叉头状转录因子01对高糖培养大鼠肾小球系膜细胞氧化应激的影响  

Effect of forkhead transcription factor O1 on oxidative stress in rat mesangial cells cultured under high glucose conditions

作　　者：吉鸿飞[1] 秦贵军[1] 张伟伟[1] 吴丽娜[1] 马晓君[1] 

机构地区：[1]郑州大学第一附属医院内分泌科,450052

出　　处：《中华糖尿病杂志》2012年第11期681-685,共5页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金资助项目（81050009）

摘　　要：目的通过激活和抑制叉头状转录因子01（Fox01）活性及表达，探讨其对高糖培养下大鼠肾小球系膜细胞（MCs）氧化应激的作用。方法采用脂质体转染法将FoxOl短发夹样RNA（Fox01shRNA）质粒载体稳定转染MCs；用高糖（30．0mmol／L）和白藜芦醇（Resv20．0txmol／L）培养稳定转染后的MCs。以5．6mmol／L葡萄糖培养的MCs为正常对照组（NG），将高糖培养的MCs分为5组：单纯高糖组（HG）、HG＋Resv组、HG＋Fox01shRNA转染组、HG＋Resv＋Fox01shRNA转染组、HG＋转染阴性对照组（shNC）。培养72h后，用荧光酶标仪检测各组MCs内活性氧（ROS）水平；逆转录聚合酶链反应（RT—PCR）检测去乙酰化酶（Sirtl）、Fox01、锰超氧化物歧化酶（MnSOD）mRNA表达；Westernblotting法检测Fox01及磷酸化Fox01（p-Fox01）水平。多组间数据比较采用单因素方差分析。结果与NG组相比，HG组SirtlmRNA表达下降，p-Fox01水平升高，MnSODmRNA表达下降，ROS水平升高，差异均有统计学意义（t=12．38、13．27、14．13、8．36，均P〈0．05）。与HG组相比，HG＋Fox01shRNA转染组Fox01mRNA表达被抑制，MnSODmRNA表达更低，ROS水平更高，差异均有统计学意义（t=20．61、13．61、10．13，均P〈0．05）；HG＋Resv组SiitlmRNA表达升高，p-Fox01水平下降，MnSODmRNA表达升高，ROS水平下降，差异均有统计学意义（t=6．02、10．69、5．39、5．37，均P〈0．05）。与HG＋Resv组比较，HG＋Resv＋Fox01shRNA转染组，Fox01mRNA表达被抑制，MnSODmRNA表达降低，ROS水平升高，差异均有统计学意义（t=22．53、15．31、14．63，均P〈0．05）。结论Fox01是调节MCs内ROS水平的重要转录因子，其可能通过激活下游抗氧化靶基因MnSOD的表达，保护MCs对抗氧化应激。Objective To study the effect and mechanism of forkhead transcription factor O1 （FoxO1） on oxidative stress in rat mesangial cells（MCs） cultured under high glucose conditions. Methods The MCs cultured in normal glucose （5.6 retool/L） medium were transfected by the plasmid vector of the FoxO1 short hairpin RNAs （FoxO1 shRNA）, and then cultured in high glucose （30. 0 mmol/L） medium and resveratrol （ Resv 20. 0 ~Lmol/L）. The MCs cultured in normal glucose medium served as normal control group （ group NG） , and the MCs cultured in high glucose medium were divided into ~＇~e groups ： simple high glucose group （group HG）, HG ＋ Resv group, HG ＋ FoxO1 shRNA group, HG ＋ Resv ＋ FoxO1 shRNA group, HG ＋ Negative control group （ shNC）. MCs in each group were cultured for 72 h. The level of reactive oxygen species （ROS） was assessed by fluorescence microplate reader. The mRNA expression of silent information regulator 1 （Sift1） , FoxO1, manganese superoxide dismutase （MnSOD） were detected by reverse transcription polymerase chain reaction （RT-RCR）. The protein expression of FoxO1 and phosphorylation FoxO1 （ p-FoxO1 ） were determined with Western blotting methods. One-way analysis of variance was applied in the data comparisons among the groups. Results Compared with group NG, the mRNA expression of Sirtl and MnSOD were significantly reduced but the p-FoxO1 and ROS were enhanced in group HG （t =12.38, 14. 13, 13.27, 8.36, allP〈0.05）. Compared with group HG, the mRNA expression levels of FoxO1 and MnSOD were obviously inhibited, but the level of ROS was enhanced in group HG ＋ FoxOlshRNA（t =20. 61, 13.61, 10. 13, all P 〈0. 05） ; and the mRNA expression of Sirtl and MnSOD increased, whereas the level of p-FoxO1 and ROS decreased significantly in group HG ＋ Resv （t = 6. 02, 5.39, 10. 69, 5.37, all P 〈 O. 05）. Compared with group HG ＋ Resv, the mRNA expression of FoxO1 and MnSOD was inhibited but the ROS was enhanced significantly in group

关 键 词：叉头转录因子类 氧化性应激 肾小球系膜细胞 高糖 大鼠 

分 类 号：R587.2[医药卫生—内分泌]