HIF-1α介导线粒体功能调控糖尿病心肌缺血再灌注损伤的作用机制研究

目的研究缺氧诱导因子1α(HIF-1α)对高糖作用下缺血再灌注(IR)损伤的心肌细胞凋亡和氧化应激,及其信号通路下游因子的调控作用。方法二甲基乙二酰基甘氨酸激活HIF-1α对高糖作用下IR心肌细胞损伤的干预。实验分四组:对照组(细胞常规培养)、高糖+缺氧复氧组(25 mmol/L葡萄糖的DMEM培养基培养24 h,缺氧2 h,复氧8 h)、HIF-1α处理组(高糖,缺氧加复氧,缺氧前3 h用100μmol/L的二甲基乙二酰基甘氨酸处理)、二甲基亚砜(DMSO)对照组(高糖,缺氧加复氧,缺氧前3 h用100μmol/L的DMSO处理)。流式细胞术测定细胞凋亡率,CCK-8法分析细胞活性; ELISA法检测细胞培养上清液中超氧化物歧化酶活性和丙二醛的表达; ATP含量试剂盒检测各组细胞ATP含量; Western Blot法检测过氧化物酶体增殖物激活受体γ辅助激活因子1α(PGC-1α)、核呼吸因子1,以及能量代谢相关的p-AMPK、AMPK蛋白表达,HIF-1α核蛋白表达。结果高糖合并IR损伤增加心肌细胞的凋亡(P<0.01),降低心肌细胞活力(P<0.01),HIF-1α表...     

软脂酸对HIT—T15细胞线粒体形态功能和胰岛素分泌的影响

目的观察软脂酸（PA）对HIT-T15细胞凋亡、线粒体结构和功能及胰岛素分泌的影响并探讨可能的机制。方法试验分对照组、0．5 mmol／LPA和1．0 mmol／LPA组,透射电镜观察细胞及线粒体形态,流式细胞仪（FC）和原位末端标记法（TUNEL）检测凋亡率,高效液相色谱法（HPLC）检测细胞ATP／ADP,RT—PCR检测过氧化物酶体增殖物激活受体γ共激活因子1（PGC-1）和核呼吸因子1（NRF-1）mRNA,放免法测基础和葡萄糖刺激后胰岛素分泌（GSIS）。结果PA能使线粒体肿胀、嵴破坏;细胞的凋亡率增加,且FC检测高浓度PA组凋亡率增加更显著;ATP／ADP比率下降;PGC-1mRNA和NRF-1mRNA表达增加,高浓度PA组增加更显著;GSIS下降（P均〈0．05）。结论PA导致HIT-T15细胞的线粒体结构和功能的损害及GSIS下降,可能与PGC-1和NRF-1的调节作用有关。     

线粒体在2型糖尿病β细胞功能障碍中的作用

胰岛β细胞线粒体对β细胞胰岛素分泌功能起着十分重要的作用,线粒体呼吸链氧化磷酸化产生ATP,与胰岛素分泌相联系。其氧化磷酸化功能受线粒体DNA和核DNA的调控,且二者通过一些相关的因子联系起来,使线粒体呼吸链亚基在转录水平协同表达。其中核呼吸因子、过氧化物酶体增殖物激活受体γ共激活因子和解偶联蛋白2等起着重要作用。     

过氧化物酶体增殖物激活受体γ及其共激活子1α与高血压的关系

过氧化物酶体增殖物激活受体γ及其共激活子1α的联合变异与高血压、2型糖尿病、肥胖、脂代谢异常及动脉粥样硬化性疾病的关系,是当今研究的热点之一,本文主要介绍其基因的单核苷酸多态性与高血压的关系。     

过氧化物酶体增殖物激活受体γ及其共激活子1α与高血压的关系

过氧化物酶体增殖物激活受体γ及其共激活子1 α的联合变异与高血压、2型糖尿病、肥胖、脂代谢异常及动脉粥样硬化性疾病的关系,是当今研究的热点之一,本文主要介绍其基因的单核苷酸多态性与高血压的关系.     

