缬沙坦对人肾小球系膜细胞糖基化终产物受体表达的影响

目的：本实验探讨缬沙坦对糖基化终产物诱导的人肾小球系膜细胞氧化应激水平及糖基化终产物受体（RAGE）表达的影响。方法：体外常规培养人肾小球系膜细胞,运用糖基化修饰的牛血清白蛋白（AGE-BSA）和缬沙坦进行干预,流式细胞术检测细胞内活性氧（ROS）,RT-PCR法检测NADPH氧化酶的亚基p47^phox的mRNA表达,RT-PCR和细胞免疫化学法检测RAGE的表达量。结果：缬沙坦干预组人肾小球系膜细胞的ROS产生量、NADPH氧化酶的亚基p47^phox mRNA表达量、RAGE表达量均低于AGE-BSA组（P〈0．05）,且缬沙坦的抑制作用呈浓度和时间依赖性。结论：缬沙坦可能通过降低氧化应激水平来抑制RAGE的表达。     

晚期糖化终产物诱导内皮细胞通透性增高

本文探讨了晚期糖化终产物(advanrced glycation end products,AGEs)修饰蛋白对内皮细胞通透性及细胞骨架肌动蛋白的形态学影响,以及特异的AGEs受体(receptors for AGEs,RAGE)、氧化应激和p38 MAPK通路在此病理过程中的作用。用不同浓度的AGEs修饰人血清白蛋白(AGE-HSA)与人脐静脉内皮细胞株ECV304在体外共同培养不同时间,并设立对照组进行比较,采用TRITC荧光标记白蛋白漏出法测定单层内皮细胞的通透系数Pa值,荧光染色法示细胞骨架的形态学改变。与对照组相比,AGE-HSA以时间和剂量依赖的方式引起单层内皮细胞通透性的升高及细胞骨架肌动蛋白F-actin形态的改变;可溶性RAGE的抗体(anti-RAGE IgG)、NADPH氧化酶抑制剂(apocynin)及p38抑制剂SB203580均可减轻AGEs对内皮细胞屏障功能和形态的影响。结果提示,AGEs修饰蛋白对单层内皮细胞通透性及骨架重排的作用可能通过与内皮细胞上的RAGE结合,引起细胞内的氧化应激,并激活p38 MAPK通路所介导。     

GLP-1拮抗活性氧对AGEs诱导乳鼠成纤维细胞凋亡的保护作用

为研究胰高血糖素样肽-1(Glucogon like peptide-1,GLP-1)对晚期糖基化终产物(Advanced Glycation End Products,AGEs)诱导成纤维细胞凋亡的影响及其机制研究。体外培养成纤维细胞,实验分为5组:正常对照组,200 mg/L AGEs试验组,AGEs(200 mg/L)+GLP-1(5 nmol/L)组,AGEs(200 mg/L)+GLP-1(10 nmol/L)以及AGEs(200 mg/L)+GLP-1(20 nmol/L)组,处理24 h。通过CCK-8比色法检测细胞存活率,相差显微镜观察细胞形态,双氯荧光素(DCFH-DA)染色荧光显微镜摄片观察细胞内活性氧(ROS)水平;运用Hoechst33258检测试剂盒及流式细胞术检测成纤维细胞的凋亡率,Western Blot检测凋亡相关蛋白Caspase-3、Bcl-2的表达量。结果表明:与正常组相比,200 mg/L AGEs组降低成纤维细胞生存率,活性氧(ROS)生成量增加;与AGEs组相比,AGEs+GLP-1组成浓度依赖性提高细胞生成率,活性氧(ROS)含量减少。与正常组相比,AGEs组导致促凋亡蛋白Caspase-3增加,抑制凋亡蛋白Bcl-2减少,细胞凋亡率升高;AGEs+GLP-1组较AGEs组下调促凋亡蛋白Caspase-3,上调抑制蛋白凋亡Bcl-2,细胞凋亡率减少。由此可见,GLP-1可以通过拮抗活性氧(ROS)发挥对AGEs诱导的成纤维细胞凋亡的保护作用。     

