金雀异黄素对终末糖基化产物刺激大鼠系膜细胞终末糖基化产物受体表达的影响

目的 研究金雀异黄素(Gen)对终末糖基化产物(AGEs)刺激下其系膜细胞受体(RAGE)表达的影响。方法 以AGEs刺激高糖培养的SD大鼠系膜细胞,采用细胞免疫组化法检测系膜细胞RAGE表达,逆转录聚合酶链式反应(RT-PCR)检测细胞内.RAGEmRNA水平;比色法检测细胞内丙二醛(MDA)水平。结果 AGEs刺激高糖培养的系膜细胞后RAGE表达及RAGEmRNA水平明显增加(P<0.05);MDA水平显著增加(P<0.01)。Gen能有效抑制RAGE及其:mRNA的表达,减少MDA水平,且呈时间和浓度依赖效应,以200μmol／L作用48 h效果最显著(P<0.01)。结论 体外模拟糖尿病环境下,AGEs刺激使。肾系膜细胞内RAGE高表达。Gen可以下调AGEs刺激后系膜细胞RAGE的异常表达,其可能的机制是阻断了氧化应激介导的AGEs-RAGE之间的正反馈途径,表现为细胞内MDA含量减低。     

糖化终末产物对人腹膜间皮细胞氧化应激的影响

目的研究不同浓度糖化终末产物（AGEs）对人腹膜间皮细胞（HPMC）氧化应激的影响。方法通过胰酶消化法从非。肾脏疾病腹腔手术患者的大网膜中得到HPMC,进行原代培养和传代,免疫组化法对培养的细胞进行鉴定。不同浓度的AGEs干预HPMC一定时间后,采用活性氧（ROS）捕获剂双氢-乙酰乙酸二氯荧光黄（DCFH-DA）孵育细胞,通过流式细胞仪检测细胞内的平均荧光强度测定细胞内ROS水平,观察不同浓度的AGEs对HPMC干预后细胞内ROS的变化。结果AGEs明显抑制HPMC活力（P〈0．05）,随着浓度和时间的增加,抑制率上升。AGEs干预HPMC后,细胞内的ROS水平明显升高。结论AGEs导致细胞发生氧化应激,其对细胞的损伤作用可能与氧自由基生成过多、ROS蓄积有关。     

人类关节滑膜细胞表达晚期糖基化终产物受体

目的 进一步探讨晚期糖基化终产物修饰的β２－微球蛋白（ＡＧＥ－β２ｍ）对关节固有细胞的生物学作用,确定人类关节滑膜细胞是否表达对ＡＧＥ特异的受体。方法 分离、培养人关节Ａ型和Ｂ型滑膜细胞,用免疫组织化学法及流式细胞仪法分别观察滑膜细胞表面ＡＧＥ受体１（ＡＧＥ－Ｒ１）,ＡＧＥ受体２（ＡＧＥ－Ｒ２）、ＡＧＥ受体３（ＡＧＥ－Ｒ３）及ＡＧＥ受体（ＲＡＧＥ）的表达,用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术     

糖基化终末产物对人外周血内皮祖细胞生物学特性的影响

目的 观察糖基化终末产物(AGEs)对体外培养的人外周血内皮祖细胞(EPCs)生物学特性的影响.方法 以密度梯度离心法获取人外周血单个核细胞(MNCs),由激光共聚焦显微镜鉴定FITC-UEA-1和Dil-acLDL双染色阳性细胞为正在分化的EPCs.加入不同浓度AGEs培养48h,然后分别采用MTT法、Boyden小室测定来观察EPCs的增殖、迁移能力;以人纤维连接蛋白(hFN)检测EPCs的黏附能力;分别采用甲醛和Dnase Ⅰ诱导EPCs凋亡作为阳性对照组,用AnnexinV-FITC/PI和TUNEL法流式细胞仪检测AGEs对EPCs凋亡率的影响.结果 高浓度AGEs可减少EPCs贴壁细胞数量(P<0.01),明显减弱EPCs的黏附(P<0.01)、增殖(P<0.001)、迁移能力(P<0.05),提高EPCs的早期凋亡率(P<0.001).结论 AGEs可减少EPCs的数昔并使EPCs的功能受损.     

