糖尿病早期大鼠视网膜神经细胞凋亡与锰超氧化物歧化酶活性及mRNA表达变化的关系

目的 探讨糖尿病早期大鼠视网膜神经细胞凋亡与锰超氧化物歧化酶(MnSOD)活性及mRNA表达变化的关系.方法 对照实验研究.健康雄性8周龄SD大鼠72只,分为正常对照组及糖尿病组.糖尿病组大鼠给予一次性腹腔注射链脲佐菌素(STZ)60 mg/kg体重,以诱导糖尿病大鼠模型;建模成功后再于建模后4、8、12周3个时间点分为3组,每组各12只大鼠.正常对照组不进行干预,与糖尿病组大鼠同时饲养,也按相同时间点分为4、8、12周3组,每组各12只大鼠.采用TdT介导DNA缺口末端的dUTP标记(TUNEL)法检测视网膜神经细胞凋亡指数,免疫组织化学方法检测半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白表达水平,黄嘌呤氧化酶法检测MnSOD及铜一锌超氧化物歧化酶(Cu-ZnSOD)活性,实时荧光定量聚合酶链反应(PCR)法检测MnSOD mRNA、Cu-ZnSODmRNA及easpase-3 mRNA表达情况.正常对照组与糖尿病组大鼠各时间点视网膜神经细胞凋亡指数、视网膜caspase-3 mRNA表达水平、Cu-ZnSOD和MnSOD活性及mRNA表达水平比较,均采用两因素方差分析,进一步两两比较采用LSD检验法.结果 (1)正常对照组4、8、12周大鼠视网膜均未见神经细胞凋亡.糖尿病组大鼠于建模后4周即出现神经细胞凋亡,凋亡指数为(0.5±1.5)%;8和12周大鼠神经细胞凋亡指数为(5.7±3.9)%和(11.8±5.1)%.糖尿病组与正常对照组大鼠各时间点视网膜神经细胞凋亡指数差异有统计学意义(F=19.5412,P＜0.05).进一步两两比较,8和12周糖尿病大鼠视网膜神经细胞凋亡指数较同期正常对照组大鼠均明显升高(均P=0.000).(2)正常对照组4、8、12周大鼠视网膜均未见caspase-3蛋白阳性表达,糖尿病组大鼠4周即可见caspase-3蛋白阳性表达,8和12周时表达增强.正常对照组4、8、12周大鼠caspase-3 mRNA表达RQ值分别为1.649±0.586、1.526±0.486及1.614±0.296;糖尿病组4、8、12周大鼠caspase-3 mRNA表达RQ值分别为5.672±1.193、12.566±2.272及14.297±2.11;正常对照组与糖尿病组大鼠各时间点视网膜caspase-3 mRNA表达RQ值差异有统计学意义(F=105.175,P＜0.05).进一步两两比较,糖尿病组大鼠各时间点视网膜caspase-3 mRNA表达RQ值均较同期正常对照组明显升高(均P=0.000).(3)正常对照组4、8、12周大鼠MnSOD活性分别为(47.118±5.018)、(46.033±6.835)及(45.813±6.859)U/mg,MnSOD mRNA表达RQ值分别为0.973±0.123、0.974±0.085及0.994±0.074,CuZnSOD活性分别为(113.884±9.07)、(112.301±5.24)及(117.52±7.982)U/mg,Cu-ZnSOD mRNA表达RQ值分别为1.067±0.109、1.055±0.119及1.092±0.180.糖尿病组4、8、12周大鼠MnSOD活性分别为(33.863±6.909)、(22.877±7.875)及(20.034±6.796)U/mg,MnSOD mRNA表达RQ值分别为0.627±0.083、0.333±0.080及0.256±0.057;8和12周大鼠Cu-ZnSOD活性分别为(98.588±9.212)和(78.168±12.180)U/mg,Cu-ZnSOD mRNA表达RQ值为0.829±0.048和0.621±0.033.正常对照组与糖尿病组大鼠各时间点视网膜MnSOD、Cu-ZnSOD活性及mRNA表达RQ值差异有统计学意义(MnSOD:活性F=19.709,P＜0.05;mRNA F=93.352,P＜0.05;Cu-ZnSOD:活性F=16.708,P＜0.05;mRNA F=16.332,P＜0.05).进一步两两比较,糖尿病组大鼠各时间点视网膜MnSOD、Cu-ZnSOD活性及mRNA表达RQ值均较同期对照组明显下降(MnSOD活性:4周P=0.002,8和12周均P=0.000;MnSOD mRNA:4、8、12周均P=0.000;Cu-ZnSOD活性:8周P=0.011,12周P=0.000;Cu-ZnSOD mRNA:8周P=0.001,12周P=0.000).结论 糖尿病早期视网膜神经细胞凋亡可能与MnSOD活性及MnSOD mRNA表达水平下降有关.     

