高糖对脑微血管内皮细胞抗氧化系统和ATP水平的影响及白藜芦醇的干预机制研究
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摘要
第一部分
高糖对脑微血管内皮细胞抗氧化系统及ATP水平的影响
糖尿病认知功能损伤与脑微血管并发症密切相关,一般认为是持续高糖对脑微小血管侵害导致脑组织缺血缺氧的结果。研究发现,糖尿病动物模型脑内ATP水平下降,是否也与认知损伤有一定关系?糖尿病微血管病变部位是否也同样存在这种能量代谢障碍以及其具体机制如何目前尚未见相关研究。氧化应激是糖尿病微血管病变产生的一个非常关键的致病机制,尽管细胞内存在铜锌超氧化物岐化酶(SOD1)、锰超氧化物岐化酶(SOD2)、过氧化氢酶(CAT)及脂质过氧化物酶(GPX)等多种抗氧化酶,然而高糖刺激时血管内皮细胞仍然会产生大量氧自由基。因此我们猜测高糖刺激时脑微血管内皮细胞,尤其氧自由基大量产生的线粒体部位某些关键的抗氧化酶可能出现了调控障碍,影响了自由基的清除,导致细胞ATP水平下降。解耦联蛋白(UCP)系统在线粒体内具有降低ATP生成和清除自由基的双重功能,线粒体内抗氧化酶的调控障碍,是否可能会引起解耦联蛋白的上调,从而进一步导致细胞ATP水平下降。
为验证我们的假设,我们通过体外培养的小鼠脑微血管内皮细胞系bEnd.3,采用H2DCF荧光法检测氧自由基(ROS)生成,RT-PCR检测高糖刺激下内皮细胞SOD1、SOD2、GPX、CAT、UCP1、UCP2、UCP3、UCP4、UCP5的表达,并通过werstern blot法检测内皮细胞内SOD2蛋白水平。结果显示高糖刺激10小时抗氧化酶SOD2、GPX、CAT的mRNA水平无明显变化,SOD1mRNA水平略有增高；SOD1、SOD2的mRNA水平在高糖刺激24小时后有明显增加,但是SOD2蛋白水平在高糖刺激48小时内并无明显增加,蛋白酶体抑制剂MG132能显著减少高糖下SOD2蛋白水平,提示高糖下SOD2的蛋白合成可能受到一定程度抑制；我们还观察到NF-kB抑制剂及显性负性突变体IkB (Dominant negtive IkB)和P13K抑制剂能上调高糖状态下的SOD2蛋白水平,而NF-kB抑制剂对高糖下SOD2 mRNA水平无明显影响,提示NF-kB可能是通过提高高糖下SOD2蛋白合成率起作用,NF-KB抑制剂对高糖下的氧自由基生成也显示出抑制作用。另外我们还观察到24小时高糖刺激下脑微血管内皮细胞能同时上调解耦联蛋白(UCP4.UCP5)mRNA水平,而对UCP1及UCP2无明显影响。NF-kB抑制剂有降低UCP5基因表达的趋势,但没有达到显著性,提示UCP5激活可能还有其他因素参与。我们也观察到高糖刺激能显著降低脑微血管内皮细胞的ATP水平,而NF-kB抑制剂有升高ATP水平的趋势但没有能达到显著性,可能与NF-kB抑制剂对UCP作用不明显有关。
我们的研究结果提示高糖确实导致了脑微血管内皮细胞内SOD2蛋白翻译障碍,NF-kB激活可能参与其中；高糖同时也引起脑微血管内皮细胞内UCP4及UCP5上调,但这种上调并非完全由SOD2调控障碍引起；高糖显著性的导致了脑微血管内皮细胞ATP水平下降,但是否与SOD2调控障碍以及UCP蛋白上调密切相关还需进一步研究。
第二部分
白藜芦醇对高糖下脑微血管内皮细胞NADPH氧化酶的影响及干预机制
近年来越来越多的证据提示,高糖刺激下还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性增高引起的氧化应激反应对糖尿病微血管和大血管病变的病情发展起重要作用。NADPH氧化酶广泛分布于血管内皮细胞,具有产生活性氧簇(ROS)的功能,尤其在细胞因子、高糖、高脂等作用下可以产生更高水平的ROS。白藜芦醇(Resveratrol)是一种常见于葡萄、虎杖等植物中的一种多酚类化合物,目前的研究显示它具有抗氧化、抗肿瘤、抗炎以及延缓衰老等多种药理功能,流行病学调查也显示白藜芦醇对心血管有显著的保护作用。白藜芦醇良好的抗氧化作用是否是能通过作用于糖尿病状态下脑微血管内皮细胞NADPH氧化酶,以降低细胞损伤水平?本课题在离体水平研究了白藜芦醇在高糖所致脑微血管内皮细胞损伤中的保护机制以及对NADPH氧化酶的影响。
我们在标准糖浓度下(5.5mM)体外培养bEnd.3细胞系并利用高糖(25mM)刺激细胞,用H2DCF-DA、MTT、Hoechst 33258及化学发光法检测高糖下脑微血管内皮细胞氧化应激水平、细胞活性、凋亡状况及NADPH氧化酶活性。RT-PCR和Western blot分别在mRNA、蛋白质水平检测高糖刺激下NADPH氧化酶亚基NOX1、NOX2、p22phox表达情况。结果发现高糖刺激下脑微血管内皮细胞氧化应激水平显著增加,细胞活性降低,凋亡增加。同时NADPH氧化酶活性增加,其亚基NOX1基因表达增加,蛋白水平升高,而NOX2及p22phox亚基mRNA水平无明显变化。NADPH氧化酶抑制香草乙酮(apocynin)及白藜芦醇能显著降低高糖刺激下脑微血管内皮细胞氧化应激水平和凋亡水平；白藜芦醇同时能降低NADPH氧化酶活性,降低NOX1 mRNA及蛋白水平。实验同时证实高糖能激活NF-kB信号通路,NF-kB抑制剂及显性负性突变体IkB (Dominant negative IkB)能显著抑制高糖下NOX1亚基的蛋白水平升高,而白藜芦醇也能抑制高糖下NF-kB的激活,提示白藜芦醇作用于NADPH氧化酶可能与NF-kB通路相关。上述结果提示NADPH氧化酶介导了高糖引起的脑微血管氧化应激反应,白藜芦醇能通过抑制NADPH氧化酶NOX1的蛋白表达从而抑制NADPH氧化酶活性,在糖尿病脑微血管病变中发挥抗氧化作用。