糖基化终末产物对人外周血内皮祖细胞生物学特性的影响

Objective In addition to be involved in the angiogenesis, endothelial progenitor cells (EPCs) have roles in endothelium repairing, wound healing, and for protecting blood vessels from restenosis, Advanced glycation end products (AGEs) facilitate the development and progression of atherosclerosis, diabetes associated vascular complications and uremia through various mechanisms such as damaging the endothelium, promoting leukocyte adhension, increasing the aggregation of platelets, and stimulating the proliferation of vascular smooth muscles. This study was designed to explore whether AGEs have effects on biological characteristics of EPCs in cultured human peripheral blood cells. Methods Total mononuclear cells (MNCs), isolated from human peripheral blood by density gradient centrifugatian and adherence cells filtration, were incuba-ted in fibronectin-coated culture dishes. Endothelial cells were identified by means of the adsorption of ulex eurepaeus-aggluti-nin- Ⅰ (UEA- Ⅰ) labelled with fluorescein isothiacyanate (FITC) and Dil-acLDL internalization. Four days later,various con-centrations of AGEs were added to the adherent cells and remained for48 hours. MTT assay and Boyden chamber were used for observing the proliferation and migration of EPCs. Human fibronectin was used to examine the adhesion ability of EPCs. Apop-tosis was induced in the EPCs with formaldehyde and Dnase Ⅰ as a positive control group. Annexin V-FITC/PI and TUNEL method of flow cytometry were used for evaluating the effects of AGEs on the rate of apeptosis in the EPCs. Results AGEs at high concentration decreased the number of EPCs independently (P < 0.01) ; reduced the proliferation (P < 0.01), migration (P<0.001) and adhesive capacity (P<0.05) of EPCs significantly,as well as increasing the apoptasis rate of EPCs in the early stage (P < 0.001). Conclusion AGEs may have adverse effects on EPCs from cultured human peripheral MNCs, such as decreasing their numbers and impairing their functions.     

糖基化终末产物对人外周血内皮祖细胞生物学特性的影响

Objective In addition to be involved in the angiogenesis, endothelial progenitor cells (EPCs) have roles in endothelium repairing, wound healing, and for protecting blood vessels from restenosis, Advanced glycation end products (AGEs) facilitate the development and progression of atherosclerosis, diabetes associated vascular complications and uremia through various mechanisms such as damaging the endothelium, promoting leukocyte adhension, increasing the aggregation of platelets, and stimulating the proliferation of vascular smooth muscles. This study was designed to explore whether AGEs have effects on biological characteristics of EPCs in cultured human peripheral blood cells. Methods Total mononuclear cells (MNCs), isolated from human peripheral blood by density gradient centrifugatian and adherence cells filtration, were incuba-ted in fibronectin-coated culture dishes. Endothelial cells were identified by means of the adsorption of ulex eurepaeus-aggluti-nin- Ⅰ (UEA- Ⅰ) labelled with fluorescein isothiacyanate (FITC) and Dil-acLDL internalization. Four days later,various con-centrations of AGEs were added to the adherent cells and remained for48 hours. MTT assay and Boyden chamber were used for observing the proliferation and migration of EPCs. Human fibronectin was used to examine the adhesion ability of EPCs. Apop-tosis was induced in the EPCs with formaldehyde and Dnase Ⅰ as a positive control group. Annexin V-FITC/PI and TUNEL method of flow cytometry were used for evaluating the effects of AGEs on the rate of apeptosis in the EPCs. Results AGEs at high concentration decreased the number of EPCs independently (P < 0.01) ; reduced the proliferation (P < 0.01), migration (P<0.001) and adhesive capacity (P<0.05) of EPCs significantly,as well as increasing the apoptasis rate of EPCs in the early stage (P < 0.001). Conclusion AGEs may have adverse effects on EPCs from cultured human peripheral MNCs, such as decreasing their numbers and impairing their functions.     

Diabetes increases formation of advanced glycation end products on Sarco(endo)plasmic reticulum Ca2+-ATPase

Prolongation of relaxation is a hallmark of diabetic cardiomyopathy. Most studies attribute this defect to decreases in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) expression and SERCA2a-to-phospholamban (PLB) ratio. Since its turnover rate is slow, SERCA2a is susceptible to posttranslational modifications during diabetes. These modifications could in turn compromise conformational rearrangements needed to translocate calcium ions, also leading to a decrease in SERCA2a activity. In the present study one such modification was investigated, namely advanced glycation end products (AGEs). Hearts from 8-week streptozotocin-induced diabetic (8D) rats showed typical slowing in relaxation, confirming cardiomyopathy. Hearts from 8D animals also expressed lower levels of SERCA2a protein and higher levels of PLB. Analysis of matrix-assisted laser desorption/ionization time-of-flight mass data files from trypsin-digested SERCA2a revealed several cytosolic SERCA2a peptides from 8D modified by single noncrosslinking AGEs. Crosslinked AGEs were also found. Lysine residues within actuator and phosphorylation domains were cross-linked to arginine residues within the nucleotide binding domain via pentosidine AGEs. Two weeks of insulin-treatment initiated after 6 weeks of diabetes attenuated these changes. These data demonstrate for the first time that AGEs are formed on SERCA2a during diabetes, suggesting a novel mechanism by which cardiac relaxation can be slowed during diabetes.     

