木犀草素对子痫前期大鼠滋养层细胞凋亡的影响及其机制

目的探讨木犀草素（LUT）对子痫前期（PE）大鼠滋养层细胞凋亡的影响及其机制。 方法取妊娠10 d SD大鼠,按随机数字表法随机分为对照组、模型组、20、40、60 mmol/L LUT （LUT-L、LUT-M、LUT-H）组,每组各12只,模型组和给药组大鼠皮下注射100 mg/（kg·d）亚硝基左旋精氨酸甲酯建立PE大鼠模型,对照组大鼠皮下注射等量生理盐水,每天1次,注射6 d。妊娠16 d的大鼠分别予以20、40、60 mmol/L LUT腹腔注射,对照组、模型组大鼠腹腔注射等量生理盐水,每天1次,注射5 d。测量各组大鼠妊娠10、16、21 d尾动脉血压及24 h尿蛋白水平;妊娠21 d,原位末端标记法（TUNEL）检测滋养层组织细胞凋亡情况,Western blot法检测滋养层组织B淋巴细胞瘤-2 （Bcl-2）、Bcl-2相关X蛋（Bax）、磷脂酰肌醇3-激酶（PI3K）、磷酸化PI3K （p-PI3K）、蛋白激酶B （Akt）、磷酸化AKT （p-Akt）、内皮型一氧化氮合酶（eNOS）和磷酸化eNOS （p-eNOS）蛋白表达量。多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。 结果妊娠10 d,各组大鼠尾动脉收缩压、舒张压、24 h尿蛋白含量差异无统计学意义;妊娠16 d,与对照组比较,模型组、LUT-L组、LUT-M组、LUT-H组大鼠尾动脉收缩压、舒张压、24 h尿蛋白含量升高（P均< 0.05）;妊娠21 d,与对照组比较,模型组、LUT-L组、LUT-M组、LUT-H组收缩压[（110.33±3.67）比（147.28±4.16）,（131.29±4.31）,（124.46±4.27）,（118.54±4.18）mmHg]、24 h蛋白尿、细胞凋亡率[（1.38±0.34）%,（43.45±3.72）%,（39.21±3.53）%,（27.86±3.41）%,（23.21±3.28）%]和Bax蛋白表达量均升高;Bcl-2、p-PI3K/PI3K （1.06±0.09比0.25±0.02,0.37±0.03,0.57±0.06,0.73±0.08）、p-Akt/Akt（0.87±0.08比0.11±0.01,0.23±0.03,0.56±0.07,0.78±0.06）和p-eNOS/eNOS蛋白表达水平（0.85±0.07比0.09±0.01,0.16±0.02,0.38±0.04,0.69±0.07）均降低（P均< 0.05）。与模型组比较,LUT-L组、LUT-M组、LUT-H组大鼠尾动脉收缩压、舒张压、滋养层组织细胞凋亡率和Bax蛋白表达量降低,Bcl-2、p-PI3K/PI3K、p-Akt/Akt和p-eNOS/eNOS蛋白表达量升高（P均< 0.05）。 结论LUT可抑制PE大鼠滋养层组织细胞凋亡,其机制可能与PI3K/Akt/eNOS信号通路激活,调控凋亡相关蛋白表达有关。     

Preeclampsia inactivates glucose-6-phosphate dehydrogenase and impairs the redox status of erythrocytes and fetal endothelial cells

Inactivation of glucose-6-phosphate dehydrogenase (G6PD) may contribute to vascular dysfunction in preeclampsia, and oxidative stress has been implicated in the pathogenesis of this disease. We have compared the susceptibility of erythrocytes and human umbilical vein endothelial cells (HUVEC) to oxidative stress in women with normotensive or preeclamptic pregnancies. The redox status of erythrocytes was also correlated with neutrophil-mediated superoxide (O₂⁻) production in women recruited to the “Vitamins in Preeclampsia” (VIP) trial. Erythrocytes and HUVEC from women with preeclampsia demonstrated impaired redox regulation and diminished response to glucose, detectable at 14–20 weeks gestation prior to onset of the clinical disease. Hexokinase and G6PD activities were decreased in erythrocytes and G6PD activity was decreased in HUVEC from preeclamptic pregnancies. Phorbol-ester-stimulated O₂⁻ was enhanced in preeclamptic neutrophils. Impaired redox regulation in erythrocytes and HUVEC in preeclampsia may be due to diminished hexokinase and G6PD activities resulting from increased release of reactive oxygen species from activated neutrophils. Our findings provide the first evidence that decreased G6PD activity in preeclampsia is associated with impaired redox regulation in erythrocytes and fetal endothelial cells. The deficiency in G6PD in preeclampsia potentially accounts for the lack of protection against oxidative stress afforded by antioxidant vitamin C/E supplementation in the VIP trial.     

