核因子E2相关因子2基因多态性与氧化应激及2型糖尿病易感性的关联研究
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摘要
2型糖尿病(type2diabetes mellitus，T2DM)正成为21世纪威胁人类健康的慢性重大性疾病之一，被世界卫生组织称作为21世纪的灾难。国际上糖尿病的患病率一直在增长,据估计,成人患糖尿病的人数从1980年的1.53亿增加到2008年的3.47亿。并且据世界卫生组织估计，到2030年全球将有3.66亿人患有糖尿病。
氧化应激在T2DM的发生、发展过程中起关键性的作用。糖尿病是由于胰岛素分泌和／或作用异常，所导致的碳水化合物、脂肪以及蛋白质代谢异常，从而引起以高血糖为主要特征的一组代谢性疾病。许多研究结果表明，慢性高血糖引起活性氧(reactive oxygen species，ROS)水平上升，超出了机体抗氧化系统的清除能力，从而使机体处于氧化应激状态。氧化应激和糖尿病的发生以及发展密切相关，氧化应激影响胰岛素合成和分泌，促进了糖尿病的发生。能够导致肌肉、脂肪及肝脏等组织细胞损伤影响胰岛素敏感性，使血糖持续升高，糖尿病又进一步加强了氧化应激。
近年来的大量研究表明，核因子E2相关因子2（NF-E2related factor2，Nrf2），在氧化应激相关疾病的发生发展中起着重要作用。在各种氧化应激的刺激下, Nrf2转入胞核内,识别并结合抗氧化反应元件（antioxidant response element，ARE），Nrf2通过ARE的转录激活能够对抗氧化损伤和炎症,通过与ARE结合, Nrf2蛋白能够诱导抗氧化基因的表达。Nrf2／ARE通路的激活，可减少高糖因素所产生的活性氧，发挥其抗氧化应激损伤以及抗慢性炎症反应的作用，改善氧化应激所诱导的胰岛素抵抗。研究表明Nrf2基因的单核苷酸多态性与氧化应激相关疾病如创伤后急性肺水肿等的易感性有明显关联。但是，Nrf2基因的单核苷酸多态性与糖尿病易感性的关系未见相关报道。
自由基主要氧化损伤DNA、蛋白以及脂质等生物分子，所以检测组织或生物体液中的氧化应激的代谢产物对于评价机体的氧化应激状态起重要作用。在抗氧化防御体系中，主要包括的抗氧化酶为谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-PX)、超氧化物歧化酶(superoxide dismutase, SOD)和过氧化氢酶(catalase,CAT)，能协助清除自由基，减轻以及消除氧化损伤。在生物膜脂质的磷脂中饱含多不饱和脂肪酸（poly-unsaturated fatty acid，PUFA），在O2存在的条件下，这些多不饱和脂肪酸极易受到自由基和其活性衍生物的攻击，引起脂质过氧化的链式反应，同时在脂质过氧化过程中所生成的LO、LOO等也能产生链引发以及链扩增反应。LOO还能通过分子内的双键加成，生成环过氧化物以及环内过氧化物自由基，最后可断裂成各种的代谢产物。过氧化脂质通过脂氧基的β断裂能产生许多醛类，如丙二醛。因此测定生物样品中的丙二醛可反映脂质过氧化程度，从而能反映糖尿病患者的氧化应激状态。
因此本研究的目的是拟采用TaqMan方法对Nrf2的基因的单核苷酸多态性位点的进行基因分型；同时检测氧化应激和抗氧化状态的相关标志物，以探讨Nrf2基因多态性与氧化应激及2型糖尿病发病风险的关联；揭示Nrf2基因多态性与2型糖尿病发病易感性的关系，为预防和治疗T2DM提供科学依据和理论指导。
第一部分氧化应激和抗氧化状态与2型糖尿病的相关性研究
目的：评价糖尿病患者、糖调节受损患者、以及正常对照组被试者的氧化应激状态和抗氧化能力。
方法：用GOD-POD法测定血浆FPG，ECLIA法测定FPI，利用稳态模式评估法进行评估HOMA-IR和HOMA-β cell。采用高铁还原法检测血浆中的TAOC水平，TBA比色法检测血浆MDA浓度，钼酸铵比色法检测血浆中的CAT活性，黄嘌呤-黄嘌呤氧化酶-亚硝酸盐方法检测红细胞SOD的活性，Beutler改良法测定红细胞的GSH含量，DNTB比色法来检测红细胞GSH-Px的活性。使用ANOVA分析和SNK进行检验，并比较氧化应激和抗氧化状态与糖调节受损和糖尿病风险之间的可能联系。
结果：本次研究总共检测2517名被试者，包括IGR306例、T2DM患者884例和NGT1327例。与正常糖耐量的对照组相比，T2DM患者血浆TAC水平（T2DM8.86±0.13U/ml vs. NGT9.57±0.17U/ml；P=0.001），CAT活性（CAT：T2DM32.04±1.04KU/L vs. NGT41.71±1.65KU/L，P <0.001），红细胞SOD活性（T2DM9997.75±164.01U/gHb vs. NGT11024.28±299.13U/gHb，P=0.002）GSH-Px的活性（T2DM129.97±2.83AU vs. NGT145.63±4.32AU，P=0.002）和GSH含量（T2DM18.94±0.29mg/gHbvs. NGT20.87±0.48mg/gHb，P=0.003）是明显降低地，而其血浆MDA浓度明显地增高（T2DM6.52±0.18nmol/L vs. NGT5.79±0.12nmol/L；P=0.001）。T2DM患者具有明显高地HOMA-IR值（T2DM4.26±0.13vs. NGT2.17±0.11；P <0.001），而HOMA-β值则显著下降（T2DM44.40±1.78vs. NGT103.37±5.01；P <0.001）。