To study the effect of hyperglycemia on antioxidase gene expression and ATP level in brain microvascular endothelial cells(BMESs). Mouse brain microvascular endothelial cells line bEnd.3 was used. Reactive oxygen species (ROS) producrion was measured using 2'7'-dichlorofluorescin diacetate (DCFH-DA); SOD1,SOD2,GPX,CAT,UCP1,UCP2,UCP3,UCP4 and UCP5 gene expression were determined in bEnd.3 under high glucose(25mM) or normal glucose(5.5mM) by reverse transcription PCR;We also determined the protein level of SOD2 and the effect of inhibitors of PI3K,NF-kB,JNK,PKC on protein level of SOD2.We found that hyperglycemia can significantly increased the mRNA levels of SOD1,SOD2,UCP4 and UCP5,while there were no increase in protein of SOD2 found under hyperglycemia condition.The inhibitor of NF-kB and PI3K can increase the protein level of SOD2 in hyperglycemia,while the inhibitors of JNK,PKC have little effect.At the same time,we found the inhibitor of NF-kB have no effect on the mRNA level of SOD2 and UCP5 under hyperglycemia.Hyperglycemia also can reduce the ATP level of bEnd.3 while the inhibitor of NF-kB could not significant increase the ATP level of bEnd.3 under hyperglyceia. These results sugget that hyperglycemia inhibit SOD2 protein synthesis maybe partial through NF-kB pathway in spite of increased mRNA level; The increased gene expression of UCP4 and UCP5 and the decreased ATP level have no significant relative with the protein level of SOD2.
Elevated oxidative stress plays an important role in diabetes-associated micro vasuclar disease in which NADPH oxidases maybe a major source of ROS generation.In this study we tested the hypothesis that high glucose induced oxidative stress was associated with changes in the expression of NADPH oxidase subunits and resveratrol can counteract this change. Fluoresence labelling with dihydroethidum,lucigenin-enhenced chemiluminescence, polymerase chain reaction,western blotting were empbyed to determine oxidative stress,NADPH oxidase activity and NADPH oxidse subunits mRNA and protein expression in cell cultures of mouse brain microvessel endothelial cells(bEnd.3).High glucose enhenced NADPH oxidase activity and ROS production in bEnd.3.High glucose also increased protein and mRNA level of NADPH catalytic isoform Noxl but had little effect on mRNA level of NOX2 and p22phox. Inhibition of NADPH oxidase activity by apocynin can significant reduced high glucose induced ROS production. The increases of NADPH oxidase activity,Noxl protein and mRNA level were significant suppressed by resveratrol which pretect endothelial cells from injury.High glucose increased NOX1 protein level was decreased by the inhibitor of NF-kB sulfasalazine and Resveratrol can inhibit the NF-kB activity by suppressing high glucose increased phosphate-IkB-alpha.These results suggest that Resveratrol could inhibite the NADPH oxidase activity through NF-kB pathway which can protect brain microvessel endothelial cells from oxidative stress damage induced by high glucose.
