AOPP在子痫前期胎盘中的表达及其对细胞凋亡通路的影响
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摘要
研究背景
妊娠期高血压疾病是妊娠期特有的全身性疾病,我国发病率为9.4%-10.4%,国外报道发病率为7%-12%。该疾病不但严重影响了母婴健康,至今为止仍然是母婴发病率及死亡率较高的主要原因。子痫前期(preeclampsia)是该疾病最常见的类型。流行病学调查结果显示,子痫前期孕产妇的死亡率约为5%-10%,且围产儿死亡率在子痫前期及子痫患者更高达33.9%-68.6%。子痫前期以妊娠20周以后首次出现高血压、蛋白尿、水肿为主要特征,并可伴有全身多脏器的损害;病情严重者可能因抽搐、脑出血、肺水肿、心功能衰竭、弥漫性血管内出血而导致死亡。临床上处理子痫前期的患者,主要以对症治疗和终止妊娠为主。由于缺乏明确的预测手段和防治对策,使子痫前期成为导致孕产妇死亡的第二大原因,也是导致围产儿发病及死亡的主要原因。其发病机制是多因素的,且目前尚不十分明确。现多数学者认为子痫前期的发生、发展的原因和过程可能与以下几种因素和学说相关：免疫失衡机制,胎盘浅着床,血管内皮细胞损伤和胎盘滋养细胞缺血,胰岛素抵抗,钙平衡失调,遗传因素、营养缺乏等。其中细胞功能损伤及凋亡均与氧化应激有着密切的关系。
现有部分研究表明氧化应激学说可能是子痫前期的重要机制之一。子痫前期患者体内氧化系统和抗氧化系统的平衡遭到了破坏；活性氧族(Reactive Oxygen Species, ROS)与抗氧化体系之间失去平衡。其中氧化作用明显加强,而抗氧化作用则相对减弱,总平衡向氧化方向增强。其实在正常妊娠孕妇体内也存在着不等量的自由基及其产物,但是由于妊娠期孕妇体内抗氧化能力相应提高,二者之间仍处于平衡状态,正常的生理性妊娠不致于形成氧化应激。据文献报道,蛋白、多肽、氨基酸对各种自由基及其相关氧化物相当敏感。在子痫前期患者体内,氧化产物增多(脂质过氧化物、蛋白氧化产物、核酸)而抗氧化能力降低,导致子痫前期患者体内呈现氧化应激状态。氧化应激在许多与细胞功能受损相关的疾病中发挥着重要作用。由于子痫前期患者的缺血、缺氧可以导致大量的自由基生成、脂质、蛋白质过氧化物产生、内皮细胞损伤等；这种过程在子痫前期疾病发生发展的过程中呈正反馈且加重了氧化应激,导致了疾病进一步恶化发展。目前研究表明,蛋白质也是氧化应激攻击的主要靶分子之一,主要表现为氨基酸残基氧化、肽链断裂或者蛋白质分子的交联聚合、蛋白质的疏水性与对水解酶的抵抗力增强,从而使蛋白质丧失生物活性并易于沉积。细胞的损伤及功能失调是子痫前期疾病特征性病理变化之一,它也是子痫前期疾病多器官功能损伤的病理生理学基础。许多子痫前期患者在临床症状出现以前,胎盘的形态与功能方面的改变就已经出现,并且临床上大部分子痫前期患者在胎盘娩出后其临床症状和体征很快就随之消失；这均表明了胎盘在子痫前期疾病的发生发展过程中可能起到了关键作用。基础研究表明,子痫前期的发病机制可归纳为胎盘浅着床和全身血管内皮细胞的激活两个阶段；第一阶段的胎盘血液灌注减少,产生过多的活性氧,造成氧化应激,导致孕妇血管内皮细胞损伤,从而引发第二阶段全身血管内皮细胞的病理性损伤。全身血管内皮损伤和系统性炎症反应又加重了机体的氧化应激失衡,最终导致了子痫前期的一系列临床症状和体征。根据目前的临床和基础研究,我们有理由相信子痫前期是一种胎盘源性疾病,胎盘的氧化-抗氧化的失衡是其发病的不可或缺的环节。
晚期氧化蛋白产物(advanced oxidation protein products, AOPP)是一种新型的蛋白类氧化应激标志物,是氧化应激引起体内各种蛋白质氧化所形成的终末产物的总称。它于1996年由Witko-Sarsat首次报道,于尿毒症患者血浆中首次发现,在慢性肾功能衰竭患者的血浆中分离出来。它是血浆白蛋白被自由基和反应性氧系氧化后形成的双酪氨酸蛋白交连产物,其主要成分来自于：血清白蛋白被自由基或反应性氧系(主要为中性粒细胞髓过氧酶产生的氯化氧化物)氧化而成。AOPP相对于既往普遍关注的脂质过氧化物标志而言,它形成早,存在时间长,更加稳定,是反映蛋白氧化应激损伤的可信指标；同时也是氧化应激时重要的促炎症因子,炎症介质是参与细胞损伤的重要物质。体外研究已经证实,AOPP可通过不同途径参与诱导肾脏足细胞凋亡、血管内皮细胞激活、胰岛素抵抗等一系列病理生理过程。目前已经证实在高龄、慢性肾脏疾病、冠状动脉疾病、糖尿病、代谢综合征、多囊卵巢综合征等等疾病患者中均存在慢性AOPP蓄积。该类人群在妊娠过程中与正常人群相比更容易出现早产、子痫前期、胎儿宫内生长受限、胎盘早剥、重复的胎儿丢失、宫内死胎等等妊娠并发症。进一步而言,对于AOPP为何沉积于上述疾病患者的体内,其机制的研究也日渐深入和明晰。Hideaki Kaneda等的研究表明慢性冠状动脉疾病与尿毒症中一样,血浆白蛋白发生了氧化,该类疾病患者的血浆中AOPP水平和尿毒症患者血浆中同样增高,并且AOPP水平与慢性冠状动脉疾病的发生以及该类疾病的严重程度相关；该实验证明了氧化应激可能与慢性冠状动脉疾病的病理生理过程密切相关。Agnieszka Piwoman等学者研究发现,如妊娠期高血压疾病、妊娠期糖尿病等均属于复杂性代谢紊乱性疾病,其发病机制与活性氧的产生及抗氧化系统功能失衡有关,在糖尿病患者中TRAP(抗酒石酸酸性磷酸酶)及SH(蚕丝水解物)水平均有不同程度的降低,而CO2、NH2及AOPP水平增高；AOPP的表达水平在Ⅱ型糖尿病患者、子痫前期患者血浆中明显增高(本课题组前期实验结果),可推测AOPP可能是一种评估糖尿病病人氧化应激介导蛋白损伤的有用指标。AOPP不仅仅是氧化应激的产物,它还可以引起单核细胞(中性粒细胞)的呼吸爆发,从而诱导单核细胞产生更多的活性氧,正反馈诱导或加重氧化应激反应,使得机体呈现出一种持续氧化应激状态。
