糖基化终末产物对角膜上皮创伤愈合的影响及机制研究
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摘要
研究背景
随着人民生活水平的提高、生活方式的改变和人口老龄化,我国糖尿病发病率呈明显的逐年增高趋势。糖尿病已经构成严重威胁人类健康的世界性问题。糖尿病患者在角膜外伤或接受角膜手术时极易出现角膜上皮愈合延迟甚至不愈,临床表现为：持续性角膜上皮损失、反复性角膜上皮糜烂、浅层角膜溃疡形成、继发严重角膜感染甚至失明。目前尚没有有效的治疗方法。因此,研究糖尿病角膜上皮创伤愈合延迟的发病机制和防治措施已成为眼科亟待解决的课题。
研究发现,持久的高血糖状态可导致糖尿病患者体内多种蛋白质、脂质甚至核酸发生糖基化反应,形成具有结构多样、高度活性的糖基化终末产物(advanced glycation end products, AGEs)。正常情况下体内AGEs的水平随年龄的增长而缓慢增加,而在糖尿病患者体内,病理性高血糖可加速糖基化反应,形成大量的AGEs,并在组织中蓄积。AGEs具有不可逆性,这使其在高血糖被纠正后也不能回复到正常水平。AGEs具有广泛的生物学活性,参与了多种糖尿病并发症的发生、发展。随着对AGEs研究的深入,人们发现AGEs与糖尿病皮肤创面愈合延迟密切相关。近年研究发现,AGEs大量蓄积在糖尿病患者及糖尿病动物模型的角膜上皮及基底膜中,但其是否参与角膜上皮创伤愈合过程而导致创伤愈合延迟?目前尚不清楚。因此,深入研究AGEs在角膜上皮创伤愈合延迟中的作用,将进一步揭示糖尿病角膜上皮创伤愈合延迟的机制,为其防治提供新的理论依据。
AGEs与细胞表面特异性受体(receptor for advanced glycation end products, RAGE)结合,能够增加细胞内活性氧(Reactive oxygen species, ROS)的生成,诱导细胞氧化应激,造成组织氧化损伤。RAGE作为信号转导受体,介导AGEs结合在细胞表面,激活细胞内各种转导信号。研究证实RAGE在正常单核巨噬细胞、血管内皮细胞、肾系膜细胞、神经细胞及平滑肌细胞等细胞中呈低水平表达,但在糖尿病条件下其表达明显增加。AGEs与RAGE表达之间存在正反馈调节机制,AGEs蓄积的病变部位往往伴随着RAGE表达的增加。ROS是指化学性质活跃的氧代谢产物或由其衍生的产物,主要有超氧阴离子、过氧化氢、羟自由基等。ROS生成过多将在细胞内形成氧化应激状态,通过氧化反应破坏许多重要生物大分子的结构和功能,导致疾病的发生、发展。
目前关于AGEs对角膜上皮创伤愈合的影响及机制研究在国内外尚未见报导。AGEs是否延迟角膜上皮创伤愈合过程?是否通过受体RAGE及ROS发挥作用?具体机制如何?回答这些问题将从根本上揭示AGEs在糖尿病角膜上皮创伤愈合延迟中的作用及机制,不仅对理解糖尿病角膜上皮创伤愈合延迟病理机制具有重要的意义,也必将为糖尿病角膜上皮创伤愈合延迟的防治提供新理论、新视角和新靶点。因此,本研究以体外制备的糖基化终末产物(AGE-BSA)作为干预因素,观察AGE-BSA对人永生化角膜上皮细胞(Human telomerase-immortalized corneal epithelial cells, THCE) RAGE、ROS表达的影响并研究其机制,探讨AGE-BSA对THCE细胞增殖、迁移以及角膜上皮创伤愈合的影响及机制。全文共分三部分：(一)AGE-BSA对THCE细胞RAGE及ROS表达的影响；(二)AGE-BSA诱导THCE细胞氧化应激机制的研究；(三)AGE-BSA对THCE细胞增殖、迁移及角膜上皮创伤愈合的影响。
第一部分糖基化终末产物对角膜上皮细胞糖基化终末产物受体及活性氧表达的影响
目的：研究AGEs对THCE细胞RAGE及ROS表达的影响。
方法：1.用D-葡萄糖和牛血清白蛋白(Bovine serum albumin, BSA)共同孵育10周制备糖基化终末产物(AGE-BSA)。
2.分别用浓度为50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA处理THCE细胞24h和浓度为2000μg/ml的AGE-BSA分别处理THCE细胞6h、12h、24h、48h。Real-time PCR和Western blot检测ⅠRAGE mRNA和蛋白的表达；
3.分别用浓度为50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA处理THCE细胞12h。预先应用anti-RAGE中和抗体处理THCE细胞1h,再用浓度为200μg/ml的AGE-BSA孵育细胞12h,激光共聚焦显微镜和流式细胞仪检测ROS的表达。
结果：1.体外制备的AGE-BSA的荧光强度为55.96荧光单位/mg蛋白,而BSA的荧光强度为1.98荧光单位/mg蛋白。
2.未经干预的THCE细胞表达少量的RAGE mRNA和蛋白,用BSA干预后RAGE mRNA和蛋白的表达与对照组无明显差异。与对照组相比,浓度为50μg/ml的AGE-BSA明显上调THCE细胞RAGE mRNA和蛋白的表达(P<0.05),随着AGE-BSA浓度的增加,RAGE mRNA和蛋白的表达逐渐增高,AGE-BSA浓度为20μg/ml时达到峰值(P<0.05)。
3.在浓度为200μg/ml的AGE-BSA作用下,AGE-BSA作用THCE细胞6h明显上调RAGE mRNA的表达(P<0.05),AGE-BSA作用THCE细胞12h明显上调RAGE蛋白的表达(P<0.05),随着AGE-BSA作用时间的延长,RAGE mRNA和蛋白的表达逐渐增高,AGE-BSA作用时间为24h时达到峰值(P<0.05)。
4.未经干预的THCE细胞表达极少量的ROS,用BSA干预后ROS的表达与对照组无明显差异。与对照组相比,浓度为100μg/ml的AGE-BSA明显上调THCE细胞ROS的表达(P<0.05),随着AGE-BSA浓度的增加,ROS的表达逐渐增高,AGE-BSA浓度为200μg/ml时达到峰值(P<0.05)。
5. anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著抑制AGE-BAS对ROS表达的上调作用(P<0.05)。
结论：1.AGE-BSA显著增加THCE细胞RAGE mRNA、蛋白及ROS的表达。
2. AGE-BSA通过与受体RAGE结合,诱导THCE细胞大量生成ROS,导致角膜上皮细胞氧化损伤,可能参与糖尿病角膜上皮创伤愈合延迟过程。
第二部分糖基化终末产物诱导角膜上皮细胞氧化应激机制的研究
目的：探讨AGEs诱导THCE细胞氧化应激的机制。
方法：1.预先应用NADPH氧化酶抑制齐apocynin及Diphenyleneiodonium (DPI)、线粒体酶复合体Ⅰ抑制齐rotenone、线粒体酶复合体Ⅱ抑制剂thenoyltrifluoroacetone (TTFA)、线粒体酶复合体Ⅲ抑制齐antimycin A、黄嘌呤氧化酶抑制齐allopurinol处理THCE细胞1h后,再用浓度为200μg/m1的AGE-BSA孵育细胞12h,应用流式细胞仪检测ROS的表达。
2.预先应用anti-RAGE中和抗体处理THCE细胞1h,再用浓度为200μg/m1的AGE-BSA孵育细胞12h,应用Real-time PCR检测NADPH氧化酶亚基p22phox、 NOX4mRNA的表达；Western blot检测p22phox、NOX4蛋白的表达。
3.预先应用anti-RAGE中和抗体处理THCE细胞1h,再用浓度为200μg/ml的AGE-BSA呼育细胞12h,检测抗氧化酶超氧化物歧化酶(Superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)的活性及丙二醛(malondialdehyde, MD A)的含量。
结果：1.与对照组相比,AGE-BSA显著上调THCE细胞ROS的表达(P<0.05),NADPH氧化酶抑制剂apocynin和DPI显著抑制AGE-BAS对ROS表达的上调作用(P<0.05),而线粒体酶复合体抑制剂、黄嘌呤氧化酶抑制剂对ROS的表达无明显影响。
2.与对照组相比,AGE-BSA明显上调THCE细胞P22phox、NOX4mRNA和蛋白的表达(P<0.05),应用anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著抑制AGE-BAS对p22phox、NOX4mRNA和蛋白表达的上调作用(P<0.05)。
3.与对照组相比,AGE-BSA显著降低THCE细胞SOD和CAT的活性(P<0.05)；应用anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著减轻AGE-BAS对SOD和CAT活性的抑制作用(P<0.05)
4.与对照组相比,AGE-BSA显著增加THCE细胞MDA的含量(P<0.05)；应用anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著抑制AGE-BAS对MDA含量的上调作用(P<0.05)
结论：1. AGE-BSA通过与受体RAGE结合,诱导THCE细胞NADPH氧化酶亚基p22phox、NOX4mRNA和蛋白高表达,促使NADPH氧化酶激活,生成大量ROS。
2. AGE-BSA通过与受体RAGE结合,降低THCE细胞抗氧化酶SOD口CAT的活性、增加MDA的含量,导致氧化／抗氧化系统失衡,促使角膜上皮细胞处于氧化应激状态。
第三部分糖基化终末产物对角膜上皮细胞增殖、迁移及角膜上皮创伤愈合的影响
目的：探讨AGEs对THCE细胞增殖、迁移及角膜上皮创伤愈合的影响。
方法：1.分别用浓度为50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA与THCE细胞共培养24h,CCK-8法检测THCE细胞增殖能力；刮痕愈合实验检测THCE细胞迁移能力。
2.预先应用anti-RAGE中和抗体、抗氧化剂乙酰半胱胺酸(N-acetyl-L-cysteine, NAC)处理THCE细胞1h,再用浓度为200μg/ml的AGE-BSA孵育细胞24h, CCK-8法检测THCE细胞增殖能力；刮痕愈合实验检测THCE细胞迁移能力。
3.体外培养猪角膜上皮创伤器官模型,在角膜中央制作直径为5mm的上皮损伤区域。预先应用anti-RAGE中和抗体、抗氧化剂NAC处理1h,再用浓度为200μg/ml的AGE-BSA5孵育48h,观察角膜上皮创伤愈合程度。
结果：1.与对照组相比,浓度为50μg/ml的AGE-BSA显著抑制THCE细胞的增殖、迁移(P<0.05),随着AGE-BSA浓度的增加,细胞增殖、迁移能力逐渐降低,AGE-BSA浓度为200μg/ml寸达到最低。anti-RAGE中和抗体、NAC显著减轻AGE-BAS对细胞增殖、迁移的抑制作用。
2.与对照组相比,浓度为200μg/ml的AGE-BSA显著延迟体外培养猪角膜上皮创伤器官模型中角膜上皮创伤愈合过程(P<0.05),anti-RAGE中和抗体、NAC可显著促进角膜上皮创伤的愈合(P<0.05)
结论：1. AGE-BSA显著抑制THCE细胞增殖、迁移,并且呈浓度依赖性。
2. AGE-BSA通过与受体RAGE结合,诱导ROS大量生成,引发氧化应激反应,进而抑制THCE细胞增殖、迁移,延迟角膜上皮创伤愈合过程。阻断AGE-BSA与RAGE结合,或清除过多的ROS,可成为防治糖尿病角膜上皮创伤愈合延迟的新途径。
Background
With the improvement of people's living standards, lifestyle changes and an aging population, the incidence of diabetes in China was increasing year by year. Diabetes constitutes a serious threat to human health worldwide. Diabetic patient's corneal trauma or corneal surgery easily occur corneal epithelial healing delay or unhealed, clinical manifestations:persistent corneal epithelial loss, recurrent corneal erosion, superficial corneal ulcer formation, secondary to severe corneal infection and even blindness. There is currently no effective treatment. Therefore, the studies of the pathogenesis and prevention measures for delayed diabetic corneal epithelial wound healing are the ophthalmic problems to be solved.
