Advanced oxidation protein products induce apoptosis of human chondrocyte through reactive oxygen species‐mediated mitochondrial dysfunction and endoplasmic reticulum stress pathways

Advanced oxidation production products (AOPPs) have been confirmed to accumulate in patients with rheumatoid arthritis (RA). Previous study demonstrated that AOPPs could accelerate cartilage destruction in rabbit arthritis model. However, the effect of AOPP stimulation on apoptosis of human chondrocyte and the underlying mechanisms remains unclear. This study demonstrated that exposure of chondrocyte to AOPPs resulted in cell apoptosis. AOPP stimulation triggered reactive oxygen species (ROS) production, which induced mitochondrial dysfunction and endoplasmic reticulum stress (ER stress) resulted in caspase activation. Furthermore, an antioxidant, N‐acetylcysteine, markedly blocked these signals. Our study demonstrated that AOPPs induce apoptosis via ROS‐related mitochondria‐ and ER‐dependent signals in human chondrocyte. Targeting AOPP‐triggered ROS generation might be as a promising option for patients with RA.     

吡哆胺对体外晚期糖基化及其终产物形成的抑制作用

目的体外观察吡哆胺对晚期糖基化和晚期糖基化终产物(AGEs)形成的抑制作用。方法应用牛血清白蛋白-葡萄糖试验和N-乙酰-甘氨酰-赖氨酸甲基酯-核糖两种体外试验,观察吡哆胺对糖基化反应及AGEs形成的抑制效果。结果牛血清白蛋白-葡萄糖试验显示,吡哆胺对糖基化反应具有明显抑制效果。在50 mmol/L和200 mmol/L葡萄糖浓度下,当吡哆胺浓度为50 mmol/L时,相对抑制率均在50%以上。200 mmol/L吡哆胺对糖基化的抑制作用最强,其相对抑制率分别达到92.12%和86.73%。N-乙酰-甘氨酰-赖氨酸甲基酯-核糖试验证明,吡哆胺能明显抑制晚期糖基化产物AGEs的形成。随着吡哆胺剂量的增大,吡哆胺对AGEs形成的抑制作用明显增强。结论吡哆胺对体外晚期糖基化反应和AGEs形成有明显抑制作用。     

Targeting the receptor for advanced glycation end products (RAGE) in type 1 diabetes

Type 1 diabetes (T1D) is one of the most common chronic diseases manifesting in early life, with the prevalence increasing worldwide at a rate of approximately 3% per annum. The prolonged hyperglycaemia characteristic of T1D upregulates the receptor for advanced glycation end products (RAGE) and accelerates the formation of RAGE ligands, including advanced glycation end products, high-mobility group protein B1, S100 calcium-binding proteins, and amyloid-beta. Interestingly, changes in the expression of RAGE and these ligands are evident in patients before the onset of T1D. RAGE signals via various proinflammatory cascades, resulting in the production of reactive oxygen species and cytokines. A large number of proinflammatory ligands that can signal via RAGE have been implicated in several chronic diseases, including T1D. Therefore, it is unsurprising that RAGE has become a potential therapeutic target for the treatment and prevention of disease. In this review, we will explore how RAGE might be targeted to prevent the development of T1D.     

