高糖致人晶状体上皮细胞中p300和NF-κB硝基化对其相互作用的影响
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摘要
目的:糖尿病(diabetes mellitus, DM)是一种常见的代谢性疾病,已严重的危害到人类健康。据世界卫生组织(WHO)报告,我国的糖尿病人数居世界第二,在未来的十年内,由糖尿病带来的经济损失,中国居世界首位。糖尿病高致残和高死亡的主要原因是糖尿病慢性并发症,而在眼部主要表现为低视力和高致盲率。糖尿病性白内障(diabetic cataract, DC)是最重要的致盲因素。由于DC的机制尚不清楚,因此,DC机制的研究对降低致盲率和提高患者的生活质量有重要的意义。
目前,氧化应激(oxidative stress)被公认为是DC发病机制的中心环节。氧化应激反应的关键酶是诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS),已证实核转录因子NF-κB(nuclear factor-kappa B,NF-κB)是调控iNOS转录最重要的反式作用因子,能增强iNOS的转录活性,产生过量的一氧化氮(nitric oxide,NO)和超氧阴离子(superoxide anion,O2-.)。NO与O2-.快速反应,生成氧化能力较H2O2大2000倍的过氧亚硝基阴离子(peroxynitrite, ONOO-),造成氧化损伤。本小组前期研究表明,ONOO-参与介导DC的发生与发展。ONOO-可引起蛋白质中酪氨酸残基硝基化,硝基化后的蛋白质其结构和功能会发生改变,而硝基酪氨酸(nitrotyrosine, NT)则是蛋白质硝基化的一个特异性检测标志。
NF-κB家族包括p50, p52, p65 (RelA), c-Rel和RelB五个成员。它们中只有p65、c-Rel和RelB含有转录激活结构域,可与共激活因子结合,激活基因转录。NF-κB普遍以p50/ p65异二聚体的形式存在于细胞浆中,激活后NF-κB转位入核,结合顺式作用元件,p65通过招募共激活因子p300共同调控基因转录。p300在不同组织含量不同,其蛋白含量的改变可调节与转录因子的相互作用,对调控基因表达有重要影响。因此,p300对NF-κB p65的激活活性有重要作用。研究表明,在血管内皮细胞中,高糖刺激增强p300与NF-κB p65的相互作用,使NF-κB的转录活性增强。在糖尿病大鼠肾脏中,p300的mRNA及蛋白表达均升高,并增强NF-κBp65的转录活性。而在眼部的晶状体中,晶状体上皮细胞是代谢、合成、转运过程的中心,也是最先受损伤的靶细胞。在DC的发病过程中,晶状体上皮细胞中的p300与NF-κB是否起着关键作用呢?已有报道,高糖致白内障大鼠晶状体内NF-κB蛋白表达量增高。而在DC中对p300蛋白作用的研究未见报道。那么,在高糖刺激的人晶状体上皮细胞(SRA01/04)中,细胞核内p300蛋白含量如何?是否能引起p300与NF-κB的硝基化?硝基化后对p300与NF-κB结合活性的影响如何?是本研究拟解决的核心问题。
本实验用高糖、SIN-1(ONOO-供体)、FeTPPS(ONOO-的分解催化剂)等因素作用于SRA01/04细胞,以观察细胞核中p300蛋白含量、p300与NF-κB的硝基化水平、及硝基化对二者结合活性的影响,探讨高糖致人晶状体上皮细胞损伤的机制,为防治DC提供新思路。
方法:
1细胞培养及收集。
SRA01/04细胞株用含10%胎牛血清、1%非必须氨基酸、低糖DMEM培养基,置于37℃、5%CO2的培养箱中培养。细胞传代后,将细胞种至培养皿内,至细胞覆盖皿底90%,加不同因素作用后,刮取,收集于EP管中,用以提取核蛋白。将细胞种于覆盖玻片的六孔板内,制细胞爬片,各因素作用后,多聚甲醛固定,用以检测细胞共定位及核转位。
2分组及检测指标。
2.1根据不同的因素和作用时间分为4组,以确定最佳浓度及时间。(1)不同浓度葡萄糖组(5,10,15,20,25,30mmol/L) (2)同浓度葡萄糖不同作用时间组(0,5,10,15,20,25,30,35,40min) (3)不同浓度SIN-1组(0,10,50,100,250,500μmol/L) (4)高糖+不同浓度FeTPPS组(0,5,10,25,50,100μmol/L)
2.1.1 SRA01/04细胞分别经2.1作用因素作用后,细胞核蛋白用Lowry法检测蛋白含量,Western-blotting检测p300的蛋白含量及硝基化水平(NT含量);NF-κB p65不同糖浓度及作用时间的蛋白含量。
2.1.2制作SRA01/04细胞爬片后,分别经2.1作用因素作用,用荧光显微镜检测NF-κB p65的核转位。
2.2以作用因素最佳浓度及时间进行以下实验。(1)正常对照组(2)高糖组(25 mmol/L,25min) (3) SIN-1组(500μmol/L,25min) (4)高糖+ FeTPPS组(25 mmol/L+50μmol/L,25min)
2.2.1 SRA01/04细胞经2.2作用因素作用后,用Lowry法定量核蛋白,检测p300与NF-κB p65的相互作用。
2.2.2 Western-blotting检测免疫共沉淀的p300和NF-κB p65蛋白含量、硝基化水平(NT含量)及硝基化对p300与NF-κB p65相互作用的影响。
2.2.3激光共聚焦显微镜检测p300与NF-κB p65的共定位。
结果:
1 SRA01/04细胞核蛋白中p300蛋白含量的Western blotting检测结果。
1.1不同浓度葡萄糖组(5,10,15,20,25,30mmol/L)与正常糖浓度5 mmol/L比较,10mmol/L(P<0.05),15mmol/L(P<0.05),20mmol/L(P<0.01),25mmol/L(P<0.01),30mmol/L(P<0.01)各组p300蛋白含量均增高,差异有统计学意义。结果显示:随着葡萄糖浓度的升高,p300蛋白含量也随之升高,25mmol/L时升高最明显,30mmol/L时下降。
1.2 25mmol/L葡萄糖不同作用时间组(0,5,10,15,20,25,30,35,40min)
与25mmol/L糖浓度作用0min比较,5min(P<0.05),10min(P<0.05),15min(P<0.01),20min(P<0.01),25min(P<0.01),30min(P<0.01),35min(P<0.01),40min(P<0.01)各组p300蛋白含量均增高,差异有统计学意义。结果显示:随着时间的延长,p300蛋白含量随之增高,25min时升高最明显,30min时下降。
1.3不同浓度SIN-1组(0,10,50,100,250,500μmol/L)与SIN-1浓度为0μmol/L即正常糖浓度5 mmol/L比较,SIN-1浓度10μmol/L(P<0.05),50μmol/L(P<0.01),100μmol/L(P<0.01),250μmol/L(P<0.01),500μmol/L(P<0.01)各组p300蛋白含量均增高,差异有统计学意义。结果显示:随SIN-1浓度的增加,p300蛋白含量随之升高,在500μmol/L时升高最明显。1.4 25mmol/L葡萄糖+不同浓度FeTPPS组(0,5,10,25,50,100μmol/L)与FeTPPS浓度为0μmol/L即25 mmol/L的糖浓度比较,FeTPPS浓度5μmol/L(P<0.05),10μmol/L(P<0.05),25μmol/L(P<0.05),50μmol/L(P<0.01),100μmol/L(P<0.01)各组p300蛋白含量均降低,差异有统计学意义。结果显示:FeTPPS浓度在5-25μmol/L时, p300蛋白含量降低。在50-100μmol/L时,p300蛋白含量降低明显。
2 SRA01/04细胞核蛋白中p300硝基化水平变化的Western blotting检测结果。
2.1不同浓度葡萄糖组作用25min(5,10,15,20,25,30mmol/L)与正常糖浓度5 mmol/L比较,10mmol/L(P>0.05),15mmol/L(P<0.05),20mmol/L(P<0.01),25mmol/L(P<0.01),30mmol/L(P<0.01)除10mmol/L外,各组p300蛋白硝基化水平均增高,差异有统计学意义。结果显示:随着葡萄糖浓度的升高,p300蛋白硝基化水平也随之升高,25mmol/L时升高最明显,30mmol/L时下降。
2.2不同浓度SIN-1组(0,10,50,100,250,500μmol/L)与SIN-1浓度为0μmol/L即正常糖浓度5 mmol/L比较,SIN-1浓度10μmol/L(P<0.05),50μmol/L(P<0.01),100μmol/L(P<0.01),250μmol/L(P<0.01),500μmol/L(P<0.01)各组p300蛋白硝基化水平均增高,差异有统计学意义。结果显示:随着SIN-1浓度的升高,p300蛋白硝基化水平也随之升高,500μmol/L时升高最明显。
2.3 25mmol/L葡萄糖+不同浓度FeTPPS组(0,5,10,25,50,100μmol/L)与FeTPPS浓度为0μmol/L即25 mmol/L的糖浓度比较,FeTPPS浓度5μmol/L(P<0.05),10μmol/L(P<0.01),25μmol/L(P<0.01),50μmol/L(P<0.01),100μmol/L(P<0.01)各组p300蛋白硝基化水平均降低,差异有统计学意义。结果显示:随着FeTPPS浓度的升高,p300蛋白硝基化水平随之降低,50-100μmol/L时,p300蛋白硝基化水平降低明显。
3 SRA01/04细胞核蛋白中NF-κB p65蛋白含量的Western blotting检测结果。
3.1不同浓度葡萄糖组(5,10,15,20,25,30mmol/L)与正常糖浓度5 mmol/L比较,10mmol/L(P<0.05),15mmol/L(P<0.01),20mmol/L(P<0.01),25mmol/L(P<0.01),30mmol/L(P<0.01)各组NF-κB p65蛋白含量均增高,差异有统计学意义。结果显示:随着葡萄糖浓度的升高,p65蛋白含量也随之升高, 25mmol/L时升高最明显,30mmol/L时下降。
3.2 25mmol/L葡萄糖不同作用时间(0,5,10,15,20,25,30,35,40min)与25mmol/L糖浓度作用0min比较,5min(P<0.05),10min(P<0.01),15min(P<0.01),20min(P<0.01),25min(P<0.01),30min(P<0.01),35min(P<0.01),40min(P<0.01)各组NF-κB p65蛋白含量均增高,差异有统计学意义。结果显示:随着时间的延长,p65蛋白含量随之增高,20min时升高最明显,25min时下降。
