黄酮类化合物对高糖环境下内皮细胞 NAD + 水平的保护作用

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Protective Pleiotropic Effect of Flavonoids on NAD+ Levels in Endothelial Cells Exposed to High Glucose


Abstract

摘要

NAD+ is important for oxidative metabolism by serving as an electron transporter. Hyperglycemia decreases NAD+ levels by activation of the polyol pathway and by overactivation of poly(ADP-ribose)-polymerase (PARP). We examined the protective role of three structurally related flavonoids (rutin, quercetin, and flavone) during high glucose conditions in an in vitro model using human umbilical vein endothelial cells (HUVECs). Additionally we assessed the ability of these flavonoids to inhibit aldose reductase enzyme activity. We have previously shown that flavonoids can inhibit PARP activation. Extending these studies, we here provide evidence that flavonoids are also able to protect endothelial cells against a high glucose induced decrease in NAD+. In addition, we established that flavonoids are able to inhibit aldose reductase, the key enzyme in the polyol pathway. We conclude that this protective effect of flavonoids on NAD+ levels is a combination of the flavonoids ability to inhibit both PARP activation and aldose reductase enzyme activity. This study shows that flavonoids, by a combination of effects, maintain the redox state of the cell during hyperglycemia. This mode of action enables flavonoids to ameliorate diabetic complications.

对唿吸作用来说很重要,因为它是一个电子传输器。高血糖可通过激活多元醇途径和过度激活多聚腺苷二磷酸核糖聚合酶(PARP)降低 NAD + 水平。在人脐静脉内皮细胞(HUVECs)体外模型中,我们研究了三种结构相关的黄酮类化合物(芦丁、槲皮素和黄酮)在高糖条件下的保护作用。此外,我们还评估了这些黄酮类化合物抑制醛糖还原酶酶活性的能力。我们之前已经证明黄酮类化合物可以抑制 PARP 的活化。延伸这些研究,我们在这里提供的证据,黄酮类化合物也能够保护内皮细胞对抗高糖诱导的 NAD + 的减少。此外,我们确定黄酮类化合物能够抑制多元醇途径中的关键酶醛糖还原酶。我们得出结论,黄酮类化合物对 NAD + 水平的保护作用是黄酮类化合物抑制 PARP 活化和醛糖还原酶酶活性能力的结合。这项研究表明,黄酮类化合物,通过联合作用,在高血糖期间维持细胞的氧化还原状态。这种作用方式使得黄酮类化合物能够改善糖尿病并发症。

1. Introduction

1. 引言

Worldwide more than 400 million people suffer from diabetes. This number will only grow due to the rapid increase in the incidence of the disease caused by population growth, aging, urbanization, and increasing prevalence of obesity and physical inactivity [1]. A hallmark of diabetes is hyperglycemia [2]. A number of epidemiological studies have shown a relationship between hyperglycemia and an increased risk of cardiovascular diseases, including microvascular pathologies in the eye, kidney, and peripheral nerves. As a consequence, diabetes is a leading cause of blindness, renal disease, and a variety of debilitating neuropathies (e.g., diabetic foot) [34].

全世界有超过4亿人患有糖尿病。由于人口增长、老龄化、城市化以及肥胖和缺乏身体活动的日益普遍导致的疾病发病率的迅速上升,这个数字只会增长。糖尿病的一个特点是高血糖。许多流行病学研究表明,高血糖与心血管疾病风险增加有关,包括眼睛、肾脏和周围神经的微血管病变。因此,糖尿病是导致失明、肾脏疾病和各种衰弱性神经病(如糖尿病足)的主要原因[3,4]。

Nicotinamide adenine dinucleotide (NAD) is found in all living cells in an oxidized form (NAD+) and a reduced form (NADH). The main function of NAD in cells is modulating cellular redox status by carrying electrons from one reaction to another. Additionally, it is also involved in other cellular processes (e.g., acting as a substrate for enzymes involved in posttranslational modification) [5]. Hyperglycemia decreases NAD+ levels by an increased flux of glucose through the polyol pathway. This pathway becomes active when intracellular glucose levels are elevated [6]. During normoglycemia only ~3% of all glucose will enter the polyol pathway. Most of the glucose will be phosphorylated to glucose-6-phosphate by hexokinase. However, under hyperglycemic conditions ten times more glucose enters the polyol pathway [7], mainly due to a saturation of hexokinase [8]. Aldose reductase, the first and rate-limiting enzyme in the pathway, reduces glucose to sorbitol using NADPH as a cofactor. Then, sorbitol is reduced to fructose by sorbitol dehydrogenase which uses NAD+ as a cofactor. The osmotic stress that accompanies sorbitol accumulation and the redox imbalance following the depletion of NADPH and NAD+ contributes to cell damage and organ injury, ultimately leading to cataract genesis, neuropathy, and other diabetic complications [911].