Expression of the blood-group-related antigens Sialyl Lewis a,Sialyl Lewis x and Lewis y in term placentas of normal,preeclampsia, IUGR- and HELLP-complicated pregnancies

Lewis antigens belong to the blood group of antigens and mediate cellular adhesion through interaction with selectins. Invasive trophoblasts use an array of adhesion molecules to facilitate cell–cell and cell–extracellular matrix interactions. Here, we examined immunohistochemically the expression of Sialyl Lewis a (sLe^a), Sialyl Lewis x (sLe^x) and Lewis y (Le^y) in term placentas obtained from cases of normal, intrauterine growth retardation (IUGR), preeclamptic (PE) and hemolysis, elevated liver enzymes and low platelets syndrome (HELLP) pregnancies. We report the expression of sLe^x in third trimester extravillous trophoblasts (EVT). sLe^x was significantly decreased in IUGR and moderately decreased in PE compared to normal placentas. sLe^x was additionally found in syncytiotrophoblast, without however any significant differences in staining intensity between normal and pathological cases. sLe^a was restricted to amnion epithelium. Finally, Le^y was expressed in cytotrophoblasts and villous endothelial cells. Le^y expression was significantly upregulated in IUGR and HELLP, whereas there was a trend toward increase in PE compared to normal placentas. The present study suggests that downregulation of sLe^x in EVT might be associated with IUGR and PE. Furthermore, Le^y, which was recently described as a potent angiogenic factor, is upregulated in placental villi in conditions associated with placental malperfusion. U. Jeschke and A. Makrigiannakis have contributed equally.     

Lipid compounds of the umbilical cord vein and their alterations in preeclampsia

The lipid composition of vascular walls changes during development, ageing and pathological processes. Preeclampsia is the most common pregnancy-associated pathological syndrome. It is accompanied by significant remodelling of the extracellular matrix, both in the umbilical cord vessels and in the surrounding Wharton's jelly. Lipids of the umbilical cord have not been extensively studied. Here we evaluate the lipid composition of the umbilical cord vein and its alteration in preeclampsia. Thin layer chromatography and high-performance liquid chromatography were employed for these analyses. It was found that the umbilical cord vein wall, as with most human tissues, contains free fatty acids, mono-, di- and triacylglycerols, free cholesterol and its esters. The characteristic feature is the presence of high amounts of monounsaturated fatty acids, mainly myristoleic acid (C14:1) and oleic acid (C18:1), and polyunsaturated fatty acids, including eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), which are rather minor lipid components of most human tissues. They exist both in a free form and in a form of acylglycerols and cholesterol esters. Preeclampsia is associated with an increase in the accumulation of free fatty acids, acylglycerols and cholesterol esters in the umbilical cord vein wall, with a proportional reduction in unsaturated fatty acid contents in all the investigated lipid fractions. Total amount of myristoleate was similar to control values. It is suggested that stimulation of lipolysis in maternal tissues increases supply of free fatty acids to foetal blood and promotes the accumulation fatty acids and their esters in some foetal vascular walls.     

子痫前期患者血清对脐静脉内皮细胞CD40 和NF-kB 表达的影响

目的：研究CD40 和NF-资B 在子痫前期患者组的脐静脉内皮细胞（HUVEC）中的表达,探讨其在子痫前期发病过程中的作 用。方法：应用子痫前期患者组的血清冲击人HUVEC 株,电镜下观察HUVEC 的形态变化,流式细胞仪下测定细胞的凋亡,用 RT-PCR 方法检测HUVEC中CD40 和NF-资B 的表达情况。结果：①电镜下正常孕妇组HUVEC呈" 铺路石" 状单层镶嵌排列, 子痫前期组HUVEC形态呈典型损伤改变,细胞稀疏,细胞核与细胞浆分界模糊不清,胞浆中有暗色颗粒。②流式细胞仪测定子 痫前期组HUVEC的细胞凋亡较正常组明显升高,差异有显著意义（P<0.05）;③ RT-PCR 测定显示子痫前期患者组HUVEC 中 CD40mRNA 及NF- BmRNA 水平均较正常组明显升高,差异有显著意义（P<0.05）;④ 子痫前期组及对照组的CD40 和NF- kB表 达均呈显著性正相关。结论：CD40和NF-资B 的激活均参与了子痫前期的发病机制,并在其中发挥重要作用。     

子痫前期发病相关高危因素的Logistic回归分析

目的:采用单因素和多因素Logistic回归方法分析本地区子痫前期发病相关高危因素。方法:选择2014年1月到2017年4月在我院进行分娩的子痫前期产妇78例作为观察组及同期在我院进行分娩的正常产妇78例作为对照组,比较两组产妇的一般情况、婚姻生育史、孕期保健情况、既往史、家族史、本次妊娠情况等。结果:两组产妇的年龄、孕次、产次、初检孕周、产前检查次数等比较差异无统计学意义(P0.05)。观察组子痫前期首次发生的平均孕周为38.26±2.63周。单因素分析显示自然流产史、子痫前期家族史、妊娠高血压、孕前BMI、孕期尿路感染与子痫前期发病明显相关(P0.05);非条件Logistic回归多因素分析结果显示自然流产史、子痫前期家族史、妊娠高血压、孕前BMI为导致子痫前期发病的主要独立危险因素(P0.05)。观察组以剖宫产为主要分娩方式,分娩孕周也明显长于对照组(P0.05);两组产妇都无死亡情况发生,但观察组的产后出血、胎盘早剥、心肝肾功能不全等并发症发生率明显高于对照组(P0.05)。结论:自然流产史、子痫前期家族史、妊娠高血压、孕前BMI为导致子痫前期发病的主要独立危险因素,可导致不良妊娠结局的增加。     

