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Glucose Metabolism-Derived Nicotinamide Adenine Dinucleotide Phosphate in Late-Onset Preeclampsia Placenta Tissue and Its Correlation with Oxidative Stress
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摘要:目的 探讨在晚发型子痫前期（late-onset preeclampsia, LOPE）胎盘氧化应激失衡中，糖代谢来源的抗氧化物质烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate, NADPH）的表达变化。方法 选取重庆医科大学第一附属医院产科于2020年11月–2021年10月行剖宫分娩的LOPE孕妇13例，正常妊娠孕妇13例，分别收集胎盘组织。采用DCFH-DA法测定LOPE孕妇和正常妊娠孕妇胎盘组织活性氧簇（reactive oxygen species, ROS）水平。分光光度法测定LOPE组及对照组胎盘组织中 NADPH、谷胱甘肽（glutathione, GSH）、葡萄糖含量，磷酸戊糖代谢途径中关键限速酶葡萄糖-6-磷酸脱氢酶（glucose-6-phosphate dehydrogenase, G6PD）、磷酸葡萄糖酸脱氢酶（phospho-gluconate dehydrogenase, PGD）的表达及活性水平。应用Western blot方法检测LOPE组及对照组胎盘组织中G6PD、PGD及糖酵解途径关键限速酶磷酸果糖激酶1（phosphofructokinase-1, PFK1）的蛋白表达变化。结果 LOPE胎盘组织中ROS水平较对照组胎盘升高（P<0.05），抗氧化物质NADPH、GSH以及葡萄糖含量较对照组胎盘组织升高（P<0.05），PFK1蛋白表达在LOPE胎盘中升高（P<0.05），而G6PD、PGD活性及其蛋白表达在LOPE组与对照组中未见明显差异。结论 晚发型子痫前期胎盘组织中葡萄糖发生代谢重编程，是抗氧化物质NADPH、GSH异常升高的原因之一。Abstract:Objective To study the changes in the expression of nicotinamide adenine dinucleotide phosphate (NADPH), a glucose metabolism-derived antioxidant, in late-onset preeclampsia (LOPE) placenta tissue and the correlation with oxidative stress.Methods A total of 13 normal pregnant women and 13 pregnant women with LOPE who were hospitalized in the Obstetrics Department, the First Affiliated Hospital of Chongqing Medical University and who underwent elective cesarean section between November 2020 and October 2021 were included in the study. Placenta tissues were collected from the subjects. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay was done to determine the ROS levels in the placenta tissues of the LOPE group and the normal control group. Spectrophotometric analysis was conducted to determine the levels of NADPH, glutathione (GSH), and glucose, and the expressions and activities of glucose-6-phosphate dehydrogenase (G6PD) and phospho-gluconate dehydrogenase (PGD), key rate-limiting enzymes of the pentose phosphate pathway (PPP), in the placenta tissues of the LOPE group and the normal control group. Western blot was done to determine changes in the protein expressions of phosphofructokinase 1 (PFK1), a key rate-limiting enzyme of the glycolytic pathway, G6PD, and PGD in the placenta tissues from the two groups.Results ROS levels in the placenta tissue of the LOPE group were significantly higher than those of the control group (P<0.05). The levels of NADPH and GSH, two antioxidants, and glucose in the LOPE placenta were significantly higher than those of the control group (P<0.05). The expression of PFK1 was significantly elevated in the LOPE group (P<0.05). However, there were no significant differences in the activities and protein expression of G6PD and PGD between the two groups.Conclusion Glucose metabolism reprogramming takes place in LOPE placenta tissue, which may be one of the causes of the abnormal elevation of NADPH and GSH.
