C肽调控NF-κB与P300防治糖尿病肾病的机制研究

发布时间：2018-09-01 05:47

【摘要】：目的:糖尿病(Diabetes mellitus,DM)是继肿瘤、心血管疾病之后第三大非传染性的慢性疾病,严重威胁着人类健康。据报道,全球已有3.82亿人患有DM,预计到2035年将达到5.92亿。我国就有近1亿人患有DM,约占全球患病人数的四分之一,患病率位居世界第一位。糖尿病肾病(Diabetic Nephropathy,DN)是1型和2型DM患者的主要并发症之一,是终末期肾脏疾病导致患者致死致残的主要原因。据统计,大约35%～50%1型、约20%2型DM患者发展成为DN。DN主要病理改变是基底膜增厚,系膜基质增多,细胞外基质增加,最终导致弥漫性或结节性肾小球硬化。目前,其发病的内在机制尚未完全阐明。近年研究确认,氧化应激是公认的DN发生发展的中心环节。其关键酶诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)转基因会引起DN 样病变。iNOS 基因转录最重要的调控因子是核因子-κB(nuclear factor kappa B,NF-κB),当受到相应刺激时被激活转位入核,调控相关基因转录。P300是细胞内最重要的共激活因子,具有核蛋白乙酰基转移酶活性,参与多种转录因子的调控。研究证实DM时,NF-κB的P65亚基与共激活因子P300结合增多,异常激活基因转录,产生大量iNOS,在DN的发生发展中起关键性作用。C肽是含31个氨基酸的多肽,在胰岛素合成过程中产生,并与胰岛素等摩尔分泌。一直以来,C肽仅作为评价胰岛β细胞功能的指标。近年来,一些研究表明对于实验动物和DM患者,外源性C肽可以防止或逆转DN。一系列研究发现生理浓度的C肽能结合胞膜G蛋白偶联受体,或进入胞质胞核发挥细胞保护效应。本课题前期研究表明高糖(25 mmol/L)刺激下,0.5 nmol/L的C肽定位于大鼠肾小球系膜细胞核,抑制iNOS基因转录及其蛋白的表达,亦能抑制高糖引起的NF-κB核转位。然而C肽是否通过调控NF-κB与P300的相互作用而发挥防治DN的作用,尚未见相关报道。本研究从细胞和动物整体两个方面进行实验。采用大鼠肾小球系膜细胞株HBZY-1进行细胞培养,用激光共聚焦显微镜(Confocal)观察C肽(0.5nmol/L)对高糖(25mmol/L)刺激的细胞中NF-κB的作用及其与P300共定位,用染色质免疫沉淀(chromatin immunoprecipitation,ChIP)技术检测C肽对NF-κB启动iNOS的调控作用,用免疫共沉淀(co-immunoprecipitation,Co-IP)技术检测C肽对NF-κB与P300相互作用的影响。用链脲佐菌素(streptozotocin,STZ)复制SD大鼠DM模型,随机分为正常组、病理组、C肽防治组、C肽治疗组和乱码C肽组,实验前后测定血糖、体重变化,测定24h尿白蛋白排泄量。取大鼠肾小球皮质于光镜及透射电镜下进行形态学观察,进一步探讨C肽对防治DN的整体功效及作用。方法:1细胞实验1.1细胞培养大鼠肾小球系膜细胞株(GMCs)HBZY-l,用含10%胎牛血清的低糖DMEM(5.5 mmol/L),0.25%EDTA-胰蛋白酶消化传代,接种于100 ml培养瓶中,在37℃、5%CO2培养箱中培养。1.2实验设计与分组(1)Confocal技术观察NF-κB P65核转位及其与P300共定位,同时确定C肽作用的最佳时间。实验分组:1)正常组(Control):GMCs(HBZY-1)用低糖DMEM(5.5mmol/L)正常培养;2)高渗对照组(HO):GMCs用L-葡萄糖 DMEM(25 mmol/L)刺激作用 24 h;3)高糖组(HG):GMCs(HBZY-1)用高糖DMEM(25 mmol/L)刺激作用24 h;4)NF-κB抑制剂(BAY11-7082)加高糖组(IH):正常培养的 GMCs 加入 BAY11-7082(10μM)孵育1 h,换高糖DMEM(25 mmol/L)刺激作用24 h;5)C肽治疗组(CP):GMCs用高糖DMEM刺激作用24 h后,换成0.5 nmol/L C肽-高糖DMEM 分别培养 10、20、30、40、50、60 min。(2)ChIP技术检测C肽对NF-κB启动iNOS的调控作用。实验分组:1)正常组(Control):GMCs 用低糖 DMEM(5.5 mmol/L)正常培养;2)高糖组(HG):GMCs用高糖DMEM(25mmol/L)刺激作用24h;3)C肽治疗组(CP):GMCs用高糖DMEM刺激作用24 h后,换成0.5 nmol/L C肽-高糖DMEM培养30 min。(3)Co-IP技术检测C肽对NF-κB与P300结合作用的影响。实验分组:1)正常组(Control):GMCs 用低糖 DMEM(5.5mmol/L)正常培养;2)高糖组(HG):GMCs用高糖DMEM(25mmol/L)刺激作用24h;3)C肽治疗组(CP):GMCs用高糖DMEM刺激作用24 h后,换成0.5 nmol/L C肽-高糖DMEM培养30 min。2动物实验2.1实验设计与分组健康雄性SD大鼠80只,随机分为2组,正常对照组(NC,8只),其余72只注射STZ溶液(STZ溶于浓度为0.1 mol/L的柠檬酸纳缓冲液,pH 4.4,新鲜配制,终浓度为10mg/mL,按45mg/kg腹腔注射)制备DM模型。注射3天后,尾静脉采血测血糖值≥16.7mmol/L,尿糖+++以上,为造模成功。造模成功68只,再随机分成4组:病理组(DM,17只),C肽防治组(DM+CP-P,17只),C肽治疗组(DM+CP-T,17只),乱码C肽治疗组(DM+scCP,17只),以上五组均于20～25℃室温下正常饮食喂养。DM+CP-P组在造模成功后,开始皮下注射人C肽(130 nmol/kg),每天2次,治疗6周后停药。DM+CP-T组和DM+scCP组在病程6周后,分别开始同剂量皮下注射人C肽或乱码C肽,每天2次,治疗6周。最后股动脉放血处死。2.2检测血糖和24 h尿液取尾静脉血,用血糖仪检测并记录注射STZ前、后第3天和第12周的血糖变化。收集24h尿液,检测尿蛋白排泄量。2.3形态学观察取大鼠肾小球皮质作病理切片,分别于光镜和透射电镜下观察。结果:1不同时间点,C肽对CP组GMCs NF-κB P65核转位及其与P300共定位的影响Confocal结果显示,高糖刺激GMCs 24 h,会引起NF-κB P65转位入核,且NF-κB P65与P300共定位于细胞核;0.5 nmol/L C肽治疗在30 min时效果显著,即NF-κB P65聚集于胞质,P300聚集于胞核;40～60 minC肽作用逐渐减弱至基本消失。因此选30 min作为C肽治疗高糖长时间刺激细胞最佳的作用时间。