糖基化终产物对U937巨噬细胞高密度脂蛋白受体蛋白表达的影响

目的　研究诱导分化及糖基化终产物 (AGEs)对人单核 /巨噬细胞 (U937细胞 )高密度脂蛋白受体 (SR BI)蛋白表达的影响 ,探讨AGEs和巨噬细胞SR BI在动脉粥样硬化中的作用。方法　U937细胞经PMA诱导分化 ,并将不同浓度或同一浓度AGEs与诱导分化 48h后的U937细胞共同孵育 ,用免疫细胞化学法和Western印迹法检测SR BI蛋白的表达。结果　诱导分化后U937细胞SR BI表达在 2 4、48h逐渐升高 ,72h下降 ;1 0 0、2 0 0和 40 0 μg/mlAGEs刺激后细胞表面SR BI蛋白表达量分别是BSA组的 1 44、2 38和 2 77倍 (P <0 0 5) ;40 0 μg/ml的AGEs作用 6、1 2、2 4、48h后 ,U937巨噬细胞SR BI蛋白表达量分别为 0h的 1 38、2 49、3 76和 4 2 5倍 (P <0 0 5)。结论　AGEs可增加U937巨噬细胞SR BI蛋白表达且呈浓度和时间依赖性。     

Ligation of RAGE with ligand S100B attenuates ABCA1 expression in monocytes

Hypothesis

ATP Binding Cassette Transporter (ABC) A1 is one of the key regulators of HDL synthesis and reverse cholesterol transport. Activation of Receptors for Advanced Glycation End products (RAGE) is involved in the pathogenesis of diabetes, and its complications. The aim of the present study is to examine the effect of RAGE ligand S100B on ABCA1 expression.

Methods

S100B mediated regulation of LXR target genes like ABCA1, ABCG1, ABCG8, LXR-α and LXR-β in THP-1 cells was analyzed by real-time PCR, RT-PCR and western blots. ABCA1 mRNA expression in monocytes from diabetic patients was studied. Effect of LXR ligand on S100B induced changes in LXR target genes was also studied. Luciferase reporter assay was used for S100B induced ABCA1 promoter regulation.

Results

S100B treatment resulted in a significant 2–3 fold reduction (p < 0.01) in ABCA1 and ABCG1 mRNA in dose and time dependent manner in THP1 cells. ABCA1 protein level was also significantly (p < 0.01) reduced. S100B-induced reduction on ABCA1 mRNA expression was blocked by treating THP-1 cell with anti-RAGE antibody. Reduced ABCA1 mRNA levels seen in peripheral blood monocytes from diabetes patients showed the in-vivo relevance of our in-vitro results. Effect of S100B on ABCA1 and ABCG1 expression was reversed by LXR ligand treatment. S100B treatment showed significant 2 fold (p < 0.01) decrease in T1317 induced ABCA1 promoter activation.

Conclusions

These results show for the first time that ligation of RAGE with S100B can attenuate the expression of ABCA1 and ABCG1 through the LXRs. This could reduce ApoA-I-mediated cholesterol efflux from monocytes.     

Advanced glycation end‐products are a risk for muscle weakness in Japanese patients with type 1 diabetes

Accumulation of advanced glycation end‐products (AGEs) is thought to contribute to muscle weakness in a diabetic animal model. Skin autofluorescence is a proposed marker for accumulation of AGEs in the skin. We aimed to investigate the relationship between AGEs accumulation, sarcopenia and muscle function of Japanese patients with type 1 diabetes. A total of 36 patients with type 1 diabetes participated in the present cross‐sectional study. Sarcopenia parameters (skeletal muscle mass index and knee extension strength) were compared with subcutaneous AGEs accumulation using skin autofluorescence. The prevalence of sarcopenia and impaired knee extension strength was 16.6% (men 0.0%, women 22.2%) and 47.2% (men 22.2%, women 55.6%), respectively. Knee extension strength was negatively correlated with skin autofluorescence (r² = 0.14, P < 0.05), but not with skeletal muscle mass index. In conclusion, the AGEs accumulation might be one of the reasons of impaired lower limb muscle function in Japanese patients with type 1 diabetes.     

