AngⅡ对腹膜间皮细胞ROS和NADPH氧化酶亚基p47phox表达的影响及黄芪的干预作用.doc

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1、AngⅡ对腹膜间皮细胞ROS和NADPH氧化酶亚基p47phox表达的影响及黄芪的干预作用作者：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

2、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p47phoxmRNA和蛋白的表达均上升，AGI可抑制AngⅡ诱导的p47phoxmRNA的表达

3、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:ObjectiveToexploretheeffectsofangiotensinⅡonthereactiveoxygenspeciesofperitoneummesothelialcellsandexp

4、ressionofnicotinamide-adeninedinucleotidephosphate(NADPH)oxidasesubunitp47phoxaswellastheroleofastragalusinjection(AGI)interventionforsecond-generationperitoneummesothelialcells.MethodsPrimaryratperitoneummesothelialcellswereculturedintothesecondgenerationinvitro.Aftersynchr

5、onizationfor24hours,thecellswererandomlyassignedtocontrolgroup(GroupA),AngⅡ(10-7mol/L)group(GroupB),AngⅡ+AGI(2g/mL)group(GroupC),andAngⅡ+AGI(1g/mL)group(GroupD).TheDCF-sensitivecellularROSwasmeasuredbyfluorometricassayandconfocalmicroscopy.RT-PCRwasemployedtodetectthemRNAexp

6、ressionofNADPHoxidasesubunitp47phox,andp47phoxproteinexpressionwasexaminedbyWesternblot.Results①AngⅡsignificantlyinducedtheproductionofintracellularROScomparedwithcontrol(P<0.05).Afterstimulationfor20minutes,ROSexpressionincreasedsignificantlycomparedwiththatincontrolgrou

7、p(P<0.05).AGIinhibitedAngⅡ-inducedROSgeneration,andtheinhibitoryeffectwaspositivelycorrelatedwithitsconcentration.7GroupsB,CandDdifferedsignificantlyfromeachother(P<0.05).②AngⅡstimulatedNADPHoxidasesubunitp47phoxmRNAandproteinoverexpressions,andAGIinhibitedtheup-regula

8、tionofp47phoxmRNAoverexpression.GroupsCandDwerecomparedwithGroupB,respectiv