4 SRA01/04细胞中NF-κB p65的核转位。
4.1 25mmol/L葡萄糖不同作用时间组(0,5,10,15,20,25,30,35,40min)
与25mmol/L糖浓度作用0min比较,5min(P>0.05),10min(P<0.05),15min(P<0.01),20min(P<0.01),25min(P<0.01),30min(P<0.01),35min(P<0.01),40min(P<0.01)除了作用5min外,各组p65核转位比率均增高,差异有统计学意义。结果显示:随着时间的延长,p65核转位也随之增多,25-30min时升高最明显,35min时下降。
4.2不同浓度SIN-1组(0,50,500μmol/L,25min)与未加SIN-1的5mmol/L正常糖浓度组比较,50μmol/L(P<0.01),500μmol/L(P<0.01)各组p65核转位比率均增高,差异有统计学意义。结果显示:随着SIN-1作用浓度的增加,p65核转位比率也随之增加。
4.3 25mmol/L葡萄糖+不同浓度FeTPPS组(0,10,50μmol/L,25min)与未加FeTPPS的25mmol/L葡萄糖组比较,10μmol/L(P<0.01),50μmol/L(P<0.01)各组p65核转位比率均降低,差异有统计学意义。结果显示:随着FeTPPS作用浓度的增加,p65核转位比率也随之下降。
5 SRA01/04细胞核蛋白中,p300与NF-κB p65的相互作用及硝基化水平。由核蛋白中p300蛋白含量及硝基化水平可确定各因素最佳作用浓度及作用时间。(1)正常组(5 mmol/L,25min) (2)高糖组(25 mmol/L,25min) (3)SIN-1组(500μmol/L,25min)(4)高糖+ FeTPPS组(25 mmol/L+50μmol/L,25min)
5.1核蛋白中p300、NF-κB p65蛋白含量
p300蛋白含量结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p300蛋白含量均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p300蛋白含量降低。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p300蛋白含量降低,差异有统计学意义。p65蛋白含量结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p65蛋白含量均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p65蛋白含量降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p65蛋白含量降低,差异有统计学意义。表明高糖组、SIN-1组中p300与NF-κB p65蛋白含量均明显增加,高糖+ FeTPPS组中p300与NF-κB p65蛋白含量较高糖组和SIN-1组明显降低。
5.2核蛋白中p300、NF-κB p65蛋白硝基化水平
p300蛋白硝基化水平结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p300蛋白硝基化水平均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p300蛋白硝基化水平降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p300蛋白硝基化水平降低,差异有统计学意义。p65蛋白硝基化水平结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p65蛋白硝基化水平均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p65蛋白硝基化水平降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p65蛋白硝基化水平降低,差异有统计学意义。表明高糖组、SIN-1组中p300与NF-κB p65蛋白硝基化水平均明显增加,高糖+ FeTPPS组中p300与NF-κB p65蛋白硝基化水平较高糖组和SIN-1组明显降低。
5.3硝基化对p300与NF-κB p65相互作用的影响。与p300蛋白相互作用的p65蛋白含量及硝基化水平结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p65蛋白含量及硝基化水平均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p65蛋白含量及硝基化水平均降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p65蛋白含量及硝基化水平均降低,差异有统计学意义。与p65蛋白相互作用的p300蛋白含量及硝基化水平结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p300蛋白含量及硝基化水平均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p300蛋白含量及硝基化水平均降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p300蛋白含量及硝基化水平均降低,差异有统计学意义。表明高糖组、SIN-1组中p300与NF-κB p65蛋白相互作用及硝基化水平均明显增加,高糖+ FeTPPS组中p300与NF-κB p65蛋白相互作用及硝基化水平较高糖组和SIN-1组明显降低。
6 SRA01/04细胞中p300与NF-κB p65蛋白的共定位
p300与NF-κB p65蛋白共定位的细胞数量,与5mmol/L正常糖浓度组比较,高糖组(P<0.01),SIN-1组(P<0.01)p300与NF-κB p65蛋白共定位的细胞数量增加,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p300与NF-κB p65蛋白共定位的细胞数量降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p300与NF-κB p65蛋白共定位的细胞数量降低,差异有统计学意义。表明高糖组、SIN-1组中p300与NF-κB p65蛋白共定位的细胞数量均明显增加,高糖+ FeTPPS组中p300与NF-κB p65蛋白共定位的细胞数量较高糖组和SIN-1组明显降低。
结论:
1高糖刺激人晶状体上皮细胞(SRA01/04),p300在核内聚集,含量增加。
2高糖刺激人晶状体上皮细胞(SRA01/04),可使p300和NF-κB蛋白硝基化。
3硝基化可增强p300与NF-κB的相互作用,抑制p300与NF-κB的硝基化可在防治DC中起关键作用。
Objective: Nowadays diabetes mellitus (DM) as a common metabolic disease has endangered the healthy of human being. According to the report of Word Health Organization (WHO), the second-largest number of DM patient exists in China. The economic losses in China due to by DM rank first in the world in the next decade. The high disability and high mortality of DM are mainly caused by chronic diabetes mellitus complications, while in the eye mainly for low vision and blindness. In addition, diabetic cataract (DC) is the most important blinding factor. Due to the pathogenesis of DC is not fully known yet, therefore, the research of DC pathogenesis present remarkable significance for reducing the rate of blindness and improve the quality of life of the patients.
Oxidative stress having been widely accepted to play an important role. The key enzyme of oxidative stress is inducible nitric oxide syntheses (iNOS), which has confirmed that the nuclear factor-kappa B (NF-κB), is the most trans-acting factor for regulating transcription of iNOS. It can enhance the transcriptional activity of iNOS, resulting in excessive nitric oxide (NO) and super oxide anion (O2-.), NO reacts rapidly with O2-. to form peroxynitrite (ONOO-) in diabetes whose oxidative ability is 2000 folds stronger than H2O2 and cause oxidative damages. The preliminary study of our reserch showed that ONOO- played a crucial role in pathogenesis and progression of DC. ONOO- can cause conspicuous nitration to tyrosine residue of proteins, after which the structure and function of the nitration of proteins will change, and nitrotyrosine (NT) is a specificity marker for nitration of proteins.