PGC-1α对高糖状态下血管内皮细胞线粒体生物合成的调节作用

目的探讨过氧化物酶增殖体激活受体γ辅激活因子1α（PGC-1α）对高糖状态下血管内皮细胞线粒体生物合成的影响。方法体外原代培养人脐静脉内皮细胞,分别放置于正常糖浓度（对照组）和高糖浓度（高糖组）培养液中。用含有编码PGC-1α蛋白基因的腺病毒表达载体及空壳载体分别感染上述两组细胞,采用Southern印记法以细胞色素b DNA为探针检测线粒体DNA拷贝数。结果与对照组相比,高糖组线粒体DNA拷贝数显著下降;通过质粒转染过度表达PGC-1α导致细胞总DNA中细胞色素b DNA升高,线粒体生物合成增加。结论内皮细胞中过量表达的PGC-1α可以显著促进线粒体生物合成,这一作用在高糖培养的内皮细胞中更加明显。     

过氧化酶体增殖物激活型受体γ对单核或巨噬细胞中抑制蛋白及基质金属蛋白酶-1表达的调控

目的 :研究过氧化酶体增殖物激活型受体 (PPAR)γ对人类单核或巨噬细胞核因子抑制蛋白 (IkBα)和基质金属蛋白酶 1 (MMP 1 )表达的调控影响 ,探讨PPARγ在抑制炎症反应和稳定动脉粥样斑块中的作用。方法 :PPARγ配体 ( 1 5 脱氧前列腺素J2和环格列酮 )干预体外培养的U93 7细胞 ,用四唑盐 (MTT)法检测细胞活力 ,细胞免疫组化法和流式细胞法检测细胞IkBα和MMP1的表达程度。结果 :PPARγ配体在低浓度时不会影响细胞活力 (存活率 >80 % ) ;在 1、3、2 4h,PPARγ激活后均能增加IkBα在细胞内的表达 ,但是随着时间推移 ,这种作用逐渐减弱 ,与佛波醇肉豆蔻酸乙酸酯阳性组相比 ,PPARγ配体组MMP1的表达水平下降 (P <0 .0 1 )。结论 :PPARγ可能通过快速增加IkBα的表达而抑制核因子(NF κB)的活性 ,同时PPARγ可通过或不通过NF κB而抑制MMP1的表达 ,从而发挥抑制炎症和增强斑块稳定作用     

PGC-1α在肺癌中的研究进展

肺癌是目前世界上最常见的恶性肿瘤,过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)是线粒体能量代谢的关键调节因子,在肿瘤的发生发展过程中起着至关重要的作用,PGC-1α合成代谢和氧化代谢可以影响肿瘤细胞的增殖、迁移和存活。本文主要就PGC-1α的基本特性及其代谢的作用机制进行综述,并阐述了PGC-1α在肺癌中...     

PGC-1α对高糖诱导的血管内皮细胞内ROS生成的影响

目的探讨过氧化物酶体增殖物激活受体辅激活因子1α（PGC-1α）对高糖诱导的血管内皮细胞内活性氧簇（ROS）生成的影响。方法将人脐带静脉内皮细胞（HUVECs）分成对照组、空病毒Ad组、d-PGC-1α感染组、PGC-1αsiRNA转染组,分别将四组细胞置于正常糖及高糖环境中进行培养。用活性氧检测试剂盒处理标本,以酶标仪检测细胞内荧光强度,显示细胞内ROS浓度。结果高糖组HUVECs内ROS浓度明显高于正常糖组（P〈0.01）;与对照组及空病毒Ad组比较,d-PGC-1α感染组ROS浓度明显降低,PGC-1αsiRNA转染组明显升高（P均〈0.01）。结论与正常糖组比较,高糖组HUVECs内ROS浓度明显升高;过度表达PGC-1α蛋白后,HUVECs内ROS浓度明显降低;抑制细胞内PGC-1α蛋白表达,则HUVECs内ROS浓度明显升高。提示PGC-1α可作为防治糖尿病血管并发症的新靶点之一。     