烟酰胺腺嘌呤二核苷酸(NAD)存在于所有的活细胞中,分别以氧化形式(NAD +)和还原形式(NADH)存在。NAD 在细胞中的主要功能是通过携带电子从一个反应到另一个反应来调节细胞的氧化还原状态。此外,它还参与其他细胞过程(例如,作为酶参与翻译后修饰的底物)。高血糖可通过多元醇途径增加葡萄糖流量降低 NAD + 水平。当细胞内葡萄糖水平升高时,这一通路就变得活跃。在正常血糖状态下,只有3% 的葡萄糖进入多元醇途径。大部分葡萄糖将被己糖激酶磷酸化为葡萄糖-6-磷酸。然而,在高血糖条件下,十倍以上的葡萄糖进入多元醇途径[7] ,主要是由于己糖激酶的饱和[8]。醛糖还原酶是这一途径中的第一个限速酶,它利用 NADPH 作为辅助因子将葡萄糖降解为山梨醇。山梨醇脱氢酶以 NAD + 为辅酶,将山梨醇还原为果糖。山梨醇蓄积和 NADPH 和 NAD + 耗尽后氧化还原失衡所引起的渗透应激会导致细胞损伤和器官损伤,最终导致白内障发生、神经病变和其他糖尿病并发症[9-11]。

Poly(ADP-ribose)-polymerase (PARP) activation can also lead to NAD+depletion. The nuclear enzyme PARP has been implicated in the regulation of many important cellular functions like DNA repair, gene transcription, cell cycle progression, cell death, chromatin function, and genomic stability [12]. PARP detects and signals single-strand DNA breaks (SSB), which can be induced by hyperglycemia. Upon detection of a SSB, PARP binds to the DNA and synthesizes a poly(ADP-ribose) (PAR) chain as a signal for DNA repair enzymes. NAD+ is required as a substrate for the synthesis of these PAR monomers. Overactivation of PARP therefore depletes cellular NAD+ stores [13]. Several studies have suggested an important role of PARP activation in the pathogenesis of diabetic complications like nephropathy, neuropathy, and retinopathy [1416].

多聚腺苷二磷酸核糖聚合酶(PARP)激活也可导致 NAD + 耗尽。核酶 PARP 参与了许多重要的细胞功能的调节,如 DNA 修复、基因转录、细胞周期进程、细胞死亡、染色质功能和基因组稳定性[12]。PARP 检测并发出单链 DNA 断裂(SSB)信号,SSB 是由高血糖引起的。检测到单链抗体后,PARP 与 DNA 结合并合成一个多聚(adp- 核糖)(PAR)链作为 DNA 修复酶的信号。NAD + 是合成这些 PAR 单体所需要的基质。因此,PARP 的过度激活会耗尽细胞内 NAD + 的存储[13]。一些研究表明 PARP 激活在糖尿病并发症如肾病、神经病变和视网膜病变的发病机制中起重要作用。

Previously we have established that dietary flavonoids inhibit PARP both in vitroand in vivo [1719]. Flavonoids are polyphenolic compounds which are found in fruits, vegetables, and plant-derived products like red wine and tea [18]. Flavonoids have been shown to display positive health effects, for example, reduced risks for cardiovascular and chronic inflammatory diseases [2023], which have been ascribed to their antioxidant and anti-inflammatory properties [2224]. We now studied the effect on NAD+ levels in endothelial cells after exposing the cells to high glucose in the presence or absence of flavonoids. In addition we determined whether three structurally related flavonoids are also able to inhibit aldose reductase, the most important enzyme of the polyol pathway.

在此之前,我们已经证实,膳食中的黄酮类化合物在体内外都能抑制 PARP [17-19]。类黄酮是一种多酚类化合物,存在于水果、蔬菜和植物衍生产品中,如红酒和茶叶[18]。黄酮类化合物已被证明具有积极的健康作用,例如,降低患心血管疾病和慢性炎症疾病的风险[20-23] ,这归功于它们的抗氧化和抗炎特性[22,24]。我们现在研究了在有无黄酮存在的情况下,将内皮细胞暴露于高糖环境下,对内皮细胞 NAD + 水平的影响。此外,我们还确定了3种结构相关的黄酮类化合物是否也能够抑制醛糖还原酶,多元醇途径中最重要的酶。

2. Material and Methods

2. 材料和方法

2.1. Chemicals
2.1. 化学品

All chemicals were purchased from Sigma-Aldrich (Steinheim, Germany) unless stated otherwise. F12K medium, Hank’s balanced salt solution (HBSS), trypsin-EDTA, non-heat-inactivated fetal calf serum (FCS), and penicillin/streptomycin were obtained from Gibco (Breda, The Netherlands). Endothelial cell growth supplement (ECGS) was obtained from BD Bioscience (Breda, The Netherlands). Heparin was purchased from Leo Pharmaceuticals (Amsterdam, The Netherlands).

