TGF-β-1对大鼠腹膜间皮细胞ROS和NADPH氧化酶亚基p67phox表达的影响及黄芪的干预作用

摘要

Objective;To explore the effects of TGF - [3, on the reactiv oxygen species of peritoneal mesothelial cells and expression of nicotinamide - adenine dinucleotide phosphate ( NADPH ) oxidase subunit p67phox as well as the role of astragalus injection ( AGI ) intervention for second generation peritoneal mesothelial cells. Methods; Primary rat peritoneal 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), AGI (2 g/ml) group ( Group B), TGF-(3,( 10 ng/ml ) group ( Group C ), TGF - |3,( 10 ng/ml) + AGI( 2 g/ml ) group ( Group D,pretreated for 1 hour with AGI before TGF - [3, stimulation ). The DCF - sensitive cellular ROS was measured by fluoromet-ric assay and confocal microscopy. RT - PCR was employed to detect the mRNA expression of NADPH oxidase subunit p67phox, and p67phox protein expression was examined by Western blot. Results; TGF - [3, 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 TGF - (3, induced ROS generation. TGF - (3, stimulated NADPH oxidase subunit p67phox mRNA and protein over expressions, and AGI inhibited the up - regulation of p67phox mRNA and protein over expression. Groups D were compared with Group C ( P <0. 05 ). Conclusion;TGF - (3, can significantly induce the production of rat peritoneal mesothelial intracellular ROS and up - regulation of NADPH oxidase subunit p67phox mRNA and protein expressions. AGI can inhibit the activity and over expression of NADPH oxidase and cellular ROS generation, which provides theoretical basis for AGI s prevention of peritoneal fibration.%目的:探讨TGF-β-1对大鼠腹膜间皮细胞(RPMCs)活性氧(ROS)和NADPH氧化酶亚基p67phox表达的影响及黄芪注射液(AGI) 对其的干预作用.方法:体外培养SD大鼠原代腹膜间皮细胞至第二代,静止24 h后,随机分为:正常对照组(A组),AGI(2 g/ml)组(B组),TGF-β-1 (10 ng/ml) 组(C组),TGF-β-1+AGI (2 g/ml) 组(D组,AGI预处理1 h).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p67phox mRNA的表达;Western印迹检测p67phox的蛋白表达.结果:TGF-β-1可显著增加大鼠腹膜间皮细胞ROS产生,刺激20 min后,ROS的表达较对照组显著上升(P< 0.05).AGI可显著抑制TGF-β-1刺激后ROS的产生 (P<0.05);大鼠腹膜间皮细胞经TGF-β-1刺激后,NADPH氧化酶亚基p67phox mRNA和蛋白的表达均上升,AGI可抑制TGF-β-1诱导的p67phox mRNA和蛋白的表达上调,差异有统计学意义(P<0.05).结论:TGF-β-1可诱导大鼠腹膜间皮细胞产生的ROS 增加、NADPH氧化酶亚基p67phox 表达上调;AGI可抑制NADPH氧化酶的表达和活性ROS的产生,从而为AGI防治腹膜纤维化提供了理论依据.