晚期糖基化终产物通过其受体促进内皮细胞分泌IL-8的研究

探讨晚期糖基化终产物(AGE)修饰蛋白对内皮细胞生成白介素8(IL-8)的作用,及晚期糖基化终产物受体(RAGE)在此病理过程中的作用.内皮细胞来自培养的人脐静脉内皮细胞(HUVEC).将内皮细胞与不同浓度的AGE修饰人血清白蛋白(AGE-HSA)在体外共同培养,或以可溶性晚期糖基化终产物受体(sRAGE)对AGE-HSA进行预处理后再与HUVEC共同培养.用蛋白质液相芯片法检测HUVEC培养上清中IL-8水平,并提取细胞RNA,进行RT-PCR反应,检测细胞中IL-8 mRNA的表达水平.结果表明,AGE-HSA以时间和剂量依赖的方式刺激HUVEC生成IL-8,未经修饰的HSA无此作用.AGE-HSA用sRAGE预处理后,刺激HUVEC生成IL-8的作用被抑制,并且此抑制作用呈剂量依赖的方式.AGE-HSA刺激HUVEC使IL-8 mRNA表达增高,未经修饰的HSA无此作用.sRAGE能够阻断AGE-HSA诱导HUVEC表达IL-8mRNA的作用.整个变化趋势与蛋白质水平一致.研究首次证实,AGE-HSA与细胞表面受体RAGE相互作用可刺激内皮细胞分泌IL-8,并上调IL-8 mRNA的表达.这为研究加速型血管病变的发病机制提供了新视角,也为治疗由AGE增多和潴留所引起的病理损害提供了新靶点.     

晚期糖基化终产物对人脐静脉内皮细胞的肝细胞生长因子表达的影响

目的探讨晚期糖基化终产物(AGEs)对人脐静脉内皮细胞的肝细胞生长因子(HGF)mRNA及蛋白表达的影响。方法体外培养人脐静脉内皮细胞,予不同浓度(100mg/L、200mg/L、400mg/L)的AGEs刺激24h及400mg/LAGEs作用6h、12h、24h及48h,采用RT-PCR及免疫细胞化学法检测内皮细胞HGFmRNA及蛋白的表达水平。结果在一定范围内随着AGEs浓度增加,内皮细胞HGF表达逐渐增高;AGEs早期作用内皮细胞,促进HGFmR-NA及蛋白的表达,随着AGEs的持续作用,HGF表达减弱。结论随着AGEs作用时间的延长,HGF对受损内皮细胞的修复作用先增强后减弱。     

氧化应激与糖尿病性心肌病的发生发展

糖尿病患者经常表现出一种独特的心脏表型,即糖尿病性心肌病（Diabetic cardiomyopathy,DCM）。由于在糖尿病患者中慢性炎症和氧化应激的发生,改变了机体代谢稳态,引发氧化还原失衡,从而诱发心肌代谢紊乱和心脏微血管病变,使心脏出现危险的氧化应激,致使了糖尿病性心肌病的发生。心肌细胞中脂肪的氧化与毒性增加以及微脉管系统中高水平的细胞血糖,让糖尿病患者体内的线粒体产生过量的活性氧（reactive oxygen species, ROS）。超氧阴离子自由基可增强己糖胺生物合成途径和多元醇通路,诱导活化蛋白激酶C(protein kinase C, PKC),增加晚期糖基化终产物（advanced glycation end products, AGEs）的形成并激活其受体（receptor for advanced glycation end products, RAGE）。这些生化级联反应同时也是ROS的其他额外来源。本文主要概述氧化应激在DCM中的作用及其涉及信号通路。同时,也简要提到了目前用于DCM的治疗方法。基于氧化应激在糖尿病性心肌病中的关键作用,新的抗氧化疗法的...     