The study found that persistent hyperglycemia of diabetes patients can lead to a variety of proteins, lipids or nucleic acid glycosylation reaction occurs, the formation of structural diversity, highly reactive advanced glycation end products (advanced glycation end products, AGEs). Normally, AGEs level in the body increase with age slowly. However, pathological hyperglycemia of diabetic patients can accelerate glycosylation reaction in vivo, the formation of a large number of AGEs in the tissues. AGEs are irreversible, so even hyperglycemia is corrected it can not return to normal levels. AGEs has a wide range of biological activity, participate in a variety of complications of diabetes development. As AGEs research depth, it was found that AGEs and diabetic skin wound healing delay is closely related. Recent studies have found that the accumulation of AGEs in corneal epithelium and basement membrane of diabetic patients and animal models of diabetes, but it is involved in corneal epithelial wound healing process and result in delayed wound healing? Now is unclear. Therefore, study the effect of AGEs in corneal epithelial wound healing will further reveal diabetic delayed corneal epithelial wound healing mechanism to provide a new theoretical basis for its prevention and control.
AGEs combination with cell surface specific receptor (receptor for advanced glycation end products, RAGE) can increase intracellular ROS (Reactive oxygen species, ROS) generation induced cellular oxidative stress, resulting in oxidative damage. RAGE as a receptor signal transduction mediated AGEs binding at the cell surface, activation of cell internal variety of signal transduction mechanisms. The study confirmed that RAGE was expressed at low levels in normal mononuclear macrophages, endothelial cells, mesangial cells, neural cells and smooth muscle cells and other cells, but its expression was significantly increased in diabetic conditions. AGEs and RAGE expression between positive feedback regulation mechanisms. The AGEs increased lesion is often accompanied by increased expression of RAGE. ROS refers to the chemical properties of active oxygen metabolites or oxygen-containing product derived therefrom, mainly superoxide anion, hydrogen peroxide, hydroxyl radicals. Excessive ROS generation will lead to cell oxidative stress and damage the structure and function of many important biological macromolecules by the oxidation reaction, leading to the occurrence and development of the disease.
Now, Effects and mechanism of AGEs in corneal epithelial wound healing has not been reported. The AGEs whether delayed comeal epithelial wound healing process? Whether through its receptor RAGE and ROS? Specific mechanism? Answers to these questions will fundamentally reveal the mechanism of AGEs in diabetic delayed corneal epithelial wound healing, is of great significance not only for the understanding of pathological mechanisms of diabetic delayed corneal epithelial wound healing, will also for the providing new theories, new perspectives and new target prevention of diabetic delayed corneal epithelial wound healing. Therefore, this study prepared in vitro AGE-BSA as an intervention factors, study the effect of AGE-BSA on RAGE, ROS expression in immortalized human corneal epithelial cells,(THCE) and its mechanism, and explore the effect of AGE-BSA on THCE cells proliferation, migration and corneal epithelial wound healing and mechanisms. This study is divided into three parts:(1) Effect of AGE-BSA on RAGE and ROS expression in THCE cell;(2) Effect of AGE-BSA on oxidative stress in THCE cell;(3) Effect of AGE-BSA on THCE cell proliferation, migration and corneal epithelial wound healing.
Part I Effect of advanced glycosylation end products on the expression of receptor for advanced glycosylation end products and reactive oxygen species in human corneal epithelial cells.
Purpose:
To investigate the effect of AGEs on the expression of RAGE and ROS in human corneal epithelial cells.
Methods and Materials:
1. D-glucose and bovine serum albumin (Bovine serum albumin, BSA) incubate for10weeks to prepare glycosylated bovine serum albumin (AGE-BSA), and that of AGEs.
2. Respectively, with a concentration of50μg/ml,100μg/ml,200μg/ml,400ug/ml of AGE-BSA treatment THCE cells for24h and the concentration of200μg/ml AGE-BSA treatment THCE cells6h,12h,24h,48h. Real-time PCR and Western blot detect RAGE mRNA and protein expression.
3. Respectively, with a concentration of50μg/ml,100μg/ml,200μg/ml,400μg/ml of AGE-BSA treatment THCE cells for12h. Pre-application of anti-RAGE antibody treatment THCE cells lh, and then the concentration of200μg/ml AGE-BSA incubated the cells for12h, confocal laser scanning microscopy and flow cytometry detect ROS expression.
Results:
1. Prepared in vitro AGE-BSA fluorescence intensity of55.96fluorescence units/mg protein, while the fluorescence intensity of the BSA1.98fluorescence units/mg protein. AGE-BSA prepared can be used for later experiments.
2. Without intervention THCE cells express a small amount of RAGE mRNA and protein, BSA intervention RAGE mRNA and protein expression was no significant difference with the control group. Compared with the control group, the concentration of50μg/ml AGE-BSA significantly unregulated THCE cell RAGE mRNA and protein expression (P<0.05), RAGE mRNA and protein expression gradually increased with the increase of the concentration of AGE-BSA, AGE-BSA concentration of200μg/ml reached the peak (P<0.05).
3. At a concentration of200μg/ml AGE-BSA significantly up-regulated expression of RAGE mRNA at6h (P<0.05), significantly up-regulated the expression of RAGE protein at12h (P<0.05), with the extension of the duration of action of AGE-BSA, RAGE mRNA and protein expression gradually increased, reached the peak at24h (P<0.05).
4. Without intervention THCE cells can express a very small amount of ROS, BSA intervention ROS expression was no significant difference with the control group. Compared with the control group,100μg/ml AGE-BSA upregulated the expression of ROS in THCE cells (P<0.05), the expression of ROS is increased gradually with the increase of the concentration of AGE-BSA,200μg/ml AGE-BSA reached a peak (P<0.05).
5. Anti-RAGE antibody blocking the interaction AGE-BSA with RAGE, significantly inhibited the AGE-BAS upregulates the expression of ROS (P<0.05).
Conclusions:
1. AGE-BSA significantly increase RAGE mRNA, protein expression and ROS in THCE cell.
2. AGE-BSA binding with its receptor RAGE induced ROS expression, resulting in oxidative damage, involved the occurrence and development of diabetic delayed corneal epithelial wound healing.