2 ChIP技术检测C肽治疗30 min时,对CP组GMCs NF-κB启动iNOS的调控作用高糖刺激GMCs 24 h后,细胞内NF-κB在iNOS启动子区近端的结合作用(4.44 ± 0.29,P0.05)与Control组相比明显升高;0.5 nmol/L C肽治疗30 min后,细胞内NF-κB在iNOS启动子区的近端结合作用(1.86 ±0.21,P0.01)与HG组相比显著下降。高糖刺激GMCs 24 h后,细胞内NF-κB在iNOS启动子区远端的结合作用(5.06 ±0.43,P0.05)与 Control 组相比明显升高;0.5 nmol/L C肽治疗30 min后,细胞内NF-κB在iNOS启动子区的近端结合作用(0.94 ±0.07,P0.05)与HG组相比明显下降。3 Co-IP技术检测C肽治疗30 min时,Control组、HG组和CP组GMCs NF-κB P65与P300的结合作用Control组、HG组和CP组细胞蛋白裂解液分别加入P300抗体沉淀处理后,Western blot结果显示:HG组GMCs P65与P300蛋白结合量(18.31 ±0.82,P0.05)比 Control 组(5.99 ± 0.07)明显升高;CP 组 GMCs P65与P300蛋白结合量(5.220±0.33,P0.01)比HG组显著下降。Control组、HG组和CP组细胞蛋白裂解液分别加入P65抗体沉淀处理后,Western blot结果显示:HG组GMCs P300与P65蛋白结合量(9.19±0.17,P0.01)比 Control 组(3.54±0.21)显著升高;CP 组 GMCsP300与P65蛋白结合量(2.98 ±0.23,P0.01)比HG组显著下降。4大鼠的一般表现,C肽对DM大鼠血糖、体重和尿白蛋白排泄量的影响造模成功的大鼠逐渐出现消瘦,皮毛疏松无光泽,反应迟钝,多饮、多尿、多食,生长发育迟缓等症状。随着实验进行,DM组和DM+scCP组病症更加明显,而DM+CP-P组和DM+CP-T组病症与NC组相似。各组大鼠入选时血糖和体重无显著性差异。整个实验中,DM+CP-P组平均血糖(29.96 ± 0.52)mmol/L,DM+CP-T 组(28.90 ± 0.74)mmol/L,DM+scCP 组(29.74 ± 0.66)mmol/L,均与 DM 组(29.23 ± 0.66)mmol/L无显著性差异。以上四组均显著高于NC组(6.40 ± 0.25)mmol/L(P0.01)。C肽治疗结束时,DM+CP-T组平均体重(271.82 ±8.76)g,DM+scCP组(248.35 ±7.19)g,均与DM组(249.0 ±7.13)g无显著性差异。以上四组均显著低于NC组(515.5 ± 14.42)g(P0.01)。C肽治疗结束时,DM组24小时尿蛋白排泄量(327.93±32.58)mg和DM+scCP 组(293.79 ±49.40)mg 均明显高于 NC 组(15.42 ± 4.06)mg(P0.05);DM+CP-P 组(55.21 ± 4.06)mg 和 DM+CP-T组(70.2 ± 9.46)mg明显低于DM组(P0.05)。5肾小球形态学观察肾小球光镜(× 400)下观察:NC组肾小球未见异常;DM组可见到肾小球结构异常、肾小囊腔增大;DM+scCP组与DM组相似,并且有肾小球坏死的现象;而DM+CP-P组和DM+CP-T组,可见肾小囊腔缩小,与病理组相比有所改善,且DM+CP-P组比DM+CP-T组的病变较轻。肾小球透射电镜(×20K)下观察:NC组肾小球未见异常。DM组可见基底膜重度增厚,局部呈丘状隆起,血管内皮细胞重度增生,部分足细胞足突融合;DM+scCP组与DM组相似,基底膜明显增厚;DM+CP-P组和DM+CP-T组可见基底膜和血管内皮细胞接近正常,足细胞足突部分轻度融合,比DM组有明显改善,且DM+CP-P组较DM+CP-T组的病理改变减轻。结论:1 C肽调控NF-κB与P300防治糖尿病肾病的机制是:(1)C肽能抑制NF-κB P65的核转位及其与P300的共定位。(2)C肽能抑制高糖引起的NF-κB核转位与iNOS启动子区远、近端均有相互作用,从而抑制NF-κB启动iNOS基因。(3)C肽能抑制高糖引起的NF-κB与P300之间的相互作用。2光镜和透射电镜下观察到,C肽能明显改善DM大鼠肾小球的功能和形态学变化。
[Abstract]:Objective: Diabetes mellitus (DM) is the third largest non-infectious chronic disease after tumor and cardiovascular disease, which seriously threatens human health. It is reported that 382 million people worldwide have DM, and it is expected to reach 592 million by 2035. Diabetic nephropathy (DN) is one of the major complications of type 1 and type 2 diabetes mellitus (DM) and the leading cause of death and disability in patients with end-stage renal disease. According to statistics, about 35%-50% of type 1 diabetes mellitus patients and 20% of type 2 diabetes mellitus patients develop into DN. The main pathological changes of DN are thickening of basement membrane, increasing of mesangial matrix and extracellular matrix. The underlying mechanism of diffuse or nodular glomerulosclerosis has not been fully elucidated. Recent studies have confirmed that oxidative stress is the central link in the genesis and development of DN. The most important regulator of gene transcription is nuclear factor kappa B (NF-kappa B), which is activated and transported into the nucleus when stimulated. P300 is the most important co-activating factor in the cell. It has the activity