| 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 tests2.1 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 nucleoproteinThe 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. |