白藜芦醇对大鼠糖尿病性白内障抗氧化损伤的保护作用

目的探讨白藜芦醇对糖尿病性白内障大鼠抗氧化损伤的保护作用。方法 90只SD大鼠随机分为正常对照组、糖尿病模型组和白藜芦醇治疗组,每组各30只。模型组及白藜芦醇治疗组采用链脲佐菌素诱发糖尿病,白藜芦醇治疗组按400mg.kg-1体质量给予白藜芦醇灌胃,灌注后4周、8周、12周观察3组大鼠晶状体混浊度的变化情况;同时检测晶状体内超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathi-one peroxidase,GSH-PX)及过氧化氢酶(catalase,CAT)的变化;采用RT-PCR检测晶状体中铜锌超氧化物歧化酶(Cu-Zn superoxide dismutase,CuZnSOD)mRNA表达水平。结果正常对照组大鼠晶状体始终透明。灌注后12周白藜芦醇治疗组晶状体混浊度高于正常对照组(χ2=8.26,P<0.05),但与糖尿病模型组相比明显减轻(χ2=14.72,P<0.05)。糖尿病模型组大鼠晶状体中SOD、GSH-PX以及CAT活性分别为:(10.39±1.25)U·mg-1、(24.36±1.05)U·mg-1、(1.62±0.24)U·mg-1,明显低于正常对照组的(40.16±1.87)U·mg-1、(67.86±1.24)U·mg-1、(5.57±1.54)U·mg-1。与糖尿病模型组相比,白藜芦醇能增强SOD、GSH-PX和CAT活性,分别为(29.06±1.33)U·mg-1、(47.58±2.35)U·mg-1、(3.26±0.47)U·mg-1。此外,白藜芦醇还能上调糖尿病大鼠晶状体内CuZnSODmRNA的表达(P<0.05)。结论白藜芦醇能增加晶状体抗氧化酶活力,能从多个环节发挥对糖尿病白内障的抗氧化作用,从而延缓白内障的发生和发展。     

HSP60在早期糖尿病大鼠晶状体上皮细胞的表达

目的观察热休克蛋白(hot shock protein,HSP)在早期糖尿病大鼠晶状体上皮细胞的表达及变化,探讨HSP60在糖尿病性白内障病变中的作用。方法鼠龄为6~8周的SD大鼠随机分成糖尿病组及对照组,每组18只,糖尿病组在禁食12 h后一次性腹腔注射10 g·L-1链脲佐菌素诱发糖尿病模型,成功诱导糖尿病模型后4周、8周、12周分别应用免疫组织化学法及Western blot分析晶状体上皮细胞上HSP60的表达变化。结果对照组大鼠晶状体上皮细胞上未发现有HSP60表达,糖尿病组大鼠在造模后4周、8周、12周均可见晶状体上皮细胞上有HSP60表达,两组间有显著差异(均为P<0.05)。糖尿病组大鼠造模后4周和8周间表达量无明显差异(P>0.05);而12周时其表达量开始减少,和4周及8周相比较,差异均有统计学意义(均为P<0.05)。结论早期糖尿病大鼠晶状体上皮细胞有大量HSP60表达,而且随着时间的延长,表达量呈下降趋势。     