Advanced glycation end products induce mitochondrial pathway apoptosis in glomerular mesangial cells

Objective To investigate whether advanced glycation end products (AGEs) can induce the expression of Ros, JC-1 and its apoptosis-related proteins in glomerular mesangial cells under high glucose environment, induce apoptosis and injury of glomerular mesangial cells. Methods Rat glomerular mesangial cell line HBZY-1 was cultured in vitro. The cells were cultured with different concentrations of AGEs for 0, 12, 24 and 48 hours respectively. MTT assay was used to observe the cell proliferation ability. After the optimal time and concentration of AGEs were selected, the caspase enzyme inhibitor Z-VAD-fmk and reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) were cultured and the apoptosis rate was detected by cell death detection apoptosis ELISA plus and Annexin V-FITC/PI kit. JC-1 staining was used to detect the changes of mitochondrial membrane potential (MMP). Cell ROX deep red flow cytometry was used to detect the total ROS level. The expression of anti-apoptotic protein Bcl-2, pro-apoptotic protein BAX, caspase-9, caspase-3 and poly ADP-ribose polymerase (PARP)-activated fragments was detected by Western blotting. Results AGEs could decrease the activity of glomerular mesangial cells in a time and concentration-dependent manner, and induce cell death. The percentage of apoptotic cells in glomerular mesangial cells was significantly increased after treatment with 250 mg/L AGEs for 24 h (P＜0.01), and Z-VAD-fmk could significantly alleviate AGEs-induced glomerular mesangial cell apoptosis (P＜0.01). Compared with the control group, AGEs increased the level of intracellular reactive oxygen species and decreased MMP in a time-dependent manner, and the two time points that AGEs significantly caused the change were 1 h and 2 h (all P＜0.01). AGEs also reduced the expression of antiapoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP (all P＜0.01). Compared with AGEs group, NAC could significantly stabilize MMP (P＜0.01), increase Bcl-2 expression (P＜0.01), and decrease the expression of BAX, cleaved caspase-9, cleaved caspase-3 and cleaved PARP (all P＜0.01). Conclusion AGEs induce mitochondrial pathway apoptosis in glomerular mesangial cells by increasing intracellular ROS level and destroying MMP.     

Preparation of advanced glycation end products in vitro.

Sir, With great interest we read the article by Chang et al. [1]on the enhancement of epithelial sodium channel mRNA expressionand protein level in renal cortical collecting duct (CCD) cellsby advanced glycation end products (AGEs). The authors showthat AGEs can increase sodium uptake in CCD cells in a     

Amino acids injure mesangial cells by advanced glycation end products, oxidative stress, and protein kinase C

BACKGROUND: In diabetes, high intake of dietary protein exacerbates responses associated with kidney damage. Increased levels of amino acids could injure cells by providing free amino groups for glycation reactions leading to advanced glycation end products (AGEs). METHODS: Rat mesangial cells were cultured with increased amino acids designed to resemble protein feeding, high glucose (30.5 mmol/L), and, the combination, amino acids/high glucose. AGEs, reactive oxygen species (ROS), protein kinase C (PKC) activity and production, and mitogen-activated protein (MAP) kinase-extracellular signal regulated kinase (ERK) 1,2 activity were measured. Inhibitors were used to determine roles of these processes in fibrosis and/or AGE formation. RESULTS: AGE immunostaining increased when cells were cultured in amino acids and was comparable to that observed with high glucose. In amino acids/high glucose, AGE immunostaining appeared even greater. Amino acids, high glucose, and amino acids/high glucose induced ROS production. Aminoguanidine and vitamin E prevented AGE accumulation and induction of protein and mRNA for fibrosis markers [transforming growth factor-beta1 (TGF-beta1), fibronectin, and collagen IV]. PKC and ERK 1,2 activity increased with amino acids, high glucose, and amino acids/high glucose. PKC-beta inhibition prevented ERK 1,2 activation and fibrosis induction. ERK 1,2 inhibition also blocked the fibrosis response. CONCLUSION: A profibrotic injury response occurred in mesangial cells exposed to amino acids, with or without high glucose, by formation of AGE, oxidative stress, and activation of the PKC-beta and MAP kinase-ERK 1,2 signal pathway. These observations provide new insight into cellular mechanisms of kidney damage produced by excess dietary protein, particularly in diabetes.     