结论：T2DM患者具有明显增加地血浆MDA浓度，然而血浆TAC和CAT活性、红细胞SOD活性、GSH-Px活性以及GSH含量出现明显地降低。这些结果表明，抗氧化能力下降和自由基产生明显增加与2型糖尿病有很强的关联性。
第二部分核因子E2相关因子2基因多态性与氧化应激的相关性研究
目的：探讨Nrf2rs6721961基因多态性与氧化应激之间的关系，检测该基因多态性与机体的抗氧化能力强度有无关联。
方法：使用TaqMan探针对Nrf2rs6721961位点进行基因分型。采用高铁还原法检测血浆中的TAOC水平，TBA比色法检测血浆MDA浓度，钼酸铵比色法检测血浆中的CAT活性，黄嘌呤-黄嘌呤氧化酶-亚硝酸盐方法检测红细胞SOD的活性，Beutler改良法测定红细胞的GSH含量，DNTB比色法来检测红细胞GSH-Px的活性。
结果：本部分病例对照研究共纳入被试者2477例，其中包括879例2型糖尿患者和1295例NGT正常对照人群。与CC基因型携带者相比，AA基因型携带者的血浆TAC水平（AA8.73±0.22vs. CC9.23±0.08U/ml，P=0.025），CAT活性（AA34.41±0.58vs. CC38.14±0.41KU/L，P=0.001），红细胞SOD活性（AA10446.01±118.61vs. CC10809.65±64.64U/gHb，P=0.042），GSH-Px活性（AA133.57±3.61vs. CC140.57±1.06AU，P=0.020），和GSH含量（AA19.49±0.31vs. CC20.10±0.10mg/gHb，P=0.042）也是明显降低的，而血浆中的丙二醛（MDA）的浓度（AA6.42±0.26vs. CC5.84±0.11nmol/ml，P=0.005）明显地增加。基因型AA携带者与基因型CC携带者相比较具有明显高的HOMA-IR值（AA2.90±0.19vs. CC2.36±0.06，P=0.003）和明显低地β细胞功能水平（AA71.37±4.05vs. CC84.37±1.59，P=0.005）。
结论：Nrf2rs6721961单核苷酸多态性的与氧化应激以及抗氧化状态密切相关。与基因型CC及基因型CA携带者相比，基因型AA携带者在血浆TAC和CAT活性，红细胞的SOD和GSH-Px活性以及红细胞GSH含量明显降低，而血浆丙二醛水平明显增高。
第三部分核因子E2相关因子2基因多态性与2型糖尿病和糖调节受损的风险关联研究
目的：探讨Nrf2rs6721961基因多态性与2型糖尿病和糖调节受损发病风险之间的关联关系。
方法：用GOD-POD法测定血浆FPG，ECLIA法测定FPI，利用稳态模式评估法进行评估HOMA-IR和HOMA-β cell。使用TaqMan探针对Nrf2rs6721961位点进行基因分型。
结果：本部分病例对照研究共纳入被试者2477例，其中包括879例2型糖尿患者，303例IGR和1295例NGT正常对照人群。Nrf2rs6721961基因多态性的等位基因的频率在2型糖尿病的病例和对照之间有显著性差异。与NGT组受试者（26.1％）相比较，A等位基因的频率（29.4％）在2型糖尿病的被试者明显地较高（P=0.019）。Nrf2rs6721961是与增加地患糖尿病的风险密切相关联。与CC基因型携带者相比较，携带AA基因型的个体有显着增加的发展2型糖尿病的风险（OR=1.77，95％CI1.26，2.49，P=0.011）。经校正年龄，性别，身体质量指数等混杂因素后，该关联仍具有统计学意义（OR=1.56，95％CI1.11，2.20，P=0.014）。与CC基因型携带者相比较，AA基因型携带者HOMA-β值显著降低(AA71.37±4.05vs. CC84.37±1.59, P=0.005)，而HOMA-IR值显著增加(AA2.90±0.19vs. CC2.36±0.06, P=0.003)。
结论： Nrf2基因rs6721961多态性与T2DM易感性存在明显关联。与CC基因型携带者相比较，AA基因型携带者患T2DM以及糖调节受损的风险明显增加。
Type2diabetes mellitus （T2DM） has been one of the biggest chronic diseases tothreaten the public health. Increases in the prevalence of diabetes have occurredinternationally, and it has been estimated that between1980and2008, the number of adultswith diabetes rose from153million to347million. Moreover, according to the estimationof the World Health Organization (WHO), there will be366million diabetic peopleworldwide by2030.