胰高血糖素样肽链-1(Glucagon-like peptide-1,GLP-1)主要是由肠道内分泌L细胞分泌的多肽,作为一种安全、有效的促胰岛素分泌剂越来越受到关注,它本是治疗Ⅱ型糖尿病的一种新途径。GLP-1除了广为人知的促进胰岛β细胞增值、抑制β细胞凋亡、促进胰岛素分泌等等作用外；近些年的研究显示其在抑制氧化应激中也承担着重要的角色。它可以降低TNF-a等诱导的炎症水平,以减少高凝状态和炎症反应对于细胞的损伤；此外,GLP-1可抑制单核细胞粘附于人的主动脉内皮细胞,减轻血管炎性损伤,组织冠状动脉粥样硬化及其相关疾病的进展。大量的临床及实验性研究资料表明,GLP-1保护细胞生理功能可能与抑制氧化应激有关,但是其机制尚未十分明确。研究表明,在胰岛β细胞、心肌细胞、胆囊细胞中,GLP-1均表现出了明显的抗氧化应激作用。那么在妊娠滋养细胞中,GLP-1是否具有抑制AOPP抗氧化应激的作用,其作用机制是否与PI13k/Akt信号传导通路有关。这些问题将在本实验中得到初步的探索性答案。
目前已发现子痫前期血浆AOPP水平明显高于正常孕妇。因此我们推测孕妇胎盘AOPP蓄积可能是诱导滋养细胞损伤、功能障碍并引起子痫前期发生过程中新的致病物质。本研究从探讨子痫前期孕妇胎盘中AOPP表达水平与子痫前期严重程度的相关性以及子痫前期患者胎盘AOPP高表达可能的临床意义,作为切入点。在体外的细胞水平进行实验探索性研究,旨在证明AOPP通过NADPH通路诱导妊娠滋养细胞凋亡；以及GLP-1抑制AOPP诱导的凋亡及其机制。为进一步寻找子痫前期的发病原因及机制提供理论基础。
第一部分AOPP在子痫前期与正常妊娠胎盘组织中的表达
【目的】通过测定AOPP在子痫前期患者和正常孕妇胎盘组织中的表达水平及其差异；探讨AOPP与子痫前期发病机制的相关性；为进一步研究氧化应激在子痫前期发病机制中的信号转导通路提供线索及切入点。
【方法】随机选取2012年9月-2013年9月南方医科大学南方医院收治并以剖宫产方式分娩的子痫前期患者共50例,其中子痫前期轻度(mildpreeclampsia, MPE)25例,子痫前期重度(severe preeclampsia, SPE)25例；选取同期足月因社会因素行剖宫产分娩的孕妇50例作为对照组。运用免疫组织化学方法检测子痫前期患者(实验组)与正常妊娠孕妇(对照组)胎盘组织中AOPP的表达水平；测定切片平均灰密度值,半定量胎盘中AOPP的表达量。采用SPSS13.0统计软件,计量资料以均数±标准差表示。组间比较采用ANOVA检验。P<0.05为差异具有统计学意义。
[结果](1)正常孕妇组胎盘组织基本无染色。子痫前期组胎盘组织中均可见棕褐色阳性染色。子痫前期轻度组AOPP主要表达于绒毛合体滋养层,偶见于绒毛细胞滋养细胞。子痫前期重度组AOPP在绒毛合体滋养层细胞以及绒毛细胞滋养细胞中均明显表达。(2)与对照组(30.746±6.99)相比,轻度子痫前期患者(64.056±9.63)胎盘组织中的AOPP表达水平明显增高,且差异具有显著性(F=415.39；P<0.05)。与对照组(30.746±6.99)相比,重度子痫前期患者(97.516±13.72)胎盘组织中的AOPP表达水平亦明显增高,差异具有显著性(F=415.39；P<0.05)。重度子痫前期患者胎盘组织中的AOPP表达水平高于轻度子痫前期患者胎盘组织中的表达量(F=415.39；P<0.05)。
[结论]AOPP在子痫前期患者的胎盘中的主要表达位置为合体滋养层细胞以及绒毛细胞滋养细胞。子痫前期患者胎盘组织中AOPP的表达水平与正常妊娠孕妇胎盘中的表达相比,具有显著性差异。本部分实验结果提示孕妇胎盘中AOPP的表达增高是子痫前期的重要病理变化,追溯其根源可能与子痫前期疾病有关,同时AOPP诱导加重了子痫前期的发生和发展；进一步说明了子痫前期的发病机制是多因素多因子共同参与,相互作用的结果。
第二部分AOPP通过NADPH通路诱导妊娠滋养细胞凋亡
[目的]通过一系列体外细胞水平的实验室基础研究,探讨AOPP诱导妊娠滋养细胞凋亡的机制。
[方法]采用Witko-Sarsat等介绍的方法制备AOPP。用ELISA试剂盒复孔检测β-HCG的含量。用侵袭小室试验检测AOPP刺激后妊娠滋养细胞侵袭能力的变化。用Hoechst33258荧光法检测凋亡。用Western Blotting检测凋亡相关蛋白的表达以及检测尼克酰胺腺嘌呤二核苷酸磷酸(Nicotinamide Adanine Dinucleotide Phosphate, NADPH)氧化酶亚基的表达。用活性氧分子(Reactive Oxygen Species,ROS)阴离子荧光探针,检测AOPP对活性氧分子的影响和作用。所有数据代表三次以上的重复试验结果,以均数±标准差表示,所有统计由统计软件SPSS13.0完成。多个样本均数的比较采用One-Way ANOVA,方差齐时两两比较采用LSD,方差不齐时采用Dunnett's T3法,P<0.05为差异具有统计学意义。