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子痫前期（preeclampsia, PE）发生在妊娠20周后，以高血压、水肿、蛋白尿为主要临床特征[1]，是围生期母婴死亡的主要原因之一[2]。妊娠34周前发病为早发型PE（early-onset PE, EOPE），妊娠34周及34周后发病为晚发型PE（late-onset PE, LOPE）[3]。
氧化应激（oxidative stress, OS）即体内的活性氧簇（reactive oxygen species, ROS）产生过多，清除减少，导致氧化/抗氧化体系失衡，引起组织损伤[4]。国内外大量研究提示LOPE发病机制与氧化应激密切相关[5-10]。机体为了免受氧化损伤，在长期的进化过程中形成了一套完善的抗氧化酶系统，包括过氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶以及硫氧还蛋白过氧化物酶等[11]。其中谷胱甘肽（glutathione, GSH）还原系统在维持PE胎盘正常氧化应激反应方面至关重要[12]。烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate, NADPH）能动态维持GSH的还原状态[13]，进而保护细胞免受氧化剂尤其是ROS的损害。磷酸戊糖途径的氧化分支（oxidative arm of the pentose phosphate pathway, oxPPP）是NADPH产生的最大通路[14-15]。近来关于应用各种抗氧化剂来预防和治疗LOPE功效方面研究甚多[16-18]，而涉及氧化应激失衡与葡萄糖代谢的研究较少。而PPP途径所产生的NADPH是维持GSH还原状态的最大通路，因此本实验通过检测两组胎盘组织葡萄糖摄取量以及PPP途径关键酶葡萄糖-6-磷酸脱氢酶（glucose-6-phosphate dehydrogenase, G6PD）、磷酸葡萄糖酸脱氢酶（phospho-gluconate dehydrogenase, PGD）的活性和表达，来判断葡萄糖代谢与胎盘组织氧化应激失衡的相关性。
1. 材料和方法
1.1 实验对象
本研究纳入对象为重庆医科大学附属第一医院产科于2020年11月–2021年10月行剖宫分娩的LOPE孕妇13例和孕周匹配的正常剖宫产孕妇13例。LOPE的诊断标准参照第九版《妇产科学》。两组研究对象按是否LOPE分组外，均需符合以下纳入标准和排除标准。纳入标准：①单胎妊娠；②年龄≤35周岁；③签署知情同意书。排除标准：①心肝肾功能不全或自身免疫性疾病；②其他妊娠合并症或并发症。该研究获得重庆医科大学附属第一医院伦理委员会批准，批准号2020793。
1.2 标本收集
所有研究对象均在剖宫产胎盘娩出后取组织样本。掀开羊膜，翻到母面，取胎盘绒毛区组织块，迅速放入预冷的生理盐水中，以冰盒进行运输，带回实验室。用预冷的生理盐水，将胎盘组织的血凝块洗净，剔除血管和钙化灶，将组织剪成约1 cm×1 cm×1 cm的大小，用无菌纱布吸干组织中的生理盐水，分装进2 mL离心管，迅速用液氮速冻后，并立即放入−80 ℃超低温冰箱中冻存备用。
1.3 实验方法
1.3.1 DCFH-DA法测定LOPE组和正常妊娠组胎盘组织ROS水平
将胎盘组织用OTC（optimal cutting temperature compound）包埋剂包埋，进行冰冻切片。将冰冻切片从−80 ℃冰箱里拿出，室温放置2 h，免疫组化笔圈出组织位置；将TBST滴在组织表面，室温放置5 min（除去制冷剂），PBS洗3次；TritonX-100滴在组织表面，室温放置10 min，PBS清洗3次；按照1∶1000用PBS稀释2,7-二氯荧光素二乙酸酯（DCFH-DA），使终浓度为10 μmol/L，滴在组织表面，孵育30 min， PBS洗涤3次，双蒸水冲洗1次；在组织上滴DAPI，室温放置5 min，吸除DAPI，用PBS洗3次；用正置荧光显微镜比较两组切片的ROS荧光强度并拍照。
1.3.2 GSH、NADPH、葡萄糖含量检测
取胎盘组织，按试剂盒（碧云天，S0053）步骤测定GSH；按试剂盒(碧云天，S0179)步骤，通过比色法来检测胎盘组织中NADPH；按试剂盒（索莱宝，BC2505）步骤，通过微量法分别检测LOPE组和对照组胎盘组织葡萄糖含量。
1.3.3 胎盘组织G6PD和PGD酶活性检测
称约0.1 g胎盘组织，放入2 mL离心管；加提取液1 mL，置冰上，电动组织匀浆器充分研磨；离心机4 ℃，10000 r/min，离心10 min，提取上清液；通过紫外分光光度法（索莱宝，BC2100， BC0260）分别检测LOPE和正常妊娠胎盘组织G6PD和PGD活性。
1.3.4 Western blot检测PFK1、G6PD、PGD的蛋白表达水平
称约0.1 g胎盘组织，用预冷的PBS清洗2次；加入200 μL含1% PMSF的蛋白裂解液，冰上充分研磨；离心机4 ℃，12000×g离心15 min，收集上清液；用BCA蛋白测定试剂盒（碧云天P0009）测定蛋白浓度后，用蛋白裂解液和4× loading buffer将蛋白浓度最终调成2 μg/μL，95 ℃蛋白变性10 min；蛋白上样量为20 μL，SDS-PAGE凝胶电泳分离蛋白，并将分离胶上蛋白电转至PVDF膜；含5%脱脂奶粉的TBST溶液室温封闭1 h，TBST溶液洗膜3次；依次孵育一抗PFK1（Proteintech，美国）、PGD（Proteintech，美国）、G6PD（Proteintech，美国）、内参β-actin（Proteintech，美国）一抗，浓度皆为1∶1000，4 ℃过夜；次日洗膜后加入相应二抗稀释液，室温孵育1 h，洗膜后，ECL显影、凝胶成像系统拍照；比较LOPE组和正常妊娠组胎盘组织PFK1、G6PD、PGD的蛋白表达水平，以各条带与内参条带灰度值的比值，为目的蛋白相对表达量。