of nuclear protein acetyltransferase and participates in the regulation of many transcription factors. In recent years, C peptide is a polypeptide containing 31 amino acids, produced during insulin synthesis and secreted with insulin and other moles. C peptide has been used only as an index to evaluate the function of islet beta cells. Some studies have shown that exogenous C peptides can prevent or reverse DN in experimental animals and DM patients. A series of studies have found that C peptides at physiological concentrations bind to membrane G protein-coupled receptors, or enter the cytoplasmic nucleus to exert cytoprotective effects. Previous studies have shown that 0.5 nmol/L C peptides are localized in rat kidneys stimulated by high glucose (25 mmol/L). The nucleus of mesangial cells inhibits the transcription of iNOS gene and the expression of iNOS protein, and also inhibits NF-kappa B nuclear translocation induced by high glucose. However, whether C peptide can prevent and treat DN by regulating the interaction between NF-kappa B and P300 has not been reported. Cell line HBZY-1 was cultured. The effect of C-peptide (0.5 nmol/L) on NF-kappa B and its co-localization with P300 in the cells stimulated by high glucose (25 mmol/L) were observed by Confocal microscopy. The regulatory effect of C-peptide on the activation of iNOS by NF-kappa B was detected by chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (co-immunoprecipitation). The effects of C-peptide on the interaction between NF-kappa B and P300 were examined by co-immunoprecipitation, Co-IP. SD rats were randomly divided into normal group, pathological group, C-peptide prevention and treatment group, C-peptide treatment group and disordered C-peptide group. Methods: The glomerular mesangial cells (GMCs) of rats were cultured in 1.1 cell experiment, digested and subcultured with low glucose DMEM (5.5 mmol/L) containing 10% fetal bovine serum and 0.25% EDTA-trypsin, and inoculated in 100 ml. The experiment design and grouping (1) Confocal technique were used to observe the nuclear translocation of NF-kappa B P65 and its co-localization with P300, and to determine the optimal time of C peptide action. High glucose group (HG): GMCs (HBZY-1) stimulated by high glucose DMEM (25 mmol/L) for 24 hours; 4) NF-kappa B inhibitor (BAY11-7082) plus high glucose group (IH): Normal GMCs were incubated with BAY11-7082 (10 mu M) for 1 hour, replaced with high glucose DMEM (25 mmol/L) for 24 hours; 5) C peptide treatment group (CP): GMCs stimulated by high glucose DMEM for 24 hours, and then replaced with high glucose DMEM (25 mmol/L) for 24 hours. (2) ChIP assay was used to detect the regulatory effect of C peptide on the initiation of iNOS by NF-kappa B. Experimental grouping: 1) Normal group (Control): GMCs were cultured with low-glucose DMEM (5.5 mmol/L); 2) High-glucose group (HG): GMCs were stimulated with high-glucose DMEM (25 mmol/L) for 24 hours; 3) C peptide treatment group (CP): GMCs were stimulated with high-glucose DMEM (25 mmol/L); 3) GMCs were treated with high-glucose DMEM (GMCs) for 24 hours. After 24 hours of stimulation, the cells were cultured with 0.5 nmol/L peptide-high glucose DMEM for 30 minutes. (3) Co-IP technique was used to detect the effect of C peptide on the binding of NF-kappa B to P300. After 24 hours of stimulation, 80 healthy male SD rats were randomly divided into 2 groups: normal control group (NC, 8 rats), and the rest 72 rats were injected with STZ solution (STZ dissolved in 0.1 mol/L sodium citrate buffer, pH 4.4, freshly prepared, the final concentration was 10 mg/mL, according to 45 mg/kg). DM model was established by intraperitoneal injection. After 3 days of injection, blood glucose (> 16.7 mmol/L) and urine glucose (+ + +) were collected from the caudal vein. Sixty-eight rats were successfully established. They were randomly divided into 4 groups: pathological group (DM, 17 rats), C peptide prevention and treatment group (DM + CP-P, 17 rats), C peptide treatment group (DM + CP-T, 17 rats) and disorderly coded C peptide treatment group (DM + scCP, 17 rats). DM+CP-T group and DM+scCP group were given subcutaneous injection of human C-peptide (130 nmol/kg) twice a day for 6 weeks. DM+CP-T group and DM+scCP group were given subcutaneous injection of human C-peptide or scrambled C-peptide at the same dose twice a day for 6 weeks. Blood glucose of caudal vein was measured and recorded by blood glucose meter before and after STZ injection. 24 hours urine was collected and urinary protein excretion was measured. Confocal results showed that high glucose stimulated GMCs for 24 hours could induce NF-kappa B P65 translocation into the nucleus, and NF-kappa B P65 and P300 co-localized in the nucleus; 0.5 nmol/L C peptide treatment had a significant effect in 30 minutes, that is, NF-kappa B P65 aggregated in the cytoplasm, P300 aggregated in the nucleus; 40-60 min C peptide effect gradually weakened to disappear. ChIP assay was used to detect the regulatory effect of C peptide on the initiation of iNOS by GMCs NF-kappa B in C P group. After 24 hours of stimulation with high glucose, the binding of NF-kappa B to the proximal end of iNOS promoter (4.44 0.29, P 0.05) was significantly higher than that in control group. After 30 minutes of treatment, the proximal binding of intracellular NF-kappa B to iNOS promoter region (1.86+0.21, P 0.01) was significantly lower than that of HG group. After 24 hours of high glucose stimulation, the binding of intracellular NF-kappa B to distal end of iNOS promoter region (5.06+0.43, P 0.05) was significantly higher than that of C ontrol group; after 30 minutes of 0.5 nmol/L C peptide treatment, intracellular NF-kappa B was significantly higher in iNOS promoter region. The proximal binding of S promoter region (0.94+0.07, P 0.05) was significantly lower than that of HG group. 