糖基化终末产物对ECV-304细胞ICAM-1及VCAM-1表达的影响

目的探讨糖基化终产物(AGEs)与其受体(RAGE)对ECV-304细胞上血管细胞黏附分子-(1ICAM-1)和细胞间黏附分子-(1VCAM-1)的影响,为研究AGEs在动脉粥样硬化发生发展中的作用提供实验依据。方法采用体外制备的糖基化终产物——糖基化的牛血清白蛋白(AGE-BSA)和反义RAGE寡核苷酸处理ECV-304细胞,通过流式细胞仪技术、Western印迹技术检测ECV-304细胞上ICAM-1、VCAM-1蛋白质的表达。结果AGEs处理ECV-304细胞后,可诱导ECV-304细胞上VCAM-1和ICAM-1的表达增加,且这种效应呈剂量依赖性。采用反义技术阻断RAGE的mRNA水平的翻译后,ECV-304细胞ICAM-1和VCAM-1的表达呈剂量依赖性降低。结论AGEs可通过与其膜受体RAGE的结合激活血管内皮细胞VCAM-1和ICAM-1的表达,这可能是AGEs促动脉粥样硬化发生发展的机制之一。     

8-溴-环磷酸腺苷对单核细胞株THP1中ABCA1及细胞间黏附分子的影响

目的 观察8-Br-cAMP刺激下,单核细胞株THP1中ABCA1、细胞间黏附分子-1(ICAM-1)及 白介素-1β(IL-1β)mRNA和蛋白质表达的变化,阐明ABCA1基因在动脉粥样硬化(AS)形成中的可能机制.方法 复苏培养THP1单核细胞,加入8-Br-cAMP(0.5 mmol/L)刺激3、6、12、24 h,设未加刺激同时孵育24 h的细胞为阴性对照组;以荧光定量RT-PCR和Western印迹法及ELISA法检测ABCA1、ICAM-1及IL-1β mRNA和蛋白质表达量;用ABCA1的反义寡核苷酸(100 nmol/L)转染THP1细胞,给予8-Br-cAMP刺激,同样的方法观察上述指标的改变.结果 予8-Br-cAMP刺激后,THP1细胞中ABCA1、ICAM-1 mRNA和蛋白质及IL-1β蛋白质表达均增高,给予反义寡核苷酸转染后氧化低密度脂蛋白(Ox-LDL)刺激3、6 h上述指标的mRNA表达降低 (P<0.01),12、24 h蛋白质表达降低 (P<0.01).结论 THP1单核细胞中,8-Br-cAMP激活ABCA1不仅可增加细胞内胆固醇外运,还具有增加炎症因子表达的作用,在AS的发生中发挥双重作用.     

ATP结合盒转运蛋白A1与动脉粥样硬化

对Tangier病病因的研究,发现ATP结合盒转运蛋白A1(ATP binding cassette transport protein A1,ABCA1)在胆固醇逆向转运(reverse cholesterol transport,RCT)中起重要作用。ABCA1主要通过核受体PPARs途径发挥作用。ABCA1基因变异影响其功能。     

Role of advanced glycation end products in cardiovascular disease

Advanced glycation end products(AGEs) are produced through the non enzymatic glycation and oxidation of proteins,lipids and nucleic acids.Enhanced formation of AGEs occurs particularly in conditions associated with hyperglycaemia such as diabetes mellitus(DM).AGEs are believed to have a key role in the development and progression of cardiovascular disease in patients with DM through the modif ication of the structure,function and mechanical properties of tissues through crosslinking intracellular as well as extracellular matrix proteins and through modulating cellular processes through binding to cell surface receptors [receptor for AGEs(RAGE)].A number of studies have shown a correlation between serum AGE levels and the development and severity of heart failure(HF).Moreover,some studies have suggested that therapies targeted against AGEs may have therapeutic potential in patients with HF.The purpose of this review is to discuss the role of AGEs in cardiovascular disease and in particular in heart failure,focussing on both cellular mechanisms of action as well as highlighting how targeting AGEs may represent a novel therapeutic strategy in the treatment of HF.     