Research suggests that oxidative stress is a major risk factor in the onset andprogression of type2diabetes mellitus (T2DM). Some of the consequences of an oxidativeenvironment may be the development of impaired glucose tolerance, β-cell dysfunction,insulin resistance, and mitochondrial dysfunction, which can contribute ultimately to thediabetic disease state. Recent evidence also suggests that NF-E2related factor2(Nrf2) is apivotal transcription factor to the antioxidant response in oxidative stress related illnesses.
Nrf2is a member of the cap ‘n’ collar (CNC) subfamily of basic leucine zippertranscription factors. Nrf2has highly conserved domains named Nrf2-erythroid-derivedCNC homology protein homology (Neh) domains. Among them, Neh1domain is CNC andbasic leucine zipper domain, which interacts with partner proteins for heterodimerization.Neh3domain, located at the extreme end of the carboxyl terminus of Nrf2, is related toNrf2transactivation. Neh4and Neh5cooperatively bind with the cyclic adenosinemonophosphate response element binding protein-binding protein. In the absence of Nrf2structural data, it is not clear how Neh4and Neh5, together with the Neh3domains, exerttransactivation activity. The Neh2domain, which is located in the N-terminus of Nrf2, is a regulatory domain that responds to oxidative stress. Neh2mediates binding with Kelch-likeerythroid-derived protein with CNC homology-associated protein1(Keap1), andnegatively regulates Nrf2function.
Keap1was originally thought to be an actin-binding protein that represses the functionof Nrf2by simply sequestering Nrf2in the cytoplasm. Recently, Keap1has also beenidentified as an adaptor protein between Nrf2and Cullin3, a component of the E3ligasecomplex. Under normal conditions, Nrf2molecules may be subjected to continuousdegradation by the proteasome. When induced by oxidative stress derived fromaccumulation of reactive oxygen species (ROS) or reactive nitrogen species, single ormultiple reactive cysteine(s) in Keap1can be modified. This conformation change causesNrf2to dissociate from Keap1. Nrf2quickly cumulates in the nucleus and elicits theantioxidant response by transactivating the antioxidant response element (ARE) in thepromoter region of many antioxidant genes. The activation of Nrf2is an important clue forthe inducible expression of cytoprotective genes. The antioxidant enzymes encoded bythese genes may play important roles in scavenging oxygen free radicals.
In the promoter region of the Nrf2gene, the promoter polymorphisms were found in aJapanese population by Yamamoto et al.. Several studies demonstrated the associations ofthe Nrf2polymorphisms with oxidative stress related illnesses, such as acute lung injuryafter major trauma and vitiligo. However, there is little research evaluating the associationbetween Nrf2polymorphisms and oxidative stress and antioxidative status. The relationshipbetween this polymorphism with β-cell function, insulin sensisity, and the risk of T2DM islargely unknown.
Therefore, the objective of the present study was to evaluate the association betweenNrf2gene polymorphisms and the susceptibility to T2DM in a relatively large Chinesepopulation. We also evaluated the functional relevance of this polymorphism by measuringβ-cell function, insulin sensitivity, oxidative stress and antioxidative status among the studypopulations.