尿毒清对晚期糖基化终末产物作用下肾小球系膜细胞影响

目的观察尿毒清对晚期糖基化终末产物（advanced glycosylation end products, AGEs）作用下肾小球系膜细胞的影响,探讨中药尿毒清在治疗糖尿病肾脏疾病（diabetic kidney dis-ease,DKD）中对肾小球系膜细胞的保护作用。方法采用冻干牛血清白蛋白和糖制备无内毒素的AGE-BSA及人工培养牛肾小球系膜细胞,培养时分为3组,在加入终浓度为0.25 mg/ml AGEs 的同时给予浓度为0.2 mg/ml的尿毒清成分溶液为尿毒清组,加入终浓度为0.25 mg/ml AGEs 为 AGEs组,同时设空白对照组,应用 MTT比色法,观察尿毒清组对体外 AGEs 培养的牛肾小球系膜细胞增殖的影响;以系膜细胞与 Alcain Blue（AB,阿利新蓝）结合量为指标,观察尿毒清组对 AGEs导致的系膜细胞膜表面电荷的影响;利用 AO/EB染料观察尿毒清组对 AGEs 诱导作用下的牛肾小球系膜细胞凋亡的影响,并利用荧光显微镜观察尿毒清的抑制作用。结果尿毒清组可以有效地抑制 AGEs对牛肾系膜细胞的促进增殖作用;对抗 AGEs 诱导的牛肾小球系膜细胞表面电荷的影响;减少 AGEs诱导的牛肾小球系膜细胞凋亡。结论尿毒清组能减少对晚期糖基化终末产物作用下肾小球系膜细胞增殖和凋亡,保护肾小球系膜细胞表面电荷。     

Identification and characteristics of a Na+/Ca2+ exchanger in cultured human mesangial cells

Excitable cells express Na+/Ca2+ exchange activity among other mechanisms modulating rapid fluctuations of cytosolic free Ca2+ ([Ca2+]i). We studied functions and regulation of a Na+/Ca2+ exchanger in cultured human glomerular mesangial cells. Fura-2-loaded confluent monolayers reacted to removal of ambient Na+ with an immediate, transient elevation of [Ca2+]i, assessed by single/dual wavelength fluorometry. Peak [Ca2+]i was inversely correlated with the extracellular Na+ concentration. Ca2+ influx was the sole mechanism implicated, as the [Ca2+]i rise was prevented by EGTA. The process was inhibited by 1 mM amiloride, but not by blockers of voltage-operated Ca2+ channels. Re-addition of Na+ resulted in a rapid decrease of [Ca2+]i, indicating bimodal operation of the exchanger. Na(+)-loading the cells with monensin and ouabain enhanced Ca2+ uptake. Prior stimulation of [Ca2+]i with the thromboxane A2 mimetic, U-46619, or angiotensin II also increased Ca2+ uptake upon subsequent Na+ removal, suggesting induction of the exchanger by vasoconstrictors. Moreover, the magnitude of agonist-induced [Ca2+]i transients was amplified by Na+ removal, indicating that the exchanger modulates the effects of vasoconstrictors. These results demonstrate that an inducible Na+/Ca2+ antiporter is operative in resting and stimulated human mesangial cells, further confirming their smooth muscle origin and potential regulatory role on glomerular hemodynamics.     

复方苍术汤对糖尿病大鼠肾脏非酶糖基化终末产物的影响

目的观察中药复方苍术汤对糖尿病大鼠肾脏非酶糖基化终末产物（AGEs）的影响。方法小剂量链脲佐菌素（STZ）注射加高糖高脂喂养建立糖尿病大鼠模型,给予复方苍术汤喂养8周,观察各组大鼠肾组织AGEs含量、肾重指数和空腹血糖的变化。结果复方苍术汤组大鼠的肾组织AGEs含量、肾重指数、血糖均较模型组降低（P〈0.01）。结论复方苍术汤降低糖尿病大鼠肾脏组织AGEs水平,其作用机制可能与降低血糖有关。     