糖基化终末产物（AGEs）诱导下SOCS基因在肾小管上皮细胞中的表达及意义

目的观察SOCS-1、SOCS-3在肾小管上皮细胞（HKC）中的基础表达及在糖基化终末产物（AGEs）诱导下的表达及意义。方法体外培养HKC细胞,随机分为正常对照组、AGEs组,倒置显微镜观察细胞形态学改变,采用流式细胞术,免疫细胞化学和检测SOCS-1、SOCS-3蛋白表达,RT-PCR法检测HKCSOCS-1、SOCS-3 mRNA表达。结果与正常组比较,AGES诱导的肾小管上皮细胞发生形态学改变;免疫细胞化学、流式细胞学发现SOCS-1、SOCS-3表达以胞浆为主,散在胞核表达;12、24、48hSOCS-1、SOCS-3蛋白表达AGEs组均高于正常对照组,差异有统计学意义,其中SOCS-1以12h表达量最大,SOCS-3以24h表达最高,RT-PCR检测SOCS-3 mRNA表达量,AGEs组高于正常组,差异有统计学意义。结论SOCS-1、SOCS-3在正常肾小管上皮细胞中有基础表达,AGEs可诱导肾小管上皮细胞SOCS-1、SOCS-3表达上调,为我们进一步研究SOCS基因与糖尿病肾病的关系提供了理论依据。     

银杏叶提取物对AGEs诱导心肌细胞内质网应激的影响

目的：探索银杏叶提取物对晚期糖基化终产物（AGEs）作用下心肌细胞损伤以及内质网应激标记性分子GRP 78和CHOP的影响。方法：原代培养SD大鼠乳鼠心肌细胞,随机分为对照组、AGEs组、AGEs＋银杏叶提取物组。MTT法检测心肌细胞存活率,Western blot法检测GRP 78和CHOP蛋白表达水平。结果：与对照组比较,AGEs组48、72 h时心肌细胞存活率降低;与AGEs组比较,银杏叶提取物处理增加心肌细胞存活率。与对照组比较,AGEs组GRP 78和CHOP蛋白表达水平显著升高;与AGEs组比较,银杏叶提取物组GRP 78和CHOP表达水平显著下调。结论：银杏叶提取物能够抑制AGEs诱导的心肌细胞损伤,其机制可能与拮抗GRP 78和CHOP的表达,减轻内质网应激有关。     

黄芪甲苷对血管紧张素Ⅱ诱导肾小球系膜细胞增殖及炎症因子表达的影响

目的：研究黄芪甲苷（As-IV）对血管紧张素Ⅱ（AngⅡ）诱导大鼠肾小球系膜细胞（GMCs）增殖及炎症因子表达的影响。方法：采用10^-6mol/L的AngⅡ刺激GMCs增殖,同时分别加入25,50,100 μmol/L的As-IV对GMCs作用48 h,运用MTT法检测各组细胞增殖状况;流式细胞术观察GMCs中细胞内活性氧（ROS）水平变化;ELISA法检测细胞上清液中单核细胞趋化蛋白-1（MCP-1）的含量;Western blot法检测细胞中转化生长因子β1（TGF-β1）蛋白的表达。结果：与AngⅡ刺激组相比,As-IV干预显著抑制GMCs细胞增殖,减少细胞内ROS水平,抑制MCP-1及TGF-β1的表达。结论：As-IV对于AngⅡ诱导GMCs的增殖具有抑制作用,且能降低相关炎症因子的表达。     

糖尿病血管病变中的RAGE可变剪接物

该文简要回顾高级糖化终产物受体（RAGE）可变剪接物[全长RAGE、截去N端型RAGE和截去C端型RAGE（esRAEG）1的结构和功能;阐述RAGE可变剪接物由于在内皮细胞中的丰度、表达水平不同,使介导的生物学效应也不同;从而有助于我们了解细胞内RAGE可变剪接物对高级糖化终产物（AGE）反应的分子机制及糖尿病血管并发症易感性的个体差异。     

Advanced Glycation End Products Induce Human Corneal Epithelial Cells Apoptosis through Generation of Reactive Oxygen Species and Activation of JNK and p38 MAPK Pathways