1.4 统计学方法
计量数据采用
$ \bar x \pm s $ 表示。两组间一般资料、生化指标及蛋白相对表达量的比较采用独立样本t检验或非参数检验，P<0.05为差异有统计学意义。2. 结果
2.1 LOPE组和正常妊娠组临床特征比较
本研究纳入孕妇共26例，其中LOPE组13例，对照组13例。两组孕妇临床资料如表1所示，两组基线匹配。LOPE孕妇舒张压和收缩压水平高于对照组（P<0.05），尿蛋白阳性水平高于对照组（P<0.05）。
表 1 两组基本情况比较Table 1. Comparison of the basic data of the two groupsCharacteristic LOPE group (n=13) Control group
(n=13)P Age/yr. 30.00±3.51 31.15±3.90 0.435 Gestational day at delivery/d 266.90±19.26 258.50±22.15 0.223 Fetal body mass/g 2995.00±531.40 3120.00±413.70 0.510 Systolic blood pressure/mmHg 158.10±9.84 116.80±9.88 0.000 Diastolic blood pressure/mmHg 97.31±9.10 69.54±8.37 0.000 Proteinuria (−) - (+) (+) - (++++) 0.007 1 mmHg=0.133 kPa. 2.2 胎盘组织中的ROS水平
免疫荧光结果显示，LOPE胎盘组织ROS水平高于对照组（图1），差异有统计学意义（P<0.05）。
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图 1 DCFH-DA法检测胎盘组织内ROS水平Figure 1. The ROS levels in the placenta tissues were determined by DCFH-DADCFH-DA: 2’,7’-dichlorodi-hydro-fluorescein diacetate. n=4.2.3 胎盘组织中NADPH、GSH、葡萄糖的含量
与对照组相比，GSH水平、NADPH水平、葡萄糖含量在LOPE胎盘组织中升高，差异有统计学意义（P均<0.05），见表2。
表 2 各组胎盘组织中GSH、NADPH和葡萄糖的含量比较Table 2. GSH, NADPH, and glucose levels in the placenta tissue of the two groupsGroup GSH/(μg/g)
(n=13)NADPH/(μmol/g)
(n=7)Glucose/(μmol/g)
(n=11)Control 7.57±3.50 0.51±0.17 2.77±1.46 LOPE 12.15±4.52 0.90±0.14 7.78±5.56 P 0.008 < 0.001 0.009 2.4 胎盘组织中G6PD、PGD的蛋白表达及其活性
G6PD、PGD的蛋白表达及活性在LOPE组及对照组中的差异无统计学意义（图2）。
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图 2 PPP途径的两个关键酶G6PD、PGD的蛋白表达及活性Figure 2. The protein expressions and activities of G6PD and PGD, two key enzymes, in the pentose phosphate pathway (PPP)A: Protein expression; B: Activity. n=7.2.5 PFK1蛋白表达在LOPE胎盘组织中明显升高
Western blot结果显示，与对照组相比，LOPE组PFK1蛋白表达水平升高，差异有统计学意义（P<0.05，图3）。
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图 3 糖酵解途径的关键限速酶PFK1蛋白表达（n=6）Figure 3. The expression of PFK1, a key rate-limiting enzyme of the glycolytic pathway (n=6)3. 讨论
自妊娠早期，机体即呈生理性氧化应激状态。随着妊娠进展，母体血流进入绒毛间隙，产生大量的ROS，同时胎盘内的抗氧化物质也开始增多，从而保护胎儿和母体免受氧化应激的损伤[6]。而在LOPE患者表现出比正常妊娠更持续、更剧烈的氧化应激状态。本研究结果意外发现在LOPE胎盘ROS升高的同时，抗氧化物NADPH、GSH含量异常升高。进一步检测生成NADPH的葡萄糖分解代谢中，PPP途径和糖酵解途径的关键限速酶的蛋白表达及胎盘组织葡萄糖摄取发现，PPP途径关键限速酶G6PD、PGD蛋白表达水平及活性在LOPE和对照组中均未见明显差异；但LOPE胎盘中葡萄糖含量较正常组增加，且糖酵解第一个关键限速酶PFK1表达水平在LOPE胎盘中升高。因此我们推测NADPH和GSH的升高可能是LOPE胎盘摄取葡萄糖增加的结果，但是这一部分葡萄糖并没有完全甚至只有少部分进入PPP途径，并不足以清除LOPE胎盘内高水平的ROS，因而LOPE患者的胎盘依旧处于过度氧化应激状态。同时，LOPE胎盘组织摄取的葡萄糖可能更多地流向另外一条分解代谢途径——糖酵解途径。因此，我们推测，通过靶向抑制糖酵解途径，使葡萄糖碳源更多地流向PPP途径，进而生成更多的NAPDH，可能是LOPE预防或者治疗的靶点。