除非另有说明,所有的化学品都是从 Sigma-Aldrich 公司购买的。从 Gibco 分离得到 F12K 培养基、 Hank 平衡盐溶液(HBSS)、胰蛋白酶 -edta、非热灭活胎牛血清(FCS)和青霉素/链霉素。内皮细胞生长补充剂(ECGS)从 BD 生物科学(布雷达,荷兰)获得。肝素是从 Leo 制药公司(荷兰阿姆斯特丹)购买的。

2.2. Cell Culture
2.2. 细胞培养

Human umbilical vein endothelial cells (HUVECs) (CRL-1730) were obtained from ATCC. HUVECs were cultured in F12K medium with 10% FCS, 1% penicillin/streptomycin, 0.05 mg/mL endothelial cell growth supplement (ECGS), and 0.1 mg/mL heparin. Cells were maintained in collagen coated flasks at 37°C in a 5% CO2 atmosphere. For experiments, cells were seeded in 6- or 96-well plates and allowed to attach overnight. Next, medium was removed and cells were washed with HBSS. Additionally, fresh medium was added containing glucose (30 mM final concentration) or vehicle (medium) and flavonoids (5 μM final concentration), sorbinil (0.5 μM final concentration), or its vehicle (DMSO).

从 ATCC 中分离培养人脐静脉内皮细胞(HUVECs)(CRL-1730)。在 F12K 培养基中加入10% FCS、1% 青霉素/链霉素、0.05 mg/mL 内皮细胞生长补充剂(ECGS)和0.1 mg/mL 肝素。细胞保持在胶原涂层烧瓶在37 ° c 的5% CO2气氛中。在实验中,将细胞种植在6孔或96孔的平板中,并让其在一夜之间附着。然后,去除培养基,用 HBSS 清洗细胞。另外,添加葡萄糖(30mm 终浓度)或载体(中等)、黄酮(5μM 终浓度)、索比尼(0.5 μm 终浓度)或载体(DMSO)的新鲜培养基。

2.3. Gene Expression Analysis
2.3. 基因表达分析

RNA was isolated from QIAzol suspended cells according to the manufacturer’s protocol and quantified spectrophotometrically with a NanoDrop. RNA (500 ng) was reverse-transcribed using iScript cDNA synthesis kit (Bio-Rad, Veenendaal, The Netherlands). Next, real time PCR was performed with a Bio-Rad MyIQ iCycler Single Color RT-PCR detection system using Sensimix Plus SYBR and Fluorescein (Quantace-Bioline, Alphen a/d Rijn, The Netherlands), 5 μL diluted (10x) cDNA, and 0.3 μM primers in a total volume of 25 μL. PCR was conducted as follows: denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 45 seconds. After PCR, a melt curve (60–95°C) was produced for product identification and purity. β-actin was included as internal control. Primer sequences are shown in Table 1. Data were analysed using the MyIQ software system (Bio-Rad) and were expressed as relative gene expression (fold change) using the  method.

根据生产商的协议,从七唑悬浮细胞中提取 RNA,并用纳滴分光光度法进行定量。用 iScript cDNA 合成试剂盒(Bio-Rad,Veenendaal,荷兰)反转录 RNA (500ng) 。其次,采用 Bio-Rad MyIQ iCycler 单色 rt-PCR 检测系统,使用 sensitimix Plus SYBR 和荧光素(Quantace-Bioline,Alphen a/d Rijn,The Netherlands)、5μL 稀释的 cDNA 和0.3 μm 引物,总体积为25μL。PCR 方法为: 95 ° c 变性10分钟,95 ° c 变性40次15秒,60 ° c 变性45秒。聚合酶链反应(PCR)后,产物的熔融曲线(60-95 ° c)可用于产品的鉴定和纯度检测。采用 β- 肌动蛋白作为内部对照。引物序列如表1所示。数据分析使用 MyIQ 软件系统(Bio-Rad) ,并表达为相关基因表达(折叠变化)使用该方法。

