Objective To investigate the role of local aldosterone system in oncofetal fibronectin ( oncofetal FN) mRNA expression and reactive oxygen species (ROS) production in human mesangial cells (HMCs) exposed to short-term intermittent high glucose and the effect of Fenofibrate.Methods The HMCs were divided into 8 groups:normal glucose(NG) ;osmotic fluctuation(OF) ;mean glucose load (MGL) ;stable high glucose (SHG),short-term intermittent high glucose (IHG) ; intermittent high glucose plus eplerenone (IHGE) ; intermittent high glucose plus fenofibrate(IHGF) ; and normal glucose plus fenofibrate (NGF) groups.The mRNA expression levels of Aldosterone synthase ( CYP11 B2 ),11 β-hydroxysteroid dehydrogenase type 2 ( 11βHSD2 ) and oncofetal FN were determined by RT-PCR.The expression of CYP11B2 protein was determined by western-blot.Aldosterone level in cell culture supernatant was detected by radioimmunoassay.The expression and translocation of mineralocorticoid receptor (MR)protein were assayed with confocal laser scanning microscopy. ROS levels were determined by Fluorescence microscopy and fluorescence microplate reader.Results ( 1 ) MGL,SHG,and IHG groups showed a 2.41,3.63,and 4.45 times increase in CYP11B2 mRNA expression,and a 1.83,2.15,and 2.78 times increase in CYP11B2 protein expression,respectively,compared with NG group (P ＜ 0.05 ).The aldosterone levels of HMCs culture supernatant in MGL,SHG,and IHG groups were also increased,being 1.49,2.04,and 2.54 times of that in NG group ( P＜0.05 ),and the degree of elevation in IHG group was more marked than that in SHG group( P＜0.05 ).MR was activated and translocated from cytosol to nucleus in MGL,SHG,and IHG groups.Quantitative analysis showed the ratioes of cytosolucleus fluorescence intensity in MGL,SHG,and IHG groups were 15％,38％,and 53％ decreased as compared with that in NG group,and the decrease was more marked in IHG group ( P＜0.05 ).(2) Oncofetal FN mRNA expression and ROS levels in MGL,SHG,and IHG groups were increased,being 1.54,2.31,3.65 and 1.26,1.91,2.48 times of those in NG group,respectively ( P＜0.05 ),and this increase was more marked in group IHG ( P＜O.05 ).Compared with IHG group,oncofetal FN mRNA expression and ROS levels in group IHGE were significantly decreased by 54％ and 53％,and in group IHGF by 45％ and 39％. ( 3 ) CYP11B2 mRNA,protein,and aldosterone levels in IHGF group were decreased by 74％,59％,and 50％,and the activation of MR in group IHGF was inhibited when the ratio of cytosoluclear fluorescence intensity was increased 1.88 fold as compared with that in group IHG ( P＜0.05 ).Conclusions Increased expressions of oncofetal FN and ROS by HMCs induced by short-term intermittent high glucose were nore marked than those induced by stable high glucose.The mechanism was associated with activation of local aldosterone system.Fenofibrate may inhibit the activation of local aldosterone system and alleviate the injury to HMCs induced by intermittent high glucose.%目的 观察短期波动高糖与持续高糖对人系膜细胞(HMCs)癌胚纤维连接蛋白(oncofetal FN) mRNA、活性氧(ROS)表达的影响,探讨局部醛固酮系统在其中的作用及非诺贝特的干预作用.方法 将培养的HMCs分为8组:正糖组(NG)、渗透压波动组(OF)、平均葡萄糖负荷组(MGL)、持续高糖组(SHG)、波动高糖组(IHG)、波动高糖+依普利酮组(IHGE)、波动高糖+非诺贝特组(IHGF)、正糖+非诺贝特组(NGF),以RT-PCR法检测醛固酮合酶(CYP11B2)、11β-羟基类固醇脱氢酶Ⅱ(11βHSD2)、oncofetal FN mRNA表达水平,Western-blot检测CYP11 B2蛋白表达水平,放射免疫法分析细胞培养液醛固酮水平,激光共聚焦显微镜观察盐皮质激素受体(MR)蛋白表达及转位,荧光显微镜和荧光酶标仪测定细胞内ROS水平.结果 (1)与NG组对比,MGL、SHG和IHG组诱导人系膜细胞CYP11 B2 mRNA及其蛋白分别为NG组的2.41倍、3.63倍、4.45倍和1.83倍、2.15倍、2.78倍,MGL、SHG和IHG组细胞上清液醛固酮含量分别为NG组的1.49倍、2.04倍、2.54倍,且IHG组增加更明显(P值均＜0.05),高糖可促进MR蛋白发生核转位,定量分析示MGL、SHG和IHG组胞浆/胞核荧光强度较正糖组分别降低15％、38％、53％,且IHG组下降更加明显(P值均＜0.05).(2)与NG组对比,MGL、SHG、IHG刺激诱导人系膜细胞oncofetal FN mRNA 及ROS表达上调,分别为NG组的1.54倍、2.31倍、3.65倍和1.26倍、1.91倍、2.48倍,IHG组作用更显著(P值均＜0.05),IHGE、IHGF组表达oncofetal FN mRNA及ROS较IHG组分别下降54％、53％和45％、39％(均P＜0.05).(3)与IHG组比较,IHGF组CYP11 B2 mRNA及蛋白水平分别下降74％和59％,培养液醛固酮水平下降50％,MR蛋白胞浆/胞核荧光强度比值为IHG组的1.88倍(均P＜0.05).结论 短期波动高糖诱导HMCs表达oncofetal FN、ROS水平较持续高糖更高,此与局部醛固酮系统活化密切相关,非诺贝特可通过抑制局部醛固酮系统的活化,而降低波动高糖诱导HMCs的损伤.
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