褪黑素对早期糖尿病大鼠视网膜锰超氧化物歧化酶和铜锌超氧化物歧化酶表达的影响

目的观察褪黑素对早期糖尿病大鼠视网膜锰超氧化物歧化酶（MnSOD）、铜锌超氧化物歧化酶（Cu/ZnSOD）活性及基因表达变化的影响,以揭示褪黑素对早期糖尿病大鼠视网膜的抗氧化作用机制。方法 54只SD大鼠按随机数字表法分为正常对照组、糖尿病组及褪黑素组,每组18只。糖尿病组及褪黑素组大鼠一次性腹腔注射链脲佐菌素（STZ）60mg/kg,升高血糖至〉16.7mmol/L,建立糖尿病大鼠模型,褪黑素组大鼠于STZ注射后第2天给予褪黑素灌胃,每日10mg/kg。于造模后4、8、12周3个时间点处死各组6只大鼠制备视网膜匀浆。应用黄嘌呤氧化酶法检测视网膜组织中总SOD、Cu/ZnSOD及MnSOD的活性;应用实时荧光定量PCR法检测视网膜组织中MnSODmRNA及Cu/ZnSODmRNA的表达量。结果模型建立后4、8、12周时糖尿病组和褪黑素组大鼠血糖水平明显高于正常对照组大鼠,差异均有统计学意义（P〈0.01）。造模后4、8、12周时各时间点糖尿病组大鼠视网膜中总SOD、MnSOD活性明显低于正常对照组,8周及12周时褪黑素组大鼠视网膜中总SOD、MnSOD活性明显高于糖尿病组,差异均有统计学意义（P〈0.01）。糖尿病组大鼠视网膜中Cu/ZnSOD活性在8周及12周时明显降低,与正常对照组比较差异均有统计学意义（P〈0.01）,12周时褪黑素组较糖尿病组有所升高,差异有统计学意义（P〈0.01）。各时间点糖尿病组大鼠MnSODmRNA在视网膜中的表达量与正常对照组相比明显下降,褪黑素组MnSODmRNA的表达明显高于糖尿病组,差异均有统计学意义（P〈0.01）;8周及12周时糖尿病组Cu/ZnSODmRNA的表达量较正常对照组明显下降,12周时褪黑素组Cu/ZnSODmRNA的表达明显高于糖尿病组,差异有统计学意义（P〈0.01）。结论糖尿病早期,褪黑素主要通过提高视网膜MnSOD的活性及增强其基因表达减轻视网膜的氧化损伤。     

ICAM-1在大鼠晶状体上皮细胞中表达的实验研究

目的:研究细胞间黏附分子-1(ICAM-1)在糖尿病性白内障发病机制中的作用及转化生长因子β-1(TGF-β1)对晶状体上皮细胞ICAM-1表达的影响。方法:SD大鼠120只随机分为对照组和糖尿病性白内障组。在糖尿病性白内障组中,建立糖尿病模型,免疫组织化学SP法观察两组晶状体上皮细胞中ICAM-1表达变化;RT-PCR法检测TGF-β1和TGF-β1中和抗体对体外培养的人晶状体上皮细胞ICAM-1mRNA表达的影响。结果:在糖尿病性白内障组大鼠晶状体上皮细胞ICAM-1的表达明显增高,且随着病程发展呈进行性增强,与对照组比较差异有统计学意义(P<0.01)。TGF-β1培养组的人晶状体上皮细胞的ICAM-1的表达则显著增强,与正常血清培养组比较差异具有统计学意义(P<0.05)。在TGF-β1抗体培养组中,ICAM-1的表达明显受到抑制,与TGF-β1培养组比较,差别具有统计学意义(P<0.05)。结论:TGF-β1可以促进晶状体上皮细胞ICAM-1的表达;ICAM-1通过促进晶状体上皮细胞的黏附、增殖而在糖尿病性白内障的发生中发挥重要作用。     

