CAJ | 학술논문

目的探讨α-玉米赤霉醇对肿瘤坏死因子α(TNF-α)刺激的人脐静脉内皮细胞中活性氧(ROS)产生及其对激活信号通路的影响.方法用小分子RNA干扰(siRNA)技术消除人脐静脉内皮细胞的NADPH氧化酶p47phox亚基;用分子探针2,7-DCF测定细胞内ROS的产生量;用RT-PCR测定p47phoxmRNA的表达以及用细胞免疫组化方法测定p47phox蛋白的表达;用Western印迹测定ERK<,2>及核因子(NF)-кB、应激蛋白(SP-1)和活化因子蛋白的表达.结果 TNF-α刺激使细胞内ROS的产生量较对照组高155.4%;α-玉米赤霉醇预处理能够剂量依赖地抑制TNF-α对ROS的诱导效应.TNF-α作用细胞24 h后,p47phox的mRNA表达水平较对照组高212.8%,蛋白的表达也明显高;α-玉米赤霉醇预处理使TNF-α诱导的p47phox mRNA表达水平低63.0%,蛋白表达水平也明显下降.p47phox的siRNA完全阻断TNF-α诱导ROS的产生.α-玉米赤霉醇预处理和p47phox的siRNA均阻断TNF-α对细胞外信号调控激酶的激活和转录因子SP-1的核转位,明显地抑制了NF-кB的核转位.结论 α-玉米赤霉醇能够抑制内皮细胞中TNF-α诱导的ROS的产生及由ROS激活的信号转导通路,主要机制是通过抑制NADPH氧化酶p47phox亚基的表达.
목적 탐토α-옥미적매순대종류배사인자α(TNF-α)자격적인제정맥내피세포중활성양(ROS)산생급기대격활신호통로적영향.방법 용소분자RNA간우(siRNA)기술소제인제정맥내피세포적NADPH양화매p47phox아기;용분자탐침2,7-DCF측정세포내ROS적산생량;용RT-PCR측정p47phoxmRNA적표체이급용세포면역조화방법측정p47phox단백적표체;용Western인적측정ERK<,2>급핵인자(NF)-кB、응격단백(SP-1)화활화인자단백적표체.결과 TNF-α자격사세포내ROS적산생량교대조조고155.4%;α-옥미적매순예처리능구제량의뢰지억제TNF-α대ROS적유도효응.TNF-α작용세포24 h후,p47phox적mRNA표체수평교대조조고212.8%,단백적표체야명현고;α-옥미적매순예처리사TNF-α유도적p47phox mRNA표체수평저63.0%,단백표체수평야명현하강.p47phox적siRNA완전조단TNF-α유도ROS적산생.α-옥미적매순예처리화p47phox적siRNA균조단TNF-α대세포외신호조공격매적격활화전록인자SP-1적핵전위,명현지억제료NF-кB적핵전위.결론 α-옥미적매순능구억제내피세포중TNF-α유도적ROS적산생급유ROS격활적신호전도통로,주요궤제시통과억제NADPH양화매p47phox아기적표체.
Objective To investigate the effects of α-zearalanol (α-ZAL) on the generation of reactive oxygen species (ROS) and ROS-activated signal transduction in the tumor necrosis factor(TNF-α)- stimulated human umbilical vein endothelial cells (HUVECs). Methods HUVECs were cultured and divided into 4 groups: (1) normal control group, (2)TNF-α stimulated group, undergoing TNF-α stimulation for 24 h, (3) α-ZAL retreatment group, undergoing re-treatment withα-ZAL of the concentrations of 1 × 10-8, 1 × 10-7, or 1 × 10-6 mol/L for 1 h, then stimulation of TNF-α for 24 h, and (4) plasmid transfection group,transfected with p47phox siRNA for 24 h to block the NADPH oxidase protein subunit p47phox in the HUVECs, or transfected with blank plasmid as control. The intracellular ROS production was detected by using 2, 7-dichlorofluorescin diacetate as probe. Semi-quantitative RT-PCR and immunocytochemistry were used to detect the mRNA and protein expression of p47phox. The activation of latory protein (SP)-1, and activator protein (AP)-1 were assessed with Western blotting. Results The ROS level in the HUVECs of the TNF-α group was higher than that of the control group by 155.4%, and α-ZAL reduced the ROS level dose-dependently. TNF-α treatment up-regulated the p47phox mRNA expression by 212. 8%, and obviously increased the p47phox protein expression; and α-ZAL pretreatment attenuated the TNF-α-induced p47phox mRNA expression by 63.0%, and also markedly inhibited the p47phox protein expression. No obvious ROS was found in the HUVECs stimulated by TNF-α after the transfection of p47phox TNF-α were abolished or markedly inhibited by α-ZAL pretreatment. Conclusion α-ZAL has a potent inhibitory effect on the ROS production and ROS-activated signaling pathway in the TNF-α stimulated endothelial cells, mainly through the inhibition of NADPH oxidase.