Concentrations of pentosidine, an advanced glycation end-product, in umbilical cord blood

Advanced glycation end-products (AGEs) are formed over several weeks to months by non-enzymatic glycation and oxidation (“glycoxidation”) reactions between carbohydrate-derived carbonyl groups and protein amino groups, known as the Maillard reaction. Pentosidine is one of the best-characterized AGEs and is accepted as a satisfactory marker for glycoxidation in vivo. The present study was intended to measure pentosidine concentrations in umbilical cord blood from newborns with various gestational ages using our recently established high-performance liquid chromatography method [Tsukahara, H. et al. (2003) Pediatr. Res. 54, 419-424]. Our study demonstrates, for the first time, that pentosidine is detected in most of the umbilical blood samples. This study also shows that the umbilical blood concentrations of pentosidine are considerably lower than normal adult values, but that they increase with gestation progression and fetal growth. Umbilical pentosidine concentrations were significantly elevated in newborns of mothers with preeclampsia compared to those of mothers without preeclampsia. We conclude that accumulation of AGEs and oxidative stress occurs in fetal tissues and organs in utero at the early stage of human life and that their accumulation is augmented in the maternal preeclampsic condition.     

L-瓜氨酸对子痫前期中的氧化应激和滋养层细胞侵袭的调控机制研究

摘要 目的：研究L-瓜氨酸（L-Cit）对子痫前期（PE）大鼠模型的治疗作用,及其对氧化应激和滋养层细胞侵袭的影响。方法：建立N-硝基-L-精氨酸甲酯（L-NAME）诱导的PE大鼠模型后,将大鼠分为Control组、PE组、PE+低、中和高剂量L-Cit组（依次为L-L-Cit、M-L-Cit和H-L-Cit组,剂量依次为100、200和500 mg/kg）,n=6,连续给药7 d。在孕第21 d时,检测各组大鼠收缩压（SBP）、24 h尿蛋白、血清超氧化物歧化酶（SOD）和丙二醛（MDA）水平,并对胎盘组织进行苏木精伊红（HE）染色。将HTR-8/Svneo细胞分为Control组、缺氧/复氧（H/R）组、H/R+L-Cit组和H/R+L-Cit+sc-221593组,分别对细胞进行H/R处理,并使用L-Cit（200 μg/mL）和特异性ERK1/2抑制剂sc-221593（10 μmol/L）培养细胞48 h。通过Transwell测定细胞侵袭。通过Western blotting检测total-ERK1/2、p-ERK1/2、total-p38、p-p38、基质金属蛋白酶（MMP）-2和MMP-9的蛋白表达。结果：与PE组比较,L-L-Cit组、M-L-Cit组和H-L-Cit组的SBP和24 h尿蛋白水平均降低（P<0.05）;胎盘组织形态明显改善;血清SOD升高,MDA降低（P<0.05）;胎盘组织中ERK1/2和p38的磷酸化水平及MMP-2和MMP-9的蛋白表达水平均升高（P<0.05）。与H/R组比较,H/R+L-Cit组的侵袭细胞数量升高（P<0.05）;ERK1/2和p38的磷酸化水平及MMP-2和MMP-9的蛋白表达水平均升高（P<0.05）。与H/R+L-Cit组比较,H/R+L-Cit+sc-221593组的上述变化均被逆转（P<0.05）。结论：L-Cit可能通过激活ERK/JNK通路减轻PE中的氧化应激并促进滋养层细胞侵袭,从而减轻PE症状。     

Fatty acid composition of triacylglycerols from Wharton's jelly determined by high-performance liquid chromatography

The improved method for HPLC determination of fatty acids was proposed. The chromatographic separation of p-bromophenacyl derivatives of fatty acids under a gradient elution was achieved at 40 °C with an RP-18 LiChroCART 5 column and organic mobile phase containing methanol, acetonitrile, water and TEAP buffer pH 5.6. The quantitative determination of those derivatives was performed at 254 nm. Preeclampsia, the most common pregnancy complication, did not affect triacylglycerol content in the umbilical cord Wharton's jelly in comparison to the control material. However, it changed the composition of fatty acids, bound to that lipid class. The method allows the determination of almost all fatty acids forming the investigated neutral lipid class, contained in a solid tissue sample. The use of TEAP buffer excluded precipitation and flow stoppage in the HPLC system. The method reduced time and costs and might be useful for all other lipid classes and different tissues.