白藜芦醇对糖尿病性白内障大鼠晶状体上皮细胞凋亡及bcl-2和bax表达的影响

目的探讨白藜芦醇对糖尿病性白内障大鼠晶状体上皮细胞(lens epithelialcell,LEC)凋亡及bcl-2和bax表达的影响。方法取90只SpragueDawley大鼠随机分为正常对照组、糖尿病模型组和白藜芦醇治疗组,每组各30只。正常对照组给予常规饲料喂养,模型组大鼠采用链脲佐菌素(60mg.kg-1)诱发糖尿病,白藜芦醇治疗组在模型组基础上按400mg.kg-1体质量给予白藜芦醇灌胃,4周、8周、12周后观察晶状体混浊度的变化情况;同时采用流式细胞术检测LEC凋亡情况,比色法检测caspase-3的活性改变情氉况。并用Western blot检测LECbcl-2和bax蛋白的表达。结果白藜芦醇治疗组12周后,晶状体混浊度Ⅰ级、Ⅱ级、Ⅲ级、Ⅳ级、Ⅴ级改变分别为66.7%、23.3%、10.0%、0.0%和0.0%,与模型组(3.3%、20.0%、53.3%、13.3%、10.0%)相比差异有统计学意义(P<0.05)。模型组4周、8周和12周时,LEC凋亡率分别为(8.24±0.88)%、(12.86±1.12)%和(19.87±1.14)%,白藜芦醇处理后,凋亡率明显降低,分别为(5.62±0.56)%、(7.05±1.09)%和(10.53±1.25)%(均为P<0.05)。此外,白藜芦醇对LEC细胞caspase-3的活性也具有一定的抑制作用。bcl-2和bax在正常LEC中均有一定表达,糖尿病模型组bcl-2表达显著降低,bax表达上调,bcl-2/bax比值下降;白藜芦醇治疗组bcl-2/bax比值明显增加。结论白藜芦醇能在一定程度上抑制大鼠LEC凋亡,从而延缓白内障的发生和发展。     

核因子-κB在糖尿病大鼠视网膜中的表达及意义

目的 观察核因子κB(nuclear factor-kappa B,NF-κB)在大鼠糖尿病早期视网膜中的表达及其在糖尿病视网膜病变(diabetic retinopathy,DR)发病过程中的作用.方法 清洁级健康成年雄性Wistar大鼠110只,随机分为正常对照组(NC组,24只)和糖尿病造模动物组(DM组,86只).大鼠腹腔注射链脲佐菌素(streptozotocin,STZ)诱发糖尿病模型,取成模的72只大鼠随机分为2周、4周、12周和20周组,每组各18只.到上述各时间点后分别取DM组大鼠6只和NC组大鼠2只并处死,免疫印记法观察NF-κB蛋白在不同病程糖尿病大鼠视网膜的表达,采用免疫组织化学法观察NF-κB在不同病程糖尿病大鼠视网膜组织和血管的表达.结果 NC组正常大鼠视网膜中NF-κB p65的蛋白质水平非常低,为17.72±8.57,DM组糖尿病诱导成功后2周NF-κB p65蛋白质表达为38.45±12.37,并随时间的延长其表达增加,4周时的免疫印记反应强度最高为89.41±19.79,各时间点蛋白质表达比NC组明显增强(均为Jp<0.01).DM组大鼠糖尿病诱导成功后2周,视网膜表层血管即散在出现NF-κB阳性染色颗粒,4周时在视网膜内核层及内核层血管均出现NF-κB阳性染色细胞,为胞核染色,并随着病程的延长,12周时在视网膜表面血管、内核层、内核层血管和外丛状层均出现大量阳性染色细胞;NC组大鼠视网膜血管铺片没有NF-κB表达.DM组大鼠糖尿病诱导成功后2周,视网膜血管壁出现散在NF-κB阳性染色颗粒,以后随病程延长,NF-κB阳性细胞逐渐增多,12周时最多,以后保持不变.结论 NF-κB在DR发生发展过程中可能起重要作用.     