PartⅠ Oxidative stress and antioxidant status and type2diabetes mellitus
Objective: To investigate oxidative stress and antioxidative status in participants withand without diabetes
Methods: The measure of plasma TAC was based on the ability of antioxidants in the samples to change Fe3+-tripyridyltriazine to Fe2+-tripyridyltriazine, a stable blue productproportional to the TAC, which was tested at593nm. Plasma CAT activity was assayed bya method of Goth. MDA as an index of lipid peroxidation was estimated by using themethod described by Beuege and Aust. The activity of SOD in erythrocyte lysates wasevaluated on the basis of its ability to inhibit the oxidation of hydroxylamine, as describedpreviously. Erythrocyte GSH-Px activity was measured by the method described by Pagliaand Valentine. Erythrocyte GSH content was measured using the method described byBeutler et al. Descriptive statistics in the clinical and laboratory characteristics of healthycontrols and patients with T2DM were calculated for the study subjects. Differencesbetween diabetes cases and controls were tested by one-way analysis of variance, followedby Chi-square (categorical variables) or t test (continuous variables).
Results: When compared with subjects with NGT, patients with T2DM had asignificant increase in plasma MDA (P=0.001) and a significant decrease in plasma TAClevels (P=0.024) and CAT activity (P <0.001). Erythrocyte SOD activity (P=0.002),GSH-Px activity (P=0.002), and GSH content (P=0.040) were also significantly lower inpatients with T2DM.
Conclusion: The results also demonstrated that patients with T2DM had an increasedfree radical production and a reduced antioxidant capacity.
PartⅡ Association of the Nrf2rs6721961polymorphism with oxidative stressand antioxidative status
Objective: To investigate oxidative stress, and antioxidative status in relation togenotypes of rs6721961polymorphism in the study populations.
Methods: The genotyping of single nucleotide polymorphisms (SNPs) of the Nrf2gene was done by using an allelic discrimination assay-by-design TaqMan method onABI7900HT. The measure of plasma TAC was based on the ability of antioxidants in thesamples to change Fe3+-tripyridyltriazine to Fe2+-tripyridyltriazine, a stable blue productproportional to the TAC, which was tested at593nm. Plasma CAT activity was assayed bya method of Goth. MDA as an index of lipid peroxidation was estimated by using themethod described by Beuege and Aust. The activity of SOD in erythrocyte lysates wasevaluated on the basis of its ability to inhibit the oxidation of hydroxylamine, as describedpreviously. Erythrocyte GSH-Px activity was measured by the method described by Paglia and Valentine. Erythrocyte GSH content was measured using the method described byBeutler et al.
Results: Compared to individuals with the CC genotype, those with the AA genotypehad a significant decrease in plasma TAC levels (P=0.025), CAT activity (P=0.001),erythrocyte SOD activity (P=0.042), GSH-Px activity (P=0.020), and GSH content (P=0.042), whereas a significant increase in plasma MDA concentration (P=0.005).
Conclusion: Polymorphisms in the Nrf2genes were significantly associated withdecreased antioxidant activity and increased oxidative stress.
Part Ⅲ Association of Nrf2rs6721961polymorphism with risk of type2diabetes mellitus
Objective: To investigate the genotype and allele frequencies of the rs6721961polymorphism in the Nrf2gene in the study populations.
Methods: The genotyping of SNPs of the Nrf2gene was done by using an allelicdiscrimination assay-by-design TaqMan method on ABI7900HT. Differences in allelic andgenotypic frequencies of the gene polymorphisms in healthy controls and patients withT2DM were compared by Chi-square test, which was also used to evaluateHardy-Weinberg equilibrium for each individual locus. We used logistic regression analysisto assess the association of diabetes events with the specific polymorphism. Odds ratios and95%confidence intervals were adjusted for known risk factors for T2DM, including age,body mass index, sex, family history of diabetes, and hypertension.
Results: There were significant differences in the allelic frequency of the rs6721961polymorphism between T2DM cases and controls. The frequency of allele A wassignificantly higher in T2DM subjects (29.4%) compared to NGT subjects (26.1%)(P=0.019). The rs6721961was associated with increased risk of diabetes. The individualscarrying the AA genotype had a significantly higher risk for developing T2DM (OR1.77;95%CI1.26,2.49; P=0.011) relative to those with the CC genotype. After adjustment forage, sex, and body mass index, this association remained statistically significant (OR1.56;95%CI1.11,2.20; P=0.014). Individuals carrying the AA genotype had a lower HOMA-β(AA71.37±4.05vs. CC84.37±1.59, P=0.005) and a higher HOMA-IR values thanthose with the CC genotype (AA2.90±0.19vs. CC2.36±0.06, P=0.003).