本研究发现LOPE胎盘ROS升高与抗氧化物NADPH、GSH升高共存。而在大部分研究中，GSH等抗氧化物质是降低的[19-20]。这是本研究与其他研究的不同之处。LOPE胎盘里高表达的NADPH可能来源于其他途径的异常激活，如苹果酸脱氢酶（malic enzyme, ME）、异柠檬酸脱氢酶（isocitrate dehydrogenase, IDH）和亚甲基四氢叶酸脱氢酶（methylenetetrahydrofolate dehydrogenase, MTHFD）等NAPDH生成途径[21]。NADPH在生物氧化还原系统中起着氢的传递体的作用[22]，参与脂类、脂肪酸和核苷酸的合成。有研究显示，在肿瘤中NADPH还参与调控表观遗传的分子机制[23]。高水平NADPH除了还原当量作用外，在LOPE发病机制中可能还参与其他非氧化还原作用，值得我们继续深入研究。此外，本研究显示胞质中的葡萄糖分解代谢很可能是NAPDH增加的主要原因，ROS主要产生于线粒体内膜[24]，而GSH主要来源于细胞质[25]。因此，LOPE胎盘中增加的ROS和NADPH、GSH等还原当量是否存在亚细胞定位不同，而胎盘线粒体转运蛋白缺失是否是导致该亚细胞定位不同的原因，值得进一步研究及探讨。
本研究样本量少，课题组后期会加大样本量，在组织和原代细胞中进行再验证。以后的研究方向将进一步深入探讨线粒体蛋白缺失对抗氧化物质不同亚细胞定位的影响。
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利益冲突 所有作者均声明不存在利益冲突
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图 1 DCFH-DA法检测胎盘组织内ROS水平
Figure 1. The ROS levels in the placenta tissues were determined by DCFH-DA
DCFH-DA: 2’,7’-dichlorodi-hydro-fluorescein diacetate. n=4.
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图 2 PPP途径的两个关键酶G6PD、PGD的蛋白表达及活性
Figure 2. The protein expressions and activities of G6PD and PGD, two key enzymes, in the pentose phosphate pathway (PPP)
A: Protein expression; B: Activity. n=7.
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图 3 糖酵解途径的关键限速酶PFK1蛋白表达（n=6）
Figure 3. The expression of PFK1, a key rate-limiting enzyme of the glycolytic pathway (n=6)
表 1 两组基本情况比较
Table 1 Comparison of the basic data of the two groups
Characteristic LOPE group (n=13) Control group
(n=13)P Age/yr. 30.00±3.51 31.15±3.90 0.435 Gestational day at delivery/d 266.90±19.26 258.50±22.15 0.223 Fetal body mass/g 2995.00±531.40 3120.00±413.70 0.510 Systolic blood pressure/mmHg 158.10±9.84 116.80±9.88 0.000 Diastolic blood pressure/mmHg 97.31±9.10 69.54±8.37 0.000 Proteinuria (−) - (+) (+) - (++++) 0.007 1 mmHg=0.133 kPa. 
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表 2 各组胎盘组织中GSH、NADPH和葡萄糖的含量比较
Table 2 GSH, NADPH, and glucose levels in the placenta tissue of the two groups
Group GSH/(μg/g)
(n=13)NADPH/(μmol/g)
(n=7)Glucose/(μmol/g)
(n=11)Control 7.57±3.50 0.51±0.17 2.77±1.46 LOPE 12.15±4.52 0.90±0.14 7.78±5.56 P 0.008 < 0.001 0.009
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