过氧化物酶体增殖物激活受体信号通路在大鼠高脂血症发生发展中的作用

目的 探讨过氧化物酶体增殖物激活受体(PPAR) γ/Caspase-8/Caspase-3信号通路在大鼠高脂血症发生发展中的作用.方法 将健康雄性SD大鼠60只〔4周龄,体质量(110±10)g〕随机分为正常对照组、高脂饮食组、叶酸组、维生索B12组、叶酸+维生素B12组.适应性喂养1周后,叶酸组、维生索B12组、叶酸+维生素B12组分别腹腔注射叶酸(0.5 mg/d)、维生素B12 (0.05 mg/d)、叶酸(0.5mg/d)+维生素B12(0.05 mg/d),同时给予高脂饲料喂养;对照组腹腔注射0.9％NaCl溶液(0.5 ml/d)同时给予正常饲料喂养;高脂饮食组给予高脂饲料喂养.第17周末取腹主动脉利用反转录-聚合酶链反应检测各组PPARγ、Caspase-8和Caspase-3 mRNA的表达.结果 叶酸组、叶酸+维生素B12组腹主动脉的PPARγ mRNA水平高于高脂饮食组;Caspase-8、Caspase-3mRNA水平低于高脂饮食组(P＜0.05),且叶酸+维生素B12组较叶酸组降低mRNA水平更明显(P＜0.05).结论 叶酸与维生素B12可以改善血管壁PPARγ、Caspase-8和Caspase-3 mRNA的水平从而防止高脂血症对血管内皮的损伤.     

Honeybees and cell lines as models of DNA methylation and aging in response to diet

DNA methylation patterns change as individuals grow older, and DNA methylation appears susceptible to modification by the diet. Thus DNA methylation may be a mechanism through which diet can affect aging and longevity. We propose that effects on DNA methylation also contribute to the extension in lifespan observed in response to dietary restriction. Relationships between diet-induced changes in DNA methylation and parallel effects on aging and/or lifespan could, of course, be purely associative. Proof of these ideas requires experimental model systems in which it is possible to manipulate genome methylation status and to measure effects on aging and/or lifespan. Commonly-used short-lived and genetically-malleable metazoan species, such as Caenorhabditis elegans and Drosophila, are not suitable for such studies; the C. elegans genome is not methylated, and DNA methylation in Drosophila is dissimilar from mammalian DNA methylation, occurring at cytosines at sites other than in CpG sequences. The honeybee provides a potentially unique and tractable model for such studies. Female larval development into the long-lived queen phenotype or short-lived worker is determined purely by diet (royal jelly) through an effect on DNA methylation, and honeybee DNA methylation mirrors that of the mammalian genome. Mammalian cell lines and biochemical approaches offer complementary tools to address specific components of hypotheses relating to effects of diet on aging through DNA methylation in a more targeted manner. Our studies using mammalian cell lines are revealing effects of Sirt1 on DNA methylation, and indicate that Sirt1 and resveratrol affect the expression of different sets of genes.     

Induction of vascular endothelial growth factor and matrix metalloproteinase-3 (stromelysin) by interleukin-1 in human articular chondrocytes and synoviocytes

We investigated the effects of interleukin (IL)-1 on vascular endothelial growth factor (VEGF) and matrix metalloproteinase-3 (MMP-3) (stromelysin) secretion from chondrocytes and synoviocytes in different clinical conditions. Specifically, cells obtained from osteoarthritic (OA) (n=7), rheumatoid arthritic (RA) (n=5), and post-traumatic (PT) (n=5) patients were stimulated in vitro with IL-1 in the presence or absence of an IL-1 receptor antagonist (IL-1ra) (anakinra). Levels of secreted MMP-3 and VEGF were measured by enzyme-linked immunosorbent assay. The VEGF mRNA expression was analyzed quantitatively. Interleukin-1 induced both VEGF and MMP-3 secretion from all of the samples tested, and VEGF mRNA expression was also upregulated. Interleukin-1ra significantly suppressed the enhancing effect of IL-1 on MMP-3 and VEGF in both cell types. In conclusion, IL-1 simultaneously induces MMP-3 and VEGF production from chondrocytes and synoviocytes in inflammatory, degenerative, and post-traumatic joints. Therefore, IL-1ra might be beneficial for protection from VEGF-mediated alterations of cartilage metabolism in pathologic and physiologic conditions.     