Gene 基因Forward (5′ to 3′) 前进(5′至3′)Reverse (5′ to 3′) 倒车(5′至3′)Beta-actin ( β- 肌动蛋白(Beta-actin)β-actin) – 肌动蛋白)CCTGGCACCCAGCACAAT 加拿大语言学家协会GCCGATCCACACGGAGTACT (美国)《电视新闻报道》季刊Aldose reductase 醛糖还原酶TACACATGGGCACAGTCGAT (英国)《独立报》月刊GGGGTTGGGTACCTGGAA (英国)《新闻日报》周刊PARP-1GCCAGTTCAGGACCTCATCAA 卡塔卡卡卡卡卡卡CGGCCTGGATCTGCCTTT 中国科学技术研究院

Table 1 表一Primer sequences for genes used for gene expression analysis. 用于基因表达分析的基因引物序列

2.4. Determination of NAD+ Levels
2.4. NAD + 水平的测定

Cells were lysed with 1% dodecyltrimethylammonium bromide (DTAB) in 0.2 N NaOH. To ensure that only NAD+ levels were measured 0.4 M HCl was added and samples were incubated at 60°C for 15 minutes. Afterwards, cells were incubated at room temperature for 10 minutes and 0.5 M Trizma base was added to the cells after which NAD+ levels were determined with the NAD+/NADH cell based assay kit from Cayman Chemical (Ann Arbor, MI, USA).

用1% 十二烷基三甲基溴化铵(DTAB)在0.2 n NaOH 溶液中对细胞进行裂解。为了确保只测定0.4 m HCl 的 NAD + 水平,样品在60 ° c 下培养15分钟。然后,在室温下培养10分钟,在细胞中加入0.5 m Trizma 碱基,用美国 Cayman 化学公司的 NAD +/NADH 检测试剂盒检测 NAD + 水平。(安阿伯)。

2.5. Preparation of Lens Aldose Reductase
2.5. 镜头醛糖还原酶的准备

Porcine lenses were used as a source of aldose reductase enzyme. Porcine eyes were obtained from a local slaughterhouse. Lenses were removed and stored at −20°C until use. Lens homogenate was freshly prepared for every experiment. Lenses were homogenized in 1.25 mL homogenization buffer (20 mM potassium phosphate buffer, pH 7.5 containing 0.5 mM EDTA and 5 mM 2-mercaptoethanol). The homogenate was centrifuged at 10.000 ×g for 10 minutes at 4°C.

猪的晶状体被用作醛糖还原酶酶的来源。猪的眼睛是从当地的一家屠宰场获得的。镜片须移除,并存放于摄氏零下20度,直至使用为止。每次实验均新鲜制备透镜匀浆。镜片均质于1.25 mL 均质缓冲液中(20mm 磷酸二氢钾缓冲液,pH 7.5,EDTA 含量0.5 mM,巯基乙醇含量5mm)。将匀浆置于10.000 × g,在4 ° c 下离心10分钟。

2.6. Aldose Reductase Assay
2.6. 醛糖还原酶分析

Aldose reductase activity was determined spectrophotometrically. The reaction mixture (0.7 mL) contained 30 mM potassium phosphate buffer (pH 6.2), 0.2 mM NADPH, 0.2 M lithium sulphate, and the substrate DL-glyceraldehyde (0–2 mM). Flavonoids (flavone, quercetin, and rutin) were added to the reaction mixture (final concentration 0.5 or 5 μM). As a positive control, the known aldose reductase inhibitor sorbinil was used in a concentration of 0.5 μM. Reaction was initiated by addition of NADPH. The consumption of NADPH was followed by the decrease in absorbance at 340 nm for 5 minutes at 37°C.

用分光光度法测定醛糖还原酶活性。反应混合物(0.7 mL)含有30mm 的磷酸二氢钾缓冲液(ph6.2) ,0.2 mM 的 NADPH,0.2 m 的硫酸锂,和底物甘油醛(0-2 mM)。黄酮类化合物(黄酮、槲皮素和芦丁)被添加到反应混合物中(最终浓度0.5或5μM)。作为阳性对照,已知的醛糖还原酶抑制剂索比尼用于浓度为0.5 μm。反应由 NADPH 的加入引发。在37 ° c 条件下,NADPH 的消耗量随着340 nm 处吸光度的降低而减少5分钟。

2.7. Statistical Analysis
2.7. 统计分析

The effect of HG incubation and effects of flavonoids were tested using Student’s -test for independent samples or the Mann-Whitney  test when not normally distributed.  values < 0.05 were considered statistically significant and  values < 0.1 were considered statistical trends. Statistical analyses were analyzed with SPSS for Windows (version 20.0; SPSS Inc., Chicago, IL, USA).