高糖对脑微血管内皮细胞抗氧化系统及ATP水平的影响
糖尿病认知功能损伤与脑微血管并发症密切相关,一般认为是持续高糖对脑微小血管侵害导致脑组织缺血缺氧的结果。研究发现,糖尿病动物模型脑内ATP水平下降,是否也与认知损伤有一定关系?糖尿病微血管病变部位是否也同样存在这种能量代谢障碍以及其具体机制如何目前尚未见相关研究。氧化应激是糖尿病微血管病变产生的一个非常关键的致病机制,尽管细胞内存在铜锌超氧化物岐化酶(SOD1)、锰超氧化物岐化酶(SOD2)、过氧化氢酶(CAT)及脂质过氧化物酶(GPX)等多种抗氧化酶,然而高糖刺激时血管内皮细胞仍然会产生大量氧自由基。因此我们猜测高糖刺激时脑微血管内皮细胞,尤其氧自由基大量产生的线粒体部位某些关键的抗氧化酶可能出现了调控障碍,影响了自由基的清除,导致细胞ATP水平下降。解耦联蛋白(UCP)系统在线粒体内具有降低ATP生成和清除自由基的双重功能,线粒体内抗氧化酶的调控障碍,是否可能会引起解耦联蛋白的上调,从而进一步导致细胞ATP水平下降。
为验证我们的假设,我们通过体外培养的小鼠脑微血管内皮细胞系bEnd.3,采用H2DCF荧光法检测氧自由基(ROS)生成,RT-PCR检测高糖刺激下内皮细胞SOD1、SOD2、GPX、CAT、UCP1、UCP2、UCP3、UCP4、UCP5的表达,并通过werstern blot法检测内皮细胞内SOD2蛋白水平。结果显示高糖刺激10小时抗氧化酶SOD2、GPX、CAT的mRNA水平无明显变化,SOD1mRNA水平略有增高；SOD1、SOD2的mRNA水平在高糖刺激24小时后有明显增加,但是SOD2蛋白水平在高糖刺激48小时内并无明显增加,蛋白酶体抑制剂MG132能显著减少高糖下SOD2蛋白水平,提示高糖下SOD2的蛋白合成可能受到一定程度抑制；我们还观察到NF-kB抑制剂及显性负性突变体IkB (Dominant negtive IkB)和P13K抑制剂能上调高糖状态下的SOD2蛋白水平,而NF-kB抑制剂对高糖下SOD2 mRNA水平无明显影响,提示NF-kB可能是通过提高高糖下SOD2蛋白合成率起作用,NF-KB抑制剂对高糖下的氧自由基生成也显示出抑制作用。另外我们还观察到24小时高糖刺激下脑微血管内皮细胞能同时上调解耦联蛋白(UCP4.UCP5)mRNA水平,而对UCP1及UCP2无明显影响。NF-kB抑制剂有降低UCP5基因表达的趋势,但没有达到显著性,提示UCP5激活可能还有其他因素参与。我们也观察到高糖刺激能显著降低脑微血管内皮细胞的ATP水平,而NF-kB抑制剂有升高ATP水平的趋势但没有能达到显著性,可能与NF-kB抑制剂对UCP作用不明显有关。
我们的研究结果提示高糖确实导致了脑微血管内皮细胞内SOD2蛋白翻译障碍,NF-kB激活可能参与其中；高糖同时也引起脑微血管内皮细胞内UCP4及UCP5上调,但这种上调并非完全由SOD2调控障碍引起；高糖显著性的导致了脑微血管内皮细胞ATP水平下降,但是否与SOD2调控障碍以及UCP蛋白上调密切相关还需进一步研究。
第二部分
白藜芦醇对高糖下脑微血管内皮细胞NADPH氧化酶的影响及干预机制
近年来越来越多的证据提示,高糖刺激下还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性增高引起的氧化应激反应对糖尿病微血管和大血管病变的病情发展起重要作用。NADPH氧化酶广泛分布于血管内皮细胞,具有产生活性氧簇(ROS)的功能,尤其在细胞因子、高糖、高脂等作用下可以产生更高水平的ROS。白藜芦醇(Resveratrol)是一种常见于葡萄、虎杖等植物中的一种多酚类化合物,目前的研究显示它具有抗氧化、抗肿瘤、抗炎以及延缓衰老等多种药理功能,流行病学调查也显示白藜芦醇对心血管有显著的保护作用。白藜芦醇良好的抗氧化作用是否是能通过作用于糖尿病状态下脑微血管内皮细胞NADPH氧化酶,以降低细胞损伤水平?本课题在离体水平研究了白藜芦醇在高糖所致脑微血管内皮细胞损伤中的保护机制以及对NADPH氧化酶的影响。
我们在标准糖浓度下(5.5mM)体外培养bEnd.3细胞系并利用高糖(25mM)刺激细胞,用H2DCF-DA、MTT、Hoechst 33258及化学发光法检测高糖下脑微血管内皮细胞氧化应激水平、细胞活性、凋亡状况及NADPH氧化酶活性。RT-PCR和Western blot分别在mRNA、蛋白质水平检测高糖刺激下NADPH氧化酶亚基NOX1、NOX2、p22phox表达情况。结果发现高糖刺激下脑微血管内皮细胞氧化应激水平显著增加,细胞活性降低,凋亡增加。同时NADPH氧化酶活性增加,其亚基NOX1基因表达增加,蛋白水平升高,而NOX2及p22phox亚基mRNA水平无明显变化。NADPH氧化酶抑制香草乙酮(apocynin)及白藜芦醇能显著降低高糖刺激下脑微血管内皮细胞氧化应激水平和凋亡水平；白藜芦醇同时能降低NADPH氧化酶活性,降低NOX1 mRNA及蛋白水平。实验同时证实高糖能激活NF-kB信号通路,NF-kB抑制剂及显性负性突变体IkB (Dominant negative IkB)能显著抑制高糖下NOX1亚基的蛋白水平升高,而白藜芦醇也能抑制高糖下NF-kB的激活,提示白藜芦醇作用于NADPH氧化酶可能与NF-kB通路相关。上述结果提示NADPH氧化酶介导了高糖引起的脑微血管氧化应激反应,白藜芦醇能通过抑制NADPH氧化酶NOX1的蛋白表达从而抑制NADPH氧化酶活性,在糖尿病脑微血管病变中发挥抗氧化作用。