Advanced Glycation End Products (AGEs) has been implicated in the progression of diabetic keratopathy. However, details regarding their function are not well understood. In the present study, we investigated the effects of intracellular reactive oxygen species (ROS) and JNK, p38 MAPK on AGE-modified bovine serum albumin (BSA) induced Human telomerase-immortalized corneal epithelial cells (HUCLs) apoptosis. We found that AGE-BSA induced HUCLs apoptosis and increased Bax protein expression, decreased Bcl-2 protein expression. AGE-BSA also induced the expression of receptor for advanced glycation end product (RAGE). AGE-BSA-RAGE interaction induced intracellular ROS generation through activated NADPH oxidase and increased the phosphorylation of p47phox. AGE-BSA induced HUCLs apoptosis was inhibited by pretreatment with NADPH oxidase inhibitors, ROS quencher N-acetylcysteine (NAC) or neutralizing anti-RAGE antibodies. We also found that AGE-BSA induced JNK and p38 MAPK phosphorylation. JNK and p38 MAPK inhibitor effectively blocked AGE-BSA-induced HUCLs apoptosis. In addition, NAC completely blocked phosphorylation of JNK and p38 MAPK induced by AGE-BSA. Our results indicate that AGE-BSA induced HUCLs apoptosis through generation of intracellular ROS and activation of JNK and p38 MAPK pathways.     

Advanced glycation end products delay corneal epithelial wound healing through reactive oxygen species generation

Delayed healing of corneal epithelial wounds is a serious complication in diabetes. Advanced glycation end products (AGEs) are intimately associated with the diabetic complications and are deleterious to the wound healing process. However, the effect of AGEs on corneal epithelial wound healing has not yet been evaluated. In the present study, we investigated the effect of AGE-modified bovine serum albumin (BSA) on corneal epithelial wound healing and its underlying mechanisms. Our data showed that AGE-BSA significantly increased the generation of intracellular ROS in telomerase-immortalized human corneal epithelial cells. However, the generation of intracellular ROS was completely inhibited by antioxidant N-acetylcysteine (NAC), anti-receptor of AGEs (RAGE) antibodies, or the inhibitor of NADPH oxidase. Moreover, AGE-BSA increased NADPH oxidase activity and protein expression of NADPH oxidase subunits, p22phox and Nox4, but anti-RAGE antibodies eliminated these effects. Furthermore, prevention of intracellular ROS generation using NAC or anti-RAGE antibodies rescued AGE-BSA-delayed epithelial wound healing in porcine corneal organ culture. In conclusion, our results demonstrated that AGE-BSA impaired corneal epithelial wound healing ex vivo. AGE-BSA increased intracellular ROS generation through NADPH oxidase activation, which accounted for the delayed corneal epithelial wound healing. These results may provide better insights for understanding the mechanism of delayed healing of corneal epithelial wounds in diabetes.     

Advanced glycation end product-induced apoptosis and overexpression of vascular endothelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells

Advanced glycation end products (AGE) have been implicated in the pathogenesis of glomerulosclerosis in diabetes. However, their involvement in the development of the early phase of diabetic nephropathy has not been fully elucidated. We investigated the effects of AGE on growth and on vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) expression in human cultured mesangial cells. We prepared three immunochemically distinct AGE by incubating bovine serum albumin (BSA) with glucose, glyceraldehyde, or glycolaldehyde. When human mesangial cells were cultured with various types of AGE-BSA, viable cell numbers as well as DNA syntheses were significantly decreased. All of the AGE-BSA were found to significantly increase p53 and Bax protein accumulations and subsequently induce apoptotic cell death in mesangial cells. An antioxidant, N-acetylcysteine, significantly prevented the AGE-induced apoptotic cell death in mesangial cells. Human mesangial cells stimulated prostacyclin production by co-cultured glomerular endothelial cells. Furthermore, various types of AGE-BSA were found to up-regulate the levels of mRNAs for VEGF and stimulate the secretion of VEGF and MCP-1 proteins in mesangial cells. The results suggest that AGE disturbed glomerular homeostasis by inducing apoptotic cell death in mesangial cells and elicited hyperfiltration and microalbuminuria by stimulating the secretion of VEGF and MCP-1 proteins, thereby being involved in the pathogenesis of the early phase of diabetic nephropathy.     

