糖尿病视网膜病变患者纤维血管膜中SIRT1的表达及其相关机制初步研究
摘要
[目的]糖尿病视网膜病变(Diabetic retinopathy, DR)是常见的糖尿病微血管并发症,为发达国家工作年龄人群中致盲率最高的疾病。SIRT1属于第Ⅲ组蛋白去乙酰化酶,与DR发病相关。本实验的目的为初步探讨SIRT1在DR纤维血管膜形成中的作用,研究SIRT1激动剂白藜芦醇是否对高糖培养的RPE细胞具有保护效应,及其相关机制。同时初步研究与白藜芦醇结构相近的二苯乙烯苷是否具有类似作用。
[方法]采集DR伴眼底纤维血管膜患者、非糖尿病伴特发性黄斑前膜患者眼部标本,石蜡包埋切片,行免疫组化检测两组患者SIRT1表达的差异。体外培养人视网膜色素上皮细胞,加入5.5mmol/L葡萄糖,5.5mmol/L葡萄糖加上甘露醇,25mmol/L葡萄糖,25mmol/L葡萄糖及不同浓度白藜芦醇、二苯乙烯苷,分别检测以下指标的变化情况：(1)CCK-8法检测细胞活力；(2)RT-PCR检测SIRT1、PGC-1α基因表达水平；(3)Western blot检测乙酰化NF-κB p65含量及AMPK活性；(4)ELISA法检测VEGF分泌量。
[结果]共收集DR患者纤维血管膜12例,非糖尿病患者黄斑前膜7例,纤维血管膜中SIRT1阳性率高于黄斑前膜组织(100%vs28.6%),去掉年龄因素影响后,差异具有统计学意义(p=0.021)。25mmol/L葡萄糖能降低人视网膜色素上皮细胞活力、抑制SIRT1及PGC-1α转录,激活NF-κB、抑制AMPK活性,促进VEGF分泌。加入一定浓度的白藜芦醇或二苯乙烯苷,可抑制高糖引起的细胞活力下降,减少SIRT1转录下降的水平,减少NF-κB的激活,VEGF分泌量也相应降低。同时,一定浓度的白藜芦醇及二苯乙烯苷可以减少高糖对PGC-1α转录的抑制作用,其幅度与对应的SIRT1、NF-κB改变水平并不完全一致,但与AMPK的活性变化量基本一致。
[结论]DR患者纤维血管膜中SIRT1表达升高,提示SIRT1与DR新生血管有一定关系。但高糖可抑制体外培养的人视网膜色素上皮细胞中SIRT1的表达,上述实验结果提示体外实验可能不能完全反映视网膜的生理情况；或者DR病情进展过程中,SIRT1的含量在不断变化。本实验数据提示二苯乙烯苷可能也具有SIRT1激动剂的作用,且白藜芦醇及二苯乙烯苷可能是通过SIRT1—NF-κB通路降低VEGF分泌。同时PGC-1α mRNA水平可能与AMPK活性相关。
Purpose:Diabetic retinopathy (DR) is a severe complication of diabetes and the leading cause of blindness among working adults worldwide. The class Ⅲ Histone deacetylases SIRT1plays crucial roles in the development of DR. We aimed to investigate the role of SIRT1in the pathogenesis of neovascularization in DR, and the protective effects of resveratrol, a SIRT1activator, on RPE and the underlying mechanisms by using an in vitro model of hyperglycemia. We also investigate whether2,3,4',5-Tetrahydroxystilbene-2-O-p-D-glucoside (TSG), a resveratrol analog, has the same effect.
Method:The expression of SIRT1was assessed via immunohistochemistry in excised neovascular membranes from DR patients and epiretinal membrane from non-diabetic patients. Retinal (ARPE-19) cells were incubated with5.5mmol/L glucose,5.5mmol/L glucose and mannitol,25mmol/L glucose,25mmol/L glucose and different concentrations of resveratrol or25mmol/L glucose and different concentrations of TSG. Cell viability was determined by the CCK-8assay. The mRNA expression of SIRTl and PGC-lα was done by real-time PCR. The protein expression of acetylated NF-κB p65and AMPK activity was done by Western blot. The level of vascular endothelial growth factor (VEGF) was determined by the enzyme-linked immunosorbent assay (ELISA).
Results:Formalin-fixed paraffin-embedded sections of neovascular membranes were excised from12patients with DR and epiretinal membranes from7non-diabetic patients. SIRT1was more frequently expressed in neovascular membranes than epiretinal membranes (100%vs28.6%). This difference was statistically significant after adjustments for age (p=0.021).25mmol/L glucose significantly induced the accumulation of VEGF, activation of NF-κB, reduction of SIRT1and PGC-la expression and AMPK activity. Incubation of ARPE-19cells with25mmol/L glucose in the presence of resveratrol or TSG reduced the inhibition effect of hyperglycemia on cell viability, SIRT1and PGC-1α expression, and AMPK activity. They also reduced the activation effect of hyperglycemia on NF-κB and VEGF. Moreover, the change of VEGF was consistent with that of SIRT1and NF-κB. The change of PGC-la was consistent with that of AMPK activity.
Discussion:SIRT1levels appear elevated in human neovascular membranes in DR eyes compared with control eyes. These data support a potential role for SIRT1in the pathogenesis of neovasculation in DR. On the other hand,hyperglycemia inhibited the expression of SIRT1in ARPE-19. These data indicate that in vitro model may be different from the in vivo physiological conditions. It is also possible that SIRT1levels fluctuate during the course of disease. Our results also suggest that TSG can function as a SIRT1activator. Resveratrol and TSG can protect the retinal pigment epithelial cells against hyperglycemia induced damage. They efficiently reduce VEGF secretion through SIRT1—NF-κB pathway. Furthermore, the level of PGC-1α may be associated with AMPK activity.