There are five members in NF-κB transcription family: p50, p52, p65 (RelA), c-Rel, and RelB. Only can p65, c-Rel and RelB which contain transcription activation domain be co-activating factor binding and activate gene transcription. Generally, NF-κB stays in the cytoplasm in the form of p50/p65 homodimers. The activated NF-κB translocation into the nucleus, combined with cis-acting element. p65 recruits coactivator p300 to regulate gene transcription. p300 has different content in different tissues; its protein content can be adjusted with the change in the interaction of transcription factors and have a major impact on the regulation of gene expression. So, p300 plays an important role on the activation of NF-κB p65 activity. According to the research, in vascular endothelial cells, high glucose induces NF-κB and p300 interaction, and then increases the transcriptional activity of NF-κB. The rats of diabetic nephropathy, the mRNA and protein expression of p300 were increased, and enhanced NF-κB p65 transcriptional activity. In the lens of the eye, the lens epithelial cells are metabolism, synthesis, transit center, but also the first injured target cells. Dose the lens epithelial cells in the p300 and NF-κB play a key role in the pathogenesis of DC? The report has been shown that high glucose-induced cataract in rat lens NF-κB protein expression has increased. However, the repot of the effect of p300 protein has not been reported in DC process. Then, in the high glucose-induced human lens epithelial cells (SRA01/04), How to p300 protein content in the nucleus? Whether it can cause p300 and NF-κB in nitration? What’s the impact of the nitration of the p300 and NF-κB on their interaction? The questions mentioned above are the core problems to be solved of this study.
In order to observe the p300 protein in the nucleus, the nitration levels of p300 and NF-κB and the impact of the nitration of the p300 and NF-κB on their interaction and investigate high glucose-induced human lens epithelial cell injury mechanism for providing new ideas for prevention and the control of DC, high-glucose, SIN-1(a peroxynitrite donor) and FeTPPS (the per- oxynitrite decomposition catalyst) acting on SRA01/04 cells were used in this study.
Methods:
1 Cell culture and collected.
SRA01/04 cell line containing 10% fetal bovine serum, 1% non-essential amino acids, DMEM medium cultured in 37℃, 5% CO2 atmosphere.
When the cells passaged, it was taken into the culture dish, which was covered by the cells for 90%. Then addde the different factors and scraped it. After that they were collected in the EP tube that was used to extract nucleoprotein.
Built-in six-well plates of six coverslips then passaged the cells. Stimulating factors were to be the role of each group then the paraformaldehyde fixed, using confocal laser scanning microscope analysis co-localization and fluorescence microscope analysis nucleus translocation.
2 Groups and tests
2.1 In accordance with different factors and acting time, four groups were divided to determine the optimal concentration and time. (1) Different concentrations of glucose groups (5, 10, 15, 20, 25, 30mmol/L) (2) The concentration of glucose with different time groups (0, 5, 10, 15, 20, 25, 30, 35, 40min) (3) Different concentrations of SIN-1 groups (0, 10, 50, 100, 250, 500μmol/L) (4) High glucose + different concentrations of FeTPPS groups (0, 5, 10, 25, 50, 100μmol/L)
2.1.1 After the cell SRA01/04 influenced by the 2.1 respectively, nucleoprotein was detected by Lowry; Western-blotting detected precipitated p300 protein content and its nitration levels (NT content); protein content of NF-κB p65 induced by different factors and acting time.
2.1.2 SRA01/04 cells lived on coverslip, influenced by the 2.1 respectively, using fluorescence microscopy NF-κB p65 nucleus translocation.
2.2 In order to make optimal concentration and time factors, the following experiment were taken. (1) The normal control group (2) High glucose group (25 mmol/L, 25min) (3) SIN-1 group (500μmol/L, 25min) (4) High glucose + FeTPPS group (25 mmol/L+50μmol/L, 25min)
2.2.1 After the cell SRA01/04 affected by the factor 2.2, nucleoprotein was detected by Lowry; Western-blotting was used to detect the reciprocity of the p300 and NF-κB p65.
2.2.2 Western-blotting detection detected precipitated p300 and NF-κB p65 protein content and its nitration levels (NT content); the influence about nitration of the p300 and NF-κB p65 on their interaction.
2.2.3 Use Laser confocal microscopy to detect p300 and NF-κB p65 on co-localizaation.
Results:
1 Western blotting detected the nucleoprotein of p300 content in SRA01/04 cell line.
1.1 Different concentrations of glucose groups (5, 10, 15, 20, 25, 30mmol/L) Compared with control group 5 mmol/L, 10mmol/L (P<0.05), 15mmol/L (P<0.05), 20mmol/L (P<0.01), 25mmol/L (P<0.01), 30mmol/L (P<0.01) each p300 protein content increased, the difference had statistical meaning. The results showed that as the rising of glucose concentration, p300 protein content rose. When it was at 25mmol/L, the rising was the most obvious. While at 30mmol/L, it dropped down.
1.2 Times of different effects of 25mmol/L glucoses (0, 5, 10, 15, 20, 25, 30, 35, 40min)
Compared with 25mmol/L glucose concentration as 0 min, 5min (P<0.05), 10min (P<0.05), 15min (P<0.01), 20min (P<0.01), 25min (P<0.01), 30min (P<0.01), 35min (P<0.01), 40min (P<0.01) p300 protein content in each group increased, the difference had statistical meaning. The results showed that as time went on, the p300 concentration content rose, and were obvious at 25min, which dropped down at 30min.
1.3 Different concentration of SIN-1 groups (0, 10, 50, 100, 250, 500μmol/L) Compared with 0μmol/L of SIN-1 control group, which equals to normal glucose concentration of 5 mmol/L, SIN-1 concentration 10μmol/L (P<0.05), 50μmol/L (P<0.01), 100μmol/L (P<0.01), 250μmol/L (P<0.01), 500μmol/L (P<0.01) p300 protein content in each group increased, the difference had statistical meaning. The results showed that as SIN-1 concentration increased, the p300 protein content rose, and were obvious at 500μmol/L.
1.4 25mmol/L glucose + different concentration FeTPPS groups (0, 5, 10, 25, 50, 100μmol/L)
Compared with 25 mmol/L glucose concentration of non-plus FeTPPS, each group of FeTPPS 5μmol/L (P<0.05), 10μmol/L (P<0.05), 25μmol/L (P<0.05), 50μmol/L (P<0.01), 100μmol/L (P<0.01) p300 protein content reduced, the difference had statistical meaning. The results showed that when FeTPPS concentration was at 5-25μmol/L, p300 protein content were few. When it was at 50-100μmol/L, p300 protein content reduced obviously.
2 Western blotting detected the nucleoprotein of nitration levels of p300 content in SRA01/04 cell line.
2.1 Different concentrations of glucose groups acted 25min (5, 10, 15, 20, 25, 30mmol/L)
Compared with 5 mmol/L control group, 10mmol/L (P>0.05), 15mmol/L (P<0.05), 20mmol/L (P<0.01), 25mmol/L (P<0.01), 30mmol/L (P<0.01) except for 10mmol/L, the nitration levels of p300 content increased averagely in each group, the difference had statistical meaning. The results showed that as the rising of glucose concentration, the nitration level of p300 protein went up as well, and were most obvious at 25mmol/L, while dropped down at 30mmol/L.
2.2 Different concentration of SIN-1 groups (0, 10, 50, 100, 250, 500μmol/L).
Compared with SIN-1 of 0μmol/L concentrations, which equals to normal glucose concentration of 5 mmol/L, SIN-1 concentration 10μmol/L (P<0.05), 50μmol/L (P<0.01), 100μmol/L (P<0.01), 250μmol/L (P<0.01), 500μmol/L (P<0.01) the nitration levels of p300 content increased averagely in each group, the difference had statistical meaning. The results showed that as the rising of SIN-1 concentration, the nitration level of p300 protein went up as well, and were most obvious at 500μmol/L.