汞孵化的效果和黄酮类化合物的效果通过独立样本的学生检验或者当不是正态分布时的 Mann-Whitney 检验进行检验。< 0.05被认为具有统计学意义,< 0.1被认为是统计学趋势。统计分析采用 SPSS for Windows (20.0版; SPSS inc. ,Chicago,IL,USA) 。

3. Results

3. 结果

In Figure 1, the effect of incubating HUVECs with 30 mM of glucose on gene expression of aldose reductase and PARP-1 is presented. It is visible that both aldose reductase and PARP-1 have a significant higher expression after 24-hour incubation compared to normal glucose. When flavonoids are coincubated during these 24 hours, there is no effect on aldose reductase expression compared to only high glucose incubation. Only quercetin seems to lower PARP-1 expression compared to high glucose incubation.

在图1中,30mm 葡萄糖孵育 HUVECs 对醛糖还原酶和 PARP-1基因表达的影响被提出。与正常葡萄糖相比,醛糖还原酶和 PARP-1在24小时培养后都有明显的高表达。当黄酮类化合物在这24小时内共孵化时,与只进行高糖孵化相比,对醛糖还原酶的表达没有影响。与高糖培养相比,仅槲皮素似乎降低了 PARP-1的表达。(a)
(a)(b)
(b)(c)
(c)(d)
(d)(a)
(a)(b)
(b)(c)
(c)(d)
(d)Figure 1 图1Effect of incubation with 30 mM glucose on the expression of aldose reductase (a) and PARP-1 (b) after several incubation times. Effect of addition of flavonoids (compared to high glucose incubation) after 24 hours incubation is shown in (c) (aldose reductase) and (d) (PARP-1). Data are expressed as mean ± standard error from three independent experiments. 30mm 葡萄糖孵育对几次孵育后醛糖还原酶和 PARP-1(b)表达的影响。培养24小时后添加黄酮类化合物(与高糖培养相比)的效果见于(c)(醛糖还原酶)和(d)(PARP-1)。数据表示为三个独立实验的平均 ± 标准误差 compared to normal glucose incubation; 与正常葡萄糖培养相比; compared to incubation with high glucose alone. 与单独使用高葡萄糖培养相比

The effect of incubation with 30 mM glucose on the NAD+ status of HUVECs is depicted in Figure 2. High glucose incubation leads to a significant decrease in NAD+ levels after 24 hours. This decrease is attenuated when the cells are coincubated with flavone or quercetin (trend) but not with rutin. Incubation with the known aldose reductase inhibitor sorbinil led to an even larger decrease in NAD+ levels.

30mm 葡萄糖孵育对人脐静脉内皮细胞 NAD + 状态的影响如图2所示。高葡萄糖培养导致24小时后 NAD + 水平显著下降。当细胞与黄酮或槲皮素共孵育时(趋势) ,而不与芦丁共孵育时,这种下降会减弱。与已知的醛糖还原酶抑制剂索比尼进行孵育可以导致 NAD + 水平的进一步下降。

Figure 2 图2Effect of 24-hour incubation with 7 (control) or 30 mM (HG) glucose on the NAD 7(对照)和30mm (HG)葡萄糖24小时孵育对 NAD 的影响+ level of HUVECs with or without coincubation with flavonoids. Data are expressed as mean ± standard error from four independent experiments. 与黄酮类化合物共孵育或不共孵育的 HUVECs 水平。数据表示为四个独立实验的平均 ± 标准误差 compared to control; 与对照组相比; compared to incubation with high glucose alone; 与单独用高葡萄糖孵育相比;## compared to incubation with high glucose alone. 与单独使用高葡萄糖培养相比

Quercetin, rutin, and flavone at a concentration of 5 μM decreased the  of the aldose reductase catalysed conversion of DL-glyceraldehyde to glycerol. Sorbinil was used as a control and decreased both the  and  at a concentration of 0.5 μM. Rutin also showed a small but significant decrease of  compared to the control (Figure 3).

槲皮素、芦丁和黄酮在5微米的浓度下降低了醛糖还原酶催化甘油醛转化为甘油的能力。用索比尼作为对照,在0.5 μm 浓度下降低血清白蛋白浓度和血清白蛋白浓度。与对照组相比,芦丁也显示出小但显著的下降(图3)。(a)
(a)(b)
(b)(a)
(a)(b)
(b)Figure 3 图3(a) Kinetics of porcine aldose reductase in the absence and presence of flavonoids. Data are expressed as mean ± standard deviation of at least three separate experiments. (a)在缺乏和存在黄酮类化合物的情况下猪醛糖还原酶的动力学。数据以至少3个独立实验的平均标准差表示 compared to control. (b) An example of a Michaelis Menten plot of aldose reductase in absence (filled circles) and presence of 5  相对于控制。(b) Michaelis Menten 醛糖还原酶缺席情节的一个例子(填满的圆圈)和存在的5μM quercetin (filled squares), 5  M 槲皮素(填充方块) ,5μM rutin (open triangles), 5  M 芦丁(开三角形) ,5μM flavone (filled triangles), or 0.5  M 黄酮(填充三角形) ,或0.5μM sorbinil (open circles). Data are expressed as mean ± standard error of at least three experiments. 索比尼(开环) ,数据表示为至少三个实验的平均标准误差