糖尿病大鼠视网膜中内质网应激蛋白的表达及意义

目的 检测内质网应激蛋白(bip)在糖尿病大鼠视网膜组织中的表达情况,探讨内质网应激在糖尿病视网膜病变(DR)中的作用和机制.方法 Wistar大鼠随机分为4组:链脲佐菌素(STZ)致糖尿病大鼠3、6、9个月模型组及正常对照组,每组10只.采用腹腔注射STZ 60 mg/kg,制备大鼠糖尿病模型;分批处死,每只大鼠左眼免疫组织化学法检测视网膜组织中bip的表达,右眼半定量RT桺CR方法 检测bip mRNA的表达.结果 bip在正常大鼠视网膜组织中少量表达,主要分布于视网膜内核层和神经节细胞层,糖尿病3个月组bip在各层的表达量升高,6个月组表达最高.结论 bip参与了DR的发病机制,在早期糖尿病大鼠视网膜组织中,细胞的应激能力随糖尿病病程延长而增强.     

硫氧还蛋白在糖尿病大鼠视网膜中的动态表达

目的 观察硫氧还蛋白( thioredoxin,Trx)在糖尿病(diabetes mellitus,DM)大鼠视网膜中的表达部位及动态变化规律.方法 成年Sprague Dawley(SD)大鼠随机分为2组,糖尿病组(DM组)一次性腹腔注射链脲佐菌素制作大鼠DM模型;对照组(NC组)注射同等剂量的柠檬酸缓冲液.在注射后4周、8周、12周,采用免疫组织化学法检测Trx在各组大鼠视网膜中的定位表达,采用Real-time PCR测定视网膜Trx mRNA表达.结果 免疫组织化学结果显示,NC组与DM组大鼠视网膜神经纤维层、神经节细胞层、内核层有Trx阳性表达,表达位于细胞浆.Real-time PCR结果显示正常大鼠视网膜有Trx mRNA的表达;与NC组相比,DM组各时间点TrxmRNA表达下降,4周时DM组Trx mRNA相对表达量为0.42±0.03,约为NC组(1.00±0.03)的0.42倍;8周时DM组Trx mRNA相对表达量为0.57±0.04,约为NC组(1.00±0.03)的0.57倍;12周时DM组Trx mRNA相对表达量为0.82±0.08,约为NC组(1.00±0.07)的0.82倍,差异均有统计学意义(均为P＜0.05).DM组视网膜组织Trx mRNA在4～12周表达逐渐升高,至第12周时仍未达到正常水平,第4周与第12周相比差异有统计学意义(P=0.00).结论 在糖尿病视网膜病变时,Trx mRNA表达下降,说明氧化应激反应产生过多的活性氧使得Trx mRNA表达量下降.随着DM病程的延长,视网膜Trx mRNA表达量逐渐升高,这可能是Trx系统功能下降、自由基增多引起的一种代偿性反应.     

单核细胞趋化蛋白-1在实验性糖尿病大鼠视网膜中的表达及意义

目的 研究单核细胞趋化蛋白（monocyte chemoattractant protem-1,MCP-1）在不同病程实验性糖尿病大鼠视网膜的表达及探讨MCP-1与糖尿病视网膜病变发生发展的关系.方法 Wistar大鼠64只随机分为正常对照组（NC组）16只和糖尿病造模组（DM组）48只,DM组腹腔注射链尿佐菌素诱发糖尿病,将造模成功DM组糖尿病大鼠随机分为2周、4周、12周和20周组,每组各12只.到各时间点后处死,采用Western Blotting和免疫组织化学法,分析MCP-1在不同病程糖尿病造模大鼠视网膜中的表达,对免疫印迹结果采用计算机图像分析系统处理.结果 免疫印迹结果显示：正常大鼠视网膜中MCP-1的表达非常微弱,糖尿病诱导成功后2周MCP-1蛋白表达比正常组明显增加（P＜0.05）,并随时间的延长表达逐渐增加,到12周时达最高.免疫组织化学结果显示：正常大鼠视网膜各层均无MCP-1表达,大鼠糖尿病诱导成功后2周,视网膜表面血管即有MCP-1阳性细胞表达,以后随病程延长而表达增多.结论 MCP-1在视网膜的表达与糖尿病视网膜病变的发生密切相关.     