2.3 25mmol/L glucose + different concentration FeTPPS groups (0, 5, 10, 25, 50, 100μmol/L) Compared with 25 mmol/L glucose concentration of non-plus FeTPPS, each group of FeTPPS 5μmol/L (P<0.05), 10μmol/L (P<0.01), 25μmol/L (P<0.01), 50μmol/L (P<0.01), 100μmol/L (P<0.01) the nitration levels of p300 protein content reduced averagely in each group, the difference had statistical meaning. The results showed that as the rising of FeTPPS concentration, the nitration level of p300 protein decreased and were most obvious at 50-100μmol/L.
3 Western blotting detected the nucleoprotein of NF-κB p65 content in SRA01/04 cell line.
3.1 Different concentrations of glucose groups (5, 10, 15, 20, 25, 30mmol/L)
Compared with normal glucose concentration 5mmol/L, 10mmol/L (P<0.05), 15mmol/L (P<0.01), 20mmol/L (P<0.01), 25mmol/L (P<0.01), 30mmol/L (P<0.01) the protein content of NF-κB p65 increased averagely in each group, the difference had statistically significant. The results showed that with the increase of glucose concentration, the protein content of NF-κB p65 went up also. It was most obvious at 25mmol/L, while dipped at 30mmol/L.
3.2 Times of different effects of 25mmol/L glucoses (0, 5, 10, 15, 20, 25, 30, 35, 40min)
Compared with 25mmol/L glucose concentration as 0 min, 5min (P<0.05), 10min (P<0.01), 15min (P<0.01), 20min (P<0.01), 25min (P<0.01), 30min (P<0.01), 35min (P<0.01), 40min (P<0.01) the protein content of NF-κB p65 increased averagely in each group, the difference had statistically significant. The results showed that as time went by, the protein content of NF-κB p65 increased, it was at 20 min, the rising was the most obvious, while at 25 min, it dropped down.
4 NF-κB p65 transfer into nucleus in SRA01/04 cells line.
4.1 Times of different effects of 25mmol/L glucoses (0, 5, 10, 15, 20, 25, 30, 35, 40min)
Compared with 25mmol/L glucose concentration as 0 min, 5min (P>0.05), 10min (P<0.05), 15min (P<0.01), 20min (P<0.01), 25min (P<0.01), 30min (P<0.01), 35min (P<0.01), 40min (P<0.01) except for 5min, the rates of nucleus translocation of NF-κB p65 increased averagely in each group, the difference had statistical meaning. The results showed that with the time prolonged, the rates of NF-κB p65 nucleus translocation also increased. In 25-30min, it increased most significant, and in 35 min, it decreased.
4.2 Different concentration of SIN-1 groups (0, 50, 500μmol/L, 25min) Compared with non-plus SIN-1 concentrations, which equals to normal glucose concentration of 5 mmol/L, 50μmol/L (P<0.01), 500μmol/L (P<0.01) the rates of nucleus translocation of NF-κB p65 increased averagely in each group, the difference had statistical meaning. The results showed that with the role of SIN-1 concentration rised, the rates of NF-κB p65 nucleus translocation ratio increased.
4.3 25mmol/L glucose + different concentration FeTPPS groups (0, 10, 50μmol/L, 25min)
Compared with 25 mmol/L glucose group of non-plus FeTPPS, 10μmol/L (P<0.01), 50μmol/L (P<0.01) the rates of nucleus translocation of NF-κB p65 reduced averagely in each group, the difference had statistical meaning. The results showed that with the role of FeTPPS concentration increased, NF-κB p65 nucleus translocation ratio is lower.
5 The interaction and nitration levels of p300 and NF-κB p65 in SRA01/04 nucleoproteins.
Make an optimal determination of the concentration and action time based on the nucleoprotein of p300 protein content and the nitration level of each factor. (1) Normal (5 mmol/L, 25min) (2) High glucose group (25 mmol/L, 25 min) (3) SIN-1 group (500μmol/L, 25min) (4) High glucose + FeTTPS group (25 mmol/L + 50μmol/L, 25min)
5.1 p300 and NF-κB p65 protein content in nucleoprotein.
The results of p300 protein content showed that, compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) each p300 protein content increased, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. In addition, compared with high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) p300 protein content reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) p300 protein content reduced, the difference had statistical meaning. The results of NF-κB p65 protein content showed that, compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) each p65 protein content increased, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) p65 protein content reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) p65 protein content reduced, the difference had statistical meaning, which showed that protein content of high glucose group and SIN-1 group, p300 and NF-κB p65 were significantly increased. In the high glucose + FeTPPS group, the protein content of p300 and NF-κB p65 were significant lower than that of high glucose and SIN-1.
5.2 The nitration level of p300, NF-κB p65 in nucleoprotein
The nitration level of p300 protein results showed that compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) the nitration levels of p300 content increased averagely in each group, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) the nitration levels of p300 content reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) the nitration levels of p300 content reduced, the difference had statistical meaning. The nitration level of NF-κB p65 protein results showed that compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) the nitration levels of p65 content increased averagely in each group, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) the nitration levels of p65 content reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) the nitration levels of p65 content reduced, the difference had statistical meaning. The results mentioned above indicated that the nitration level of p300 and NF-κB p65 protein in SIN-1 group went up obviously. p300 and NF-κB p65 protein nitration in high glucose + FeTPPS group were significantly lower than that in glucose group and SIN-1 group.
5.3 The interaction effect of nitration to the p300 and NF-κB.
The NF-κB p65 protein content which interacts with p300 protein and its nitration level showed that compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) protein interactions and nitration level increased averagely in each group, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) protein interactions and nitration level reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) protein interactions and nitration level reduced, the difference had statistical meaning. The p300 protein content which interact with NF-κB p65 protein and its nitration level showed that compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) protein interactions and nitration level increased averagely in each group, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) protein interactions and nitration level reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) protein interactions and nitration level reduced, the difference had statistical meaning. The conclusion stated that protein interactions and nitration level of p300 and NF-κB p65 in high glucose group and SIN-1 group increased significantly. While the contrary situation is in high glucose + FeTPPS group, the protein interactions and nitration level of p300 and NF-κB p65 were significantly lower.
6 p300 and NF-κB p65 protein co-localization in SRA01/04 cells line. Compared with 5mmol/L normal glucose concentration group, the cell number of p300 and NF-κB p65 protein co-localization, high glucose group (P<0.01), SIN-1 group (P<0.01) the cell number of co-localization increased in each group, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) the cell number of co-localization reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (p<0.01) the cell number of co-localization reduced, the difference had statistical meaning. The results showed that the cell number of co-localization of p300 and NF-κB p65 in high glucose group and SIN-1 group increased obviously, which is obviously higher than that in high glucose + FeTPPS group.
Conclusions:
1 p300 content increased and gathered in the nucleus, when high glucose-induced human lens epithelial cells (SRA01/04).
2 p300 and NF-κB protein can be nitration, when high glucose-induced human lens epithelial cells (SRA01/04).
3 Nitration can enhance the interaction of p300 and NF-κB, p300 and NF-κB inhibition of nitration may play a key role in prevention and control of DC.
