糖基化终末产物对大鼠肾脏微血管内皮细胞的损伤及丙丁酚的保护作用
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摘要
前言
糖尿病肾病(Diabetic nephropathy,DN)作为糖尿病的特征性并发症是终末期肾衰常见原因之一。糖尿病肾病主要是微血管病变,其机制可能与糖基化终末产物(advanced glyciation end products,AGEs)诱导的肾脏微血管内皮细胞损伤有关。AGEs是机体长期高血糖状态下体内蛋白质的氨基与醛基之间发生的非酶性糖基化反应的终产物的总称。糖尿病肾病微血管病变时,循环中AGEs与受体结合诱导氧化应激并影响内皮细胞内信号传导。丙丁酚(Probucol,普罗布考),化学名:4,4-[(1.甲基亚乙基)双(硫)]双[2,6-双(1,1-二甲基乙基)苯酚]。20世纪70年代就作为一种降脂药物应用于临床,最近的研究发现它不仅具有调血脂、抗氧化、抗炎、改善内皮细胞功能、抑制血管内膜增生等作用,在防治心血管疾病和延缓糖尿病肾病发展和改善肾功能等方面有广阔的应用前景。
本实验通过建立糖尿病肾病的体外模型探讨AGEs导致大鼠肾脏微血管内皮细胞的损伤的机制,以及抗氧化剂丙丁酚的保护作用的分子机制。
方法
体外分离培养大鼠肾脏微血管(RMEC)内皮细胞,将其分为正常对照组、AGE-BSA损伤组,丙丁酚干预组;应用TBA法、硝酸还原酶法检测各组细胞丙二醛(MDA)和NO浓度;采用RT-PCR法及Western-blot检测细胞内皮型一氧化氮合酶(eNOS)mRNA水平和还原型辅酶Ⅱ(NADPH)氧化酶蛋白表达;应用荧光显微镜观察核转录因子-κB(NF-κB)活化。
结果
与正常对照组相比,损伤组内皮细胞MDA浓度上升(p＜0.05或p＜0.01)、eNOS mRNA水平降低、细胞质NADPH氧化酶蛋白表达减少。AGE-BSA作用时间在30min和6h时培养液中NO浓度升高(p＜0.05或p＜0.01),而12h后NO浓度随着AGEs作用时间延长降低(p＜0.05或p＜0.01)。丙丁酚浓度在(10、20、50、100μmol/L)范围内以剂量依赖方式降低细胞MDA水平(p＜0.05或p＜0.01),抑制NADPH氧化酶蛋白活化,上调NO和eNOS mRNA水平。AGE-BSA刺激微血管内皮细胞后NF-κB表达部位由细胞质移到核内,作用30min时核内表达达高峰,而后持续高表达。
讨论
本实验中我们观察到从肾皮质分离并体培养细胞呈典型的铺路石外观,Ⅷ因子相关抗原阳性和Matrigel上形成管腔样结构,根据这些特征鉴定其为内皮细胞。多项研究表明氧化应激在糖尿病血管并发症的发病过程中具有重要作用循环中AGEs水平的增加是导致活性氧增加的直接原因。本实验中观察到AGEs能导致RMEC损伤和氧化应激在RMEC损伤中具有重要作用。此外,为了进一步研究氧化应激发生的机制,本实验分别检测了两种与氧化应激密切相关的基因NADPH氧化酶和eNOS,结果发现AGEs能导致NADPH氧化酶蛋白表达增加和eNOSmRNA水平异常以及抗氧化剂丙丁酚干预能够抑制AGEs能导致NADPH氧化酶蛋白表达增加和eNOSmRNA水平。NF-κB是一种重要转录因子,广泛参与了炎症、增殖等病理过程。本实验中发现,AGE-BSA作用于RMEC后,NF-κB从细胞质内转移至细胞核,从而验证了AGE能够激活NF-κB这一重要通路;probucol抗炎作用的机制可能与其抑制NF-κB的活化作用有关。
结论
1.糖基化终末产物导致的大鼠RMEC损伤和功能下降的机制可能包括①引发脂质过氧化;②活化NADPH氧化酶,增加活性氧的生成;③eNOSmRNA水平异常,影响NO生成;④激活NF-κB。
2.丙丁酚的作用机制可能是通过拮抗AGE-BSA导致的脂质过氧化,NF-κB、NADPH氧化酶活化和eNOS mRNA表达异常来保护RMEC功能。
Introduction
Diabetic nephropathy is a leading cause of end-stage renal failure.Glucose and other reducing sugars can react nonenzymatically with the free amino groups of proteins to form reversible Schiff bases followed by Amadori rearrangement.These early glycation products undergo further complex reactons such as dehydtraton,condersation,and cross-linking to yield irreversible fluorescent derivatives termed advanced glycation end products(AGEs).Many studies reveal ed that AGEs have been implicated in the initiation and development of diabetic microvascular complication primarily by inter with their intracellular signal transducing receptor for advanced glycation end products(RAGE).The engagement of RAGE on endothelial cells by AGEs results in the induction of intracellular reactive oxygen species(ROS),which seems to be linked to the activaton of the NADPH-oxidase system and the mitochondrial electron transport system.The increased ROS in turn activates acomplex cascade of signal transduction pathways and sbusequentlly enhances the expression of many genes that are highly relevant for inflammation,and atherosclerosis.Probucol,an old-line anticholesterol medication,has powerful antioxidant properties also.In clinical studies,probucol improved the function of diabetic kidney.In this study we aimed to explore the effect of AGEs on injuring of rat renal microvascular endothelial cells and protective effect of probucol.