晚期糖基化终产物诱导大鼠腹膜间皮细胞上皮-间叶转化

目的探讨晚期糖基化终产物对大鼠腹膜间皮细胞上皮-间叶转化(EMT)的影响,建立大鼠腹膜间皮细胞EMT模型。方法体外培养的大鼠腹膜间皮细胞经含40 mmol/L晚期糖基化终产物(AGEs)、80 mmol/L AGEs的M199培养基分别培养48、72 h;以正常M199培养基和含80 mmol/LBSA的M199培养基为对照。采用相差显微镜观察细胞形态学改变,应用实时定量PCR法检测间皮细胞E-cadherin、α-SMA、Collagen I、TGF-β1、VEGF mRNA的表达;应用Western blot检测E-cadher-in、α-SMA蛋白的表达;应用ELISA法检测间皮细胞TGF-β1、VEGF蛋白表达水平。结果①AGEs(40 mmol/L、80 mmol/L)刺激后,大鼠腹膜间皮细胞由典型的上皮细胞形态逐渐变为梭形及不规则形,类似肌成纤维细胞;②AGEs(40 mmol/L、80mmol/L)刺激后,大鼠腹膜间皮细胞α-SMA、Collagen I、TGF-β1、VEGF mRNA和α-SMA、TGF-β、VEGF蛋白表达水平显著增加(P<0.05);③AGEs(40 mmol/L、80 mmol/L)刺激后,大鼠腹膜间皮细胞E-cadherin mRNA和E-cadherin蛋白表达水平显著下降(P<0.05)。结论 AGEs能上调α-SMA、Collagen I、TGF-β、VEGF表达和下调E-cadherin表达,AGEs诱导大鼠腹膜间皮细胞EMT。     

晚期糖基化终产物在β样淀粉蛋白25-35诱导PC12细胞氧化损伤中的作用

目的探讨晚期糖基化终产物(AGEs)在β样淀粉蛋白25-35(Aβ25-35)诱导PC12细胞氧化损伤中的作用。方法将实验对象分为五组:10、20、304、0μmol/L四种浓度的Aβ25-35干预组和正常对照组。采用MTT法测定细胞生存率,采用ELISA法测定AGEs的含量。结果Aβ25-35诱导PC12细胞,细胞生存率为50%时,Aβ25-35浓度约为30μmol/L;与正常对照组相比,30μmol/L Aβ25-35干预组细胞生存率明显降低(P<0.001)、细胞内AGEs含量明显升高(P<0.05)。结论AGEs参与了β样淀粉蛋白25-35诱导PC12细胞氧化损伤的过程。     

糖尿病前期患者晚期糖基化终末产物受体与氧化应激的关系

目的 通过测定糖耐量正常者、糖尿病前期及糖尿病患者血浆可溶性晚期糖基化终末产物受体(sRAGE)、内源性分泌型晚期糖基化终末产物受体(esRAGE)及氧化应激的水平,探讨糖尿病前期血浆sRAGE、esRAGE水平的变化与氧化应激的相关性,为深入了解糖尿病前期血管并发症的发病机制提供依据.方法 研究对象均行75 g标准口服葡萄糖耐量实验(OGTT)和胰岛素释放试验,按血糖水平分为糖耐量正常(NGT)组、糖尿病前期(Pre-DM)组、糖尿病(DM)组,采用酶联免疫吸附检测(ELISA)法检测血浆sRAGE、esRAGE、8-异前列腺素F2α(8-isoPGF2α);硫代巴比妥酸(TBA)法测定丙二醛(MDA),羟胺法测定超氧物岐化酶(SOD),比色法测定血浆总抗氧化能力(TAOC).结果 Pre-DM组和DM组的sRAGE、esRAGE、SOD水平明显低于NGT组,Pre-DM组和DM组的8-iso-PGF2α、MDA水平明显高于NGT组,差异均有统计学意义(P＜0.05);Pre-DM组和NGT组的TAOC水平明显高于DM组(P＜0.05);血浆esRAGE水平与sRAGE、TAOC呈正相关(r=0.39、0.64,P＜0.05),与MDA呈负相关(r =-0.45,P＜0.05),与年龄、体质量指数(BMI)、收缩压、舒张压、空腹血糖、空腹胰岛素、糖化血红蛋白、甘油三酯、低密度脂蛋白、高密度脂蛋白、8-iso PGF2α无明显相关性(P＜0.05).结论 糖尿病前期患者已发生sRAGE、esRAGE及氧化应激水平的改变,sRAGE、esRAGE在机体氧化应激与抗氧化防御平衡间可能发挥重要作用.     