目前,氧化应激(oxidative stress)被公认为是DC发病机制的中心环节。氧化应激反应的关键酶是诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS),已证实核转录因子NF-κB(nuclear factor-kappa B,NF-κB)是调控iNOS转录最重要的反式作用因子,能增强iNOS的转录活性,产生过量的一氧化氮(nitric oxide,NO)和超氧阴离子(superoxide anion,O2-.)。NO与O2-.快速反应,生成氧化能力较H2O2大2000倍的过氧亚硝基阴离子(peroxynitrite, ONOO-),造成氧化损伤。本小组前期研究表明,ONOO-参与介导DC的发生与发展。ONOO-可引起蛋白质中酪氨酸残基硝基化,硝基化后的蛋白质其结构和功能会发生改变,而硝基酪氨酸(nitrotyrosine, NT)则是蛋白质硝基化的一个特异性检测标志。
NF-κB家族包括p50, p52, p65 (RelA), c-Rel和RelB五个成员。它们中只有p65、c-Rel和RelB含有转录激活结构域,可与共激活因子结合,激活基因转录。NF-κB普遍以p50/ p65异二聚体的形式存在于细胞浆中,激活后NF-κB转位入核,结合顺式作用元件,p65通过招募共激活因子p300共同调控基因转录。p300在不同组织含量不同,其蛋白含量的改变可调节与转录因子的相互作用,对调控基因表达有重要影响。因此,p300对NF-κB p65的激活活性有重要作用。研究表明,在血管内皮细胞中,高糖刺激增强p300与NF-κB p65的相互作用,使NF-κB的转录活性增强。在糖尿病大鼠肾脏中,p300的mRNA及蛋白表达均升高,并增强NF-κBp65的转录活性。而在眼部的晶状体中,晶状体上皮细胞是代谢、合成、转运过程的中心,也是最先受损伤的靶细胞。在DC的发病过程中,晶状体上皮细胞中的p300与NF-κB是否起着关键作用呢?已有报道,高糖致白内障大鼠晶状体内NF-κB蛋白表达量增高。而在DC中对p300蛋白作用的研究未见报道。那么,在高糖刺激的人晶状体上皮细胞(SRA01/04)中,细胞核内p300蛋白含量如何?是否能引起p300与NF-κB的硝基化?硝基化后对p300与NF-κB结合活性的影响如何?是本研究拟解决的核心问题。
本实验用高糖、SIN-1(ONOO-供体)、FeTPPS(ONOO-的分解催化剂)等因素作用于SRA01/04细胞,以观察细胞核中p300蛋白含量、p300与NF-κB的硝基化水平、及硝基化对二者结合活性的影响,探讨高糖致人晶状体上皮细胞损伤的机制,为防治DC提供新思路。
方法:
1细胞培养及收集。
SRA01/04细胞株用含10%胎牛血清、1%非必须氨基酸、低糖DMEM培养基,置于37℃、5%CO2的培养箱中培养。细胞传代后,将细胞种至培养皿内,至细胞覆盖皿底90%,加不同因素作用后,刮取,收集于EP管中,用以提取核蛋白。将细胞种于覆盖玻片的六孔板内,制细胞爬片,各因素作用后,多聚甲醛固定,用以检测细胞共定位及核转位。
2分组及检测指标。
2.1根据不同的因素和作用时间分为4组,以确定最佳浓度及时间。(1)不同浓度葡萄糖组(5,10,15,20,25,30mmol/L) (2)同浓度葡萄糖不同作用时间组(0,5,10,15,20,25,30,35,40min) (3)不同浓度SIN-1组(0,10,50,100,250,500μmol/L) (4)高糖+不同浓度FeTPPS组(0,5,10,25,50,100μmol/L)
2.1.1 SRA01/04细胞分别经2.1作用因素作用后,细胞核蛋白用Lowry法检测蛋白含量,Western-blotting检测p300的蛋白含量及硝基化水平(NT含量);NF-κB p65不同糖浓度及作用时间的蛋白含量。
2.1.2制作SRA01/04细胞爬片后,分别经2.1作用因素作用,用荧光显微镜检测NF-κB p65的核转位。
2.2以作用因素最佳浓度及时间进行以下实验。(1)正常对照组(2)高糖组(25 mmol/L,25min) (3) SIN-1组(500μmol/L,25min) (4)高糖+ FeTPPS组(25 mmol/L+50μmol/L,25min)
2.2.1 SRA01/04细胞经2.2作用因素作用后,用Lowry法定量核蛋白,检测p300与NF-κB p65的相互作用。
2.2.2 Western-blotting检测免疫共沉淀的p300和NF-κB p65蛋白含量、硝基化水平(NT含量)及硝基化对p300与NF-κB p65相互作用的影响。
2.2.3激光共聚焦显微镜检测p300与NF-κB p65的共定位。
结果:
1 SRA01/04细胞核蛋白中p300蛋白含量的Western blotting检测结果。
1.1不同浓度葡萄糖组(5,10,15,20,25,30mmol/L)与正常糖浓度5 mmol/L比较,10mmol/L(P<0.05),15mmol/L(P<0.05),20mmol/L(P<0.01),25mmol/L(P<0.01),30mmol/L(P<0.01)各组p300蛋白含量均增高,差异有统计学意义。结果显示:随着葡萄糖浓度的升高,p300蛋白含量也随之升高,25mmol/L时升高最明显,30mmol/L时下降。
1.2 25mmol/L葡萄糖不同作用时间组(0,5,10,15,20,25,30,35,40min)
与25mmol/L糖浓度作用0min比较,5min(P<0.05),10min(P<0.05),15min(P<0.01),20min(P<0.01),25min(P<0.01),30min(P<0.01),35min(P<0.01),40min(P<0.01)各组p300蛋白含量均增高,差异有统计学意义。结果显示:随着时间的延长,p300蛋白含量随之增高,25min时升高最明显,30min时下降。
1.3不同浓度SIN-1组(0,10,50,100,250,500μmol/L)与SIN-1浓度为0μmol/L即正常糖浓度5 mmol/L比较,SIN-1浓度10μmol/L(P<0.05),50μmol/L(P<0.01),100μmol/L(P<0.01),250μmol/L(P<0.01),500μmol/L(P<0.01)各组p300蛋白含量均增高,差异有统计学意义。结果显示:随SIN-1浓度的增加,p300蛋白含量随之升高,在500μmol/L时升高最明显。1.4 25mmol/L葡萄糖+不同浓度FeTPPS组(0,5,10,25,50,100μmol/L)与FeTPPS浓度为0μmol/L即25 mmol/L的糖浓度比较,FeTPPS浓度5μmol/L(P<0.05),10μmol/L(P<0.05),25μmol/L(P<0.05),50μmol/L(P<0.01),100μmol/L(P<0.01)各组p300蛋白含量均降低,差异有统计学意义。结果显示:FeTPPS浓度在5-25μmol/L时, p300蛋白含量降低。在50-100μmol/L时,p300蛋白含量降低明显。
2 SRA01/04细胞核蛋白中p300硝基化水平变化的Western blotting检测结果。
2.1不同浓度葡萄糖组作用25min(5,10,15,20,25,30mmol/L)与正常糖浓度5 mmol/L比较,10mmol/L(P>0.05),15mmol/L(P<0.05),20mmol/L(P<0.01),25mmol/L(P<0.01),30mmol/L(P<0.01)除10mmol/L外,各组p300蛋白硝基化水平均增高,差异有统计学意义。结果显示:随着葡萄糖浓度的升高,p300蛋白硝基化水平也随之升高,25mmol/L时升高最明显,30mmol/L时下降。
2.2不同浓度SIN-1组(0,10,50,100,250,500μmol/L)与SIN-1浓度为0μmol/L即正常糖浓度5 mmol/L比较,SIN-1浓度10μmol/L(P<0.05),50μmol/L(P<0.01),100μmol/L(P<0.01),250μmol/L(P<0.01),500μmol/L(P<0.01)各组p300蛋白硝基化水平均增高,差异有统计学意义。结果显示:随着SIN-1浓度的升高,p300蛋白硝基化水平也随之升高,500μmol/L时升高最明显。
2.3 25mmol/L葡萄糖+不同浓度FeTPPS组(0,5,10,25,50,100μmol/L)与FeTPPS浓度为0μmol/L即25 mmol/L的糖浓度比较,FeTPPS浓度5μmol/L(P<0.05),10μmol/L(P<0.01),25μmol/L(P<0.01),50μmol/L(P<0.01),100μmol/L(P<0.01)各组p300蛋白硝基化水平均降低,差异有统计学意义。结果显示:随着FeTPPS浓度的升高,p300蛋白硝基化水平随之降低,50-100μmol/L时,p300蛋白硝基化水平降低明显。
3 SRA01/04细胞核蛋白中NF-κB p65蛋白含量的Western blotting检测结果。
3.1不同浓度葡萄糖组(5,10,15,20,25,30mmol/L)与正常糖浓度5 mmol/L比较,10mmol/L(P<0.05),15mmol/L(P<0.01),20mmol/L(P<0.01),25mmol/L(P<0.01),30mmol/L(P<0.01)各组NF-κB p65蛋白含量均增高,差异有统计学意义。结果显示:随着葡萄糖浓度的升高,p65蛋白含量也随之升高, 25mmol/L时升高最明显,30mmol/L时下降。
3.2 25mmol/L葡萄糖不同作用时间(0,5,10,15,20,25,30,35,40min)与25mmol/L糖浓度作用0min比较,5min(P<0.05),10min(P<0.01),15min(P<0.01),20min(P<0.01),25min(P<0.01),30min(P<0.01),35min(P<0.01),40min(P<0.01)各组NF-κB p65蛋白含量均增高,差异有统计学意义。结果显示:随着时间的延长,p65蛋白含量随之增高,20min时升高最明显,25min时下降。
4 SRA01/04细胞中NF-κB p65的核转位。
4.1 25mmol/L葡萄糖不同作用时间组(0,5,10,15,20,25,30,35,40min)