NO/PKG对THP-1巨噬细胞ABCA1基因mRNA表达和胆固醇含量的影响

目的:探讨THP-1巨噬细胞泡沫化过程中NO/PKG的变化,以及NO/PKG对THP-1巨噬细胞ATP结合盒转运体A1(ABCA1)基因mRNA表达和胆固醇含量的影响。方法:THP-1细胞诱导分化为巨噬细胞,用氧化低密度脂蛋白(ox-LDL)处理,油红O染色观察巨噬细胞脂滴,酶标仪测定一氧化氮(NO)的释放;用一氧化氮供体L-精氨酸和硝普钠(SNP)、乙二醇二乙醚二胺四乙酸(EGTA)、PKG激动剂处理巨噬细胞,RT-PCR法测定细胞ABCA1基因mRNA的表达,高效液相色谱法(HPLC)测定细胞内胆固醇的含量变化。结果:ox-LDL可导致THP-1巨噬细胞脂质的蓄积和NO释放的增加,实验组NO释放量较对照组显著升高(P<0.05),实验组中50mg/L组、100mg/L组、200mg/L组与25mg/L组相比显著升高(P<0.05)。用PKG激动剂处理巨噬细胞后促进ABCA1基因mRNA表达上调(P<0.05);用PKG激动剂、L-精氨酸、SNP处理后的巨噬细胞内胆固醇较对照组显著减少(P<0.05)。EGTA能显著增加细胞内胆固醇含量(P<0.05)。结论:NO/PKG能降低细胞内胆固醇,其机制可能是PK...     

体外制备的晚期糖基化终产物对内皮祖细胞数量的影响

目的观察体外制备的晚期糖基化终产物对内皮祖细胞数量的影响。方法人血清白蛋白与葡萄糖共同孵育90d后,行Bradford检测法蛋白定量和荧光值测定。从正常成人外周血中分离提取单个核细胞,培养7d后,流式细胞仪鉴定细胞表型,分别加入浓度为2mg/L、20mg/L和200mg/L的晚期糖基化终产物并设置对照组,干预不同的时间(0h、24h、48h和72h),200倍光学显微镜下随机取10个视野计数。结果该方法制备的晚期糖基化终产物具有荧光性。细胞培养7d,CD34/CD133/KDR单克隆抗体流式细胞仪鉴定细胞,结果发现表达KDR达78.60%±2.20%、CD34为8.60%±2.00%和CD1332.80%±0.60%,提示大部分细胞为内皮祖细胞。以不同时间(0h、24h、48h和72h)和浓度(2mg/L、20mg/L和200mg/L)晚期糖基化终产物干预内皮祖细胞生长,对比0h组每个视野下细胞数量(186.0±6.7),干预24h细胞数量减少(146.67±4.98),72h降至最低(73.67±3.76)(P<0.001),呈现明显的时间效应(r=0.9658,P<0.01);终浓度为2mg/L或20mg/L晚期糖基化终产物干预内皮祖细胞72h后,与同时培养72h的对照组无显著差异(P>0.05),但随着浓度加大,浓度效明显(r=0.9988,P<0.01)。结论此方法制备的晚期糖基化终产物符合文献所述的特性,晚期糖基化终产物能抑制内皮祖细胞生长,具有时间效应和浓度效应。     

斑块内泡沫细胞外迁功能障碍的研究进展

泡沫细胞的形成及外迁受抑是动脉壁斑块恶性重塑的关键环节,糖基化终末产物(AGE)则是糖尿病加速动脉粥样硬化进展的重要推手。基于国内外最新进展及本课题组已有成果,文章阐述了清道夫受体CD36在介导AGE关键活性成分羧甲基赖氨酸抑制泡沫细胞外迁中的作用及机制,希冀为今后靶向泡沫细胞外迁机制的治疗策略提供新的切入点。     

Advanced glycation end products depress function of endothelial progenitor cells via p38 and ERK 1/2 mitogen-activated protein kinase pathways