罗格列酮对内皮细胞单核细胞趋化蛋白-1和血管细胞黏附分子-1表达的影响

目的观察罗格列酮(RGZ)对高糖及C反应蛋白(CRP)诱导的人脐静脉内皮细胞(HUVECs)的单核细胞趋化蛋白-1(MCP-1)及血管细胞黏附分子-1(VCAM-1)mRNA和蛋白表达的影响。方法体外培养HUVECs,细胞传至5代,随机分为7组,正常对照组(C组),高糖组(HG组),高糖+CRP组(HGC组),CRP组,高糖+RGZ组(HGR组),高糖+CRP+RGZ组(HGCR组),CRP+RGZ组(CRPR组)。采用RGZ 5.0μmol/L干预HUVECs24 h,RT-PCR、Western blot法分别检测干预前后MCP-1、VCAM-1 mRNA和蛋白的表达水平。结果HG组、HGC组、CRP组HUVECs中MCP-1、VCAM-1的mRNA和蛋白水平较C组显著升高(P<0.01);RGZ干预后,HGR组、HGCR组、CRPR组分别较HG组、HGC组、CRP组MCP-1、VCAM-1的mRNA和蛋白水平显著降低(P<0.01)。结论RGZ通过降低HUVECs中MCP-1、VCAM-1的表达,延缓糖尿病动脉粥样硬化的进程。     

Arginine bioavailability and endothelin-1 system in the regulation of vascular function of umbilical vein endothelial cells from intrauterine growth restricted newborns

Background and aims

Intrauterine growth restriction (IUGR) is a major risk factor for perinatal morbidity and mortality, leading to long-term adverse cardiovascular outcomes. The present study aimed to investigate the potential mechanisms in IUGR-associated vascular endothelial dysfunction.

Relationship between protective effects of rosiglitazone on endothelium and endogenous nitric oxide synthase inhibitor in streptozotocin-induced diabetic rats and cultured endothelial cells

BACKGROUND: Previous investigations have indicated that the level of asymmetric dimethylarginine (ADMA) is increased in diabetic patients and animals, and rosiglitazone has a protective effect on the endothelium. In the present study, we tested the relationship between protective effects of rosiglitazone and ADMA in streptozotocin (STZ)-induced diabetic rats and cultured endothelial cells. METHODS: Blood samples were collected from carotid artery. Vasodilator responses to acetylcholine (ACh) in the isolated aortic rings were measured, and serum concentrations of glucose, lipid, nitrite/nitrate, ADMA and tumour necrosis factor-alpha (TNF-alpha) were determined. Cultured endothelial cells were treated with ADMA, and the concentrations of intercellular adhesion molecule (ICAM-1), TNF-alpha, and the activity of nuclear factor-kappaB (NF-kappaB) were determined. RESULTS: Vasodilator responses to ACh were decreased markedly and the serum concentrations of TNF-alpha, nitrite/nitrate and ADMA were increased significantly in diabetic rats. Rosiglitazone (3, 10 or 30 mg/kg) produced a significant reduction of the inhibition of vasodilator responses to ACh, but had no effect on the serum concentrations of glucose, lipid, nitrite/nitrate and ADMA in diabetic rats. ADMA (30 microM) significantly increased the activity of NF-kappaB and elevated the levels of ICAM-1 and TNF-alpha, and pre-treatment with rosiglitazone (10 or 30 microM) markedly inhibited the increased activity of NF-kappaB and reduced the elevated levels of TNF-alpha and ICAM-1 induced by ADMA in cultured endothelial cells. CONCLUSIONS: Rosiglitazone improves endothelial function in diabetic rats, which is related to the reduction of the inflammatory response induced by ADMA.     

丝裂原激活蛋白激酶磷酸酶-1和p38在内膜损伤后血管平滑肌细胞表型转化过程中的表达变化

目的:观察动脉内膜损伤后血管平滑肌细胞(VSMC)表型转化和p38MAPK及丝裂原激活蛋白激酶磷酸酶-1(MKP-1)表达的动态变化。方法:分别用免疫组化、免疫印迹(Westernblot)和逆转录-聚合酶链反应方法检测假损伤组(S组)和损伤组损伤后不同时间点血管壁中增殖细胞核抗原(PCNA)、平滑肌α肌动蛋白(SMα-actin)、p38蛋白和MKP-1mRNA及蛋白表达的变化。结果:①S组中膜VSMC及内皮细胞PCNA为阴性表达;中膜于损伤后1~14d,新生内膜(NI)于5~14d阳性细胞率逐渐增加,28d后开始逐渐减少,NI阳性率高于中膜。②S组中膜SMα-actin表达为阳性,内皮为阴性;中膜阳性表达于损伤后1d开始减少,3d最为明显,5d后开始逐渐增加,NI阳性表达弱于中膜。③S组中膜p38呈阴性或弱阳性;损伤后1~35d呈持续高表达,NI阳性表达强于中膜。p38与PCNA表达变化呈正相关。④S组中膜MKP-1呈弱阳性或阳性表达;损伤后1d即开始下降,14~28d稍有回升,至35d仍未回到S组水平,NI阳性表达稍弱于中膜。MKP-1与PCNA表达变化呈负相关。结论:VSMC增殖能力与其表型转化密切相关,p38MAPK和MKP-1参与了损伤后VSMC表型转化的信号转导及其调节。     