3 The binding of GMCs NF-kappa B P65 to P300 in control group, HG group and CP group was detected by Co-IP after 30 minutes of treatment with control group, HG group and CP group, respectively. The results of Western blot showed that GMCs P in HG group was precipitated by P300 antibody. The binding capacity of GMCs P65 to P300 protein (18.31+0.82, P 0.05) in control group was significantly higher than that in control group (5.99+0.07), and the binding capacity of GMCs P65 to P300 protein (5.220+0.33, P 0.01) in CP group was significantly lower than that in HG group. The combined dose of GMCsP300 and P65 protein (2.98+0.23, P 0.01) in CP group was significantly lower than that in HG group. The symptoms of DM + CP - P group and DM + CP - T group were similar to those of NC group. There was no significant difference in blood glucose and body weight between DM + CP - P group and DM + CP - T group. There was no significant difference between DM + scCP group and DM group (29.23 + 0.66) mmol / L. All the above four groups were significantly higher than NC group (6.40 + 0.25) mmol / L (P 0.01). At the end of C peptide treatment, the average body weight of DM + CP-T group (271.82 + 8.76) g, DM + scCP group (248.35 + 7.19) g, and DM group (249.0 + 7.13) g had no significant difference. At the end of C-peptide treatment, 24-hour urinary protein excretion in DM group (327.93 (+32.58) mg and DM+scCP group (293.79 (+49.40) mg were significantly higher than those in NC group (15.42 (+4.06) mg (P 0.05), DM+CP-P group (55.21 (+4.06) mg and DM+CP-T group (70.2 (+9.46) mg) significantly lower than those in DM group (P 0.05). The glomeruli of NC group were not abnormal; the glomeruli of DM group were abnormal and the cysts of DM group were enlarged; the glomeruli of DM + scCP group were similar to DM group and glomerular necrosis was observed; while the glomeruli of DM + CP - P group and DM + CP - T group were smaller, and the pathological changes of DM + CP - P group were less than DM + CP - T group. The glomeruli of NC group were normal. In DM group, the basement membrane was thickened severely, the vascular endothelial cells were proliferated severely, and some podocytes were fused. In DM + scCP group, the basement membrane was thickened obviously, and the basement membrane and vascular endothelial cells were close to normal in DM + CP - P group and DM + CP - T group. Conclusion: The mechanism of C peptide regulating NF-kappa B and P300 in preventing and treating diabetic nephropathy is: (1) C peptide can inhibit the nuclear translocation of NF-kappa B P65 and its co-location with P300. (2) C peptide can inhibit the nuclear translocation of NF-kappa B induced by high glucose far from the iNOS promoter region. (3) C peptide could inhibit the interaction between NF-kappa B and P300 induced by high glucose. 2 Light and transmission electron microscopy showed that C peptide could significantly improve the function and morphological changes of glomeruli in DM rats.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692.9

【相似文献】