罗格列酮对糖基化终产物诱导的大鼠心肌成纤维细胞及胶原蛋白合成的影响

目的探讨罗格列酮(RGZ)对糖基化终产物(AGEs)诱导的大鼠心肌成纤维细胞(CFs)及胶原蛋白合成的影响。方法采用胰酶消化法和差速贴壁分离法获取CFs,经AGEs诱导,并给予不同浓度RGZ干预48 h,收集培养上清液,应用四甲基偶氮唑盐比色法(MTT)检测不同浓度RGZ对AGEs作用下的CFs增殖的影响;应用酶联免疫吸附试验(ELISA)检测干预前后胶原蛋白Ⅰ、Ⅲ含量。结果与AGEs 0 mg/L组比较,经不同浓度AGEs诱导后,各组CFs OD490 nm值有所升高,且随AGEs浓度的增加而呈递增趋势;与未干预组比较,各干预组OD490 nm值、胶原蛋白Ⅰ、Ⅲ含量均显著降低,且随RGZ浓度的增加呈递减趋势(P0.05或P0.01)。结论 RGZ对AGEs诱导的CFs增殖及促进胶原蛋白Ⅰ、Ⅲ合成作用具有明显的抑制效应,且抑制程度呈浓度依赖性,可有效改善心肌纤维化状态。     

Advanced glycation end product is implicated in amyloid‐related kidney complications

Background. Kidney failure is a common complication in familial amyloidotic polyneuropathy (FAP). It has been suggested that advanced glycation end products (AGEs) play an important role in the development and pathogenesis of FAP. Material and methods. To evaluate the impact of AGEs on FAP patients' kidney dysfunction, we measured AGE in serum and urine of 28 FAP patients and 18 healthy controls by AGE‐specific enzyme‐linked immunosorbent assay (ELISA). Immunohistochemistry utilizing antibodies to AGE and the receptor for AGE (RAGE) were used on kidney tissue from 3 FAP patients and 3 diabetic patients to disclose a correlation between amyloid deposits and AGE–RAGE. Results. The glomeruli of FAP patients were heavily deposited with amyloid and the glomerular size was enlarged. The space between Bowman's capsule and glomerulus was totally covered by the enlarged glomerulus. AGE and RAGE were deposited in glomeruli and tubuli and correlated with amyloid deposits. Decreased AGE levels in the liver‐transplanted FAP patients' serum compared with that of non‐transplanted patients were noted, and AGE concentration in serum tended to be higher in non‐transplanted FAP patients compared with normal control subjects. There were no differences in the AGE urine levels in FAP patients compared with controls. No correlation could be found between AGE in urine and serum compared with serum albumin, serum creatinine and creatinine clearance. Conclusions. The accumulation of AGE, RAGE and amyloid in the kidney of FAP patients suggests that these molecules play an important role in the origin and pathogenesis of renal failure in FAP patients.     

糖基化终产物对大鼠视网膜超微结构及凋亡的影响

目的观察外源性非酶糖基化终产物(AGEs)对大鼠视网膜超微结构以及细胞凋亡的影响。方法应用鼠血清白蛋白(RSA)体外孵育AGEs修饰蛋白,并将其注入健康大鼠体内,每日一次,连续两周。TUNEL法检测大鼠视网膜凋亡细胞,透射电镜下观察内核层神经细胞的超微变化。结果AGEs组大鼠视网膜内核层可见凋亡阳性细胞,透射电镜下深染细胞明显增多,细胞核缩小、不规则,染色质形成大小不等的团块凝结、边聚于核膜处。胞质深染,细胞器损害。结论AGEs可使大鼠视网膜内核层神经细胞凋亡,超微结构改变。     