Expression modification of uncoupling proteins and MnSOD in retinal endothelial cells and pericytes induced by high glucose: the role of reactive oxygen species in diabetic retinopathy

Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. They belong to the family of anion mitochondrial carriers. UCPs could act as proton carriers activated by metabolites and create a shunt between complexes of the respiratory chain and ATP synthase. The increased leakiness of the mitochondrial inner membrane to protons may be to minimize superoxide production by limiting the maximum Deltamu(H+). The purpose of this study was to detect UCP expression in retinal capillary cells and their modification in high levels of glucose. The role of reactive oxygen species (ROS) of mitochondria and UCPs in pathogenesis of diabetic retinopathy was investigated. Bovine retinal capillary endothelial cells and pericytes were cultured with selective culture media, respectively. Passage cells were cultured in three different glucose concentrations (5, 23, 30 mM) until passage four. ROS changes in mitochondria of these cells in different glucose concentrations were detected with scanning laser confocal microscopy (SLCM). The mitochondria membrane potential (Deltapsi), cell death rate and apoptosis rate were measured with flowing cytometry. UCP expression in retinal capillary cells was detected by immunocytochemistry. Expression and modification of MnSOD and uncoupling proteins (UCPs) in different concentrations of glucose were detected by means of semi-quantitative RT-PCR. ROS in mitochondria of both endothelial cells and pericytes increased as the glucose concentration of media increased. Deltapsi and cell death rate of endothelial cells increased also. ROS was correlated to Deltapsi and cell death rate positively in endothelial cells. No difference in Deltapsi and cell death rate among different glucose levels was found in pericytes. Apoptosis rate of endothelial cells and pericytes in high glucose levels was higher than that in lower glucose levels. UCP1 and UCP2 were expressed in cultured retinal capillary cells whereas UCP3 was not. At high levels of glucose, expression of UCP1, UCP2 and MnSOD increased to accommodate ROS production compensatively. The compensative mechanism disappeared when glucose concentration was too high (30 mM). The results of this study showed that increasing mitochondrial ROS could be induced by high glucose concentration. Those proteins related to antioxidation mechanism, such as MnSOD and UCPs, could exert compensative action to a certain extent. This compensative action was insufficient when the glucose concentration was too high.     

糖尿病早期大鼠视网膜神经细胞凋亡与caspase-3表达的关系

目的:观察糖尿病早期大鼠,视网膜神经细胞凋亡与caspase-3表达变化的关系。方法:健康雄性8周龄SD大鼠36只,建立糖尿病大鼠模型18只,正常对照18只。再各自分为4,8,12wk3组,每组6只大鼠。TdT介导DNA缺口末端的dUTP标记(TUNEL)法检测视网膜神经细胞的凋亡程度;免疫组织化学方法检测半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白表达水平,实时荧光定量聚合酶链反应(PCR)法检测caspase-3mRNA表达情况。结果:正常对照组4,8,12wk大鼠均未见视网膜神经细胞凋亡。糖尿病组大鼠于建模后4wk即出现视网膜神经细胞凋亡,8wk和12wk大鼠视网膜神经凋亡程度较同期正常对照组大鼠均明显增强。神经细胞凋亡主要位于视网膜神经节细胞层及内核层。正常对照组4,8,12wk大鼠视网膜均未见caspase-3蛋白阳性表达,糖尿病组大鼠4wk即可见caspase-3蛋白阳性表达,8和12wk时表达增强。正常对照组4,8,12wk大鼠caspase-3mRNA表达RQ值分别为1.6±0.6,1.5±0.5,1.6±0.3;糖尿病组4,8,12wk大鼠caspase-3mRNA表达RQ值分别为5.7±1.2,12.6±2.3,14.3±2.1;较同期对照组均明显升高,差异有统计学意义(P<0.05)。结论:糖尿病早期视网膜神经细胞凋亡可能与capase-3表达增强有关。     