4. Discussion

4. 讨论

In epidemiological studies, the intake of flavonoids has been related to a reduced risk for various diseases, including diabetes [232526]. Many complications that arise from diabetes are attributed to a redox imbalance. In previous studies we established that flavonoids were able to attenuate NAD+depletion by inhibiting PARP overactivation both in vitro and in vivo [1719]. Extending these studies, we here provide evidence that flavonoids are also able to protect endothelial cells against a decrease in NAD+ due to high glucose. In addition we show that flavonoids are able to inhibit the key enzyme of the polyol pathway, aldose reductase. From previous (unpublished) experiments we know that flavonoids at the concentration used in this study (5 μM) do not influence glucose uptake. The flavonoids’ concentration needs to be at least 10-fold higher to influence the uptake of glucose.

在流行病学研究中,黄酮类化合物的摄入与降低各种疾病的风险有关,包括糖尿病[23,25,26]。糖尿病引起的许多并发症都归因于氧化还原失衡。在以前的研究中,我们确定黄酮类化合物能够通过抑制 PARP 的过度激活在体外和体内抑制 NAD + 的损耗[17-19]。延伸这些研究,我们在这里提供的证据,黄酮类化合物也能够保护内皮细胞对抗由于高糖导致的 NAD + 的减少。此外,我们还发现黄酮类化合物能够抑制多元醇途径的关键酶醛糖还原酶。从以前的(未发表的)实验中我们知道,在这项研究中使用的浓度(5微米)的黄酮类化合物不影响葡萄糖的摄取。黄酮类化合物的浓度至少需要提高10倍才能影响葡萄糖的摄取。

In this study three structurally related flavonoids were studied: flavone, the core structure of the flavonoid subgroup flavones, a compound that is present in many cereal grains as well as in dill weed [27]; quercetin, one of the most prominent dietary flavonoids present in many foods including citrus fruit and berries [28]; and rutin, a glycoside of quercetin which is found in buckwheat [29]. These compounds are usually conjugated to sugar moieties but are certainly of interest as protectors during inflammatory conditions when the pH and glucuronidase appear favorable for deconjugation as has been shown by [30].

在这项研究中,我们研究了三种结构相关的黄酮类化合物: 黄酮类化合物黄酮的核心结构,这是一种存在于许多谷物和莳萝中的化合物[27] ; 槲皮素,在许多食物中存在的最突出的膳食黄酮类化合物之一,包括柑橘类水果和浆果[28] ; 以及芦丁,发现于荞麦中的槲皮素的一种糖苷[29]。这些化合物通常与糖分子偶联,但在炎症条件下,当 pH 和葡萄糖醛酸酶显示有利于去除浓缩物时,这些化合物当然是感兴趣的保护剂[30]。

Gene expression of aldose reductase and PARP was investigated in endothelial cells exposed to 30 mM glucose. A higher expression of aldose reductase in peripheral blood mononuclear cells has been linked to an increased risk for kidney disease in diabetic patients [31]. Furthermore, in transgenic mice, it was found that human aldose reductase expression increased atherosclerosis lesion size which could be attenuated by aldose reductase inhibitors [3233]. An increase in PARP mRNA expression was found in patients with type 2 diabetes and microangiopathy [34]. We found an increase in the expression of both genes when endothelial cells were exposed to 30 mM glucose for 24 hours. Coincubation with flavone, rutin, or sorbinil did not affect this increase.