[结果]可实现无内毒素的AOPP的制备。AOPP抑制妊娠滋养细胞β-HCG的分泌(MSA刺激组F=0.913, P=0.477;AOPP刺激组F=20.603,P=0.000)。AOPP抑制妊娠滋养细胞的侵袭能力(F=197.593;P=0.000)。AOPP组妊娠滋养细胞凋亡率明显升高(不同浓度刺激组,F=37.813,P=0.000;不同时间刺激组,F=24.318,P=0.000),apocynin阻断AOPP此效应。AOPP诱导了体外的NADPH氧化酶及其相关亚基的活化(NOX2:F=21.318,P=0.000;NOX4:F=73.643,P=0.000;p47phox: F=126.678, P=0.000;p22phox:F=4.323, P=0.000),NADPH氧化酶的抑制剂apocynin可阻断AOPP此效应。AOPP诱导凋亡相关蛋白的表达(p53:F=40.979,P=0.00;Bax:F=274.420, P=0.00;Bcl-2:F=38.881, P=0.00)。 AOPP在体外刺激了ROS的表达(F=38.277,P=0.00),apocynin可阻断AOPP此效应。
[结论]这一部分的实验研究证实,AOPP抑制P-HCG的分泌和妊娠滋养细胞的侵袭能力可能与胎盘浅着床(子痫前期发病机制中比较受肯定和公认的理论之一)的理论相符合,因此AOPP的高表达是不利于妊娠顺利进行的。AOPP以时间和剂量的方式诱导了妊娠滋养细胞的凋亡,其途径主要是通过NADPH通路。P53、Bax蛋白参与AOPP诱导的妊娠滋养细胞凋亡,P53、Bax蛋白在NAPDH氧化酶下游。NADPH氧化酶抑制剂apocynin干预阻断了AOPP诱导的细胞损伤。因此,AOPP诱导的妊娠滋养细胞NADPH氧化酶的活化以及产生的大量ROS是导致细胞损伤的重要因素之一,这一结论为临床防治子痫前期等妊娠期相关病变提供了一定程度上的理论依据。
第三部分GLP-1抑制AOPP所诱导的凋亡及其机制
[目的]在本课题第二部分初步证实了AOPP诱导妊娠滋养细胞凋亡的研究基础上,通过GLP-1干预AOPP诱导妊娠滋养细胞凋亡的作用,并检验其对凋亡相关分子及PI3K/Akt通路的影响,初步探讨GLP-1对AOPP诱导细胞凋亡的影响及其分子机制。
[方法]CCK-8(cell counting kit-8)比色法测定细胞活力及增值能力。荧光显微镜检测活性氧(ROS)的表达。ELISA法检测细胞凋亡蛋白酶caspase-9,-3的活性。Western-blot检测p-Akt等通路相关蛋白的表达。ELISA法检测凋亡相关蛋白Bcl-2、Bax、Cyto-C的表达。所有数据代表三次以上的重复试验结果,实验数据计量资料以均数±标准差表示,所有统计由统计软件SPSS13.0完成。多个样本均数的比较采用One-Way ANOVA,方差齐时两两比较采用LSD,方差不齐时采用Dunnet's T3法,P<0.05为差异具有统计学意义。
[结果]在一定浓度范围内(50-150nmol/L),GLP-1处理AOPP诱导的妊娠滋养细胞后进行实验,结果表明GLP-1抑制AOPP诱导的妊娠滋养细胞内ROS的生成(P=86.222P=0.000)。GLP-1可部分减轻AOPP诱导caspase-9、 caspase-3活性的增加(caspase-9:F=10.321P=0.004; caspase-3:F=20.989,P=0.000)。LY294002作为P13K的抑制剂,可拮抗GLP-1对AOPP的以下作用：GLP-1抑制AOPP诱导的细胞活力下降(F=71.07,P=0.000)、GLP-1抑制AOPP诱导细胞p-Akt水平低表达(不同浓度刺激时F=10.679,P=0.004；不同时间刺激时F=45.886P=0.000),GLP-1抑制AOPP诱导细胞Bax(F=33.017P=0.00)、 cyto-c(F=49.483,P=0.00)高表达以及Bcl-2的低表达(F=45.258P=0.00)。
[结论]本实验发现GLP-1在一定的浓度范围内有拮抗AOPP抑制妊娠滋养细胞活力的作用,而且减少AOPP诱导的细胞损伤。AOPP作用于妊娠滋养细胞后,ROS水平升高,凋亡蛋白酶caspase-9、-3的活性增加,GLP-1可部分性减少AOPP诱导的上述效应。因此推测氧化应激、caspase-9、-3可能参与了GLP-1拮抗AOPP诱导的细胞凋亡的作用。GLP-1至少部分通过PI3k/Akt信号转导通路发挥抗凋亡作用,减轻AOPP诱导的细胞凋亡。GLP-1可调节AOPP诱导妊娠滋养细胞内Bcl-2、Bax、cyto-c表达水平,且此调节作用与PI3K/Akt信号通路有关。
Background
