AngⅡ对腹膜间皮细胞ROS和NADPH氧化酶亚基p47phox表

【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餾 prevention of peritoneal fibration.KEY WORES: peritoneum mesothelial cell; angiotensin Ⅱ; reaction oxygen species; nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase; astragalus

　　腹膜纤维化是长期腹膜透析(peritoneum dialysis, PD)患者的重要并发症之一，最终导致腹膜结构异常、丧失超滤功能而退出腹膜透析。高糖腹膜透析液可引起腹膜发生氧化应激反应，导致腹膜结构和功能异常。近年研究证实，血管紧张素Ⅱ(angiotensin Ⅱ, AngⅡ)参与了腹膜间皮细胞的转分化和细胞外基质的积聚，与腹膜纤维化的发生发展密切相关[1-2];烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide-adenine dinucleotide phosphate, NADPH)氧化酶作为活性氧(reactive oxygen species, ROS)的调控酶，其胞浆亚基p47phox在多种组织的氧化应激反应中起着枢纽性作用[3-4]。调节NADPH氧化酶亚基p47phox的表达及活性成为改善氧化应激状态、防治腹膜纤维化的重要切入点。色谱指纹图谱证实黄芪注射液有稳定的清除自由基、抗氧化的色谱峰[5]，超微结构证实黄芪注射液能拮抗高糖环境对腹膜间皮细胞的氧化应激损伤[6-7]。为此，本研究应用黄芪注射液来观察AngⅡ对腹膜间皮细胞的影响，以探讨AngⅡ对腹膜间皮细胞ROS和NADPH氧化酶亚基p47phox等表达的影响、及黄芪的干预作用，为腹膜纤维化防治提供新的思路。

　　1材料与方法

　　1.1主要试剂胎牛血清(FBS)、DMEM/F12干粉培养基(GIBCO公司);胰蛋白酶(Amerasco公司);AngiotensinⅡ(Sigma, USA);黄芪注射液(正大青春宝药业有限公司生产，国药准字Z33020179，每支10mL相当于原材料20g);H2-DCFDA(Molecular Probes, USA);Trizol试剂(Invitrogen公司);逆转录试剂盒(Fermentas公司);Taq DNA聚合酶(Fermentas公司);RNase抑制剂(GIBCO BRL公司);焦碳酸二乙酯(DEPC，博彩公司);DNA Marker(DL2000，TaKaRa公司);琼脂糖(Biolabs公司);兔抗大鼠p47phox抗体(Upstate公司，USA);兔抗大鼠GAPDH抗体(Dako公司，Denmark);BCA法蛋白浓度测定试剂盒(Pierce公司);TEMED(Sigma公司)，其余化学试剂为国产分析纯。

　　1.2大鼠腹膜间皮细胞的分离与培养[8]健康清洁级雄性SD大鼠，体重180～220g，由河南科技大学机能实验中心提供，每只肌肉注射100mL/L水合氯醛约0.6～1.0mL。待麻醉后向大鼠腹腔内注射20～30mL 2.5g/L胰蛋白酶+0.2g/L EDTA消化液。2h后断颈处死大鼠，750mL/L酒精全身浸泡消毒5min。将大鼠仰面放置于超净工作台上，沿腹白线依次剪开腹壁皮肤及肌肉，吸出腹腔内液体，移入15mL离心管中。1500r/min离心15min，弃上清，加入含100mL/L FBS的DMEM/F12培养液，轻轻用吸管吹打使之成为细胞混悬液，分装于25cm2的细胞培养瓶中。 再加入培养基使之总体积达到4～5mL，放入条件为37℃、950mL/L空气、50mL/L CO2的细胞培养箱中培养。每3d更换一次培养基。免疫组织化学方法鉴定结果示细胞角蛋白(cytokeratin)阳性，将2代腹膜细胞随机分为以下4组：正常对照组，AngⅡ(10-7mol/L)刺激组，两组用无血清的DMEM/F12培养基培养;黄芪注射液高浓度(2g/mL)干预组，黄芪注射液低浓度(1g/mL)干预组，两组提前孵育1h进行干预处理。在合适的时间点，观察以上各组各指标的变化情况。

　　1.3细胞内ROS的测定ROS的测定按文献报道方法[9]。具体如下：将细胞传至激光共聚焦专用培养皿(Corning公司)，静止24h同步化后，KRH缓冲液清洗3次，将各组细胞用10mg/L H2-DCFDA避光孵育37℃ 15min，KRH缓冲液再清洗3次，