PartⅡ Effect of advanced glycosylation end products on oxidative stress in human corneal epithelial cells.
Purpose:
To investigate the effect of AGEs on oxidative stress in human corneal epithelial cells.
Methods and Materials:
1. Pre-application NADPH oxidase inhibitor apocynin and diphenyleneiodonium (DPI), mitochondrial enzyme complex I inhibitor rotenone, mitochondrial enzyme complex Ⅱ inhibitor thenoyltrifluoroacetone (TTFA), mitochondrial enzyme complexthe body Ⅲ inhibitor antimycin A, xanthine oxidase inhibitor allopurinol treatment THCE cells for1h, and then200μg/ml AGE-BSA incubated cells for12hours and analyzed the expression of ROS by flow cytometry.
2. Pre-application of anti-RAGE antibody treatment THCE cell for1h, and then200μg/ml AGE-BSA incubated cells for12h, Real-time PCR detect p22phox, NOX4mRNA expression; Western blot detect p22phox, NOX4protein expression.
3. Pre-application of anti-RAGE antibody treatment THCE cell for1h, then200μg/ml AGE-BSA incubated cells for12h, detection of the antioxidant enzyme superoxide dismutase (SOD), catalase (CAT) activity and malondialdehyde (MDA) content.
Results:
1. Compared with the control group, AGE-BSA significantly up-regulated the expression of ROS in THCE cells (P<0.05), NADPH oxidase inhibitors apocynin and DPI significantly inhibited AGE-BAS upregulates the expression of ROS (P<0.05), mitochondrial enzyme complex inhibitor and xanthine oxidase inhibitor had no significant effect on the expression of ROS.
2. Compared with the control group, AGE-BSA significantly increased p22phox, NOX4mRNA and protein expression inTHCE cells (P<0.05), application of anti-RAGE antibody blocking AGE-BSA interaction with RAGE, significantly inhibite p22phox, NOX4mRNA and protein expression (P<0.05).
3. Compared with the control group, AGE-BSA significantly reduce the activity of SOD and CAT in THCE cells (P<0.05); application of anti-RAGE antibody blocking AGE-BSA interaction with RAGE, significantly reduce AGE-BAS inhibition of SOD and CAT activities (P<0.05).
4. Compared with the control group, AGE-BSA significantly increase the content of MDA in THCE cells (P<0.05); application of anti-RAGE antibody blocking AGE-BSA interaction RAGE could significantly inhibit AGE-BAS upregulation of MDA content (P<0.05).
Conclusions:
1. AGE-BSA combination its receptor RAGE, increase of NADPH oxidase subunit p22phox, NOX4mRNA and protein expression in THCE cells, prompting the activation of NADPH oxidase, to generate a large number of ROS.
2. AGE-BSA combination its receptor RAGE, reduce antioxidant enzymes SOD.and CAT activity, increased MDA content in THCE cell, leading to the imbalance of the antioxidant system and oxidative system, prompting the corneal epithelial cells to a state of oxidative stress.
Part Ⅲ Effect of advanced glycosylation end products on human corneal epithelial cells proliferation, migration and corneal epithelial wound healing
Purpose:
To investigate the effect of AGEs on human corneal epithelial cells proliferation, migration and corneal epithelial wound healing.
Methods and Materials:
1. Concentration of50μg/ml,100μg/ml,200μg/ml,400μg/ml AGE-BS A cultured with THCE cells for24h, CCK-8method detect THCE cell proliferative capacity; scratch healing assay detect THCE cell migration.
2. Pre-applied anti-RAGE antibody, the ROS scavenger (NAC) process THCE cells for lh, and then200μg/ml AGE-BS A incubated cells for24h, CCK-8method detect THCE cell proliferation ability; scratch healing assay detect THCE cell migration.
3. In vitro cultured the pig corneal epithelial organ trauma model, produced a diameter of5mm damage area in the central corneal epithelial. Pre-applied anti-RAGE antibody, ROS scavenger (NAC) treatment the pig corneal epithelial wound organ models for1h, and then200μg/ml AGE-BSA were incubated for48hours to observe the degree of corneal epithelial wound healing.
Results:
1. Compared with the control group,50μg/ml of AGE-BSA significantly inhibit THCE cell proliferation, migration (P<0.05), cell proliferation, migration ability is gradually reduced with the increase of the concentration of AGE-BSA,200mg/ml AGE-BSA reach to the minimum. anti-RAGE antibodies, ROS scavenger NAC can significantly increased cell proliferation, migration.
2. Compared with the control group,200ug/ml of AGE-BSA could significantly delay the corneal epithelial wound healing process (P<0.05), but the anti-RAGE antibody, ROS scavenger NAC can significantly promote corneal epithelial wound healing (P<0.05).
Conclusions:
1. AGE-BSA suppress THCE cell proliferation, migration, in a dose-dependent manner.
2. AGE-BSA binding with RAGE, increased ROS generation, induced oxidative stress, thereby inhibit THCE cell proliferation, migration.
3. AGE-BSA binding with RAGE, increased ROS generation, induced oxidative stress, delayed corneal epithelial wound healing process. Blocking the binding of AGE-BSA RAGE or clear excess ROS may delay a new approach to the regulation and reversal of diabetes corneal epithelial wound healing.
随着人民生活水平的提高、生活方式的改变和人口老龄化,我国糖尿病发病率呈明显的逐年增高趋势。糖尿病已经构成严重威胁人类健康的世界性问题。糖尿病患者在角膜外伤或接受角膜手术时极易出现角膜上皮愈合延迟甚至不愈,临床表现为：持续性角膜上皮损失、反复性角膜上皮糜烂、浅层角膜溃疡形成、继发严重角膜感染甚至失明。目前尚没有有效的治疗方法。因此,研究糖尿病角膜上皮创伤愈合延迟的发病机制和防治措施已成为眼科亟待解决的课题。
研究发现,持久的高血糖状态可导致糖尿病患者体内多种蛋白质、脂质甚至核酸发生糖基化反应,形成具有结构多样、高度活性的糖基化终末产物(advanced glycation end products, AGEs)。正常情况下体内AGEs的水平随年龄的增长而缓慢增加,而在糖尿病患者体内,病理性高血糖可加速糖基化反应,形成大量的AGEs,并在组织中蓄积。AGEs具有不可逆性,这使其在高血糖被纠正后也不能回复到正常水平。AGEs具有广泛的生物学活性,参与了多种糖尿病并发症的发生、发展。随着对AGEs研究的深入,人们发现AGEs与糖尿病皮肤创面愈合延迟密切相关。近年研究发现,AGEs大量蓄积在糖尿病患者及糖尿病动物模型的角膜上皮及基底膜中,但其是否参与角膜上皮创伤愈合过程而导致创伤愈合延迟?目前尚不清楚。因此,深入研究AGEs在角膜上皮创伤愈合延迟中的作用,将进一步揭示糖尿病角膜上皮创伤愈合延迟的机制,为其防治提供新的理论依据。
AGEs与细胞表面特异性受体(receptor for advanced glycation end products, RAGE)结合,能够增加细胞内活性氧(Reactive oxygen species, ROS)的生成,诱导细胞氧化应激,造成组织氧化损伤。RAGE作为信号转导受体,介导AGEs结合在细胞表面,激活细胞内各种转导信号。研究证实RAGE在正常单核巨噬细胞、血管内皮细胞、肾系膜细胞、神经细胞及平滑肌细胞等细胞中呈低水平表达,但在糖尿病条件下其表达明显增加。AGEs与RAGE表达之间存在正反馈调节机制,AGEs蓄积的病变部位往往伴随着RAGE表达的增加。ROS是指化学性质活跃的氧代谢产物或由其衍生的产物,主要有超氧阴离子、过氧化氢、羟自由基等。ROS生成过多将在细胞内形成氧化应激状态,通过氧化反应破坏许多重要生物大分子的结构和功能,导致疾病的发生、发展。