Hypertensive disorder complicating pregnancy is the systemic disease, which is idiopathic in the gestational period; the morbidity in China is9.4%-10.4%, while it is7%-12%abroad. The disease is not only a serious impact on maternal and child health. So far it also remains high maternal and child morbidity and mortality of the main reasons. Preeclampsia (PE) is the most common type of the disease. Epidemiological investigations showed that maternal mortality of preeclampsia is about5%to10%. As well as, perinatal mortality rate was as high as33.9%～68.6%in preeclampsia and eclampsia. The main feature of preeclampsia is the first occurrence of hypertension after20weeks gestation, proteinuria and edema. It also can be accompanied by systemic multi-organ damage; severe cases may be dead due to convulsions, cerebral hemorrhage, pulmonary edema, heart failure, and disseminated intravascular hemorrhage. Clinically we treated patients with pre-eclampsia mainly basing on symptomatic treatment and termination of pregnancy. Due to the lack of a clear prediction methods and countermeasures that preeclampsia become the second leading cause of maternal mortality. Furthermore, it is the main reasons of perinatal morbidity and mortality. Its pathogenesis is multifactorial and the pathogenesis is not very clear. Most scholars now believe that the incidence of preeclampsia, the causes and course of development may be related to several factors and theories related to:immune imbalance mechanism, shallow placental implantation, vascular endothelial cell injury and ischemic placental trophoblastic cells, insulin resistance, calcium balance disorders, genetic factors, nutritional deficiencies and so on. Among the factor mentioned above, endothelial dysfunction and oxidative stress in cells is closely related.
Some studies indicate that oxidative stress existing doctrine may be one of the main mechanisms of preeclampsia. Oxidative balance system and antioxidant systems in preeclampsia have been destroyed and balance lose between reactive oxygen species (ROS) and antioxidant system. Oxidation strengthened and the relative weakening of antioxidant enhance the overall balance of the oxidation direction. In normal pregnant women also have the amount of free radicals and their products, but because of pregnancy corresponding increase in vivo antioxidant capacity, it is still in the balance between the two and prevented the formation of oxidative stress. According to the literature, proteins, peptides and amino