Effects of a nonnutritive sweetener on body adiposity and energy metabolism in mice with diet-induced obesity

Objective

Nonnutritive sweeteners (NNSs) have been studied in terms of their potential roles in type 2 diabetes, obesity, and related metabolic disorders. Several studies have suggested that NNSs have several specific effects on metabolism such as reduced postprandial hyperglycemia and insulin resistance. However, the detailed effects of NNSs on body adiposity and energy metabolism have not been fully elucidated. We investigated the effects of an NNS on energy metabolism in mice with diet-induced obesity (DIO).

Methods

DIO mice were divided into NNS-administered (4% NNS in drinking water), sucrose-administered (33% sucrose in drinking water), and control (normal water) groups. After supplementation for 4 weeks, metabolic parameters, including uncoupling protein (UCP) levels and energy expenditure, were assessed.

Results

Sucrose supplementation increased hyperglycemia, body adiposity, and body weight compared to the NNS-administered and control groups (P < 0.05 for each). In addition, NNS supplementation decreased hyperglycemia compared to the sucrose-administered group (P < 0.05). Interestingly, NNS supplementation increased body adiposity, which was accompanied by hyperinsulinemia, compared to controls (P < 0.05 for each). NNS also increased leptin levels in white adipose tissue and triglyceride levels in tissues compared to controls (P < 0.05 for each). Notably, compared to controls, NNS supplementation decreased the UCP1 level in brown adipose tissue and decreased O₂ consumption in the dark phase.

Conclusions

NNSs may be good sugar substitutes for people with hyperglycemia, but appear to influence energy metabolism in DIO mice.     

Gly482Ser polymorphism of the peroxisome proliferator-activated receptor-gamma coactivator-1 gene might be a risk factor for diabetic retinopathy in Slovene population (Caucasians) with type 2 diabetes and the Pro12Ala polymorphism of the PPARgamma gene is not

BACKGROUND: The peroxisome proliferator-activated receptor-gamma (PPARgamma) gene has been recently associated with type 2 diabetes, obesity and traits depending on VEGF expression (e.g. retinopathy). The PPARgamma gene and its coactivator, the peroxisome proliferator-activated receptor-gamma coactivator-1 (PPARGC1) gene, have been implicated to be involved in glucose uptake and altered lipid oxidation. We therefore hypothesized that the Gly482Ser polymorphism of the PPARGC1 gene and Pro12Ala polymorphism of the PPARgamma gene might confer susceptibility to diabetic retinopathy in type 2 diabetes. The aim of this study was to investigate the association between the Pro12Ala polymorphism in the PPARgamma gene and Gly482Ser polymorphism in the PPARGC1 gene and the development of diabetic retinopathy in the Slovene population (Caucasians) with type 2 diabetes. METHODS: One hundred and sixty subjects with type 2 diabetes and diabetic retinopathy were compared with 101 diabetic subjects without diabetic retinopathy. Chi-square test was used to compare discrete variables, and continuous clinical data were compared by unpaired students t - test. RESULTS: A significantly higher frequency of the AA genotype of the Gly482Ser polymorphism of the PPARGC1 gene was found in the patients with diabetic retinopathy compared to the patients without diabetic retinopathy (14.4% vs 5.9%; p = 0.035), whereas the Pro12Ala polymorphism of the PPARgamma gene failed to yield an association with diabetic retinopathy. CONCLUSIONS: The present study demonstrates that the AA genotype of the Gly482Ser polymorphism in the PPARGC1 gene might be a risk factor for diabetic retinopathy in the Slovene population (Caucasians) with type 2 diabetes (odds ratio 2.7, 95% confidence interval 1.0-6.8), whereas the Pro12Ala polymorphism of the PPARgamma gene failed to confer susceptibility to diabetic retinopathy.