与25mmol/L糖浓度作用0min比较,5min(P>0.05),10min(P<0.05),15min(P<0.01),20min(P<0.01),25min(P<0.01),30min(P<0.01),35min(P<0.01),40min(P<0.01)除了作用5min外,各组p65核转位比率均增高,差异有统计学意义。结果显示:随着时间的延长,p65核转位也随之增多,25-30min时升高最明显,35min时下降。
4.2不同浓度SIN-1组(0,50,500μmol/L,25min)与未加SIN-1的5mmol/L正常糖浓度组比较,50μmol/L(P<0.01),500μmol/L(P<0.01)各组p65核转位比率均增高,差异有统计学意义。结果显示:随着SIN-1作用浓度的增加,p65核转位比率也随之增加。
4.3 25mmol/L葡萄糖+不同浓度FeTPPS组(0,10,50μmol/L,25min)与未加FeTPPS的25mmol/L葡萄糖组比较,10μmol/L(P<0.01),50μmol/L(P<0.01)各组p65核转位比率均降低,差异有统计学意义。结果显示:随着FeTPPS作用浓度的增加,p65核转位比率也随之下降。
5 SRA01/04细胞核蛋白中,p300与NF-κB p65的相互作用及硝基化水平。由核蛋白中p300蛋白含量及硝基化水平可确定各因素最佳作用浓度及作用时间。(1)正常组(5 mmol/L,25min) (2)高糖组(25 mmol/L,25min) (3)SIN-1组(500μmol/L,25min)(4)高糖+ FeTPPS组(25 mmol/L+50μmol/L,25min)
5.1核蛋白中p300、NF-κB p65蛋白含量
p300蛋白含量结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p300蛋白含量均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p300蛋白含量降低。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p300蛋白含量降低,差异有统计学意义。p65蛋白含量结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p65蛋白含量均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p65蛋白含量降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p65蛋白含量降低,差异有统计学意义。表明高糖组、SIN-1组中p300与NF-κB p65蛋白含量均明显增加,高糖+ FeTPPS组中p300与NF-κB p65蛋白含量较高糖组和SIN-1组明显降低。
5.2核蛋白中p300、NF-κB p65蛋白硝基化水平
p300蛋白硝基化水平结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p300蛋白硝基化水平均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p300蛋白硝基化水平降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p300蛋白硝基化水平降低,差异有统计学意义。p65蛋白硝基化水平结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p65蛋白硝基化水平均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p65蛋白硝基化水平降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p65蛋白硝基化水平降低,差异有统计学意义。表明高糖组、SIN-1组中p300与NF-κB p65蛋白硝基化水平均明显增加,高糖+ FeTPPS组中p300与NF-κB p65蛋白硝基化水平较高糖组和SIN-1组明显降低。
5.3硝基化对p300与NF-κB p65相互作用的影响。与p300蛋白相互作用的p65蛋白含量及硝基化水平结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p65蛋白含量及硝基化水平均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p65蛋白含量及硝基化水平均降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p65蛋白含量及硝基化水平均降低,差异有统计学意义。与p65蛋白相互作用的p300蛋白含量及硝基化水平结果显示,与正常组相比,高糖组(P<0.01),SIN-1组(P<0.01)p300蛋白含量及硝基化水平均增高,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p300蛋白含量及硝基化水平均降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p300蛋白含量及硝基化水平均降低,差异有统计学意义。表明高糖组、SIN-1组中p300与NF-κB p65蛋白相互作用及硝基化水平均明显增加,高糖+ FeTPPS组中p300与NF-κB p65蛋白相互作用及硝基化水平较高糖组和SIN-1组明显降低。
6 SRA01/04细胞中p300与NF-κB p65蛋白的共定位
p300与NF-κB p65蛋白共定位的细胞数量,与5mmol/L正常糖浓度组比较,高糖组(P<0.01),SIN-1组(P<0.01)p300与NF-κB p65蛋白共定位的细胞数量增加,差异有统计学意义;高糖+ FeTPPS组(P>0.05)差异无统计学意义。与高糖组相比,SIN-1组(P>0.05)差异无统计学意义,高糖+ FeTPPS组(P<0.01)p300与NF-κB p65蛋白共定位的细胞数量降低,差异有统计学意义。与SIN-1组相比,高糖+ FeTPPS组(P<0.01)p300与NF-κB p65蛋白共定位的细胞数量降低,差异有统计学意义。表明高糖组、SIN-1组中p300与NF-κB p65蛋白共定位的细胞数量均明显增加,高糖+ FeTPPS组中p300与NF-κB p65蛋白共定位的细胞数量较高糖组和SIN-1组明显降低。
结论:
1高糖刺激人晶状体上皮细胞(SRA01/04),p300在核内聚集,含量增加。
2高糖刺激人晶状体上皮细胞(SRA01/04),可使p300和NF-κB蛋白硝基化。
3硝基化可增强p300与NF-κB的相互作用,抑制p300与NF-κB的硝基化可在防治DC中起关键作用。
Objective: Nowadays diabetes mellitus (DM) as a common metabolic disease has endangered the healthy of human being. According to the report of Word Health Organization (WHO), the second-largest number of DM patient exists in China. The economic losses in China due to by DM rank first in the world in the next decade. The high disability and high mortality of DM are mainly caused by chronic diabetes mellitus complications, while in the eye mainly for low vision and blindness. In addition, diabetic cataract (DC) is the most important blinding factor. Due to the pathogenesis of DC is not fully known yet, therefore, the research of DC pathogenesis present remarkable significance for reducing the rate of blindness and improve the quality of life of the patients.
Oxidative stress having been widely accepted to play an important role. The key enzyme of oxidative stress is inducible nitric oxide syntheses (iNOS), which has confirmed that the nuclear factor-kappa B (NF-κB), is the most trans-acting factor for regulating transcription of iNOS. It can enhance the transcriptional activity of iNOS, resulting in excessive nitric oxide (NO) and super oxide anion (O2-.), NO reacts rapidly with O2-. to form peroxynitrite (ONOO-) in diabetes whose oxidative ability is 2000 folds stronger than H2O2 and cause oxidative damages. The preliminary study of our reserch showed that ONOO- played a crucial role in pathogenesis and progression of DC. ONOO- can cause conspicuous nitration to tyrosine residue of proteins, after which the structure and function of the nitration of proteins will change, and nitrotyrosine (NT) is a specificity marker for nitration of proteins.