红杉醇对高糖诱导的人脐静脉内皮细胞NOX4、eNOS表达的影响

目的：观察红杉醇（Scq）对高糖诱导的人脐静脉内皮细胞（HUVECs）损伤的保护作用及机制。方法：原代培养HUVECs,红杉醇（0．1,1,10μmol/L）预处理1h后,30mmol/L葡萄糖诱导内皮细胞损伤。5-溴脱氧尿嘧啶核苷（BrdU）掺入法检测细胞增殖,流式细胞术检测细胞周期,2’7’-二乙酰二氯荧光素（DCFH-DA）免疫荧光法检测细胞内活性氧簇（R0s）水平,比色法检测细胞-氧化氮（NO）、丙二醛（MDA）及过氧化氢（H202）水平,real-timePCR和Westernblot检测细胞内皮型一氧化氮合酶（eNos）及NADPH氧化酶4（NOX4）mRNA和蛋白表达。结果：Seq预处理1h后能明显减轻高糖诱导的血管内皮细胞损伤,促进细胞增殖,降低胞内NOX4的表达及ROS、MDA及H202水平,上调eNOS的表达及NO水平。结论：Seq对高糖诱导的内皮细胞损伤具有一定的保护作用,其机制可能与其抗氧化、上调eNOS的表达有关。     

低氧诱导大鼠内脏脂肪组织炎症状态对SePP1的影响

目的：既往研究显示SePP1具有一定的抗氧化作用,而随着年龄的增加机体逐步出现一个慢性低氧、炎症状态,我们通过4％O2浓度体外培养大鼠脂肪前体细胞模拟其体内的低氧状态,进而观察常氧（21％O2）及低氧（4％02）状态下大鼠脂肪前体细胞中炎症因子（IL-6,MCP-1,SePP1）水平的变化及不同状态下硒蛋白SePP1水平的变化。方法：取6—8周SD大鼠肾周脂肪前体细胞,分别于常氧（21％O2）及低氧（4％O2）状态下进行体外培养,诱导分化后采用油红0染色进行鉴定,至第三代后,分别采用PCR及Westem Blot技术检测两种状态下脂肪前体细胞中1L-6,MCP-1,SePPl基因及蛋白表达的不同变化,同时观察不同氧浓度对脂肪前体细胞增殖的影响。结果：4％氧浓度状态下培养的脂肪前体细胞中IL-6,MCP-1的基因及蛋白表达均明显高于正常氧浓度下的脂肪前体细胞,而SePP1的基因及蛋白表达均下降,且低氧状态下脂肪前体细胞增殖较常氧状态下加快。结论：低氧培养可进一步使机体内脏脂肪组织堆积加重,造成脂肪前体细胞的炎症状态,并且可导致SePP1的表达下降,而SePP1具有一定的抗氧化作用,与机体动脉粥样硬化等心血管疾病的发生、发展有一定的关联,本实验结论为通过干预体内SePP1的水平为靶点治疗动脉粥样硬化提供了一定的实验依据,为进一步研究SePP1在低氧状态下对动脉粥样硬化的影响及作用机制提供了一定的试验基础。     

RAGE and αVβ3-integrin are essential for suPAR signaling in podocytes

The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in the pathogenesis of kidney diseases including primary and recurrent focal and segmental glomerulosclerosis (FSGS), diabetic nephropathy, and acute kidney injuries (AKI). Elevated serum suPAR concentration is a negative prognostic indicator in multiple critical clinical conditions. This study has examined the initial transduction steps used by suPAR in cultured mouse podocytes. We now report that the receptor for advanced glycation end-products (RAGE) co-immunoprecipitates with αV and β3 integrin subunits, which have been previously shown to initiate suPAR signal transduction at the podocyte cell surface. siRNA knock-down of RAGE attenuated Src phosphorylation evoked by either suPAR or by glycated albumin (AGE-BSA), a prototypical RAGE agonist. suPAR effects on Src phosphorylation were also blocked by the structurally dissimilar RAGE antagonists FPS-ZM1 and azeliragon, as well as by cilengitide, an inhibitor of outside-in signaling through αV-integrins. FPS-ZM1 also blocked Src phosphorylation evoked by AGE-BSA. FPS-ZM1 blocked increases in cell surface TRPC6 abundance, cytosolic reactive oxygen species (ROS) and activation of the small GTPase Rac1 evoked by either suPAR or AGE-BSA. In addition, FPS-ZM1 inhibited Src phosphorylation evoked by serum collected from a patient with recurrent FSGS during a relapse. The magnitude of this inhibition was indistinguishable from the effect produced by a neutralizing antibody against suPAR. These data suggest that orally bioavailable small molecule RAGE antagonists could represent a useful therapeutic strategy for a wide range of clinical conditions associated with elevated serum suPAR, including primary FSGS and AKI.     