目前关于AGEs对角膜上皮创伤愈合的影响及机制研究在国内外尚未见报导。AGEs是否延迟角膜上皮创伤愈合过程?是否通过受体RAGE及ROS发挥作用?具体机制如何?回答这些问题将从根本上揭示AGEs在糖尿病角膜上皮创伤愈合延迟中的作用及机制,不仅对理解糖尿病角膜上皮创伤愈合延迟病理机制具有重要的意义,也必将为糖尿病角膜上皮创伤愈合延迟的防治提供新理论、新视角和新靶点。因此,本研究以体外制备的糖基化终末产物(AGE-BSA)作为干预因素,观察AGE-BSA对人永生化角膜上皮细胞(Human telomerase-immortalized corneal epithelial cells, THCE) RAGE、ROS表达的影响并研究其机制,探讨AGE-BSA对THCE细胞增殖、迁移以及角膜上皮创伤愈合的影响及机制。全文共分三部分：(一)AGE-BSA对THCE细胞RAGE及ROS表达的影响；(二)AGE-BSA诱导THCE细胞氧化应激机制的研究；(三)AGE-BSA对THCE细胞增殖、迁移及角膜上皮创伤愈合的影响。
第一部分糖基化终末产物对角膜上皮细胞糖基化终末产物受体及活性氧表达的影响
目的：研究AGEs对THCE细胞RAGE及ROS表达的影响。
方法：1.用D-葡萄糖和牛血清白蛋白(Bovine serum albumin, BSA)共同孵育10周制备糖基化终末产物(AGE-BSA)。
2.分别用浓度为50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA处理THCE细胞24h和浓度为2000μg/ml的AGE-BSA分别处理THCE细胞6h、12h、24h、48h。Real-time PCR和Western blot检测ⅠRAGE mRNA和蛋白的表达；
3.分别用浓度为50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA处理THCE细胞12h。预先应用anti-RAGE中和抗体处理THCE细胞1h,再用浓度为200μg/ml的AGE-BSA孵育细胞12h,激光共聚焦显微镜和流式细胞仪检测ROS的表达。
结果：1.体外制备的AGE-BSA的荧光强度为55.96荧光单位/mg蛋白,而BSA的荧光强度为1.98荧光单位/mg蛋白。
2.未经干预的THCE细胞表达少量的RAGE mRNA和蛋白,用BSA干预后RAGE mRNA和蛋白的表达与对照组无明显差异。与对照组相比,浓度为50μg/ml的AGE-BSA明显上调THCE细胞RAGE mRNA和蛋白的表达(P<0.05),随着AGE-BSA浓度的增加,RAGE mRNA和蛋白的表达逐渐增高,AGE-BSA浓度为20μg/ml时达到峰值(P<0.05)。
3.在浓度为200μg/ml的AGE-BSA作用下,AGE-BSA作用THCE细胞6h明显上调RAGE mRNA的表达(P<0.05),AGE-BSA作用THCE细胞12h明显上调RAGE蛋白的表达(P<0.05),随着AGE-BSA作用时间的延长,RAGE mRNA和蛋白的表达逐渐增高,AGE-BSA作用时间为24h时达到峰值(P<0.05)。
4.未经干预的THCE细胞表达极少量的ROS,用BSA干预后ROS的表达与对照组无明显差异。与对照组相比,浓度为100μg/ml的AGE-BSA明显上调THCE细胞ROS的表达(P<0.05),随着AGE-BSA浓度的增加,ROS的表达逐渐增高,AGE-BSA浓度为200μg/ml时达到峰值(P<0.05)。
5. anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著抑制AGE-BAS对ROS表达的上调作用(P<0.05)。
结论：1.AGE-BSA显著增加THCE细胞RAGE mRNA、蛋白及ROS的表达。
2. AGE-BSA通过与受体RAGE结合,诱导THCE细胞大量生成ROS,导致角膜上皮细胞氧化损伤,可能参与糖尿病角膜上皮创伤愈合延迟过程。
第二部分糖基化终末产物诱导角膜上皮细胞氧化应激机制的研究
目的：探讨AGEs诱导THCE细胞氧化应激的机制。
方法：1.预先应用NADPH氧化酶抑制齐apocynin及Diphenyleneiodonium (DPI)、线粒体酶复合体Ⅰ抑制齐rotenone、线粒体酶复合体Ⅱ抑制剂thenoyltrifluoroacetone (TTFA)、线粒体酶复合体Ⅲ抑制齐antimycin A、黄嘌呤氧化酶抑制齐allopurinol处理THCE细胞1h后,再用浓度为200μg/m1的AGE-BSA孵育细胞12h,应用流式细胞仪检测ROS的表达。
2.预先应用anti-RAGE中和抗体处理THCE细胞1h,再用浓度为200μg/m1的AGE-BSA孵育细胞12h,应用Real-time PCR检测NADPH氧化酶亚基p22phox、 NOX4mRNA的表达；Western blot检测p22phox、NOX4蛋白的表达。
3.预先应用anti-RAGE中和抗体处理THCE细胞1h,再用浓度为200μg/ml的AGE-BSA呼育细胞12h,检测抗氧化酶超氧化物歧化酶(Superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)的活性及丙二醛(malondialdehyde, MD A)的含量。
结果：1.与对照组相比,AGE-BSA显著上调THCE细胞ROS的表达(P<0.05),NADPH氧化酶抑制剂apocynin和DPI显著抑制AGE-BAS对ROS表达的上调作用(P<0.05),而线粒体酶复合体抑制剂、黄嘌呤氧化酶抑制剂对ROS的表达无明显影响。
2.与对照组相比,AGE-BSA明显上调THCE细胞P22phox、NOX4mRNA和蛋白的表达(P<0.05),应用anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著抑制AGE-BAS对p22phox、NOX4mRNA和蛋白表达的上调作用(P<0.05)。
3.与对照组相比,AGE-BSA显著降低THCE细胞SOD和CAT的活性(P<0.05)；应用anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著减轻AGE-BAS对SOD和CAT活性的抑制作用(P<0.05)
4.与对照组相比,AGE-BSA显著增加THCE细胞MDA的含量(P<0.05)；应用anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著抑制AGE-BAS对MDA含量的上调作用(P<0.05)
结论：1. AGE-BSA通过与受体RAGE结合,诱导THCE细胞NADPH氧化酶亚基p22phox、NOX4mRNA和蛋白高表达,促使NADPH氧化酶激活,生成大量ROS。
2. AGE-BSA通过与受体RAGE结合,降低THCE细胞抗氧化酶SOD口CAT的活性、增加MDA的含量,导致氧化／抗氧化系统失衡,促使角膜上皮细胞处于氧化应激状态。
第三部分糖基化终末产物对角膜上皮细胞增殖、迁移及角膜上皮创伤愈合的影响
目的：探讨AGEs对THCE细胞增殖、迁移及角膜上皮创伤愈合的影响。
方法：1.分别用浓度为50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA与THCE细胞共培养24h,CCK-8法检测THCE细胞增殖能力；刮痕愈合实验检测THCE细胞迁移能力。
2.预先应用anti-RAGE中和抗体、抗氧化剂乙酰半胱胺酸(N-acetyl-L-cysteine, NAC)处理THCE细胞1h,再用浓度为200μg/ml的AGE-BSA孵育细胞24h, CCK-8法检测THCE细胞增殖能力；刮痕愈合实验检测THCE细胞迁移能力。
3.体外培养猪角膜上皮创伤器官模型,在角膜中央制作直径为5mm的上皮损伤区域。预先应用anti-RAGE中和抗体、抗氧化剂NAC处理1h,再用浓度为200μg/ml的AGE-BSA5孵育48h,观察角膜上皮创伤愈合程度。
结果：1.与对照组相比,浓度为50μg/ml的AGE-BSA显著抑制THCE细胞的增殖、迁移(P<0.05),随着AGE-BSA浓度的增加,细胞增殖、迁移能力逐渐降低,AGE-BSA浓度为200μg/ml寸达到最低。anti-RAGE中和抗体、NAC显著减轻AGE-BAS对细胞增殖、迁移的抑制作用。
2.与对照组相比,浓度为200μg/ml的AGE-BSA显著延迟体外培养猪角膜上皮创伤器官模型中角膜上皮创伤愈合过程(P<0.05),anti-RAGE中和抗体、NAC可显著促进角膜上皮创伤的愈合(P<0.05)
结论：1. AGE-BSA显著抑制THCE细胞增殖、迁移,并且呈浓度依赖性。
2. AGE-BSA通过与受体RAGE结合,诱导ROS大量生成,引发氧化应激反应,进而抑制THCE细胞增殖、迁移,延迟角膜上皮创伤愈合过程。阻断AGE-BSA与RAGE结合,或清除过多的ROS,可成为防治糖尿病角膜上皮创伤愈合延迟的新途径。
Background
With the improvement of people's living standards, lifestyle changes and an aging population, the incidence of diabetes in China was increasing year by year. Diabetes constitutes a serious threat to human health worldwide. Diabetic patient's corneal trauma or corneal surgery easily occur corneal epithelial healing delay or unhealed, clinical manifestations:persistent corneal epithelial loss, recurrent corneal erosion, superficial corneal ulcer formation, secondary to severe corneal infection and even blindness. There is currently no effective treatment. Therefore, the studies of the pathogenesis and prevention measures for delayed diabetic corneal epithelial wound healing are the ophthalmic problems to be solved.
The study found that persistent hyperglycemia of diabetes patients can lead to a variety of proteins, lipids or nucleic acid glycosylation reaction occurs, the formation of structural diversity, highly reactive advanced glycation end products (advanced glycation end products, AGEs). Normally, AGEs level in the body increase with age slowly. However, pathological hyperglycemia of diabetic patients can accelerate glycosylation reaction in vivo, the formation of a large number of AGEs in the tissues. AGEs are irreversible, so even hyperglycemia is corrected it can not return to normal levels. AGEs has a wide range of biological activity, participate in a variety of complications of diabetes development. As AGEs research depth, it was found that AGEs and diabetic skin wound healing delay is closely related. Recent studies have found that the accumulation of AGEs in corneal epithelium and basement membrane of diabetic patients and animal models of diabetes, but it is involved in corneal epithelial wound healing process and result in delayed wound healing? Now is unclear. Therefore, study the effect of AGEs in corneal epithelial wound healing will further reveal diabetic delayed corneal epithelial wound healing mechanism to provide a new theoretical basis for its prevention and control.
