The Role Of High Glucose On Nf-кb In Human Lens Epithelial Cells

Objective: Diabetic cataract is one of the major factors of blindness in diabetic patients. Clinical observations show that there is a higher incidence of senile cataract in diabetic patients, and age of onset is more advanced than non-diabetic patients, but also mature more rapidly, namely, diabetes can make cataract occur earlier or accelerate its development. However, at present the pathogenesis of diabetic cataract is not fully understood yet. In recent years, a series of basic and clinical studies have shown that there exists the obvious oxidative stress in the diabetic situation; oxidative stress is an important induced factor for the lens epithelial cell damage.Our preliminary study showed that inducible nitric oxide synthase (iNOS) mRNA and protein expression were significantly increased in rat lens epithelial cells of diabetes mellitus, and iNOS activity was obviously increased, increasing the synthesized nitric oxide (NO) notably. Once excessive NO meets superoxide anion (O2-), peroxynitrite (ONOO-) will be generated at a diffusion coefficient limited rate. ONOO- can induce nitration of tyrosine residues to form 3-nitrotyrosine (3-NT) effectively. 3-NT levels have been recognized as a biomarker of ONOO- activity. When proteins are nitrated by ONOO-, their structures and functions will be affected.Our previous study proved that ONOO- can cause nitration of iNOS and cyclooxygenase-2 (COX-2) protein in diabetes, aggravating the diabetic vascular damages. It is well known that NF-кB is the upstream regulation factor of iNOS. The NF-кB family of transcription factors consists of five members, p65(Rel-A), c-Rel, Rel-B, p50 (precursor protein p105) and p52(precursor protein p100). Almostly NF-кB family proteins can form homodimers or heterodimers. The term NF-кB commonly refers specifically to a p50-p65 heterodimer, which is one of the most avidly forming dimers and is the major Rel/NF-кB complex in most cells. The activity of NF-кB is primarily regulated by interaction with inhibitory kappa B proteins (IкBs). When the cell is in a resting state, p50-p65 dimer and IкB exsit in the form of trimer , so that NF-кB remains in a non-active state in the cytoplasm. When the cell is stimulated by high glucose, I-kappa B kinase complex (IKK) is activated, the N-terminal of IкBs is phosphorylated. Then the freed NF-кB dimers translocate to the nucleus, where they bind to specificкB sequences in the promoter or enhancer regions, then regulate gene transcription activity. However, until now, it has not been reported that how is NF-кB nuclear translocation, nuclear protein content and its nitration in the lens epithelial cells with high glucose.3-morpholinosydnonimine (SIN-1) is the specific donor of ONOO-.5,10,15,20-Tetrakis-(4-sulfonatophenyl)porphyrinato-iron(III) (FeTPPS) is the decomposition catalyst of ONOO-. In this experiment, human lens epithelial cell line SRA01/04 was used, and diabetic model was induced with high glucose. We utilized different concerntrations of glucose, ONOO- donor SIN-1 and ONOO- decomposition catalyst FeTPPS to experiment. NF-кB nuclear translocation, nuclear protein content and its nitration were detected in the experiment, so as to lay foundation for the further study of effect of nitration on the NF-кB-DNA bounding activity and on the activity of its target genes (such as iNOS and COX-2).Methods:1 Cell cultureThe SRA01/04 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, with 5.5 mmol/L glucose) including 10% newborn calf serum, 1% non-essential amino acid, 100 IU/ml penicillin and 100 IU/ml streptomycin at 37℃in a 5% CO2 humidified atmosphere.2 Experimental design and groups2.1 Detection of the effect of high glucose (25mmol/L) on the NF-кBp65 nuclear translocation and nuclear protein content in the SRA01/04 cells. The SRA01/04 cells were incubated in DMEM with 25mmol/L glucose. According to different time, cells were divided into 9 groups: 0, 5, 10, 15, 20, 25, 30, 35, 40min groups.2.2 Detection of the effect of different concentrations of glucose on the NF-кBp65 nuclear translocation, nuclear protein content and its nitration level in the SRA01/04 cells. Cells were divided into 6 groups: 5.5, 10, 15, 20, 25, 30mmol/L groups. The reaction time was within 20 min.2.3 Detection of the effect of ONOO- on the NF-кBp65 nuclear translocation, nuclear protein content and its nitration level in the SRA01/04 cells. According to different concentrations of the ONOO- donor SIN-1, cells were divided into 5 groups: 0, 10, 50, 100, 250, 500μmol/L groups. The reaction time was within 20 min.2.4 Detection of the effect of eliminating ONOO- on the NF-кBp65 nuclear translocation, nuclear protein content and its nitration level in the SRA01/04 cells. The cells were incubated in DMEM with 25mmol/L glucose. According to different concentrations of the ONOO- decomposition catalyst FeTPPS, cells were divided into 5 groups: 0, 5, 10, 25, 50, 100μmol/L groups.The reaction time was within 20 min.3 Morphological observations of the NF-кBp65 nuclear translocation. Immunofluorescence staining was used to detect whether the NF-кBp65 was translocated into the nucleus, in order to confirm whether NF-кB was activated.4 Analysis of the nuclear protein content and nitration level of NF-кB.After nucleoprotein was extracted, protein concentration was determined by Lowry method. Western-blotting detected the the nuclear protein content and nitration level of NF-кB (NT content).Results:1 The effect of high glucose (25mmol/L) on the NF-кBp65 nuclear translocation and nuclear protein content in the SRA01/04 cells. Immunofluorescence