acids are quite sensitive to a variety of related oxide radicals. In preeclampsia, increased oxidation products (lipid peroxidation, protein oxidation products, nucleic acids) and reduced antioxidant capacity, resulted in patients showing oxidative stress. Because ischemia and hypoxia of preeclampsia patients, that can cause a lot of free radical production, lipid, protein peroxides, endothelial cell injury. This process occurs in the process of developing preeclampsia will increase oxidative stress CKS feedback, resulting in a further development of the disease. The present study showed also the major target of oxidative stress proteins attack, mainly for the oxidation of an amino acid residue, a peptide or protein molecule strand cross-linking polymerization of hydrophobic protein with increased resistance to enzymatic hydrolysis increased, so that it will resulted in the loss of biological activity and protein tend to accumulate. Injury and dysfunction of endothelial cells is one of the pathological changes characteristic of preeclampsia disease, it is also pathophysiology foundation of multiple organ dysfunction in preeclampsia. Many patients before clinical symptoms of preeclampsia, placental morphological and functional aspects of the change have occurred, and after the placenta delivery, clinical symptoms and clinical signs soon disappeared. This indicates that the placenta may play a critical role in the development of preeclampsia disease. Basic research showed that the pathogenesis of preeclampsia can be summarized as the formation of the placenta and systemic adverse vascular endothelial cells activated in two stages;first stage reduced placental perfusion, excessive ROS cause oxidative stress, resulting in pregnant women vascular endothelial cell injury, causing damage to the second phase of pathological systemic vascular endothelial cells. Systemic vascular endothelial injury and systemic inflammation and oxidative stress increased the imbalance in the body, eventually leading to a series of pre-eclampsia clinical signs and symptoms. Based on current clinical and basic research, we have reason to believe that pre-eclampsia is a placental-borne disease, placental oxidation-antioxidant imbalance is an indispensable part of its incidence.