AGEs combination with cell surface specific receptor (receptor for advanced glycation end products, RAGE) can increase intracellular ROS (Reactive oxygen species, ROS) generation induced cellular oxidative stress, resulting in oxidative damage. RAGE as a receptor signal transduction mediated AGEs binding at the cell surface, activation of cell internal variety of signal transduction mechanisms. The study confirmed that RAGE was expressed at low levels in normal mononuclear macrophages, endothelial cells, mesangial cells, neural cells and smooth muscle cells and other cells, but its expression was significantly increased in diabetic conditions. AGEs and RAGE expression between positive feedback regulation mechanisms. The AGEs increased lesion is often accompanied by increased expression of RAGE. ROS refers to the chemical properties of active oxygen metabolites or oxygen-containing product derived therefrom, mainly superoxide anion, hydrogen peroxide, hydroxyl radicals. Excessive ROS generation will lead to cell oxidative stress and damage the structure and function of many important biological macromolecules by the oxidation reaction, leading to the occurrence and development of the disease.
Now, Effects and mechanism of AGEs in corneal epithelial wound healing has not been reported. The AGEs whether delayed comeal epithelial wound healing process? Whether through its receptor RAGE and ROS? Specific mechanism? Answers to these questions will fundamentally reveal the mechanism of AGEs in diabetic delayed corneal epithelial wound healing, is of great significance not only for the understanding of pathological mechanisms of diabetic delayed corneal epithelial wound healing, will also for the providing new theories, new perspectives and new target prevention of diabetic delayed corneal epithelial wound healing. Therefore, this study prepared in vitro AGE-BSA as an intervention factors, study the effect of AGE-BSA on RAGE, ROS expression in immortalized human corneal epithelial cells,(THCE) and its mechanism, and explore the effect of AGE-BSA on THCE cells proliferation, migration and corneal epithelial wound healing and mechanisms. This study is divided into three parts:(1) Effect of AGE-BSA on RAGE and ROS expression in THCE cell;(2) Effect of AGE-BSA on oxidative stress in THCE cell;(3) Effect of AGE-BSA on THCE cell proliferation, migration and corneal epithelial wound healing.
Part I Effect of advanced glycosylation end products on the expression of receptor for advanced glycosylation end products and reactive oxygen species in human corneal epithelial cells.
Purpose:
To investigate the effect of AGEs on the expression of RAGE and ROS in human corneal epithelial cells.
Methods and Materials:
1. D-glucose and bovine serum albumin (Bovine serum albumin, BSA) incubate for10weeks to prepare glycosylated bovine serum albumin (AGE-BSA), and that of AGEs.
2. Respectively, with a concentration of50μg/ml,100μg/ml,200μg/ml,400ug/ml of AGE-BSA treatment THCE cells for24h and the concentration of200μg/ml AGE-BSA treatment THCE cells6h,12h,24h,48h. Real-time PCR and Western blot detect RAGE mRNA and protein expression.
3. Respectively, with a concentration of50μg/ml,100μg/ml,200μg/ml,400μg/ml of AGE-BSA treatment THCE cells for12h. Pre-application of anti-RAGE antibody treatment THCE cells lh, and then the concentration of200μg/ml AGE-BSA incubated the cells for12h, confocal laser scanning microscopy and flow cytometry detect ROS expression.
Results:
1. Prepared in vitro AGE-BSA fluorescence intensity of55.96fluorescence units/mg protein, while the fluorescence intensity of the BSA1.98fluorescence units/mg protein. AGE-BSA prepared can be used for later experiments.
2. Without intervention THCE cells express a small amount of RAGE mRNA and protein, BSA intervention RAGE mRNA and protein expression was no significant difference with the control group. Compared with the control group, the concentration of50μg/ml AGE-BSA significantly unregulated THCE cell RAGE mRNA and protein expression (P<0.05), RAGE mRNA and protein expression gradually increased with the increase of the concentration of AGE-BSA, AGE-BSA concentration of200μg/ml reached the peak (P<0.05).
3. At a concentration of200μg/ml AGE-BSA significantly up-regulated expression of RAGE mRNA at6h (P<0.05), significantly up-regulated the expression of RAGE protein at12h (P<0.05), with the extension of the duration of action of AGE-BSA, RAGE mRNA and protein expression gradually increased, reached the peak at24h (P<0.05).
4. Without intervention THCE cells can express a very small amount of ROS, BSA intervention ROS expression was no significant difference with the control group. Compared with the control group,100μg/ml AGE-BSA upregulated the expression of ROS in THCE cells (P<0.05), the expression of ROS is increased gradually with the increase of the concentration of AGE-BSA,200μg/ml AGE-BSA reached a peak (P<0.05).
5. Anti-RAGE antibody blocking the interaction AGE-BSA with RAGE, significantly inhibited the AGE-BAS upregulates the expression of ROS (P<0.05).
Conclusions:
1. AGE-BSA significantly increase RAGE mRNA, protein expression and ROS in THCE cell.
2. AGE-BSA binding with its receptor RAGE induced ROS expression, resulting in oxidative damage, involved the occurrence and development of diabetic delayed corneal epithelial wound healing.
PartⅡ Effect of advanced glycosylation end products on oxidative stress in human corneal epithelial cells.
Purpose:
To investigate the effect of AGEs on oxidative stress in human corneal epithelial cells.
Methods and Materials:
1. Pre-application NADPH oxidase inhibitor apocynin and diphenyleneiodonium (DPI), mitochondrial enzyme complex I inhibitor rotenone, mitochondrial enzyme complex Ⅱ inhibitor thenoyltrifluoroacetone (TTFA), mitochondrial enzyme complexthe body Ⅲ inhibitor antimycin A, xanthine oxidase inhibitor allopurinol treatment THCE cells for1h, and then200μg/ml AGE-BSA incubated cells for12hours and analyzed the expression of ROS by flow cytometry.
2. Pre-application of anti-RAGE antibody treatment THCE cell for1h, and then200μg/ml AGE-BSA incubated cells for12h, Real-time PCR detect p22phox, NOX4mRNA expression; Western blot detect p22phox, NOX4protein expression.
3. Pre-application of anti-RAGE antibody treatment THCE cell for1h, then200μg/ml AGE-BSA incubated cells for12h, detection of the antioxidant enzyme superoxide dismutase (SOD), catalase (CAT) activity and malondialdehyde (MDA) content.
Results:
1. Compared with the control group, AGE-BSA significantly up-regulated the expression of ROS in THCE cells (P<0.05), NADPH oxidase inhibitors apocynin and DPI significantly inhibited AGE-BAS upregulates the expression of ROS (P<0.05), mitochondrial enzyme complex inhibitor and xanthine oxidase inhibitor had no significant effect on the expression of ROS.
2. Compared with the control group, AGE-BSA significantly increased p22phox, NOX4mRNA and protein expression inTHCE cells (P<0.05), application of anti-RAGE antibody blocking AGE-BSA interaction with RAGE, significantly inhibite p22phox, NOX4mRNA and protein expression (P<0.05).
3. Compared with the control group, AGE-BSA significantly reduce the activity of SOD and CAT in THCE cells (P<0.05); application of anti-RAGE antibody blocking AGE-BSA interaction with RAGE, significantly reduce AGE-BAS inhibition of SOD and CAT activities (P<0.05).
4. Compared with the control group, AGE-BSA significantly increase the content of MDA in THCE cells (P<0.05); application of anti-RAGE antibody blocking AGE-BSA interaction RAGE could significantly inhibit AGE-BAS upregulation of MDA content (P<0.05).
Conclusions:
1. AGE-BSA combination its receptor RAGE, increase of NADPH oxidase subunit p22phox, NOX4mRNA and protein expression in THCE cells, prompting the activation of NADPH oxidase, to generate a large number of ROS.
2. AGE-BSA combination its receptor RAGE, reduce antioxidant enzymes SOD.and CAT activity, increased MDA content in THCE cell, leading to the imbalance of the antioxidant system and oxidative system, prompting the corneal epithelial cells to a state of oxidative stress.
Part Ⅲ Effect of advanced glycosylation end products on human corneal epithelial cells proliferation, migration and corneal epithelial wound healing
Purpose:
To investigate the effect of AGEs on human corneal epithelial cells proliferation, migration and corneal epithelial wound healing.
Methods and Materials:
1. Concentration of50μg/ml,100μg/ml,200μg/ml,400μg/ml AGE-BS A cultured with THCE cells for24h, CCK-8method detect THCE cell proliferative capacity; scratch healing assay detect THCE cell migration.
2. Pre-applied anti-RAGE antibody, the ROS scavenger (NAC) process THCE cells for lh, and then200μg/ml AGE-BS A incubated cells for24h, CCK-8method detect THCE cell proliferation ability; scratch healing assay detect THCE cell migration.
3. In vitro cultured the pig corneal epithelial organ trauma model, produced a diameter of5mm damage area in the central corneal epithelial. Pre-applied anti-RAGE antibody, ROS scavenger (NAC) treatment the pig corneal epithelial wound organ models for1h, and then200μg/ml AGE-BSA were incubated for48hours to observe the degree of corneal epithelial wound healing.