Methods
Microvascular endothelial cells isolated and cultured from rat renal were divided into 3 groups:normal control group,AGEs group and probucol group.The levels of MDA and NO were determined by the assay TBA and nitrate reductase method respectively.The expression of eNOS mRNA and NADPH oxidase protein was detected by RT-PCR and Western-blot respectively.The intracellular disposition of NF-κB was observed by immunostaining microscopy.
Results
In AGEs group,the level of MDA and the expression of NADPH oxidase protein increased but and the expression of eNOS mRNA decreased,as compared with control. The level of NO in culture medium was increased after exposure to AGE-BSA for 30 min and 6h,but after 12h,NO level was decreased.MDA level and the expression of NADPH oxidase protein were decreased but the expression of eNOS mRNA and NO level were upregulated by probucol with a dose-dependent effect in the concentrations of 10,20,50,100μmol/L.The localization of NF-κB shifted from cytoplasm to nucleus after microvascular endothelial cells were exposed to AGE-BSA.After exposed for 30 min,the expression in nucleus reached the peaked and then maintained high level.
Discussion
The classical criteria for identification of microvascuar endothelial cells included cobblestone appearance in vitro culture,synthesis of v Wf by cells,and tubular structure by matrigel in vitro.In the present study,Microvascular endothelial cells isolated and cultured from rat renal were identificated by the classical criteria successfully. Numerous reports have demonstrated that oxidative stress induced by diabetes plays an important role in the development and progression of diabetic vascular complications including nephropathy.Indeed,there is emerging evidence that the formation of reactive oxygen species(ROS)is a direct consequence of high level of advanced glyciation end products.In the present study,we clearly demonstrated that AGEs could cause dysfunction of rat renal microvascular cells.Our result confirmed Oxidative Stress played an important role in Injuring of Rat Renal Microvascular Endothelial Cells.In the present study,we demonstrated in AGEs group the expression of NADPH oxidase protein increased but the expression of eNOSmRNA decreased,resulting in an enhancement of Oxidative Stress in RMEC.The enhancement oxidative stress was further confirmed by normalization of level of MDA,expression of NADPH oxidase protein and in RMEC treated with probucol in parallel with the decrease in oxidative stress.The transcription factors of the NF-kB/Rel family form dimeric complexes that control expression of various genes involved in inflammation and proliferation.In our study,we observed that localization of NF-κB shifted from cytoplasm to nucleus after microvascular endothelial cells were exposed to AGE-BSA and probucol could prevent activation of NF-κB.Protective Effect of Probucol on microvascular entothelial cells probably via antagonisting AGE-induced activating of NF-κB.
Conclusion
AGEs can cause the injury and dysfunction of renal microvascular endothelial cells via the following possible mechanisms:(1)inducing lipid preoxidation(2)activating NADPH oxidase and increasing the production of ROS;(3)abnormal expression of eNOS mRNA and further affecting NO production;(4)activatin of NF-κB.Probucol can protect microvascular entothelial cells probably via antagonisting the lipid preoxidation,activating of NF-κB and NADPH oxidase,and increasing the AGE-induced abnormal expression of eNOS caused by AGEs.