staining indicated that in control group (0min) NF-кBp65 mainly distributed in the cytoplasm, and there was little in the nucleus (NF-кBp65 was inactivated in the cytoplasm while activated in the nuclei). After SRA01/04 cells were incubated with high glucose (25mmol/L) for different time, nuclear translocation of NF-кBp65 increased time-dependent in cells, of which the most obviously was after 20min.Western blotting results showed that the nuclear protein contents of NF-кBp65 after 5min (2.38±0.39), 10min (2.23±0.45), 15min (2.33±0.41), 20min (2.99±0.62), 25min (2.37±0.35) increased significantly comparing to the control group (1.50±0.32) (P<0.05). Among them, the nuclear protein content of NF-кBp65 after 20min increased the most obviously. Internal control histone expressed almost the same result. (Fig.1 and Fig.2)2 The effect of different concentrations of glucose on the NF-кBp65 nuclear translocation, nuclear protein content and its nitration level in the SRA01/04 cells.Immunofluorescence staining indicated that in control group (5.5mmol/L) NF-кBp65 mainly distributed in the cytoplasm, and there was little in the nucleus. After SRA01/04 cells were incubated with different concentrations of glucose for 20min, nuclear translocation of NF-кBp65 increased concentration-dependent, of which the most obviously was 20mmol/L and 25mmol/L groups.Western blotting results showed that the nuclear protein content of NF-кBp65 (2.29±0.29) and nitration level (3.18±0.46) in 10mmol/L group and the nuclear protein content of NF-кBp65 (2.42±0.36) and nitration level (3.27±0.47) in 15mmol/L group increased significantly comparing to the nuclear protein content of NF-кBp65 (1.67±0.31) and nitration level (2.02±0.48) in the control group (P<0.05). There was no obvious difference between the 10mmol/L group and the 15mmol/L group. The nuclear protein content of NF-кBp65 (2.50±0.41) and nitration level (4.62±0.62) in 20mmol/L group and the nuclear protein content of NF-кBp65 (2.69±0.37) and nitration level (4.17±0.57) in 25mmol/L group increased the most significantly comparing to the nuclear protein content of NF-кBp65 and nitration level in other groups (P<0.05). There were no obvious differences between the 20mmol/L group and the 25mmol/L group. It suggested that the effect of 20mmol/L and 25mmol/L glucose on the nuclear protein content of NF-кBp65 and nitration level was the most obviously. Internal control histone expressed almost the same result. (Fig.3 and Fig.4)3 The effect of different concentrations of SIN-1 on the NF-кBp65 nuclear translocation, nuclear protein content and its nitration level in the SRA01/04 cells.Immunofluorescence staining indicated that in control group (0μmol/L SIN-1) NF-кBp65 mainly distributed in the cytoplasm, and there was little in the nucleus. After SRA01/04 cells were incubated with different concentrations of SIN-1 for 20min, nuclear translocation of NF-кBp65 increased concentration-dependent, of which the most obviously were the 50μmol/L group and the 100μmol/L SIN-1 group.Western blotting results showed that the nuclear protein content of NF-кBp65 (1.34±0.37) and nitration level (0.66±0.11) in 10μmol/L SIN-1 group increased significantly comparing to the nuclear protein content of NF-кBp65 (0.68±0.19) and nitration level (0.28±0.07 in the control group (P<0.05). The nuclear protein content of NF-кBp65 (1.87±0.35) and nitration level (0.95±0.18) in 50μmol/L SIN-1 group and the nuclear protein content of NF-кBp65 (2.40±0.63) and nitration level (1.08±0.19) in 100μmol/L SIN-1 group increased the most significantly comparing to the nuclear protein content of NF-кBp65 and nitration level in other groups (P<0.05). There were no obvious differences between the 50μmol/L SIN-1 group and the 100μmol/L SIN-1 group. There were no obvious differences among the 0μmol/L SIN-1 group, the 250μmol/L SIN-1 group and the 500μmol/L SIN-1 group. It suggested that 50~100μmol/L was the most effective concentration range of SIN-1. Internal control histone expressed almost the same result. (Fig.5 and Fig.6)4 The effect of different concentrations of FeTPPS on the NF-кBp65 nuclear translocation,nuclear protein content and its nitration level in the SRA01/04 cells.Immunofluorescence staining indicated that in control group (0μmol/L FeTPPS) NF-кBp65 mainly distributed in the nucleus. After SRA01/04 cells were incubated with different concentrations of FeTPPS+25mmol/L glucose for 20min, nuclear translocation of NF-кBp65 decreased obviously in 25μmol/L, 50μmol/L and 100μmol/L FeTPPS groups.Western blotting results showed that the nuclear protein content of NF-кBp65 (1.63±0.28) and nitration level (0.38±0.09) in 25μmol/L FeTPPS group, the nuclear protein content of NF-кBp65 (1.62±0.42) and nitration level (0.44±0.12) in 50μmol/L FeTPPS group and the nuclear protein content of NF-кBp65 (1.35±0.38) and nitration level (0.34±0.09) in 100μmol/L FeTPPS group decreased the most significantly comparing to the nuclear protein content of NF-кBp65 (2.51±0.26) and nitration level (0.88±0.17) in control group (P<0.05). There were no obvious differences among the three groups. It suggested that 25~100μmol/L was the most effective concentration range of FeTPPS. There were no obvious differences among 0μmol/L, 5μmol/L and 10μmol/L FeTPPS groups. Internal control histone expressed almost the same result. (Fig.7 and Fig.8)Conclusions:1 High glucose can cause NF-кB nuclear translocation in the human lens epithelial cells.2 In the situation of high glucose, the nuclear protein content of NF-кB in the human lens epithelial cells increased.3 High glucose can induce the nitration of NF-кB in the human lens epithelial cells.