To study the effect of hyperglycemia on antioxidase gene expression and ATP level in brain microvascular endothelial cells(BMESs). Mouse brain microvascular endothelial cells line bEnd.3 was used. Reactive oxygen species (ROS) producrion was measured using 2'7'-dichlorofluorescin diacetate (DCFH-DA); SOD1,SOD2,GPX,CAT,UCP1,UCP2,UCP3,UCP4 and UCP5 gene expression were determined in bEnd.3 under high glucose(25mM) or normal glucose(5.5mM) by reverse transcription PCR;We also determined the protein level of SOD2 and the effect of inhibitors of PI3K,NF-kB,JNK,PKC on protein level of SOD2.We found that hyperglycemia can significantly increased the mRNA levels of SOD1,SOD2,UCP4 and UCP5,while there were no increase in protein of SOD2 found under hyperglycemia condition.The inhibitor of NF-kB and PI3K can increase the protein level of SOD2 in hyperglycemia,while the inhibitors of JNK,PKC have little effect.At the same time,we found the inhibitor of NF-kB have no effect on the mRNA level of SOD2 and UCP5 under hyperglycemia.Hyperglycemia also can reduce the ATP level of bEnd.3 while the inhibitor of NF-kB could not significant increase the ATP level of bEnd.3 under hyperglyceia. These results sugget that hyperglycemia inhibit SOD2 protein synthesis maybe partial through NF-kB pathway in spite of increased mRNA level; The increased gene expression of UCP4 and UCP5 and the decreased ATP level have no significant relative with the protein level of SOD2.
Elevated oxidative stress plays an important role in diabetes-associated micro vasuclar disease in which NADPH oxidases maybe a major source of ROS generation.In this study we tested the hypothesis that high glucose induced oxidative stress was associated with changes in the expression of NADPH oxidase subunits and resveratrol can counteract this change. Fluoresence labelling with dihydroethidum,lucigenin-enhenced chemiluminescence, polymerase chain reaction,western blotting were empbyed to determine oxidative stress,NADPH oxidase activity and NADPH oxidse subunits mRNA and protein expression in cell cultures of mouse brain microvessel endothelial cells(bEnd.3).High glucose enhenced NADPH oxidase activity and ROS production in bEnd.3.High glucose also increased protein and mRNA level of NADPH catalytic isoform Noxl but had little effect on mRNA level of NOX2 and p22phox. Inhibition of NADPH oxidase activity by apocynin can significant reduced high glucose induced ROS production. The increases of NADPH oxidase activity,Noxl protein and mRNA level were significant suppressed by resveratrol which pretect endothelial cells from injury.High glucose increased NOX1 protein level was decreased by the inhibitor of NF-kB sulfasalazine and Resveratrol can inhibit the NF-kB activity by suppressing high glucose increased phosphate-IkB-alpha.These results suggest that Resveratrol could inhibite the NADPH oxidase activity through NF-kB pathway which can protect brain microvessel endothelial cells from oxidative stress damage induced by high glucose.
引文
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