糖尿病肾病(Diabetic nephropathy,DN)作为糖尿病的特征性并发症是终末期肾衰常见原因之一。糖尿病肾病主要是微血管病变,其机制可能与糖基化终末产物(advanced glyciation end products,AGEs)诱导的肾脏微血管内皮细胞损伤有关。AGEs是机体长期高血糖状态下体内蛋白质的氨基与醛基之间发生的非酶性糖基化反应的终产物的总称。糖尿病肾病微血管病变时,循环中AGEs与受体结合诱导氧化应激并影响内皮细胞内信号传导。丙丁酚(Probucol,普罗布考),化学名:4,4-[(1.甲基亚乙基)双(硫)]双[2,6-双(1,1-二甲基乙基)苯酚]。20世纪70年代就作为一种降脂药物应用于临床,最近的研究发现它不仅具有调血脂、抗氧化、抗炎、改善内皮细胞功能、抑制血管内膜增生等作用,在防治心血管疾病和延缓糖尿病肾病发展和改善肾功能等方面有广阔的应用前景。
本实验通过建立糖尿病肾病的体外模型探讨AGEs导致大鼠肾脏微血管内皮细胞的损伤的机制,以及抗氧化剂丙丁酚的保护作用的分子机制。
方法
体外分离培养大鼠肾脏微血管(RMEC)内皮细胞,将其分为正常对照组、AGE-BSA损伤组,丙丁酚干预组;应用TBA法、硝酸还原酶法检测各组细胞丙二醛(MDA)和NO浓度;采用RT-PCR法及Western-blot检测细胞内皮型一氧化氮合酶(eNOS)mRNA水平和还原型辅酶Ⅱ(NADPH)氧化酶蛋白表达;应用荧光显微镜观察核转录因子-κB(NF-κB)活化。
结果
与正常对照组相比,损伤组内皮细胞MDA浓度上升(p＜0.05或p＜0.01)、eNOS mRNA水平降低、细胞质NADPH氧化酶蛋白表达减少。AGE-BSA作用时间在30min和6h时培养液中NO浓度升高(p＜0.05或p＜0.01),而12h后NO浓度随着AGEs作用时间延长降低(p＜0.05或p＜0.01)。丙丁酚浓度在(10、20、50、100μmol/L)范围内以剂量依赖方式降低细胞MDA水平(p＜0.05或p＜0.01),抑制NADPH氧化酶蛋白活化,上调NO和eNOS mRNA水平。AGE-BSA刺激微血管内皮细胞后NF-κB表达部位由细胞质移到核内,作用30min时核内表达达高峰,而后持续高表达。
讨论
本实验中我们观察到从肾皮质分离并体培养细胞呈典型的铺路石外观,Ⅷ因子相关抗原阳性和Matrigel上形成管腔样结构,根据这些特征鉴定其为内皮细胞。多项研究表明氧化应激在糖尿病血管并发症的发病过程中具有重要作用循环中AGEs水平的增加是导致活性氧增加的直接原因。本实验中观察到AGEs能导致RMEC损伤和氧化应激在RMEC损伤中具有重要作用。此外,为了进一步研究氧化应激发生的机制,本实验分别检测了两种与氧化应激密切相关的基因NADPH氧化酶和eNOS,结果发现AGEs能导致NADPH氧化酶蛋白表达增加和eNOSmRNA水平异常以及抗氧化剂丙丁酚干预能够抑制AGEs能导致NADPH氧化酶蛋白表达增加和eNOSmRNA水平。NF-κB是一种重要转录因子,广泛参与了炎症、增殖等病理过程。本实验中发现,AGE-BSA作用于RMEC后,NF-κB从细胞质内转移至细胞核,从而验证了AGE能够激活NF-κB这一重要通路;probucol抗炎作用的机制可能与其抑制NF-κB的活化作用有关。
结论
1.糖基化终末产物导致的大鼠RMEC损伤和功能下降的机制可能包括①引发脂质过氧化;②活化NADPH氧化酶,增加活性氧的生成;③eNOSmRNA水平异常,影响NO生成;④激活NF-κB。
2.丙丁酚的作用机制可能是通过拮抗AGE-BSA导致的脂质过氧化,NF-κB、NADPH氧化酶活化和eNOS mRNA表达异常来保护RMEC功能。
Introduction
Diabetic nephropathy is a leading cause of end-stage renal failure.Glucose and other reducing sugars can react nonenzymatically with the free amino groups of proteins to form reversible Schiff bases followed by Amadori rearrangement.These early glycation products undergo further complex reactons such as dehydtraton,condersation,and cross-linking to yield irreversible fluorescent derivatives termed advanced glycation end products(AGEs).Many studies reveal ed that AGEs have been implicated in the initiation and development of diabetic microvascular complication primarily by inter with their intracellular signal transducing receptor for advanced glycation end products(RAGE).The engagement of RAGE on endothelial cells by AGEs results in the induction of intracellular reactive oxygen species(ROS),which seems to be linked to the activaton of the NADPH-oxidase system and the mitochondrial electron transport system.The increased ROS in turn activates acomplex cascade of signal transduction pathways and sbusequentlly enhances the expression of many genes that are highly relevant for inflammation,and atherosclerosis.Probucol,an old-line anticholesterol medication,has powerful antioxidant properties also.In clinical studies,probucol improved the function of diabetic kidney.In this study we aimed to explore the effect of AGEs on injuring of rat renal microvascular endothelial cells and protective effect of probucol.