There are five members in NF-κB transcription family: p50, p52, p65 (RelA), c-Rel, and RelB. Only can p65, c-Rel and RelB which contain transcription activation domain be co-activating factor binding and activate gene transcription. Generally, NF-κB stays in the cytoplasm in the form of p50/p65 homodimers. The activated NF-κB translocation into the nucleus, combined with cis-acting element. p65 recruits coactivator p300 to regulate gene transcription. p300 has different content in different tissues; its protein content can be adjusted with the change in the interaction of transcription factors and have a major impact on the regulation of gene expression. So, p300 plays an important role on the activation of NF-κB p65 activity. According to the research, in vascular endothelial cells, high glucose induces NF-κB and p300 interaction, and then increases the transcriptional activity of NF-κB. The rats of diabetic nephropathy, the mRNA and protein expression of p300 were increased, and enhanced NF-κB p65 transcriptional activity. In the lens of the eye, the lens epithelial cells are metabolism, synthesis, transit center, but also the first injured target cells. Dose the lens epithelial cells in the p300 and NF-κB play a key role in the pathogenesis of DC? The report has been shown that high glucose-induced cataract in rat lens NF-κB protein expression has increased. However, the repot of the effect of p300 protein has not been reported in DC process. Then, in the high glucose-induced human lens epithelial cells (SRA01/04), How to p300 protein content in the nucleus? Whether it can cause p300 and NF-κB in nitration? What’s the impact of the nitration of the p300 and NF-κB on their interaction? The questions mentioned above are the core problems to be solved of this study.
In order to observe the p300 protein in the nucleus, the nitration levels of p300 and NF-κB and the impact of the nitration of the p300 and NF-κB on their interaction and investigate high glucose-induced human lens epithelial cell injury mechanism for providing new ideas for prevention and the control of DC, high-glucose, SIN-1(a peroxynitrite donor) and FeTPPS (the per- oxynitrite decomposition catalyst) acting on SRA01/04 cells were used in this study.
Methods:
1 Cell culture and collected.
SRA01/04 cell line containing 10% fetal bovine serum, 1% non-essential amino acids, DMEM medium cultured in 37℃, 5% CO2 atmosphere.
When the cells passaged, it was taken into the culture dish, which was covered by the cells for 90%. Then addde the different factors and scraped it. After that they were collected in the EP tube that was used to extract nucleoprotein.
Built-in six-well plates of six coverslips then passaged the cells. Stimulating factors were to be the role of each group then the paraformaldehyde fixed, using confocal laser scanning microscope analysis co-localization and fluorescence microscope analysis nucleus translocation.
2 Groups and tests
2.1 In accordance with different factors and acting time, four groups were divided to determine the optimal concentration and time. (1) Different concentrations of glucose groups (5, 10, 15, 20, 25, 30mmol/L) (2) The concentration of glucose with different time groups (0, 5, 10, 15, 20, 25, 30, 35, 40min) (3) Different concentrations of SIN-1 groups (0, 10, 50, 100, 250, 500μmol/L) (4) High glucose + different concentrations of FeTPPS groups (0, 5, 10, 25, 50, 100μmol/L)
2.1.1 After the cell SRA01/04 influenced by the 2.1 respectively, nucleoprotein was detected by Lowry; Western-blotting detected precipitated p300 protein content and its nitration levels (NT content); protein content of NF-κB p65 induced by different factors and acting time.
2.1.2 SRA01/04 cells lived on coverslip, influenced by the 2.1 respectively, using fluorescence microscopy NF-κB p65 nucleus translocation.
2.2 In order to make optimal concentration and time factors, the following experiment were taken. (1) The normal control group (2) High glucose group (25 mmol/L, 25min) (3) SIN-1 group (500μmol/L, 25min) (4) High glucose + FeTPPS group (25 mmol/L+50μmol/L, 25min)
2.2.1 After the cell SRA01/04 affected by the factor 2.2, nucleoprotein was detected by Lowry; Western-blotting was used to detect the reciprocity of the p300 and NF-κB p65.
2.2.2 Western-blotting detection detected precipitated p300 and NF-κB p65 protein content and its nitration levels (NT content); the influence about nitration of the p300 and NF-κB p65 on their interaction.
2.2.3 Use Laser confocal microscopy to detect p300 and NF-κB p65 on co-localizaation.
Results:
1 Western blotting detected the nucleoprotein of p300 content in SRA01/04 cell line.
1.1 Different concentrations of glucose groups (5, 10, 15, 20, 25, 30mmol/L) Compared with control group 5 mmol/L, 10mmol/L (P<0.05), 15mmol/L (P<0.05), 20mmol/L (P<0.01), 25mmol/L (P<0.01), 30mmol/L (P<0.01) each p300 protein content increased, the difference had statistical meaning. The results showed that as the rising of glucose concentration, p300 protein content rose. When it was at 25mmol/L, the rising was the most obvious. While at 30mmol/L, it dropped down.
1.2 Times of different effects of 25mmol/L glucoses (0, 5, 10, 15, 20, 25, 30, 35, 40min)
Compared with 25mmol/L glucose concentration as 0 min, 5min (P<0.05), 10min (P<0.05), 15min (P<0.01), 20min (P<0.01), 25min (P<0.01), 30min (P<0.01), 35min (P<0.01), 40min (P<0.01) p300 protein content in each group increased, the difference had statistical meaning. The results showed that as time went on, the p300 concentration content rose, and were obvious at 25min, which dropped down at 30min.
1.3 Different concentration of SIN-1 groups (0, 10, 50, 100, 250, 500μmol/L) Compared with 0μmol/L of SIN-1 control group, which equals to normal glucose concentration of 5 mmol/L, SIN-1 concentration 10μmol/L (P<0.05), 50μmol/L (P<0.01), 100μmol/L (P<0.01), 250μmol/L (P<0.01), 500μmol/L (P<0.01) p300 protein content in each group increased, the difference had statistical meaning. The results showed that as SIN-1 concentration increased, the p300 protein content rose, and were obvious at 500μmol/L.
1.4 25mmol/L glucose + different concentration FeTPPS groups (0, 5, 10, 25, 50, 100μmol/L)
Compared with 25 mmol/L glucose concentration of non-plus FeTPPS, each group of FeTPPS 5μmol/L (P<0.05), 10μmol/L (P<0.05), 25μmol/L (P<0.05), 50μmol/L (P<0.01), 100μmol/L (P<0.01) p300 protein content reduced, the difference had statistical meaning. The results showed that when FeTPPS concentration was at 5-25μmol/L, p300 protein content were few. When it was at 50-100μmol/L, p300 protein content reduced obviously.
2 Western blotting detected the nucleoprotein of nitration levels of p300 content in SRA01/04 cell line.
2.1 Different concentrations of glucose groups acted 25min (5, 10, 15, 20, 25, 30mmol/L)
Compared with 5 mmol/L control group, 10mmol/L (P>0.05), 15mmol/L (P<0.05), 20mmol/L (P<0.01), 25mmol/L (P<0.01), 30mmol/L (P<0.01) except for 10mmol/L, the nitration levels of p300 content increased averagely in each group, the difference had statistical meaning. The results showed that as the rising of glucose concentration, the nitration level of p300 protein went up as well, and were most obvious at 25mmol/L, while dropped down at 30mmol/L.
2.2 Different concentration of SIN-1 groups (0, 10, 50, 100, 250, 500μmol/L).
Compared with SIN-1 of 0μmol/L concentrations, which equals to normal glucose concentration of 5 mmol/L, SIN-1 concentration 10μmol/L (P<0.05), 50μmol/L (P<0.01), 100μmol/L (P<0.01), 250μmol/L (P<0.01), 500μmol/L (P<0.01) the nitration levels of p300 content increased averagely in each group, the difference had statistical meaning. The results showed that as the rising of SIN-1 concentration, the nitration level of p300 protein went up as well, and were most obvious at 500μmol/L.
2.3 25mmol/L glucose + different concentration FeTPPS groups (0, 5, 10, 25, 50, 100μmol/L) Compared with 25 mmol/L glucose concentration of non-plus FeTPPS, each group of FeTPPS 5μmol/L (P<0.05), 10μmol/L (P<0.01), 25μmol/L (P<0.01), 50μmol/L (P<0.01), 100μmol/L (P<0.01) the nitration levels of p300 protein content reduced averagely in each group, the difference had statistical meaning. The results showed that as the rising of FeTPPS concentration, the nitration level of p300 protein decreased and were most obvious at 50-100μmol/L.
3 Western blotting detected the nucleoprotein of NF-κB p65 content in SRA01/04 cell line.
3.1 Different concentrations of glucose groups (5, 10, 15, 20, 25, 30mmol/L)
Compared with normal glucose concentration 5mmol/L, 10mmol/L (P<0.05), 15mmol/L (P<0.01), 20mmol/L (P<0.01), 25mmol/L (P<0.01), 30mmol/L (P<0.01) the protein content of NF-κB p65 increased averagely in each group, the difference had statistically significant. The results showed that with the increase of glucose concentration, the protein content of NF-κB p65 went up also. It was most obvious at 25mmol/L, while dipped at 30mmol/L.