Objective  Advanced glycation end products (AGEs) and endothelial progenitor cells (EPCs) play divergent roles in the process of atherosclerosis. We investigated the effects of AGE-human serum albumin (AGE-HSA) on receptor expression for AGEs (RAGE) and EPCs apoptosis. Methods  The human mononuclear cells were obtained by Ficoll density gradient centrifugation and cultured in M199 medium containing rh-VEGF (30 ng/ml), rh-b-FGF(6 ng/ml) and 20% NBCS for 8 days. The adhesive EPCs were sequentially harvested after 24 h synchronization and challenged with AGE-HSA (concentration range from 0 to 300 μg/ml) for 24 h and 200 μg/ml AGE-HSA (time range from 0 to 36 h). EPCs apoptosis and migration were determined, expressions of RAGE, phosphorylated ERK1/2, JNK and p38 mitogen-activated protein kinase (MAPK) of EPCs were quantified by fluorescent quantitation RT-PCR and Western-blot, effect of AGE-HSA on NF-κB activtiy was determined by EMSA (electrophoretic mobility shift assay) in the presence and absence of special MAPK pathways pathway inhibitors. Results  AGE-HSA upregulated the expression of RAGE, this effect could be significantly inhibited by p38 MAPK and ERK MAPK inhibitor, but not by JNK MAPK inhibitor. AGE-HSA also promoted EPCs apoptosis and inhibited EPCs migration and increased NF-κB activity, these effects could be significantly attenuated by the anti-RAGE neutralizing antibody as well as by p38 and ERK MAPK inhibitors. Conclusion  AGE-HSA could promote atherosclerosis by upregulating EPCs RAGE expressions and promoting EPCs apoptosis via p38, ERK MAPK pathways, activation of NF-κB might also play a role in this process. C. Sun and C. Liang contributed equally to this work. Returned for 1. Revision: 13 December 2007 1. Revision received: 20 February 2008 Returned for 2. Revision: 7 March 2008 2. Revision received: 9 June 2008     

The advanced glycation end product pentosidine correlates to IL-6 and other relevant inflammatory markers in rheumatoid arthritis

Objective Oxidative stress and inflammatory processes accelerate the formation of advanced glycation end products (AGE), e.g. of pentosidine. The aim of this study was to investigate the relationships between levels of pentosidine in serum and synovial fluid, proinflammatory cytokines, other markers of inflammatory activity, and the state of radiologically visible bone destruction in patients with rheumatoid arthritis (RA).Objectives One hundred thirty-three nondiabetic RA patients and 56 age-matched, healthy subjects were included. Serum and synovial fluid pentosidine, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and rheumatoid factor levels were determined. In 30 patients, the proinflammatory cytokines interleukin (IL)-1, IL-6, and TNF- and the soluble receptors sIL-2R, sIL-6R, sTNF-, and RI/RII were also measured.Results Serum levels of pentosidine were on average significantly higher in RA patients than in healthy subjects and correlated significantly to ESR, CRP, and serum levels of IL-6. Serum and synovial fluid pentosidine did not show any differences. Rheumatoid factor-positive RA patients had higher pentosidine levels in the synovial fluid than rheumatoid factor-negative patients. Correlations could not be found between pentosidine and the other cytokines or cytokine receptors measured.Conclusion The binding of AGE on cell receptors induces activation of nuclear factor kappa B, resulting in enhanced synthesis of proinflammatory cytokines. Moreover, AGE generation may also lead to the formation of new, immunologically relevant epitopes at synovial proteins. Both mechanisms could contribute to initiation and perpetuation of the inflammatory and destructive processes in RA.     

血红素加氧酶1在高糖和糖基化终产物致人单核细胞氧化应激中的作用

目的探讨血红素加氧酶1（HO-1）高表达在对抗高糖和糖基化终产物（AGEs）诱导的人单核细胞（THP-1）氧化应激中的作用。方法THP-1细胞分为对照（NC）组、GLU＋AGEs组、钴原卟啉（CoPP）＋GLU4-AGEs组和CoPP组4组,孵育24h后收集细胞及培养液上清,检测各组细胞的活性氧（ROS）产量、培养液上清丙二醛（MDA）和肿瘤坏死因子α（TNF-α）水平及HO-1表达。结果GLU4-AGEs组的ROS产量、MDA和TNF-α水平均显著高于NC组（P〈0．05）;CoPP4-GLU4-AGEs组的ROS产量和MDA水平均显著低于GLU4-AGEs组（P〈0．05）。GLU4-AGES组的HO-1基因和蛋白表达均显著高于NC组（P％0．01）,CoPP4-GLU＋AGEs组的HO-1蛋白表达显著高于GLU4-AGEs组（P〈0．01）。结论CoPP通过诱导HO-1蛋白高表达拮抗高糖和AGEs导致的THP-1细胞氧化应激,通过诱导HO-1高表达可能拮抗糖尿病所致单核细胞氧化应激。     