AOPP is a new class of protein markers of oxidative stress; oxidative stress is caused by a variety of proteins in vivo oxidation of AGEs formed in general. It was first reported in1996by the Witko-Sarsat, in uremic patients plasma and it is separated from the plasma in patients with chronic renal failure. It is a double cross-linked product of tyrosine protein is serum albumin and the reactive oxygen radicals formed by oxidation of the system, the main component of serum albumin or reactive oxygen free radical system (mainly neutrophils myeloperoxidase IDO chloride produced oxide). AOPP general concern with respect to previous mark of lipid peroxides, which formed early, existence of long time, more stable, is a reflection of the credibility of protein oxidative stress indicators. Simultaneously, it also important pro-inflammatory cytokines, inflammatory mediators when oxidative stress happened, which is an important substance involved in endothelial injury. In vitro studies have demonstrated that, AOPP through different pathways involved in a series of pathophysiological processes, such as the induction of apoptosis in renal podocytes, endothelial cell activation, insulin resistance, etc. Now it has been confirmed in elderly patients with chronic kidney disease, coronary artery disease, diabetes, metabolic syndrome, polycystic ovary syndrome, etc. are present in chronic AOPP accumulation. Such people are more prone to preterm labor, preeclampsia, intrauterine growth restriction, placental abruption, repeated fetal loss, intrauterine fetal death, and so pregnancy complications compared to the normal population during pregnancy. Further, why AOPP easy the deposition to these diseases the patient's body, to study of the mechanism increasing depth and clarity. Hideaki Kaneda and other studies have shown that chronic coronary artery disease, the same as uremia, plasma albumin oxidation occurs. In patients with such diseases, AOPP levels in plasma and plasma uremic patients also increased. AOPP levels and the incidence of chronic coronary artery disease, as well as, severity of these diseases have close relationship. The experiment proved that oxidative stress might be associated with the pathophysiology of chronic coronary artery disease. Agnieszka Piwoman scholars found that diabetes is a complex metabolic disorders, which produces and antioxidant system function in the pathogenesis and reactive oxygen species imbalance, in diabetic patients TRAP (tartrate-resistant acid phosphatase) and SH (silk hydrolyzate) lower levels of varying degrees, while the CO2, NH2and AOPP levels increased. AOPP expression levels in patients with type II diabetes was significantly increased, AOPP presumably may be a useful indicator for diabetes oxidative stress-mediated protein damage. AOPP is not just a product of oxidative stress, it can cause monocytes neutrophil respiratory burst, thereby inducing monocytes to produce more reactive oxygen species, a positive feedback to induce or aggravate oxidative stress, making body showing a sustained oxidative stress. Glucagon-like peptide-1(Glucagon-like peptide-1, GLP-1) is mainly secreted by the intestinal endocrine L-cells of a polypeptide. As a safe and effective insulin secretion agent more and more attention, it is a new way to treat this type II diabetes. In addition to promoting cell proliferation of islet cell β, inhibit cell β apoptosis, insulin secretion promoting, recent studies show it also plays an important role in the inhibition of micro vascular lesions. It can reduce the induction of TNF-a and other inflammatory molecule level. In order to reduce hypercoagulable state and inflammation damage to endothelial cells. Besides, GLP-1inhibits monocyte adhesion to human aortic endothelial cells, reducing vascular inflammatory injury, the progress of atherosclerosis and its related organizations coronary artery disease.A large number of clinical and experimental research data shows, GLP-1protects endothelial cells by inhibiting oxidative stress, but its mechanism is not very clear. GLP-1receptor agonist exendin-4activation PKA-PI13k/Akt-eNOS signaling pathways to stimulate endothelial cell proliferation, in order to facilitate the rapid repair of vascular injury. Research shows that in pancreatic β cells, cardiomyocytes, gallbladder cells, GLP-1exhibited significant anti-oxidative stress. So in gestational trophoblastic cells, whether GLP-1inhibition effect of AOPP or not, what its mechanism may be related to, these issues will have a preliminary exploratory answers in this experiment.