Conclusion: The rs6721961polymorphism of the Nrf2gene was associated with an increased risk of T2DM. The polymorphisms were also associated with impaired β-cellfunction and increasing insulin resistance.
氧化应激在T2DM的发生、发展过程中起关键性的作用。糖尿病是由于胰岛素分泌和／或作用异常，所导致的碳水化合物、脂肪以及蛋白质代谢异常，从而引起以高血糖为主要特征的一组代谢性疾病。许多研究结果表明，慢性高血糖引起活性氧(reactive oxygen species，ROS)水平上升，超出了机体抗氧化系统的清除能力，从而使机体处于氧化应激状态。氧化应激和糖尿病的发生以及发展密切相关，氧化应激影响胰岛素合成和分泌，促进了糖尿病的发生。能够导致肌肉、脂肪及肝脏等组织细胞损伤影响胰岛素敏感性，使血糖持续升高，糖尿病又进一步加强了氧化应激。
近年来的大量研究表明，核因子E2相关因子2（NF-E2related factor2，Nrf2），在氧化应激相关疾病的发生发展中起着重要作用。在各种氧化应激的刺激下, Nrf2转入胞核内,识别并结合抗氧化反应元件（antioxidant response element，ARE），Nrf2通过ARE的转录激活能够对抗氧化损伤和炎症,通过与ARE结合, Nrf2蛋白能够诱导抗氧化基因的表达。Nrf2／ARE通路的激活，可减少高糖因素所产生的活性氧，发挥其抗氧化应激损伤以及抗慢性炎症反应的作用，改善氧化应激所诱导的胰岛素抵抗。研究表明Nrf2基因的单核苷酸多态性与氧化应激相关疾病如创伤后急性肺水肿等的易感性有明显关联。但是，Nrf2基因的单核苷酸多态性与糖尿病易感性的关系未见相关报道。
自由基主要氧化损伤DNA、蛋白以及脂质等生物分子，所以检测组织或生物体液中的氧化应激的代谢产物对于评价机体的氧化应激状态起重要作用。在抗氧化防御体系中，主要包括的抗氧化酶为谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-PX)、超氧化物歧化酶(superoxide dismutase, SOD)和过氧化氢酶(catalase,CAT)，能协助清除自由基，减轻以及消除氧化损伤。在生物膜脂质的磷脂中饱含多不饱和脂肪酸（poly-unsaturated fatty acid，PUFA），在O2存在的条件下，这些多不饱和脂肪酸极易受到自由基和其活性衍生物的攻击，引起脂质过氧化的链式反应，同时在脂质过氧化过程中所生成的LO、LOO等也能产生链引发以及链扩增反应。LOO还能通过分子内的双键加成，生成环过氧化物以及环内过氧化物自由基，最后可断裂成各种的代谢产物。过氧化脂质通过脂氧基的β断裂能产生许多醛类，如丙二醛。因此测定生物样品中的丙二醛可反映脂质过氧化程度，从而能反映糖尿病患者的氧化应激状态。
因此本研究的目的是拟采用TaqMan方法对Nrf2的基因的单核苷酸多态性位点的进行基因分型；同时检测氧化应激和抗氧化状态的相关标志物，以探讨Nrf2基因多态性与氧化应激及2型糖尿病发病风险的关联；揭示Nrf2基因多态性与2型糖尿病发病易感性的关系，为预防和治疗T2DM提供科学依据和理论指导。
第一部分氧化应激和抗氧化状态与2型糖尿病的相关性研究
目的：评价糖尿病患者、糖调节受损患者、以及正常对照组被试者的氧化应激状态和抗氧化能力。
方法：用GOD-POD法测定血浆FPG，ECLIA法测定FPI，利用稳态模式评估法进行评估HOMA-IR和HOMA-β cell。采用高铁还原法检测血浆中的TAOC水平，TBA比色法检测血浆MDA浓度，钼酸铵比色法检测血浆中的CAT活性，黄嘌呤-黄嘌呤氧化酶-亚硝酸盐方法检测红细胞SOD的活性，Beutler改良法测定红细胞的GSH含量，DNTB比色法来检测红细胞GSH-Px的活性。使用ANOVA分析和SNK进行检验，并比较氧化应激和抗氧化状态与糖调节受损和糖尿病风险之间的可能联系。
结果：本次研究总共检测2517名被试者，包括IGR306例、T2DM患者884例和NGT1327例。与正常糖耐量的对照组相比，T2DM患者血浆TAC水平（T2DM8.86±0.13U/ml vs. NGT9.57±0.17U/ml；P=0.001），CAT活性（CAT：T2DM32.04±1.04KU/L vs. NGT41.71±1.65KU/L，P <0.001），红细胞SOD活性（T2DM9997.75±164.01U/gHb vs. NGT11024.28±299.13U/gHb，P=0.002）GSH-Px的活性（T2DM129.97±2.83AU vs. NGT145.63±4.32AU，P=0.002）和GSH含量（T2DM18.94±0.29mg/gHbvs. NGT20.87±0.48mg/gHb，P=0.003）是明显降低地，而其血浆MDA浓度明显地增高（T2DM6.52±0.18nmol/L vs. NGT5.79±0.12nmol/L；P=0.001）。T2DM患者具有明显高地HOMA-IR值（T2DM4.26±0.13vs. NGT2.17±0.11；P <0.001），而HOMA-β值则显著下降（T2DM44.40±1.78vs. NGT103.37±5.01；P <0.001）。