云芝糖肽在ROS、p38信号通路中对巨噬细胞Raw264．7的免疫调节作用

目的：探究云芝糖hk（PSP）对静息和脂多糖（LPS）过度刺激两种不同状态的巨噬细胞Raw264．7产生活性氧簇（ROS）TLp38、p-p38的影响及其相关机制。方法：采用流式细胞术方法检测ROS的表达;Westernblot方法检测MAPK信号通路中p38磷酸化及非磷酸化的相对表达量。结果：100g／mlPSP下调1g／ml LPS单独作用于巨噬细胞时ROS及p-p38的表达量;100g／mlPSP上调正常巨噬细胞p-p38的表达量;p38表达量基本不变。结论：PSP通过ROS,p38两条通路参与巨噬细胞的免疫调节作用。     

番茄红素对大鼠乳鼠心肌细胞缺氧复氧的保护作用以及其机制

目的：探讨番茄红素对心肌细胞缺氧复氧的保护作用以及其分子机制。方法：采用原代培养心肌细胞建立缺氧/复氧损伤模型,实验分8组：正常对照组,H/R组,H/R＋番茄红素（1,2,4,8,16,32μmol/L）剂量组。观察各组细胞经H/R损伤后,细胞内天冬氨酸氨基转移酶（AST）、肌酸激酶（CK）、乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量的变化情况,选择正常对照组,H/R组,最佳番茄红素剂量组做MTT分析细胞凋亡,Western检测TRL 4以及NF-κB的表达。结果：番茄红素（16,8,4,2μmol/L）剂量组可显著降低缺氧/复氧损伤心肌细胞内AST、CK、LDH释放量及MDA的生成,并能提高SOD活性。此外番茄红素可减少心肌细胞缺氧/复氧损伤后的心肌凋亡,减少TRL 4受体以及NF-κB的表达。结论：番茄红素具有抗缺氧/复氧损伤,保护心肌细胞的作用,其机制可能是通过抑制TRL 4通路来实现的。     

白藜芦醇对氧糖剥夺／再灌注损伤的PCI2细胞的保护作用及其作用机制

目的：探讨白藜芦醇对氧糖剥夺／再灌注（OGD／R）损伤的PCI2细胞的保护作用及其机制。方法：体外培养PCI2细胞,分为对照组,白藜芦醇组,OGD／R组及OGD／R＋白藜芦醇组。以改良的噻唑蓝法测定细胞活性,采用AnnexinV—FITC／PI双染法检测细胞的凋亡率,用双氯罗丹明（DHR）检测细胞内活性氧簇（Ros）的水平,采用蛋白印迹法（westemblot）分析SIRTl的蛋白表达情况。结果：与对照组相比,经过OGD／R损伤后,细胞活力显著降低。而在OGD／R的同时给予10μmol／L的白藜芦醇处理。可以明显提高细胞活力。流式细胞仪检测发现,10μmol／L的白藜芦醇可以显著地减少OGD／R引起的细胞凋亡,抑制细胞内的ROS产生。westemblot的结果提示,与对照组比较,白藜芦醇可提高SIRTl的蛋白表达水平。结论：白藜芦醇可以通过抑制ROS的产生和上调SIRTl的表达等机制而发挥其对抗氧糖剥夺／再灌注损伤的神经保护性作用。