Results:
1. Compared with the control group,50μg/ml of AGE-BSA significantly inhibit THCE cell proliferation, migration (P<0.05), cell proliferation, migration ability is gradually reduced with the increase of the concentration of AGE-BSA,200mg/ml AGE-BSA reach to the minimum. anti-RAGE antibodies, ROS scavenger NAC can significantly increased cell proliferation, migration.
2. Compared with the control group,200ug/ml of AGE-BSA could significantly delay the corneal epithelial wound healing process (P<0.05), but the anti-RAGE antibody, ROS scavenger NAC can significantly promote corneal epithelial wound healing (P<0.05).
Conclusions:
1. AGE-BSA suppress THCE cell proliferation, migration, in a dose-dependent manner.
2. AGE-BSA binding with RAGE, increased ROS generation, induced oxidative stress, thereby inhibit THCE cell proliferation, migration.
3. AGE-BSA binding with RAGE, increased ROS generation, induced oxidative stress, delayed corneal epithelial wound healing process. Blocking the binding of AGE-BSA RAGE or clear excess ROS may delay a new approach to the regulation and reversal of diabetes corneal epithelial wound healing.
引文
1. Zhao D, Zhao F, Li Y, Zheng Z. Projected and observed diabetes epidemics in China and beyond. Curr Cardiol Rep 2012,14:106-111.
2. Imam K. Management and treatment of diabetes mellitus. Adv Exp Med Biol 2012,771:356-380.
3. Klocek MS, Sassani JW, McLaughlin PJ, Zagon IS. Naltrexone and insulin are independently effective but not additive in accelerating corneal epithelial healing in type I diabetic rats. Exp Eye Res 2009,89:686-692.
4. Yap FY, Kantharidis P, Coughlan MT, Slattery R, Forbes JM. Advanced glycation end products as environmental risk factors for the development of type 1 diabetes. Curr Drug Targets 2012,13:526-540.
5. Li J, Liu D, Sun L, Lu Y, Zhang Z. Advanced glycation end products and neurodegenerative diseases:mechanisms and perspective. J Neurol Sci 2012,317:1-5.
6. Prasad A, Bekker P, Tsimikas S. Advanced glycation end products and diabetic cardiovascular disease. Cardiol Rev 2012,20:177-183.
7. Thomas MC. Advanced glycation end products. Contrib Nephrol 2011,170:66-74.
8. Pietropaoli D, Monaco A, Del Pinto R, Cifone MG, Marzo Q Giannoni M. Advanced glycation end products:possible link between metabolic syndrome and periodontal diseases. Int J Immunopathol Pharmacol 2012,25:9-17.
9. Sukkar MB, Ullah MA, Gan WJ, Wark PA, Chung KF, Hughes JM, et al. RAGE:a new frontier in chronic airways disease. Br J Pharmacol 2012,167:1161-1176.
10. Sorci G, Riuzzi F, Giambanco I, Donato R. RAGE in tissue homeostasis, repair and regeneration. Biochim Biophys Acta 2013,1833:101-109.
11. Kang P, Tian C, Jia C. Association of RAGE gene polymorphisms with type 2 diabetes mellitus, diabetic retinopathy and diabetic nephropathy. Gene 2012,500:1-9.
12. Ramasamy R, Yan SF, Schmidt AM. Receptor for AGE (RAGE):signaling mechanisms in the pathogenesis of diabetes and its complications. Ann N Y Acad Sci 2011,1243:88-102.
13. Win MT, Yamamoto Y, Munesue S, Saito H, Han D, Motoyoshi S, et al. Regulation of RAGE for attenuating progression of diabetic vascular complications. Exp Diabetes Res 2012,2012:894605.
14. Alves M, Calegari VC, Cunha DA, Saad MJ, Velloso LA, Rocha EM. Increased expression of advanced glycation end-products and their receptor, and activation of nuclear factor kappa-B in lacrimal glands of diabetic rats. Diabetologia 2005,48:2675-2681.
15. Ge J, Jia Q, Liang C, Luo Y, Huang D, Sun A, et al. Advanced glycosylation end products might promote atherosclerosis through inducing the immune maturation of dendritic cells. Arterioscler Thromb Vasc Biol 2005,25:2157-2163.
16. Wilkinson-Berka JL, Rana I, Armani R, Agrotis A. Reactive oxygen species, Nox and angiotensin Ⅱ in angiogenesis:implications for retinopathy. Clin Sci (Lond) 2013,124:597-615.
17. Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Mol Cell 2012,48:158-167.
18. Bryan N, Ahswin H, Smart N, Bayon Y, Wohlert S, Hunt JA. Reactive oxygen species (ROS)--a family of fate deciding molecules pivotal in constructive inflammation and wound healing. Eur Cell Mater 2012,24:249-265.
19. Alfadda AA, Sallam RM. Reactive oxygen species in health and disease. J Biomed Biotechnol 2012,2012:936486.
20. Kaji Y, Amano S, Usui T, Oshika T, Yamashiro K, Ishida S, et al. Expression and function of receptors for advanced glycation end products in bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 2003,44:521-528.
21. Maeda S, Matsui T, Takeuchi M, Yoshida Y, Yamakawa R, Fukami K. et al. Pigment epithelium-derived factor (PEDF) inhibits proximal tubular cell injury in early diabetic nephropathy by suppressing advanced glycation end products (AGEs)-receptor (RAGE) axis. Pharmacol Res 2011,63:241-248.
22. Chen J, Song M, Yu S, Gao P, Yu Y, Wang H, et al. Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress. Mol Cell Biochem 2010,335:137-146.
23. Kim J, Kim CS, Sohn E, Jeong IH, Kim H, Kim JS. Involvement of advanced glycation end products, oxidative stress and nuclear factor-kappaB in the development of diabetic keratopathy. Graefes Arch Clin Exp Ophthalmol 2011,249:529-536.
24. Kim J, Kim CS, Kim H, Jeong IH, Sohn E, Kim JS. Protection against advanced glycation end products and oxidative stress during the development of diabetic keratopathy by KIOM-79. J Pharm Pharmacol 2011,63:524-530.
25. Kaji Y, Usui T, Oshika T, Matsubara M, Yamashita H, Araie M, et al. Advanced glycation end products in diabetic corneas. Invest Ophthalmol Vis Sci 2000,41:362-368.
26. Sato E, Mori F, Igarashi S, Abiko T, Takeda M, Ishiko S, et al. Corneal advanced glycation end products increase in patients with proliferative diabetic retinopathy. Diabetes Care 2001,24:479-482.
27. McDermott AM, Xiao TL, Kern TS, Murphy CJ. Non-enzymatic glycation in corneas from normal and diabetic donors and its effects on epithelial cell attachment in vitro. Optometry 2003,74:443-452.
28. Yucel I, Yucel G, Akar Y, Demir N, Gurbuz N, Aslan M. Transmission electron microscopy and autofluorescence findings in the cornea of diabetic rats treated with aminoguanidine. Can J Ophthalmol 2006,41:60-66.
29. Gul M, Emre S, Esrefoglu M, Vard N. Protective effects of melatonin and aminoguanidine on the cornea in streptozotocin-induced diabetic rats. Cornea 2008,27:795-801.
30. Liu Y, Ma Y, Wang R, Xia C, Zhang R, Lian K, et al. Advanced glycation end products accelerate ischemia/reperfusion injury through receptor of advanced end product/nitrative thioredoxin inactivation in cardiac microvascular endothelial cells. Antioxid Redox Signal 2011,15:1769-1778.
31. Ginter E, Simko V. Type 2 diabetes mellitus, pandemic in 21st century. Adv Exp Med Biol 2012,771:42-50.
32. Shikata K, Ninomiya T, Kiyohara Y. Diabetes mellitus and cancer risk:review of the epidemiological evidence. Cancer Sci 2013,104:9-14.
33. Yamagishi S, Matsui T. Advanced glycation end products (AGEs), oxidative stress and diabetic retinopathy. Curr Pharm Biotechnol 2011,12:362-368.
34. Yamagishi S, Maeda S, Matsui T, Ueda S, Fukami K, Okuda S. Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications in diabetes. Biochim Biophys Acta 2012,1820:663-671.
35. Yamagishi S, Matsui T. Advanced glycation end products, oxidative stress and diabetic nephropathy. Oxid Med Cell Longev 2010,3:101-108.
36. Luevano-Contreras C, Chapman-Novakofski K. Dietary advanced glycation end products and aging. Nutrients 2010,2:1247-1265.
37. Ramasamy R, Yan SF, Schmidt AM. Advanced glycation endproducts:from precursors to RAGE:round and round we go. Amino Acids 2012,42:1151-1161.
38. D'Agati V, Schmidt AM. RAGE and the pathogenesis of chronic kidney disease. Nat Rev Nephrol 2010,6:352-360.
39. Barlovic DP, Thomas MC, Jandeleit-Dahm K. Cardiovascular disease:what's all the AGE/RAGE about? Cardiovasc Hematol Disord Drug Targets 2010,10:7-15.