结论：T2DM患者具有明显增加地血浆MDA浓度，然而血浆TAC和CAT活性、红细胞SOD活性、GSH-Px活性以及GSH含量出现明显地降低。这些结果表明，抗氧化能力下降和自由基产生明显增加与2型糖尿病有很强的关联性。
第二部分核因子E2相关因子2基因多态性与氧化应激的相关性研究
目的：探讨Nrf2rs6721961基因多态性与氧化应激之间的关系，检测该基因多态性与机体的抗氧化能力强度有无关联。
方法：使用TaqMan探针对Nrf2rs6721961位点进行基因分型。采用高铁还原法检测血浆中的TAOC水平，TBA比色法检测血浆MDA浓度，钼酸铵比色法检测血浆中的CAT活性，黄嘌呤-黄嘌呤氧化酶-亚硝酸盐方法检测红细胞SOD的活性，Beutler改良法测定红细胞的GSH含量，DNTB比色法来检测红细胞GSH-Px的活性。
结果：本部分病例对照研究共纳入被试者2477例，其中包括879例2型糖尿患者和1295例NGT正常对照人群。与CC基因型携带者相比，AA基因型携带者的血浆TAC水平（AA8.73±0.22vs. CC9.23±0.08U/ml，P=0.025），CAT活性（AA34.41±0.58vs. CC38.14±0.41KU/L，P=0.001），红细胞SOD活性（AA10446.01±118.61vs. CC10809.65±64.64U/gHb，P=0.042），GSH-Px活性（AA133.57±3.61vs. CC140.57±1.06AU，P=0.020），和GSH含量（AA19.49±0.31vs. CC20.10±0.10mg/gHb，P=0.042）也是明显降低的，而血浆中的丙二醛（MDA）的浓度（AA6.42±0.26vs. CC5.84±0.11nmol/ml，P=0.005）明显地增加。基因型AA携带者与基因型CC携带者相比较具有明显高的HOMA-IR值（AA2.90±0.19vs. CC2.36±0.06，P=0.003）和明显低地β细胞功能水平（AA71.37±4.05vs. CC84.37±1.59，P=0.005）。
结论：Nrf2rs6721961单核苷酸多态性的与氧化应激以及抗氧化状态密切相关。与基因型CC及基因型CA携带者相比，基因型AA携带者在血浆TAC和CAT活性，红细胞的SOD和GSH-Px活性以及红细胞GSH含量明显降低，而血浆丙二醛水平明显增高。
第三部分核因子E2相关因子2基因多态性与2型糖尿病和糖调节受损的风险关联研究
目的：探讨Nrf2rs6721961基因多态性与2型糖尿病和糖调节受损发病风险之间的关联关系。
方法：用GOD-POD法测定血浆FPG，ECLIA法测定FPI，利用稳态模式评估法进行评估HOMA-IR和HOMA-β cell。使用TaqMan探针对Nrf2rs6721961位点进行基因分型。
结果：本部分病例对照研究共纳入被试者2477例，其中包括879例2型糖尿患者，303例IGR和1295例NGT正常对照人群。Nrf2rs6721961基因多态性的等位基因的频率在2型糖尿病的病例和对照之间有显著性差异。与NGT组受试者（26.1％）相比较，A等位基因的频率（29.4％）在2型糖尿病的被试者明显地较高（P=0.019）。Nrf2rs6721961是与增加地患糖尿病的风险密切相关联。与CC基因型携带者相比较，携带AA基因型的个体有显着增加的发展2型糖尿病的风险（OR=1.77，95％CI1.26，2.49，P=0.011）。经校正年龄，性别，身体质量指数等混杂因素后，该关联仍具有统计学意义（OR=1.56，95％CI1.11，2.20，P=0.014）。与CC基因型携带者相比较，AA基因型携带者HOMA-β值显著降低(AA71.37±4.05vs. CC84.37±1.59, P=0.005)，而HOMA-IR值显著增加(AA2.90±0.19vs. CC2.36±0.06, P=0.003)。
结论： Nrf2基因rs6721961多态性与T2DM易感性存在明显关联。与CC基因型携带者相比较，AA基因型携带者患T2DM以及糖调节受损的风险明显增加。
Type2diabetes mellitus （T2DM） has been one of the biggest chronic diseases tothreaten the public health. Increases in the prevalence of diabetes have occurredinternationally, and it has been estimated that between1980and2008, the number of adultswith diabetes rose from153million to347million. Moreover, according to the estimationof the World Health Organization (WHO), there will be366million diabetic peopleworldwide by2030.