晚期糖基化终末产物对高血压及其合并早期肾损害相关性分析

目的:通过测定高血压病(EH)患者与正常人血清晚期糖基化终末产物(AGEs)及尿微量白蛋白(mALB)的水平,探讨AGEs与EH及其早期肾功能损害的关系,为高血压发病机制及其早期肾功能损害防治研究提供理论依据。方法:筛选EH患者100例,其中EH mALB正常组42例,EH mALB异常组58例。所有入选者分别用放免法、ELISA法测晨尿微量白蛋白(mALB)、AGEs水平。结果:与对照组比较,EH mALB正常组、EH mALB异常组血清AGEs显著增高(P<0.001);与EHmALB正常组比较,EHmALB异常组血清AGEs显著增高(P<0.001)。相关分析AGEs与SBP、PP、mALB之间有显著的正相关性(r=0.448-0.687,P<0.001);mALB与SBP、PP之间有显著的正相关性(r=0.271-0.354,P<0.001)。结论:AGEs、SBP共同参与高血压病肾脏功能的损害,SBP随AGEs水平升高而增高。     

肝纤维化大鼠晚期糖基化终末产物变化及氨基胍的干预作用

目的研究四氯化碳诱导的实验性大鼠肝纤维化不同阶段体内晚期糖基化终末产物的变化情况，以及晚期糖基化终末产物抑制剂氨基胍对肝纤维化的干预作用。方法sD大鼠分为3组：对照组、模型组、氨基胍干预组。检测四氯化碳诱导的大鼠肝纤维化及氨基胍干预后不同阶段血清及肝组织匀浆晚期糖基化终末产物含量。血清透明质酸水平，并对照观察肝脏的组织病理学改变进行肝纤维化程度评分。结果血清及肝组织匀浆晚期糖基化终末产物含量、透明质酸水平以及肝纤维化评分，肝纤维化l2周组高于对照组（t值分别为3．26、3．49、4．70和25．4，P值均〈0．01），氨基胍干预组低于肝纤维化组（t值分别为2．11，2．11、2．93和2．47，P值均〈0．05）。血清及肝组织匀浆晚期糖基化终末产物含量与血清透明质酸水平呈线性相关。结论肝纤维化晚期血清及肝组织匀浆晚期糖基化终末产物含量是增高的。一定程度上，体内晚期糖基化终末产物水平可以反映肝纤维化情况。氨基胍对四氯化碳诱导的实验性大鼠肝纤维化有一定的干预作用。     

Glycated hemoglobin and its spinoffs: Cardiovascular disease markers or risk factors?

Atherosclerosis is a major complication of diabetes, increasing the risk of cardiovascular related morbidities and mortalities. The hallmark of diabetes is hyperglycemia which duration is best predicted by elevated glycated haemoglobin A_1C (HbA_1C) levels. Diabetic complications are usually attributed to oxidative stress associated with glycation of major structural and functional proteins. This non-enzymatic glycation of long lived proteins such as collagen, albumin, fibrinogen, liver enzymes and globulins result in the formation of early and advanced glycation end products (AGEs) associated with the production of myriads of free radicles and oxidants that have detrimental effects leading to diabetic complications. AGEs have been extensively discussed in the literature as etiological factors in the advancement of atherogenic events. Mechanisms described include the effects of glycation on protein structure and function that lead to defective receptor binding, impairment of immune system and enzyme function and alteration of basement membrane structural integrity. Hemoglobin (Hb) is a major circulating protein susceptible to glycation. Glycated Hb, namely HbA_1C is used as a useful tool in the diagnosis of diabetes progression. Many studies have shown strong positive associations between elevated HbA_1C levels and existing cardiovascular disease and major risk factors. Also, several studies presented HbA_1C as an independent predictor of cardiovascular risk. In spite of extensive reports on positive associations, limited evidence is available considering the role of glycated Hb in the etiology of atherosclerosis. This editorial highlights potential mechanisms by which glycated hemoglobin may contribute, as a causative factor, to the progression of atherosclerosis in diabetics.