40. Deane RJ. Is RAGE still a therapeutic target for Alzheimer's disease? Future Med Chem 2012,4:915-925.
41. Cohen MB, Rokhlin OW. Mechanisms of prostate cancer cell survival after inhibition of AR expression. J Cell Biochem 2009,106:363-371.
42. Fernyhough ME, Hausman GJ, Guan LL, Okine E, Moore SS, Dodson MV. Mature adipocytes may be a source of stem cells for tissue engineering. Biochem Biophys Res Commun 2008,368:455-457.
43. Ramasamy R, Schmidt AM. Receptor for advanced glycation end products (RAGE) and implications for the pathophysiology of heart failure. Curr Heart Fail Rep 2012,9:107-116.
44. Maczurek A, Shanmugam K, Munch G. Inflammation and the redox-sensitive AGE-RAGE pathway as a therapeutic target in Alzheimer's disease. Ann N Y Acad Sci 2008,1126:147-151.
45. Yan HD, Li XZ, Xie JM, Li M. Effects of advanced glycation end products on renal fibrosis and oxidative stress in cultured NRK-49F cells. Chin Med J (Engl) 2007,120:787-793.
46. Mizumoto S, Takahashi J, Sugahara K. Receptor for advanced glycation end products (RAGE) functions as receptor for specific sulfated glycosaminoglycans, and anti-RAGE antibody or sulfated glycosaminoglycans delivered in vivo inhibit pulmonary metastasis of tumor cells. J Biol Chem 2012,287:18985-18994.
1. Chen K, Keaney JF, Jr. Evolving concepts of oxidative stress and reactive oxygen species in cardiovascular disease. Curr Atheroscler Rep 2012,14:476-483.
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7. Yap FY, Kantharidis P, Coughlan MT, Slattery R, Forbes JM. Advanced glycation end products as environmental risk factors for the development of type 1 diabetes. Curr Drug Targets 2012,13:526-540.
8. Prasad A, Bekker P, Tsimikas S. Advanced glycation end products and diabetic cardiovascular disease. Cardiol Rev 2012,20:177-183.
9. Thomas MC. Advanced glycation end products. Contrib Nephrol 2011,170:66-74.
10. Sukkar MB, Ullah MA, Gan WJ, Wark PA, Chung KF, Hughes JM, et al. RAGE:a new frontier in chronic airways disease. Br J Pharmacol 2012,167:1161-1176.
11. Wilkinson-Berka JL, Rana I, Armani R, Agrotis A. Reactive oxygen species, Nox and angiotensin Ⅱ in angiogenesis:implications for retinopathy. Clin Sci (Lond) 2013,124:597-615.
12. Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Mol Cell 2012,48:158-167.
13. Bryan N, Ahswin H, Smart N, Bayon Y, Wohlert S, Hunt JA. Reactive oxygen species (ROS)--a family of fate deciding molecules pivotal in constructive inflammation and wound healing. Eur Cell Mater 2012,24:249-265.
14. Alfadda AA, Sallam RM. Reactive oxygen species in health and disease. J Biomed Biotechnol 2012,2012:936486.
15. Basta G, Lazzerini G, Del Turco S, Ratto GM, Schmidt AM, De Caterina R. At least 2 distinct pathways generating reactive oxygen species mediate vascular cell adhesion molecule-1 induction by advanced glycation end products. Arterioscler Thromb Vase Biol 2005,25:1401-1407.
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1. Tavakoli M, Boulton AJ, Efron N, Malik RA. Increased Langerhan cell density and corneal nerve damage in diabetic patients:role of immune mechanisms in human diabetic neuropathy. Cont Lens Anterior Eye 2011,34:7-11.
2. Abdelkader H, Patel DV, McGhee C, Alany RG. New therapeutic approaches in the treatment of diabetic keratopathy:a review. Clin Experiment Ophthalmol 2011,39:259-270.
3. Yap FY, Kantharidis P, Coughlan MT, Slattery R, Forbes JM. Advanced glycation end products as environmental risk factors for the development of type 1 diabetes. Curr Drug Targets 2012,13:526-540.
4. Prasad A, Bekker P, Tsimikas S. Advanced glycation end products and diabetic cardiovascular disease. Cardiol Rev 2012,20:177-183.
5. Pietropaoli D, Monaco A, Del Pinto R, Cifone MG, Marzo G. Giannoni M. Advanced glycation end products:possible link between metabolic syndrome and periodontal diseases. Int J Immunopathol Pharmacol 2012,25:9-17.
6. Sukkar MB, Ullah MA, Gan WJ, Wark PA, Chung KF, Hughes JM, et al. RAGE:a new frontier in chronic airways disease. Br J Pharmacol 2012,167:1161-1176.
7. Kaji Y, Amano S, Usui T, Oshika T, Yamashiro K, Ishida S, et al. Expression and function of receptors for advanced glycation end products in bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 2003,44:521-528.
8. Maeda S, Matsui T, Takeuchi M, Yoshida Y, Yamakawa R, Fukami K, et al. Pigment epithelium-derived factor (PEDF) inhibits proximal tubular cell injury in early diabetic nephropathy by suppressing advanced glycation end products (AGEs)-receptor (RAGE) axis. Pharmacol Res 2011,63:241-248.
9. Peppa M, Brem H, Ehrlich P, Zhang JG, Cai W, Li Z, et al. Adverse effects of dietary glycotoxins on wound healing in genetically diabetic mice. Diabetes 2003,52:2805-2813.
10. Zhu Y, Lan F, Wei J, Chong B, Chen P, Huynh L, et al. Influence of dietary advanced glycation end products on wound healing in nondiabetic mice. J Food Sci 2011,76:T5-10.
11. Goova MT, Li J, Kislinger T, Qu W, Lu Y, Bucciarelli LG, et al. Blockade of receptor for advanced glycation end-products restores effective wound healing in diabetic mice. Am J Pathol 2001,159:513-525.
12. Kim J, Kim CS, Kim H, Jeong IH, Sohn E, Kim JS. Protection against advanced glycation end products and oxidative stress during the development of diabetic keratopathy by KIOM-79. J Pharm Pharmacol 2011,63:524-530.
13. Kim J, Kim CS, Sohn E, Jeong IH, Kim H, Kim JS. Involvement of advanced glycation end products, oxidative stress and nuclear factor-kappaB in the development of diabetic keratopathy. Graefes Arch Clin Exp Ophthalmol 2011,249:529-536.
14. Zou C, Wang S, Huang F, Zhang YA. Advanced glycation end products and ultrastructural changes in corneas of long-term streptozotocin-induced diabetic monkeys. Cornea 2012,31:1455-1459.
15. Kaji Y, Usui T, Oshika T, Matsubara M, Yamashita H, Araie M, et al. Advanced glycation end products in diabetic corneas. Invest Ophthalmol Vis Sci 2000,41:362-368.
16. Sato E, Mori F, Igarashi S, Abiko T, Takeda M, Ishiko S, et al. Corneal advanced glycation end products increase in patients with proliferative diabetic retinopathy. Diabetes Care 2001,24:479-482.
17. McDermott AM, Xiao TL, Kern TS, Murphy CJ. Non-enzymatic glycation in corneas from normal and diabetic donors and its effects on epithelial cell attachment in vitro. Optometry 2003,74:443-452.
18. Yucel I, Yucel G, Akar Y, Demir N, Gurbuz N, Aslan M. Transmission electron microscopy and autofluorescence findings in the cornea of diabetic rats treated with aminoguanidine. Can J Ophthalmol 2006,41:60-66.
19. Gul M, Emre S, Esrefoglu M, Vard N. Protective effects of melatonin and aminoguanidine on the cornea in streptozotocin-induced diabetic rats. Cornea 2008,27:795-801.
20. Masson E, Troncy L, Ruggiero D, Wiernsperger N, Lagarde M, El Bawab S. a-Series gangliosides mediate the effects of advanced glycation end products on pericyte and mesangial cell proliferation:a common mediator for retinal and renal microangiopathy? Diabetes 2005,54:220-227.
21. Wang SH, Guo YJ, Yuan Y, Li L, Li FF, Ye KP, et al. PPARgamma-mediated advanced glycation end products regulate neural stem cell proliferation but not neural differentiation through the BDNF-CREB pathway. Toxicol Lett 2011,206:339-346.
22. Jacobsen JN, Steffensen B, Hakkinen L, Krogfelt KA, Larjava HS. Skin wound healing in diabetic beta6 integrin-deficient mice. APMIS 2010,118:753-764.
23. Sun C, Liang C, Ren Y, Zhen Y, He Z, Wang H, et al. Advanced glycation end products depress function of endothelial progenitor cells via p38 and ERK 1/2 mitogen-activated protein kinase pathways. Basic Res Cardiol 2009,104:42-49.
24. Liu Y, Ma Y, Wang R, Xia C, Zhang R, Lian K, et al. Advanced glycation end products accelerate ischemia/reperfusion injury through receptor of advanced end product/nitrative thioredoxin inactivation in cardiac microvascular endothelial cells. Antioxid Redox Signal 2011,15:1769-1778.