Research suggests that oxidative stress is a major risk factor in the onset andprogression of type2diabetes mellitus (T2DM). Some of the consequences of an oxidativeenvironment may be the development of impaired glucose tolerance, β-cell dysfunction,insulin resistance, and mitochondrial dysfunction, which can contribute ultimately to thediabetic disease state. Recent evidence also suggests that NF-E2related factor2(Nrf2) is apivotal transcription factor to the antioxidant response in oxidative stress related illnesses.
Nrf2is a member of the cap ‘n’ collar (CNC) subfamily of basic leucine zippertranscription factors. Nrf2has highly conserved domains named Nrf2-erythroid-derivedCNC homology protein homology (Neh) domains. Among them, Neh1domain is CNC andbasic leucine zipper domain, which interacts with partner proteins for heterodimerization.Neh3domain, located at the extreme end of the carboxyl terminus of Nrf2, is related toNrf2transactivation. Neh4and Neh5cooperatively bind with the cyclic adenosinemonophosphate response element binding protein-binding protein. In the absence of Nrf2structural data, it is not clear how Neh4and Neh5, together with the Neh3domains, exerttransactivation activity. The Neh2domain, which is located in the N-terminus of Nrf2, is a regulatory domain that responds to oxidative stress. Neh2mediates binding with Kelch-likeerythroid-derived protein with CNC homology-associated protein1(Keap1), andnegatively regulates Nrf2function.
Keap1was originally thought to be an actin-binding protein that represses the functionof Nrf2by simply sequestering Nrf2in the cytoplasm. Recently, Keap1has also beenidentified as an adaptor protein between Nrf2and Cullin3, a component of the E3ligasecomplex. Under normal conditions, Nrf2molecules may be subjected to continuousdegradation by the proteasome. When induced by oxidative stress derived fromaccumulation of reactive oxygen species (ROS) or reactive nitrogen species, single ormultiple reactive cysteine(s) in Keap1can be modified. This conformation change causesNrf2to dissociate from Keap1. Nrf2quickly cumulates in the nucleus and elicits theantioxidant response by transactivating the antioxidant response element (ARE) in thepromoter region of many antioxidant genes. The activation of Nrf2is an important clue forthe inducible expression of cytoprotective genes. The antioxidant enzymes encoded bythese genes may play important roles in scavenging oxygen free radicals.
In the promoter region of the Nrf2gene, the promoter polymorphisms were found in aJapanese population by Yamamoto et al.. Several studies demonstrated the associations ofthe Nrf2polymorphisms with oxidative stress related illnesses, such as acute lung injuryafter major trauma and vitiligo. However, there is little research evaluating the associationbetween Nrf2polymorphisms and oxidative stress and antioxidative status. The relationshipbetween this polymorphism with β-cell function, insulin sensisity, and the risk of T2DM islargely unknown.
Therefore, the objective of the present study was to evaluate the association betweenNrf2gene polymorphisms and the susceptibility to T2DM in a relatively large Chinesepopulation. We also evaluated the functional relevance of this polymorphism by measuringβ-cell function, insulin sensitivity, oxidative stress and antioxidative status among the studypopulations.
