| BackgroundWith 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 healingPurpose: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. |
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