Methods
Microvascular endothelial cells isolated and cultured from rat renal were divided into 3 groups:normal control group,AGEs group and probucol group.The levels of MDA and NO were determined by the assay TBA and nitrate reductase method respectively.The expression of eNOS mRNA and NADPH oxidase protein was detected by RT-PCR and Western-blot respectively.The intracellular disposition of NF-κB was observed by immunostaining microscopy.
Results
In AGEs group,the level of MDA and the expression of NADPH oxidase protein increased but and the expression of eNOS mRNA decreased,as compared with control. The level of NO in culture medium was increased after exposure to AGE-BSA for 30 min and 6h,but after 12h,NO level was decreased.MDA level and the expression of NADPH oxidase protein were decreased but the expression of eNOS mRNA and NO level were upregulated by probucol with a dose-dependent effect in the concentrations of 10,20,50,100μmol/L.The localization of NF-κB shifted from cytoplasm to nucleus after microvascular endothelial cells were exposed to AGE-BSA.After exposed for 30 min,the expression in nucleus reached the peaked and then maintained high level.
Discussion
The classical criteria for identification of microvascuar endothelial cells included cobblestone appearance in vitro culture,synthesis of v Wf by cells,and tubular structure by matrigel in vitro.In the present study,Microvascular endothelial cells isolated and cultured from rat renal were identificated by the classical criteria successfully. Numerous reports have demonstrated that oxidative stress induced by diabetes plays an important role in the development and progression of diabetic vascular complications including nephropathy.Indeed,there is emerging evidence that the formation of reactive oxygen species(ROS)is a direct consequence of high level of advanced glyciation end products.In the present study,we clearly demonstrated that AGEs could cause dysfunction of rat renal microvascular cells.Our result confirmed Oxidative Stress played an important role in Injuring of Rat Renal Microvascular Endothelial Cells.In the present study,we demonstrated in AGEs group the expression of NADPH oxidase protein increased but the expression of eNOSmRNA decreased,resulting in an enhancement of Oxidative Stress in RMEC.The enhancement oxidative stress was further confirmed by normalization of level of MDA,expression of NADPH oxidase protein and in RMEC treated with probucol in parallel with the decrease in oxidative stress.The transcription factors of the NF-kB/Rel family form dimeric complexes that control expression of various genes involved in inflammation and proliferation.In our study,we observed that localization of NF-κB shifted from cytoplasm to nucleus after microvascular endothelial cells were exposed to AGE-BSA and probucol could prevent activation of NF-κB.Protective Effect of Probucol on microvascular entothelial cells probably via antagonisting AGE-induced activating of NF-κB.
Conclusion
AGEs can cause the injury and dysfunction of renal microvascular endothelial cells via the following possible mechanisms:(1)inducing lipid preoxidation(2)activating NADPH oxidase and increasing the production of ROS;(3)abnormal expression of eNOS mRNA and further affecting NO production;(4)activatin of NF-κB.Probucol can protect microvascular entothelial cells probably via antagonisting the lipid preoxidation,activating of NF-κB and NADPH oxidase,and increasing the AGE-induced abnormal expression of eNOS caused by AGEs.
引文
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