PartⅠ Oxidative stress and antioxidant status and type2diabetes mellitus
Objective: To investigate oxidative stress and antioxidative status in participants withand without diabetes
Methods: The measure of plasma TAC was based on the ability of antioxidants in the samples to change Fe3+-tripyridyltriazine to Fe2+-tripyridyltriazine, a stable blue productproportional to the TAC, which was tested at593nm. Plasma CAT activity was assayed bya method of Goth. MDA as an index of lipid peroxidation was estimated by using themethod described by Beuege and Aust. The activity of SOD in erythrocyte lysates wasevaluated on the basis of its ability to inhibit the oxidation of hydroxylamine, as describedpreviously. Erythrocyte GSH-Px activity was measured by the method described by Pagliaand Valentine. Erythrocyte GSH content was measured using the method described byBeutler et al. Descriptive statistics in the clinical and laboratory characteristics of healthycontrols and patients with T2DM were calculated for the study subjects. Differencesbetween diabetes cases and controls were tested by one-way analysis of variance, followedby Chi-square (categorical variables) or t test (continuous variables).
Results: When compared with subjects with NGT, patients with T2DM had asignificant increase in plasma MDA (P=0.001) and a significant decrease in plasma TAClevels (P=0.024) and CAT activity (P <0.001). Erythrocyte SOD activity (P=0.002),GSH-Px activity (P=0.002), and GSH content (P=0.040) were also significantly lower inpatients with T2DM.
Conclusion: The results also demonstrated that patients with T2DM had an increasedfree radical production and a reduced antioxidant capacity.
PartⅡ Association of the Nrf2rs6721961polymorphism with oxidative stressand antioxidative status
Objective: To investigate oxidative stress, and antioxidative status in relation togenotypes of rs6721961polymorphism in the study populations.
Methods: The genotyping of single nucleotide polymorphisms (SNPs) of the Nrf2gene was done by using an allelic discrimination assay-by-design TaqMan method onABI7900HT. The measure of plasma TAC was based on the ability of antioxidants in thesamples to change Fe3+-tripyridyltriazine to Fe2+-tripyridyltriazine, a stable blue productproportional to the TAC, which was tested at593nm. Plasma CAT activity was assayed bya method of Goth. MDA as an index of lipid peroxidation was estimated by using themethod described by Beuege and Aust. The activity of SOD in erythrocyte lysates wasevaluated on the basis of its ability to inhibit the oxidation of hydroxylamine, as describedpreviously. Erythrocyte GSH-Px activity was measured by the method described by Paglia and Valentine. Erythrocyte GSH content was measured using the method described byBeutler et al.
Results: Compared to individuals with the CC genotype, those with the AA genotypehad a significant decrease in plasma TAC levels (P=0.025), CAT activity (P=0.001),erythrocyte SOD activity (P=0.042), GSH-Px activity (P=0.020), and GSH content (P=0.042), whereas a significant increase in plasma MDA concentration (P=0.005).
Conclusion: Polymorphisms in the Nrf2genes were significantly associated withdecreased antioxidant activity and increased oxidative stress.
Part Ⅲ Association of Nrf2rs6721961polymorphism with risk of type2diabetes mellitus
Objective: To investigate the genotype and allele frequencies of the rs6721961polymorphism in the Nrf2gene in the study populations.
Methods: The genotyping of SNPs of the Nrf2gene was done by using an allelicdiscrimination assay-by-design TaqMan method on ABI7900HT. Differences in allelic andgenotypic frequencies of the gene polymorphisms in healthy controls and patients withT2DM were compared by Chi-square test, which was also used to evaluateHardy-Weinberg equilibrium for each individual locus. We used logistic regression analysisto assess the association of diabetes events with the specific polymorphism. Odds ratios and95%confidence intervals were adjusted for known risk factors for T2DM, including age,body mass index, sex, family history of diabetes, and hypertension.
Results: There were significant differences in the allelic frequency of the rs6721961polymorphism between T2DM cases and controls. The frequency of allele A wassignificantly higher in T2DM subjects (29.4%) compared to NGT subjects (26.1%)(P=0.019). The rs6721961was associated with increased risk of diabetes. The individualscarrying the AA genotype had a significantly higher risk for developing T2DM (OR1.77;95%CI1.26,2.49; P=0.011) relative to those with the CC genotype. After adjustment forage, sex, and body mass index, this association remained statistically significant (OR1.56;95%CI1.11,2.20; P=0.014). Individuals carrying the AA genotype had a lower HOMA-β(AA71.37±4.05vs. CC84.37±1.59, P=0.005) and a higher HOMA-IR values thanthose with the CC genotype (AA2.90±0.19vs. CC2.36±0.06, P=0.003).
Conclusion: The rs6721961polymorphism of the Nrf2gene was associated with an increased risk of T2DM. The polymorphisms were also associated with impaired β-cellfunction and increasing insulin resistance.
引文
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