3.2 Times of different effects of 25mmol/L glucoses (0, 5, 10, 15, 20, 25, 30, 35, 40min)
Compared with 25mmol/L glucose concentration as 0 min, 5min (P<0.05), 10min (P<0.01), 15min (P<0.01), 20min (P<0.01), 25min (P<0.01), 30min (P<0.01), 35min (P<0.01), 40min (P<0.01) the protein content of NF-κB p65 increased averagely in each group, the difference had statistically significant. The results showed that as time went by, the protein content of NF-κB p65 increased, it was at 20 min, the rising was the most obvious, while at 25 min, it dropped down.
4 NF-κB p65 transfer into nucleus in SRA01/04 cells line.
4.1 Times of different effects of 25mmol/L glucoses (0, 5, 10, 15, 20, 25, 30, 35, 40min)
Compared with 25mmol/L glucose concentration as 0 min, 5min (P>0.05), 10min (P<0.05), 15min (P<0.01), 20min (P<0.01), 25min (P<0.01), 30min (P<0.01), 35min (P<0.01), 40min (P<0.01) except for 5min, the rates of nucleus translocation of NF-κB p65 increased averagely in each group, the difference had statistical meaning. The results showed that with the time prolonged, the rates of NF-κB p65 nucleus translocation also increased. In 25-30min, it increased most significant, and in 35 min, it decreased.
4.2 Different concentration of SIN-1 groups (0, 50, 500μmol/L, 25min) Compared with non-plus SIN-1 concentrations, which equals to normal glucose concentration of 5 mmol/L, 50μmol/L (P<0.01), 500μmol/L (P<0.01) the rates of nucleus translocation of NF-κB p65 increased averagely in each group, the difference had statistical meaning. The results showed that with the role of SIN-1 concentration rised, the rates of NF-κB p65 nucleus translocation ratio increased.
4.3 25mmol/L glucose + different concentration FeTPPS groups (0, 10, 50μmol/L, 25min)
Compared with 25 mmol/L glucose group of non-plus FeTPPS, 10μmol/L (P<0.01), 50μmol/L (P<0.01) the rates of nucleus translocation of NF-κB p65 reduced averagely in each group, the difference had statistical meaning. The results showed that with the role of FeTPPS concentration increased, NF-κB p65 nucleus translocation ratio is lower.
5 The interaction and nitration levels of p300 and NF-κB p65 in SRA01/04 nucleoproteins.
Make an optimal determination of the concentration and action time based on the nucleoprotein of p300 protein content and the nitration level of each factor. (1) Normal (5 mmol/L, 25min) (2) High glucose group (25 mmol/L, 25 min) (3) SIN-1 group (500μmol/L, 25min) (4) High glucose + FeTTPS group (25 mmol/L + 50μmol/L, 25min)
5.1 p300 and NF-κB p65 protein content in nucleoprotein.
The results of p300 protein content showed that, compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) each p300 protein content increased, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. In addition, compared with high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) p300 protein content reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) p300 protein content reduced, the difference had statistical meaning. The results of NF-κB p65 protein content showed that, compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) each p65 protein content increased, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) p65 protein content reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) p65 protein content reduced, the difference had statistical meaning, which showed that protein content of high glucose group and SIN-1 group, p300 and NF-κB p65 were significantly increased. In the high glucose + FeTPPS group, the protein content of p300 and NF-κB p65 were significant lower than that of high glucose and SIN-1.
5.2 The nitration level of p300, NF-κB p65 in nucleoprotein
The nitration level of p300 protein results showed that compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) the nitration levels of p300 content increased averagely in each group, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) the nitration levels of p300 content reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) the nitration levels of p300 content reduced, the difference had statistical meaning. The nitration level of NF-κB p65 protein results showed that compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) the nitration levels of p65 content increased averagely in each group, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) the nitration levels of p65 content reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) the nitration levels of p65 content reduced, the difference had statistical meaning. The results mentioned above indicated that the nitration level of p300 and NF-κB p65 protein in SIN-1 group went up obviously. p300 and NF-κB p65 protein nitration in high glucose + FeTPPS group were significantly lower than that in glucose group and SIN-1 group.
5.3 The interaction effect of nitration to the p300 and NF-κB.
The NF-κB p65 protein content which interacts with p300 protein and its nitration level showed that compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) protein interactions and nitration level increased averagely in each group, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) protein interactions and nitration level reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) protein interactions and nitration level reduced, the difference had statistical meaning. The p300 protein content which interact with NF-κB p65 protein and its nitration level showed that compared with the normal group, high glucose group (P<0.01), SIN-1 group (P<0.01) protein interactions and nitration level increased averagely in each group, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) protein interactions and nitration level reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (P<0.01) protein interactions and nitration level reduced, the difference had statistical meaning. The conclusion stated that protein interactions and nitration level of p300 and NF-κB p65 in high glucose group and SIN-1 group increased significantly. While the contrary situation is in high glucose + FeTPPS group, the protein interactions and nitration level of p300 and NF-κB p65 were significantly lower.
6 p300 and NF-κB p65 protein co-localization in SRA01/04 cells line. Compared with 5mmol/L normal glucose concentration group, the cell number of p300 and NF-κB p65 protein co-localization, high glucose group (P<0.01), SIN-1 group (P<0.01) the cell number of co-localization increased in each group, the difference had statistical meaning; high glucose + FeTPPS group (P>0.05) the difference had not statistical meaning. Compared with the high glucose group, SIN-1 group (P>0.05) the difference had not statistical meaning, high glucose + FeTPPS group (P<0.01) the cell number of co-localization reduced, the difference had statistical meaning. Compared with SIN-1 group, high glucose + FeTPPS group (p<0.01) the cell number of co-localization reduced, the difference had statistical meaning. The results showed that the cell number of co-localization of p300 and NF-κB p65 in high glucose group and SIN-1 group increased obviously, which is obviously higher than that in high glucose + FeTPPS group.
Conclusions:
1 p300 content increased and gathered in the nucleus, when high glucose-induced human lens epithelial cells (SRA01/04).
2 p300 and NF-κB protein can be nitration, when high glucose-induced human lens epithelial cells (SRA01/04).
3 Nitration can enhance the interaction of p300 and NF-κB, p300 and NF-κB inhibition of nitration may play a key role in prevention and control of DC.
引文
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2巩秋红,李光伟.2005北京国际糖尿病预防高层论坛纪要.中华内分泌代谢杂志,2006,22(1):94-95
3 Blanco M, Rodriguez-Yanez M, Sobrino T, et al. Platelets, inflammation, and atherothrombotic neurovascular disease: the role of endothelial dysfunction. Cerebrovasc Dis, 2005. 20(2): 32-39
4 Kiritoshi S, Nishikawa T, Sonoda K, et al. Reactive oxygen species from mitochondria induce cyclooxygenase-2 gene expression in human mesangial cells: potential role in diabetic nephropathy. Diabetes, 2003. 52(10): 2570-2577
5 Sareila O, Korhonen R, Auvinen H, et al. Effects of levo- and dextrosimendan on NF-kappaB-mediated transcription, iNOS expression and NO production in response to inflammatory stimuli. Br J Pharmacol, 2008. 155(6): 884-895
6 Guo H, Mi Z, and Kuo P.C. Characterization of short range DNA looping in endotoxin-mediated transcription of the murine inducible nitric-oxide synthase (iNOS) gene. J Biol Chem, 2008. 283(37): 25209-25217
7 Dzurik R and Spustova V. Nitric oxide and the kidneys. Vnitr Lek, 2001. 47(2): 101-105
8 Freeman B. Free radical chemistry of nitric oxide. Looking at the dark side. Chest, 1994. 105(3): 79-84
9 Upmacis R.K, Deeb R.S and Hajjar D.P. Oxidative alterations of cyclooxygenase during atherogenesis. Prostaglandins Other Lipid Mediat, 2006. 80(1-2): 1-14
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