葡萄糖调节蛋白在糖尿病性白内障发病机制中的作用

目的探讨葡萄糖调节蛋白（78 kd glucose-regu-lated protein,grp78）在糖尿病性白内障发病机制中的作用。方法32只Wistar大鼠随机分为4组：链脲佐菌素（strepto-zotocin，STZ）糖尿病大鼠1、2、3个月模型组及正常对照组，每组8只。对大鼠采用STZ60mg／kg的剂量一次性腹腔注射制备糖尿病模型。各时段分批处死大鼠，每只大鼠左眼采用免疫组织化学法．右眼用半定量RT-PCR的方法检测晶状体上皮细胞中grp78的表达情况。结果Grp78在正常大鼠晶状体上皮细胞中少量表达,免疫组化光密度值为28．66±5．90，RT-PCR检测光密度比值为0．889±0．006；糖尿病大鼠1、2、3个月组免疫组化光密度值分别为49．56±6．54、63．74±9．05、78．15±9．05，RT-PCR光密度比值分别为0．920±0．011、0．948±0．004、0．976±0．006。经统计学分析,糖尿病组大鼠晶状体上皮细胞中grp78的表达较正常组高（P〈0．01），并随着病程的延长而表达增加（P〈0．05）。结论葡萄糖调节蛋白参与了糖尿病性白内障的发生、发展。     

The presence of the -866A/55Val/Ins haplotype in the uncoupling protein 2 (UCP2) gene is associated with decreased UCP2 gene expression in human retina

Uncoupling protein 2 (UCP2) is a mitochondrial transporter present in the inner membrane of mitochondria, and it uncouples substrate oxidation from ATP synthesis, thereby dissipating the membrane potential energy and consequently decreasing ATP production by mitochondrial respiratory chain. As a consequence of the uncoupling, UCP2 decreases the reactive oxygen species (ROS) formation by mitochondria. ROS overproduction is related to diabetic retinopathy (DR), a chronic complication of diabetes mellitus (DM). Recently, our group reported that the -866A/55Val/Ins haplotype (-866G/A, Ala55Val and Ins/Del polymorphisms) of the UCP2 gene was associated with increased risk for DR in patients with DM. The purpose of this study was to analyze the effect of this haplotype on UCP2 gene expression in human retina. In addition, MnSOD2 gene expression was also investigated according to different UCP2 haplotypes. This cross-sectional study included 188 cadaveric cornea donors. In a subset of 91 retinal samples differentiated according to the presence of the mutated UCP2 haplotype and risk alleles of the -866G/A and Ins/Del polymorphisms, UCP2 and MnSOD2 gene expressions were measured by semi-quantitative RT-qPCR. Mutated UCP2 haplotype carriers (homozygous?+?heterozygous) had a lower UCP2 gene expression than reference haplotype carriers (8.4?±?7.6 vs. 18.8?±?23.7 arbitrary units; P?=?0.046). Accordingly, UCP2 gene expression was decreased in -866A carriers when compared with G/G carriers (P?=?0.010). UCP2 gene expression did not differ between Ins allele carriers and Del/Del carriers (P?=?0.556). Interestingly, subjects carrying the heterozygous UCP2 haplotype showed increased MnSOD2 gene expression (P?=?0.025). This is the first report suggesting that the presence of the -866A/55Val/Ins haplotype is associated with decreased UCP2 gene expression in human retina. Possibly, MnSOD2 expression might influence the UCP2 effect in the protection against oxidative stress.     