骨形成蛋白-7对晚期糖基化终产物诱导大鼠腹膜间皮细胞上皮-间叶转化的影响

目的探讨骨形成蛋白-7(BMP-7)对晚期糖基化终产物诱导大鼠腹膜间皮细胞上皮-间叶转化(EMT)的影响。方法晚期糖基化终产物诱导发生上皮-间叶转化的体外培养大鼠腹膜间皮细胞,分别经含5 ng/mL及80 mmol/L晚期糖基化终产物的M199培养基和含10 ng/mL BMP-7及80 mmol/L晚期糖基化终产物的M199培养基培养48 h,以含80 mmol/L晚期糖基化终产物的M199培养基为对照,应用实时定量PCR法检测间皮细胞E-cadherin、α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(Collagen I)、转化生长因子β1(TGF-β1)、血管内皮生长因子(VEGF)mRNA的表达;应用Western印迹法检测E-cadherin、α-SMA蛋白的表达;应用ELISA法检测间皮细胞TGF-β1、VEGF蛋白表达水平。结果 BMP-7作用后,上皮-间叶转化的大鼠腹膜间皮细胞E-cadherin mRNA和E-cadherin蛋白表达水平显著增加(P<0.05)。BMP-7作用后,上皮-间叶转化的大鼠腹膜间皮α-SMA、Collagen I、TGF-β1、VEGFmRNA和α-SMA、TGF-β、VEGF蛋白表达水平显著下降(P<0.05)。结论 BMP-7能上调上皮-间叶转化的大鼠腹膜间皮细胞E-cadherin表达和下调α-SMA、Collagen I、TGF-β、VEGF表达,BMP-7能逆转晚期糖基化终产物诱导大鼠腹膜间皮细胞EMT。     

microRNA-192在晚期糖基化终产物诱导人腹膜间皮细胞上皮-间叶转化中的作用

目的探讨microRNA-192(miR-192)对晚期糖基化终产物(AGEs)诱导人腹膜间皮细胞上皮-间叶转化(EMT)的调控作用。方法人腹膜间皮细胞、转染miR-192抑制物后人腹膜间皮细胞和转染miR-192抑制物阴性对照的人腹膜间皮细胞在含AGEs的培养基培养72 h,以M199培养基和含80 m M BSA的M199培养基为对照,然后运用实时荧光定量PCR法检测miR-192和mRNA的表达情况,运用蛋白质印迹法检测蛋白的表达情况。结果在AGEs刺激后,人腹膜间皮细胞miR-192、胶原蛋白I(Collagen I)mRNA、α-平滑肌肌动蛋白(α-SMA)mRNA和蛋白表达水平显著上升(P0.05),而E-钙黏蛋白(E-cadherin)mRNA和蛋白表达水平显著下降(P0.05)。与转染miR-192抑制物阴性对照的人腹膜间皮细胞相比,转染miR-192抑制物的人腹膜间皮细胞miR-192、Collagen I mRNA、α-SMAmRNA和蛋白表达水平显著下降(P0.05),而E-cadherin mRNA和蛋白表达水平显著上升(P0.05)。结论 AGEs可能通过上调miR-192表达诱导人腹膜间皮细胞EMT。miR-192抑物可能通过下调miR-192表达阻止AGEs诱导人腹膜间皮细胞EMT。miR-192在AGEs诱导人腹膜间皮细胞EMT中起重要调控作用。     

脂质体槲皮素对糖尿病大鼠视网膜糖基化终产物生成及受体表达的影响

目的观察脂质体槲皮素（LQ）对糖尿病（DM）大鼠视网膜糖基化终产物（AGEs）生成及受体（RAGEmRNA）表达的影响。方法采用蒸发-高压均质法制备LQ悬浊液,链脲佐菌素（STZ）法诱导DM大鼠模型,并随机分为DM模型组、LQ低、中、高剂量及氨基胍灌胃组;连续给药12周后,免疫荧光法、RT—PCR方法分别检测DM大鼠视网膜中AGEs沉积情况及RAGEmRNA的表达。结果与DM模型组结果（AGEs：0．922±0．005;RAGEmRNA：1．192±0．098）比较,LQ各组及氨基胍组视网膜中AGEs沉积减少,RAGEmRNA表达也降低（P〈0．05）,以LQ中剂量组（AGEs：0．734±0．007;RAGEmRNA：0．724±0．028）降低最为明显（P〈0．01）。结论LQ可通过有效减少DM大鼠视网膜中AGEs的沉积,下调RAGEmRNA的表达。     

Soluble receptor for advanced glycation end products predicts 28-day mortality in critically ill patients with sepsis

