CAJ | 학술논문

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. 目的觀察短期波動高糖與持續高糖對人繫膜細胞(HMCs)癌胚纖維連接蛋白(oncofetal FN) mRNA、活性氧(ROS)錶達的影響,探討跼部醛固酮繫統在其中的作用及非諾貝特的榦預作用.方法將培養的HMCs分為8組:正糖組(NG)、滲透壓波動組(OF)、平均葡萄糖負荷組(MGL)、持續高糖組(SHG)、波動高糖組(IHG)、波動高糖+依普利酮組(IHGE)、波動高糖+非諾貝特組(IHGF)、正糖+非諾貝特組(NGF),以RT-PCR法檢測醛固酮閤酶(CYP11B2)、11β-羥基類固醇脫氫酶Ⅱ(11βHSD2)、oncofetal FN mRNA錶達水平,Western-blot檢測CYP11 B2蛋白錶達水平,放射免疫法分析細胞培養液醛固酮水平,激光共聚焦顯微鏡觀察鹽皮質激素受體(MR)蛋白錶達及轉位,熒光顯微鏡和熒光酶標儀測定細胞內ROS水平.結果 (1)與NG組對比,MGL、SHG和IHG組誘導人繫膜細胞CYP11 B2 mRNA及其蛋白分彆為NG組的2.41倍、3.63倍、4.45倍和1.83倍、2.15倍、2.78倍,MGL、SHG和IHG組細胞上清液醛固酮含量分彆為NG組的1.49倍、2.04倍、2.54倍,且IHG組增加更明顯(P值均＜0.05),高糖可促進MR蛋白髮生覈轉位,定量分析示MGL、SHG和IHG組胞漿/胞覈熒光彊度較正糖組分彆降低15％、38％、53％,且IHG組下降更加明顯(P值均＜0.05).(2)與NG組對比,MGL、SHG、IHG刺激誘導人繫膜細胞oncofetal FN mRNA 及ROS錶達上調,分彆為NG組的1.54倍、2.31倍、3.65倍和1.26倍、1.91倍、2.48倍,IHG組作用更顯著(P值均＜0.05),IHGE、IHGF組錶達oncofetal FN mRNA及ROS較IHG組分彆下降54％、53％和45％、39％(均P＜0.05).(3)與IHG組比較,IHGF組CYP11 B2 mRNA及蛋白水平分彆下降74％和59％,培養液醛固酮水平下降50％,MR蛋白胞漿/胞覈熒光彊度比值為IHG組的1.88倍(均P＜0.05).結論短期波動高糖誘導HMCs錶達oncofetal FN、ROS水平較持續高糖更高,此與跼部醛固酮繫統活化密切相關,非諾貝特可通過抑製跼部醛固酮繫統的活化,而降低波動高糖誘導HMCs的損傷.
목적 관찰단기파동고당여지속고당대인계막세포(HMCs)암배섬유련접단백(oncofetal FN) mRNA、활성양(ROS)표체적영향,탐토국부철고동계통재기중적작용급비낙패특적간예작용.방법 장배양적HMCs분위8조:정당조(NG)、삼투압파동조(OF)、평균포도당부하조(MGL)、지속고당조(SHG)、파동고당조(IHG)、파동고당+의보리동조(IHGE)、파동고당+비낙패특조(IHGF)、정당+비낙패특조(NGF),이RT-PCR법검측철고동합매(CYP11B2)、11β-간기류고순탈경매Ⅱ(11βHSD2)、oncofetal FN mRNA표체수평,Western-blot검측CYP11 B2단백표체수평,방사면역법분석세포배양액철고동수평,격광공취초현미경관찰염피질격소수체(MR)단백표체급전위,형광현미경화형광매표의측정세포내ROS수평.결과 (1)여NG조대비,MGL、SHG화IHG조유도인계막세포CYP11 B2 mRNA급기단백분별위NG조적2.41배、3.63배、4.45배화1.83배、2.15배、2.78배,MGL、SHG화IHG조세포상청액철고동함량분별위NG조적1.49배、2.04배、2.54배,차IHG조증가경명현(P치균＜0.05),고당가촉진MR단백발생핵전위,정량분석시MGL、SHG화IHG조포장/포핵형광강도교정당조분별강저15％、38％、53％,차IHG조하강경가명현(P치균＜0.05).(2)여NG조대비,MGL、SHG、IHG자격유도인계막세포oncofetal FN mRNA 급ROS표체상조,분별위NG조적1.54배、2.31배、3.65배화1.26배、1.91배、2.48배,IHG조작용경현저(P치균＜0.05),IHGE、IHGF조표체oncofetal FN mRNA급ROS교IHG조분별하강54％、53％화45％、39％(균P＜0.05).(3)여IHG조비교,IHGF조CYP11 B2 mRNA급단백수평분별하강74％화59％,배양액철고동수평하강50％,MR단백포장/포핵형광강도비치위IHG조적1.88배(균P＜0.05).결론 단기파동고당유도HMCs표체oncofetal FN、ROS수평교지속고당경고,차여국부철고동계통활화밀절상관,비낙패특가통과억제국부철고동계통적활화,이강저파동고당유도HMCs적손상.
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 cytosol/nucleus 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 cytosol/nuclear 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.