25. Xu K, McDermott M, Villanueva L, Schiffman RM, Hollander DA. Ex vivo corneal epithelial wound healing following exposure to ophthalmic nonsteroidal anti-inflammatory drugs. Clin Ophthalmol 2011,5:269-274.
26. Kapetanaki MG, Mora AL, Rojas M. Influence of age on wound healing and fibrosis. J Pathol 2013,229:310-322.
27. Choi JS, Joo CK. Wakayama symposium:new therapies for modulation of epithelialization in corneal wound healing. Ocul Surf 2013,11:16-18.
28. Klocek MS, Sassani JW, McLaughlin PJ, Zagon IS. Naltrexone and insulin are independently effective but not additive in accelerating corneal epithelial healing in type I diabetic rats. Exp Eye Res 2009,89:686-692.
29. Thomas MC. Advanced glycation end products. Contrib Nephrol 2011,170:66-74.
30. Kang P, Tian C, Jia C. Association of RAGE gene polymorphisms with type 2 diabetes mellitus, diabetic retinopathy and diabetic nephropathy. Gene 2012,500:1-9.
31. Win MT, Yamamoto Y, Munesue S, Saito H, Han D, Motoyoshi S, et al. Regulation of RAGE for attenuating progression of diabetic vascular complications. Exp Diabetes Res 2012,2012:894605.
32. Yamamoto N, Katakami C, Yamamoto M. [Proliferation of corneal epithelial cells in diabetic rats]. Nihon Ganka Gakkai Zasshi 1998,102:475-480.
33. Piehl M, Gilotti A, Donovan A, DeGeorge G, Cerven D. Novel cultured porcine corneal irritancy assay with reversibility endpoint. Toxicol In Vitro 2010,24:231-239.
34. Huo Y, Qiu WY, Pan Q, Yao YF, Xing K, Lou MF. Reactive oxygen species (ROS) are essential mediators in epidermal growth factor (EGF)-stimulated corneal epithelial cell proliferation, adhesion, migration, and wound healing. Exp Eye Res 2009,89:876-886.
35. Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Mol Cell 2012,48:158-167.
36. Bryan N, Ahswin H, Smart N, Bayon Y, Wohlert S, Hunt JA. Reactive oxygen species (ROS)--a family of fate deciding molecules pivotal in constructive inflammation and wound healing. Eur Cell Mater 2012,24:249-265.
2. Imam K. Management and treatment of diabetes mellitus. Adv Exp Med Biol 2012,771:356-380.
3. Klocek MS, Sassani JW, McLaughlin PJ, Zagon IS. Naltrexone and insulin are independently effective but not additive in accelerating corneal epithelial healing in type I diabetic rats. Exp Eye Res 2009,89:686-692.
4. Yap FY, Kantharidis P, Coughlan MT, Slattery R, Forbes JM. Advanced glycation end products as environmental risk factors for the development of type 1 diabetes. Curr Drug Targets 2012,13:526-540.
5. Li J, Liu D, Sun L, Lu Y, Zhang Z. Advanced glycation end products and neurodegenerative diseases:mechanisms and perspective. J Neurol Sci 2012,317:1-5.
6. Prasad A, Bekker P, Tsimikas S. Advanced glycation end products and diabetic cardiovascular disease. Cardiol Rev 2012,20:177-183.
7. Thomas MC. Advanced glycation end products. Contrib Nephrol 2011,170:66-74.
8. Pietropaoli D, Monaco A, Del Pinto R, Cifone MG, Marzo Q Giannoni M. Advanced glycation end products:possible link between metabolic syndrome and periodontal diseases. Int J Immunopathol Pharmacol 2012,25:9-17.
9. Sukkar MB, Ullah MA, Gan WJ, Wark PA, Chung KF, Hughes JM, et al. RAGE:a new frontier in chronic airways disease. Br J Pharmacol 2012,167:1161-1176.
10. Sorci G, Riuzzi F, Giambanco I, Donato R. RAGE in tissue homeostasis, repair and regeneration. Biochim Biophys Acta 2013,1833:101-109.
11. Kang P, Tian C, Jia C. Association of RAGE gene polymorphisms with type 2 diabetes mellitus, diabetic retinopathy and diabetic nephropathy. Gene 2012,500:1-9.
12. Ramasamy R, Yan SF, Schmidt AM. Receptor for AGE (RAGE):signaling mechanisms in the pathogenesis of diabetes and its complications. Ann N Y Acad Sci 2011,1243:88-102.
13. Win MT, Yamamoto Y, Munesue S, Saito H, Han D, Motoyoshi S, et al. Regulation of RAGE for attenuating progression of diabetic vascular complications. Exp Diabetes Res 2012,2012:894605.
14. Alves M, Calegari VC, Cunha DA, Saad MJ, Velloso LA, Rocha EM. Increased expression of advanced glycation end-products and their receptor, and activation of nuclear factor kappa-B in lacrimal glands of diabetic rats. Diabetologia 2005,48:2675-2681.
15. Ge J, Jia Q, Liang C, Luo Y, Huang D, Sun A, et al. Advanced glycosylation end products might promote atherosclerosis through inducing the immune maturation of dendritic cells. Arterioscler Thromb Vasc Biol 2005,25:2157-2163.
16. Wilkinson-Berka JL, Rana I, Armani R, Agrotis A. Reactive oxygen species, Nox and angiotensin Ⅱ in angiogenesis:implications for retinopathy. Clin Sci (Lond) 2013,124:597-615.
17. Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Mol Cell 2012,48:158-167.
18. Bryan N, Ahswin H, Smart N, Bayon Y, Wohlert S, Hunt JA. Reactive oxygen species (ROS)--a family of fate deciding molecules pivotal in constructive inflammation and wound healing. Eur Cell Mater 2012,24:249-265.
19. Alfadda AA, Sallam RM. Reactive oxygen species in health and disease. J Biomed Biotechnol 2012,2012:936486.
20. Kaji Y, Amano S, Usui T, Oshika T, Yamashiro K, Ishida S, et al. Expression and function of receptors for advanced glycation end products in bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 2003,44:521-528.
21. Maeda S, Matsui T, Takeuchi M, Yoshida Y, Yamakawa R, Fukami K. et al. Pigment epithelium-derived factor (PEDF) inhibits proximal tubular cell injury in early diabetic nephropathy by suppressing advanced glycation end products (AGEs)-receptor (RAGE) axis. Pharmacol Res 2011,63:241-248.
22. Chen J, Song M, Yu S, Gao P, Yu Y, Wang H, et al. Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress. Mol Cell Biochem 2010,335:137-146.
23. Kim J, Kim CS, Sohn E, Jeong IH, Kim H, Kim JS. Involvement of advanced glycation end products, oxidative stress and nuclear factor-kappaB in the development of diabetic keratopathy. Graefes Arch Clin Exp Ophthalmol 2011,249:529-536.
24. Kim J, Kim CS, Kim H, Jeong IH, Sohn E, Kim JS. Protection against advanced glycation end products and oxidative stress during the development of diabetic keratopathy by KIOM-79. J Pharm Pharmacol 2011,63:524-530.
25. Kaji Y, Usui T, Oshika T, Matsubara M, Yamashita H, Araie M, et al. Advanced glycation end products in diabetic corneas. Invest Ophthalmol Vis Sci 2000,41:362-368.
26. Sato E, Mori F, Igarashi S, Abiko T, Takeda M, Ishiko S, et al. Corneal advanced glycation end products increase in patients with proliferative diabetic retinopathy. Diabetes Care 2001,24:479-482.
27. McDermott AM, Xiao TL, Kern TS, Murphy CJ. Non-enzymatic glycation in corneas from normal and diabetic donors and its effects on epithelial cell attachment in vitro. Optometry 2003,74:443-452.
28. Yucel I, Yucel G, Akar Y, Demir N, Gurbuz N, Aslan M. Transmission electron microscopy and autofluorescence findings in the cornea of diabetic rats treated with aminoguanidine. Can J Ophthalmol 2006,41:60-66.
29. Gul M, Emre S, Esrefoglu M, Vard N. Protective effects of melatonin and aminoguanidine on the cornea in streptozotocin-induced diabetic rats. Cornea 2008,27:795-801.
30. Liu Y, Ma Y, Wang R, Xia C, Zhang R, Lian K, et al. Advanced glycation end products accelerate ischemia/reperfusion injury through receptor of advanced end product/nitrative thioredoxin inactivation in cardiac microvascular endothelial cells. Antioxid Redox Signal 2011,15:1769-1778.
31. Ginter E, Simko V. Type 2 diabetes mellitus, pandemic in 21st century. Adv Exp Med Biol 2012,771:42-50.
32. Shikata K, Ninomiya T, Kiyohara Y. Diabetes mellitus and cancer risk:review of the epidemiological evidence. Cancer Sci 2013,104:9-14.
33. Yamagishi S, Matsui T. Advanced glycation end products (AGEs), oxidative stress and diabetic retinopathy. Curr Pharm Biotechnol 2011,12:362-368.
34. Yamagishi S, Maeda S, Matsui T, Ueda S, Fukami K, Okuda S. Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications in diabetes. Biochim Biophys Acta 2012,1820:663-671.
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