[方法]采集DR伴眼底纤维血管膜患者、非糖尿病伴特发性黄斑前膜患者眼部标本,石蜡包埋切片,行免疫组化检测两组患者SIRT1表达的差异。体外培养人视网膜色素上皮细胞,加入5.5mmol/L葡萄糖,5.5mmol/L葡萄糖加上甘露醇,25mmol/L葡萄糖,25mmol/L葡萄糖及不同浓度白藜芦醇、二苯乙烯苷,分别检测以下指标的变化情况：(1)CCK-8法检测细胞活力；(2)RT-PCR检测SIRT1、PGC-1α基因表达水平；(3)Western blot检测乙酰化NF-κB p65含量及AMPK活性；(4)ELISA法检测VEGF分泌量。
[结果]共收集DR患者纤维血管膜12例,非糖尿病患者黄斑前膜7例,纤维血管膜中SIRT1阳性率高于黄斑前膜组织(100%vs28.6%),去掉年龄因素影响后,差异具有统计学意义(p=0.021)。25mmol/L葡萄糖能降低人视网膜色素上皮细胞活力、抑制SIRT1及PGC-1α转录,激活NF-κB、抑制AMPK活性,促进VEGF分泌。加入一定浓度的白藜芦醇或二苯乙烯苷,可抑制高糖引起的细胞活力下降,减少SIRT1转录下降的水平,减少NF-κB的激活,VEGF分泌量也相应降低。同时,一定浓度的白藜芦醇及二苯乙烯苷可以减少高糖对PGC-1α转录的抑制作用,其幅度与对应的SIRT1、NF-κB改变水平并不完全一致,但与AMPK的活性变化量基本一致。
[结论]DR患者纤维血管膜中SIRT1表达升高,提示SIRT1与DR新生血管有一定关系。但高糖可抑制体外培养的人视网膜色素上皮细胞中SIRT1的表达,上述实验结果提示体外实验可能不能完全反映视网膜的生理情况；或者DR病情进展过程中,SIRT1的含量在不断变化。本实验数据提示二苯乙烯苷可能也具有SIRT1激动剂的作用,且白藜芦醇及二苯乙烯苷可能是通过SIRT1—NF-κB通路降低VEGF分泌。同时PGC-1α mRNA水平可能与AMPK活性相关。
Purpose:Diabetic retinopathy (DR) is a severe complication of diabetes and the leading cause of blindness among working adults worldwide. The class Ⅲ Histone deacetylases SIRT1plays crucial roles in the development of DR. We aimed to investigate the role of SIRT1in the pathogenesis of neovascularization in DR, and the protective effects of resveratrol, a SIRT1activator, on RPE and the underlying mechanisms by using an in vitro model of hyperglycemia. We also investigate whether2,3,4',5-Tetrahydroxystilbene-2-O-p-D-glucoside (TSG), a resveratrol analog, has the same effect.
Method:The expression of SIRT1was assessed via immunohistochemistry in excised neovascular membranes from DR patients and epiretinal membrane from non-diabetic patients. Retinal (ARPE-19) cells were incubated with5.5mmol/L glucose,5.5mmol/L glucose and mannitol,25mmol/L glucose,25mmol/L glucose and different concentrations of resveratrol or25mmol/L glucose and different concentrations of TSG. Cell viability was determined by the CCK-8assay. The mRNA expression of SIRTl and PGC-lα was done by real-time PCR. The protein expression of acetylated NF-κB p65and AMPK activity was done by Western blot. The level of vascular endothelial growth factor (VEGF) was determined by the enzyme-linked immunosorbent assay (ELISA).
Results:Formalin-fixed paraffin-embedded sections of neovascular membranes were excised from12patients with DR and epiretinal membranes from7non-diabetic patients. SIRT1was more frequently expressed in neovascular membranes than epiretinal membranes (100%vs28.6%). This difference was statistically significant after adjustments for age (p=0.021).25mmol/L glucose significantly induced the accumulation of VEGF, activation of NF-κB, reduction of SIRT1and PGC-la expression and AMPK activity. Incubation of ARPE-19cells with25mmol/L glucose in the presence of resveratrol or TSG reduced the inhibition effect of hyperglycemia on cell viability, SIRT1and PGC-1α expression, and AMPK activity. They also reduced the activation effect of hyperglycemia on NF-κB and VEGF. Moreover, the change of VEGF was consistent with that of SIRT1and NF-κB. The change of PGC-la was consistent with that of AMPK activity.
Discussion:SIRT1levels appear elevated in human neovascular membranes in DR eyes compared with control eyes. These data support a potential role for SIRT1in the pathogenesis of neovasculation in DR. On the other hand,hyperglycemia inhibited the expression of SIRT1in ARPE-19. These data indicate that in vitro model may be different from the in vivo physiological conditions. It is also possible that SIRT1levels fluctuate during the course of disease. Our results also suggest that TSG can function as a SIRT1activator. Resveratrol and TSG can protect the retinal pigment epithelial cells against hyperglycemia induced damage. They efficiently reduce VEGF secretion through SIRT1—NF-κB pathway. Furthermore, the level of PGC-1α may be associated with AMPK activity.
引文
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[2]Wild S, Roglic G, Green A, et al. Global prevalence of diabetes:estimates for the year 2000 and projections for 2030 [J]. Diabetes care,2004,27(5):1047-1053.
[3]Brownlee M. Biochemistry and molecular cell biology of diabetic complications [J]. Nature,2001,414(6865):813-820.
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