Abstract

Objective. Multiple biomarkers are used to assess sepsis severity and prognosis. Increased levels of the soluble receptor for advanced glycation end products (sRAGE) were previously observed in sepsis but also in end-organ injury without sepsis. We evaluated associations between sRAGE and (i) 28-day mortality, (ii) sepsis severity, and (iii) individual organ failure. Traditional biomarkers procalcitonin (PCT), C-reactive protein (CRP) and lactate served as controls. Methods. sRAGE, PCT, CRP, and lactate levels were observed on days 1 (D1) and 3 (D3) in 54 septic patients. We also assessed the correlation between the biomarkers and acute respiratory distress syndrome (ARDS), acute kidney injury (AKI) and acute heart failure. Results. There were 38 survivors and 16 non-survivors. On D1, non-survivors had higher sRAGE levels than survivors (p = 0.027). On D3, sRAGE further increased only in non-survivors (p < 0.0001) but remained unchanged in survivors. Unadjusted odds ratio (OR) for 28-day mortality was 8.2 (95% CI: 1.02–60.64) for sRAGE, p = 0.048. Receiver operating characteristic analysis determined strong correlation with outcome on D3 (AUC = 0.906, p < 0.001), superior to other studied biomarkers. sRAGE correlated with sepsis severity (p < 0.00001). sRAGE showed a significant positive correlation with PCT and CRP on D3. In patients without ARDS, sRAGE was significantly higher in non-survivors (p < 0.0001) on D3. Conclusion. Increased sRAGE was associated with 28-day mortality in patients with sepsis, and was superior compared to PCT, CRP and lactate. sRAGE correlated with sepsis severity. sRAGE was increased in patients with individual organ failure. sRAGE could be used as an early biomarker in prognostication of outcome in septic patients.     

氧化应激和糖基化终末产物在2型糖尿病心肌病变中的作用及其相关性

目的研究氧化应激因素和糖基化终末产物(AGEs)在2型糖尿病(T2DM)心肌病变(DCM)的作用及其相关性。方法选择泰安地区的T2DM患者78例,根据超声心动图、冠状动脉造影等情况,分为单纯T2DM组40例,DCM组38例,另选取正常对照组(NC)40例。测定超氧化物歧化酶(SOD)、丙二醛(MDA)、一氧化氮(NO)以及谷胱甘肽过氧化物酶(GHS-PX)等氧化应激指标和糖基化终末产物(AGEs),进行数据统计,并进行相关性分析。结果 T2DM组SOD、NO、GHS-PX较NC组均明显下降,MDA、AGEs则较NC组均明显升高;而DCM组较T2DM组SOD、GHS-PX明显下降,MDA、AGEs明显升高,差异均有统计学意义(P<0.01),而血清NO水平T2DM组较NC组下降,DCM组再次升高,且高于NC组,差异均有统计学意义(P<0.01)。结论氧化应激因素和AGEs在DCM的进展中均有重要作用,促进DCM的发生和发展,且二者之间有相关性,SOD的下降和MDA的升高是AGEs的独立相关因素。     

拮抗晚期糖基化终末产物受体对烫伤延迟复苏大鼠多器官功能及死亡率的影响

目的 观察拮抗晚期糖基化终末产物受体(RAGE)对烫伤延迟复苏动物多器官功能及死亡率的影响,并探讨其作用机制.方法 选取雄性Wistar大鼠,采用重度烫伤延迟复苏模型(30%总体表面积Ⅲ度烫伤).实验分为两部分.死亡率观察:130只大鼠被随机分为假手术组(n=10)、烫伤组(n=60)及RAGE抗体治疗组(n=60),观察伤后7 d内每日动物的存活率.器官功能观察:72只大鼠被随机分为假手术组(n=24)、烫伤组(n=24)及RAGE抗体治疗组(n=24),于伤后1、3、5和7 d各活杀6只动物,取血,用全自动生化分析仪测定肝、肾和心脏器官功能指标.结果 烫伤组伤后1～7 d,大鼠血中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、肌酐(cr)、尿素氮(BUN)及肌酸激酶同工酶(CK-MB)水平均显著高于假手术组(P<0.05或P<0.01),其中ALT、AST、Cr及BUN水平均于伤后3 d达峰值;给予RAGE抗体治疗可不同程度降低烫伤动物血中各指标水平,其中AST在伤后1～7 d显著下降,余指标在伤后1～5 d均明显改善(P<0.05或P<0.01).RAGE抗体治疗组伤后7 d内每日大鼠存活率显著高于烫伤组(P<0.05或P<0.01).结论 RAGE抗体干预能明显改善重度烫伤延迟复苏大鼠的预后,并对重要器官功能具有显著保护作用.     