研究了30mm 葡萄糖作用下内皮细胞中醛糖还原酶和 PARP 的基因表达。醛糖还原酶在外周血单个核细胞中的高表达与糖尿病患者肾脏疾病的风险增加有关。此外,在转基因小鼠中,发现人类醛糖还原酶表达增加了动脉粥样硬化病灶的大小,而醛糖还原酶抑制剂可以减小这种大小[32,33]。在2型糖尿病和微血管病变患者中发现 PARP mRNA 表达增加[34]。我们发现当内皮细胞暴露于30mm 的葡萄糖24小时时,这两个基因的表达都增加。与黄酮、芦丁或索比尼共孵育不影响这种增加。

NAD+ is a cofactor in numerous critical oxidation reactions. Because of the involvement in redox signalling, NAD+NADH is regarded as one of the most important redox couples of the cells and therefore an important determinant of redox status of cells. We found a slight decrease in NAD+ levels after incubating HUVECs with 30 mM glucose for 24 hours. This change is most likely a combination of the two previously described pathways: a decrease in NAD+ due to activation of the polyol pathway and overactivation of PARP-1. Therefore we also investigated the potential of flavonoids to inhibit aldose reductase. The flavonoids’ ability to inhibit aldose reductase has been described previously [35]. In our study, it was found that all tested flavonoids were able to inhibit aldose reductase enzyme activity at a concentration of 5 μM. Quercetin and flavone appear to be noncompetitive inhibitors because only the  of the reaction is decreased. Conversely, not only did rutin decrease the , but it also decreased the  slightly. This would indicate a slightly higher reaction rate at very low substrate concentrations but a much lower rate at higher substrate concentrations. Rutin contains rutinose, which is a disaccharide composed of rhamnose and glucose. The latter is a substrate of aldose reductase; however the affinity of aldose reductase for DL-glyceraldehyde is higher [7]. Rutin as a competitive inhibitor is further supported by the results of sorbinil, which is a known competitive inhibitor of aldose reductase [36]. Sorbinil was tested at a lower concentration (0.5 μM) but shows the same results as rutin, a decrease in both  and . Of the tested flavonoids, rutin showed the strongest inhibition, while flavone had the least effect. This is contrary to their capacity to inhibit PARP overactivation, where flavone is the most potent inhibitor and rutin is not able to inhibit PARP (Table 2). In both reactions quercetin is an intermediate inhibitor compared to rutin and flavone.

NAD + 是许多临界氧化反应中的一个辅因子。由于 nad + nadh 参与了氧化还原信号的传递,因此被认为是细胞中最重要的氧化还原配对之一,是决定细胞氧化还原状态的重要因素。我们发现在 HUVECs 与30mm 葡萄糖孵育24小时后,NAD + 水平略有下降。这种变化很可能是以前描述的两种途径的结合: NAD + 的减少是由于多元醇途径的激活和 PARP-1的过度激活。因此,我们也研究了黄酮类化合物抑制醛糖还原酶的潜力。黄酮类化合物抑制醛糖还原酶的能力已经在前面被描述过。在我们的研究中,发现所有测试的黄酮类化合物在5微米的浓度下都能抑制醛糖还原酶酶活性。槲皮素和黄酮是非竞争性抑制剂,因为只有反应减弱。相反地,芦丁不仅降低了血清白蛋白含量,而且还略有下降。这表明在非常低的底物浓度下反应速率略高,但在较高的底物浓度下反应速率低得多。芦丁含有芦丁糖,它是一种由鼠李糖和葡萄糖组成的双糖。后者是醛糖还原酶的基质,然而醛糖还原酶对甘油醛的亲和力更高[7]。芦丁作为一种竞争性抑制剂,索比尼的结果进一步支持,索比尼是一种已知的竞争性抑制醛糖还原酶[36]。在较低浓度(0.5 μm)下测定了索比尼,但结果与芦丁相同,两者均有降低。黄酮类化合物中芦丁的抑制作用最强,黄酮类化合物的抑制作用最弱。这与它们抑制 PARP 过度激活的能力相反,其中黄酮是最有效的抑制剂,而芦丁不能抑制 PARP (表2)。与芦丁和黄酮相比,槲皮素在这两个反应中都是中间抑制剂。

Name姓名Flavone 黄酮Quercetin 槲皮素Rutin 女名女子名Structure结构PARP inhibiting capacity PARP 抑制能力Strong, also at low concentrations 浓度高,同样在低浓度下Strong, less at low concentrations 浓度较高,低浓度时较少No inhibiting capacity 无抑制能力

Table 2 表二Overview of structure and PARP inhibiting capacity of flavonoids used in this study [ 本研究所用黄酮类化合物的结构和 PARP 抑制能力概述[1718].