晚期糖基化终末产物与其受体对糖尿病创面氧化应激反应的影响

目的 观察糖尿病患者皮肤和创面中晚期糖基化终末产物(AGE)的蓄积和炎症反应,并结合体外干预实验推测两者之间可能存在的关系. 方法 采集10例2型糖尿病患者(糖尿病组)和12例非糖尿病患者(非糖对照组)的皮肤和创面组织标本,部分于光学显微镜下观察胶原排列、细胞分布(HE染色),AGE及其受体(RAGE)表达(免疫组织化学染色);部分匀浆后采用ELISA法检测丙二醛含量.体外采集、分离健康人外周血中性粒细胞,并分为正常对照组(添加RPMI-1640培养液培养)、低浓度干预组、中浓度干预组和高浓度干预组,3个干预组分别在RPMI-1640培养液中添加AGE修饰的人血清白蛋白,其中AGE的浓度依次为0.315、0.625、1.250 mg/mL;噻唑蓝法检测细胞存活率,荧光探针二氢二氯荧光黄双乙酸钠测定细胞内活性氧水平.对数据进行t检验. 结果 与非糖对照组比较,糖尿病组皮肤组织中胶原萎缩,排列紊乱;创面组织中炎性细胞弥散分布,胶原排列稀疏紊乱.糖尿病组皮肤组织中AGE和RAGE表达均多于非糖对照组;2组创面组织中RAGE呈阳性表达的细胞数量均多于所在组皮肤组织,以糖尿病组更显著,其创面内可见大量RAGE呈阳性表达的炎性细胞.糖尿病组患者皮肤和创面组织中每毫克蛋白丙二醛含量分别为(6 3±1.0)、(7.1±2.4)nmol,均明显高于非糖对照组相应组织[(2.9±1.0)、(3.6±1.4)nmol,t值分别为8.017、4.349,P ＜0.05或P＜0.01].与正常对照组[(34±5)％]比较,低浓度干预组、中浓度干预组、高浓度干预组中性粒细胞存活率均明显上升[(59±8)％、(77±5)％、( 67±6)％,t值分别为7.195、14.890、11.130,P＜0.05或P＜0.01].与正常对照组(0.58±0.06)比较,低浓度干预组、中浓度干预组、高浓度干预组中性粒细胞内活性氧水平均升高(1.67±0.14、2.13±0.17、3 48±0.48,t值分别为20.195、24.905、16.864,P＜0.05或P＜0.01). 结论 异常的氧化应激水平导致糖尿病皮肤具有异常的创伤修复起点,创面愈合过程中AGE-RAGE效应是影响糖尿病创面氧化应激水平的重要因素.     

Nitric oxide inhibits the formation of advanced glycation end products

BACKGROUND: Advanced glycation end products (AGEs) are elevated in renal failure and have been implicated in the pathogenesis of several uremic complications. Their formation is closely associated with oxidative stress. The recent observation that nitric oxide (NO) has an antioxidant effect led us to examine the possible role of NO in the generation of AGEs. METHODS: We examined the effect of NO donors, 2, 2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC18) and S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the in vitro formation of pentosidine, which was used as a surrogate marker for AGEs. Bovine serum albumin was incubated under air at 37 degrees C in a medium containing either several AGE precursors or uremic plasma. To elucidate further the mechanism of the NO effect on AGE formation, we examined the generation of free radicals and carbonyls in pentose-driven pentosidine formation. RESULTS: NO donors significantly inhibit the formation of pentosidine in a dose-dependent manner. The effect is abolished by the addition of a NO scavenging agent, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). The inhibitory effect results from NO but not from the NO donor molecule. It is best explained by the ability of NO to scavenge carbon-centered radicals, hydroxyl radical, and carbonyl compounds. CONCLUSIONS: NO inhibits pentosidine formation by scavenging free radicals and by inhibiting carbonyl compound formation. NO might be implicated in the atherogenic and inflammatory effects of AGEs: Reduced NO production and increased oxidative stress associated with atherosclerotic lesions may accelerate AGE formation and, thus, exacerbate endothelial dysfunction and accelerate the development of atherosclerosis in uremia.     

肝素抑制晚期糖基化终产物诱导的单核细胞促炎症细胞因子释放

目的：研究晚期糖基化终产物（AGE）对血透患者单核细胞产生肿瘤坏死因子（TNF－α）和白细胞介素－1β（IL-1β）的作用以及肝素对这一作用的影响。方法：用密度梯度离心法分离正常人和血透患者外周血单核细胞,在体外与AGE修饰的白蛋白（AGE－HSA）共同孵育,用ELISA法检测培养上清中TNF－α和IL－1β水平。结果：AGE－HSA刺激人单核细胞产生TNF－α和IL－1β。在相同剂量AGE－HSA刺激下,血透患者单核细胞产生的TNF－α和IL－1β水平较正常人明显增高。在培养体系中加入肝素明显减少血透患者单核细胞由AGE－HSA诱导的TNF－α和IL－1β的分泌,但低分子量肝素无此作用。结论：血透患者单核细胞在AGE修饰白蛋白刺激下能产生较正常人更多的促炎症细胞因子,这一机制可能是血透患者循环促炎症细胞因子水平升高的原因之一。肝素可以部分阻断这一机制,具有潜在的治疗价值。