ROS和NADPH氧化酶亚基p47phox表达的影响及二代腹膜间皮细胞经黄芪注射液(AGI)预处理后的干预作用。方法体外培养SD大鼠原代腹膜间皮细胞至2代，静止24h后，随机分为：正常对照组(A组)，AngⅡ(10-7mol/L)组(B组)，AngⅡ+AGI(2g/mL)组(C组)，AngⅡ+AGI(1g/mL)组(D组)。用荧光染料(DCF)及激光共聚焦显微镜检测细胞内活性氧(ROS)。RT-PCR检测NADPH氧化酶亚基p47phox的表达。Western印迹检测p47phox的蛋白表达。结果①AngⅡ可显著增加大鼠腹膜间皮细胞ROS产生，刺激20min后，ROS的表达较对照组显著上升(P<0.05)。AGI可显著抑制AngⅡ刺激后ROS的产生,且AGI对ROS的抑制程度与AGI的浓度呈正相关，B组、C组、D组三组比较后差异显著(P<0.05);②大鼠腹膜间皮细胞经AngⅡ刺激后，NADPH氧化酶亚基p47phox mRNA和蛋白的表达均上升，AGI可抑制AngⅡ诱导的p47phox mRNA的表达上调，C、D两组分别与B组比较后差异显著(P<0.05)。结论AngⅡ可诱导大鼠腹膜间皮细胞产生的ROS增加、NADPH氧化酶亚基p47phox表达上调;AGI可抑制NADPH氧化酶的表达和活性及ROS的产生，从而为AGI防治腹膜纤维化提供了理论依据。
【关键词】 腹膜间皮细胞;血管紧张素Ⅱ;活性氧;NADPH氧化酶;黄芪
ABSTRACT: ObjectiveTo explore the effects of angiotensin Ⅱ on the reactive oxygen species of peritoneum mesothelial cells and expression of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase subunit p47phox as well as the role of astragalus injection (AGI) intervention for second-generation peritoneum mesothelial cells.MethodsPrimary rat peritoneum mesothelial cells were cultured into the second generation in vitro. After synchronization for 24 hours, the cells were randomly assigned to control group (Group A), AngⅡ (10-7mol/L) group (Group B), AngⅡ +AGI (2g/mL) group (Group C), and AngⅡ +AGI (1g/mL) group (Group D). The DCF-sensitive cellular ROS was measured by fluorometric assay and confocal microscopy. RT-PCR was employed to detect the mRNA expression of NADPH oxidase subunit p47phox, and p47phox protein expression was examined by Western blot.Results① AngⅡ significantly induced the production of intracellular ROS compared with control (P<0.05). After stimulation for 20 minutes, ROS expression increased significantly compared with that in control group (P<0.05). AGI inhibited AngⅡ-induced ROS generation, and the inhibitory effect was positively correlated with its concentration. Groups B, C and D differed significantly from each other (P<0.05). ② AngⅡ stimulated NADPH oxidase subunit p47phox mRNA and protein overexpressions, and AGI inhibited the up-regulation of p47phox mRNA overexpression. Groups C and D were compared with Group B, respectively (P<0.05). ConclusionAngⅡ can significantly induce the production of rat peritoneum mesothelial intracellular ROS and up-overexpression of NADPH oxidase and cellular ROS generation, which provides theoretical basis for AGI?s prevention of peritoneal fibration.KEY WORES: peritoneum mesothelial cell; angiotensin Ⅱ; reaction oxygen species; nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase; astragalus