儿茶素对糖尿病性白内障大鼠晶状体热休克蛋白27表达的影响

目的　检测糖尿病性白内障大鼠晶状体中热休克蛋白（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ２７, ＨＳＰ２７）的表达,探讨儿茶素在糖尿病性白内障大鼠病程发生发展过程中的作用。方法　取６０只雄性ＳＤ大鼠,随机分为３组,每组２０只。正常组大鼠腹腔注射枸橼酸缓冲液;糖尿病对照组、儿茶素治疗组大鼠先行造模,大鼠腹腔注射链脲佐菌素溶液（６５ｍｇ·ｋｇ－１体质量）诱发糖尿病性白内障,造模成功后,治疗组大鼠给予儿茶素２００ｍｇ·ｋｇ－１（用蒸馏水配制）灌胃。正常组及对照组大鼠给予等量蒸馏水灌胃。于造模成功后２周、４周、８周３个时间点用免疫组织化学法检测ＨＳＰ２７表达,计算平均ＩＨＳ评分值。结果正常组晶状体中ＨＳＰ２７阳性上皮细胞及纤维细胞较少,染色较浅,在造模成功后２周、４周、８周无明显变化。对照组造模成功后２周时晶状体ＨＳＰ２７阳性细胞增多,染色加深,４周时表现最明显,８周时下降,但仍然高于正常组。治疗组在造模成功后２周、４周、８周ＨＳＰ２７阳性细胞较对照组明显增多、染色加深。正常组ＨＳＰ２７阳性上皮细胞ＩＨＳ评分值在实验各时间点无明显变化。对照组ＨＳＰ２７阳性上皮细胞ＩＨＳ评分值在造模成功后２周、４周及８周时均高于正常组,差异均有统计学意义（Ｆ２周＝９．２９４,Ｐ＜０．０５;Ｆ４周＝３８．４００及Ｆ８周＝１６．０００,均为Ｐ＜０．０１）。治疗组在相应时间点ＨＳＰ２７阳性上皮细胞ＩＨＳ评分值在相应时间点高于对照组,在２周及４周时差异均有统计学意义（Ｆ２周＝９．２９４,Ｐ＜０．０５,Ｆ４周＝３８．４００,Ｐ＜０．０１）。结论　大鼠糖尿病性白内障晶状体组织中ＨＳＰ２７的表达增强,全身应用儿茶素可提高晶状体组织中ＨＳＰ２７表达。     

Effect of instillation of aldose reductase inhibitor FR74366 on diabetic cataract.

The authors investigate the effect of aldose reductase inhibitor FR74366 on diabetic cataract. Streptozocin (STZ)-induced diabetic rats were treated with eye drops of FR74366 (0.03%, 0.1%, and 0.3%) for 16 weeks. Lenses were examined using a slit lamp, and the score of lens opacity was determined on a scale of from 0 (normal lens) to 4 (matured nuclear cataract). Diabetic placebo control rats developed lens opacity linearly, beginning at 3 weeks and reaching a maximum at 8 weeks after STZ injection. Instillation of FR74366 to diabetic rats delayed the cataract formation and inhibited lens sorbitol accumulation in a dose-dependent manner. At 16 weeks after STZ injection, the score of lens opacity was more than 3 (diffuse central opacities) in diabetic placebo control rats, whereas it was less than 2 (peripheral vesicles and cortical opacities) and the lenses remained clear in animals treated with 0.3% of FR74366. Measurement of tissue drug concentrations indicated that FR74366 penetrated into the lens, where its levels were increased in a dose- and time-dependent manner. These three parameters (score of lens opacity and sorbitol and FR74366 levels) were well correlated with each other. Instillation of FR74366 also reduced the sorbitol levels in the retina. However, the sorbitol levels in the sciatic nerve and renal cortex were not changed by instillation of FR74366. Instillation or oral administration of FR74366 has not shown serious side effects in animal toxicity studies. These results suggested that instillation of FR74366 may be a useful therapeutic agent against diabetic cataract and retinopathy.