晚期糖基化终产物与冠心病发病机制的研究进展

晚期糖基化终产物(AGEs)密切参与了血管平滑肌细胞(VSMCs)分化与增殖以及冠心病等心血管疾病的病理生理过程。AGEs能通过单核细胞趋化蛋白(ERK)、丝氨酸/苏氨酸蛋白激酶(Akt)信号通路来诱导VSMCs的自噬作用,可依赖骨髓基质细胞衍生因子-1(SDF-1)/趋化因子受体CXCR4轴信号通路促进心肌微血管内皮细胞(CMECs)的增生。AGEs-2和AGEs-3上调了单核细胞AGEs受体(RAGE)的表达。AGEs能抑制内皮祖细胞(EPCs)的增殖、迁移和黏附功能并诱导EPCs凋亡;能增加平滑肌细胞结缔组织因子(CTGF)mRNA和蛋白质的表达,刺激心肌成纤维细胞增殖并分泌转化生长因子-β1(TGF-β1),同时诱导Smad2及Smad4的表达。羧甲基赖氨酸(CML)/RAGE轴通过主动脉平滑肌成骨细胞的分化,诱导巨噬细胞凋亡,从而使AGEs在糖尿病动脉粥样硬化中发挥重要作用。可溶性RAGE(sRAGE)可作为RAGE配体的诱饵来防止动脉粥样硬化,其灵敏度和阴性预测值在判定冠状动脉介入治疗(PCI)术后再狭窄方面均高于AGEs/sRAGE比值。ALT-711是一种AGEs的裂解剂,能明显抑制AGEs介导的活性氧(ROS)产生、细胞外信号调节激酶磷酸化及环氧合酶-2的表达;色素上皮衍生因子(PEDF)能抑制AGEs诱导的血小板CD40配体(CD40L)表达,从而有可能成为预防冠心病的一个治疗靶点。他汀类药物亦能抑制AGEs诱导主动脉平滑肌细胞的增殖及ROS的产生。通过以上诸多因素的研究,可揭示冠心病的某些发病机制,为相应干预药物的研究及调整临床治疗策略提供依据和方向。     

2型糖尿病大鼠骨折愈合障碍与体内晚期糖基化终末产物的变化

背景：近年来,晚期糖基化终末产物在骨组织领域的作用日益受到重视,而糖代谢紊乱是引起晚期糖基化终末产物增加的主要原因之一。 目的：观察2型糖尿病大鼠体内晚期糖基化终末产物表达的变化,并探讨其与糖尿病骨折愈合障碍的关系。方法：30只SD大鼠随机均分为2组,实验组制备2型糖尿病模型,对照组正常饲养。糖尿病模型制备成功后,所有大鼠建立左胫骨骨折牵引成骨模型,胫骨延长0.3 mm/d,持续14 d。 结果与结论：牵引结束后,X 射线摄片显示实验组糖尿病模型大鼠骨折断端之间牵引骨痂形成较对照组明显减少;骨痂组织学检查表现为微骨柱排列紊乱,初始基质前沿浅染。ELISA 法检测实验组血清和双侧骨痂组织中晚期糖基化终末产物水平较对照组明显升高（P 〈0.01）,骨钙素明显降低（P 〈0.01）。提示2型糖尿病大鼠骨折牵引骨痂生成障碍,而骨组织中晚期糖基化终末产物水平增高可能是导致2型糖尿病骨折愈合障碍的原因。