These findings indicate that the overactivation of PARP-1 plays a larger role than the polyol pathway in the decrease of NAD+ levels in HUVECs. When cells were coincubated with flavonoids, we observed that flavone was able to attenuate the decrease in NAD+ concentration. Flavone is the most potent PARP-1 inhibitor but did not have a large effect on aldose reductase activity. This finding is also supported by the observation that rutin, the most potent aldose reductase activity inhibitor, did not show an effect on NAD+ levels. Quercetin, an average inhibitor of both pathways, showed a trend towards increasing NAD+ levels to normal. The influence of the polyol pathway on the lower NAD+ level seems to be small. Most likely the activation of this pathway has a more pronounced effect on the levels of NADPH. By lowering the levels of this essential cofactor for glutathione, the cells get more susceptible to oxidative stress [37]. This in turn can lead to more reactive oxygen species that can damage DNA, inducing activation of PARP-1, which subsequently can lead to a decrease in NAD+ levels as we observed in HUVECs. This might also be the reason why coincubation with sorbinil leads to an extra decrease in NAD+ levels in HUVECs. By inhibiting the aldose reductase almost completely, unlike the flavonoids which show a mild inhibition, other pathways involved in the pathogenesis of diabetic complications may become more activated (e.g., activation PKC); this then can lead to more oxidative stress and activation of PARP-1 [37].

这些结果表明,PARP-1的过度激活在降低人脐静脉内皮细胞 NAD + 水平中起着比多元醇通路更重要的作用。当细胞与黄酮类化合物共孵育时,黄酮类化合物能够减弱 NAD + 浓度的下降。黄酮是最有效的 PARP-1抑制剂,但对醛糖还原酶活性没有很大的影响。这一发现也得到了观察结果的支持,即最有效的醛糖还原酶活性抑制剂芦丁对 NAD + 水平没有影响。槲皮素是两种途径的平均抑制剂,呈现出增加 NAD + 水平至正常的趋势。多元醇通路对低 NAD + 水平的影响似乎较小。最有可能的是,这一通路的激活对 NADPH 水平有更明显的影响。通过降低谷胱甘肽必需辅助因子的水平,细胞更容易受到氧化应激的影响。这反过来又会导致更多的活性氧类损伤 DNA,诱导 PARP-1的激活,随后导致 NAD + 水平的下降,正如我们在 HUVECs 中观察到的那样。这也可能是与索比尼共孵育导致人脐静脉内皮细胞 NAD + 水平额外下降的原因。通过几乎完全地抑制醛糖还原酶,不像黄酮类化合物那样表现出轻微的抑制作用,与糖尿病并发症发病机制有关的其他通路可能变得更加活跃(例如,激活 PKC) ,这样就可以导致更多的氧化应激和 PARP-1的激活。

5. Conclusions

5. 结论

We conclude that flavonoids are able to exert pleiotropic protective effects under high glucose conditions (Figure 4). We observed that flavonoids were able to inhibit overactivation of PARP-1, thereby preventing a fall in NAD+levels. Furthermore we observed that flavonoids are able to inhibit aldose reductase activity, preventing an additional decrease in NAD+ levels. Moreover, because of the known antioxidant properties of flavonoids they are also able to prevent the deleterious effects of reactive oxygen species which can be formed when a redox imbalance is present. In conclusion, the combination of all these effects is most likely the reason why flavonoids were able to protect endothelial cells against a high glucose induced drop in NAD+ levels in an in vitro system.

我们认为黄酮类化合物在高糖条件下能够发挥多效性保护作用(图4)。我们观察到黄酮类化合物能够抑制 PARP-1的过度活化,从而防止 NAD + 水平的下降。此外,我们观察到黄酮类化合物能够抑制醛糖还原酶活性,防止 NAD + 水平的额外下降。此外,由于黄酮类化合物已知的抗氧化特性,它们也能够防止氧化还原失衡时形成的活性氧类的有害影响。总之,所有这些作用的结合很可能是黄酮类化合物能够保护内皮细胞免受高糖诱导的体外系统 NAD + 水平下降的原因。

Figure 4 图4Flavonoids can protect cells under hyperglycemic stress in several ways. First, flavonoids are able to inhibit overactivation of PARP-1, preventing a decrease in NAD 黄酮类化合物可以通过多种途径保护细胞免受高血糖应激。首先,黄酮类化合物能够抑制 PARP-1的过度活化,防止 NAD 的减少+ levels. Furthermore, flavonoids are able to inhibit aldose reductase activity, preventing an additional decrease in NAD 此外,黄酮类化合物能够抑制醛糖还原酶活性,防止 NAD 的额外减少+ and NADH levels. Also, because of their antioxidant properties, flavonoids are able to prevent damaging effects of oxidative stress. By a combination of all these effects flavonoids are able to protect cells against high glucose induced damage. 和 NADH 水平。同时,由于它们的抗氧化特性,类黄酮可以防止氧化应激的破坏作用。通过所有这些作用的结合,黄酮类化合物能够保护细胞免受高糖诱导的损伤

Conflict of Interests

利益冲突

The authors declare that there is no conflict of interests regarding the publication of this paper.

作者声明,在发表这篇论文方面没有利益冲突。

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