Preeclampsia has been found in plasma AOPP levels were significantly higher than normal pregnant women. Therefore, we hypothesized that placental trophoblastic AOPP, as a new pathogenic substances, its accumulation could be induced cell damage and dysfunction caused by in the process of preeclampsia occurs. This study investigates AOPP expression and severity of preeclampsia correlation in the placenta from pregnant women with preeclampsia. It also investigates AOPP high expression of possible clinical significance in preeclampsia placenta. That is the stating point of the assay. Experimental exploratory research at the cellular level in vitro is to study AOPP induced by NADPH gestational trophoblastic apoptosis pathway. Beside, GLP-1inhibited AOPP-induced apoptosis and its mechanism. The purpose of this study is providing a theoretical basis for further search and mechanisms of pathogenesis of preeclampsia.
PART1AOPP expression in normal pregnancy and preeclampsia placenta
[Objective] by measuring the expression levels and their differences of AOPP in placental tissue form patients with preeclampsia and normal pregnant women, explore the correlation between AOPP and pathogenesis of preeclampsia. This can provide clues and the starting point for further study of oxidative stress in the pathogenesis of preeclampsia in the signal transduction pathway.
[Method] This study was performed in50cases of pregnant women with preeclampsia in the obstetrical department of NanFang Hospital from September2012to September2013, in which25cases were mild preeclampsia (MPE) and25 cases were severe preeclampsia (SPE).50cases the same period in term pregnant women delivered by cesarean section due to social factors as a control group. Immunohistochemistry was used to detect preeclampsia and normal pregnant women in AOPP (control group) placental tissue expression levels. The average ash density values measured, semi-quantitative expression of the placenta AOPP. Using SPSS13.0statistical software, data were expressed as mean±standard. Groups comparing using ANOVA test. P<0.05was considered statistically significant.
[Results](1) Normal pregnant group placentas almost no staining. Preeclampsia placentas were visible brown staining. Mild preeclampsia group AOPP will mainly expressed in the villous syncytiotrophoblastic, occasionally in villous trophoblastic cells. Severe preeclampsia group AOPP in villous syncytiotrophoblastic and villous trophoblastic cells were significantly in expression.(2) Compared with the control group (30.746±6.99),mild preeclampsia (64.056±9.63) placenta AOPP expression levels were significantly higher, and the difference was significant (F=415.39;P <0.05). Compared with the control group (30.746±6.99), patients with severe preeclampsia placenta (97.516±13.72) AOPP expression level was also significantly increased, the difference was significant (F=415.39;P<0.05). The patient with severe preeclampsia will have higher AOPP placenta expression levels compare with mild preeclampsia placenta tissue (F=415.39; P<0.05).
[Conclusion] The mainly expressed position of AOPP in the placenta from preeclampsia is syncytiotrophoblastic and villous trophoblastic cells. There is a significant difference
When we mentioned AOPP expression levels between preeclampsia patients and normal pregnant women in placenta, the results of this part showed, expression in the placenta of pregnant women increased AOPP is an important pre-eclampsia pathological changes. Trace its roots, that may be associated with pre-eclampsia, while AOPP induced increased incidence of preeclampsia and development; further illustrate the pathogenesis of preeclampsia is multifactorial joint participation and the results of the interaction.
PART2AOPP-induced apoptosis in gestational trophoblastic pathway through NADPH
[Objective] Through a series of in vitro cell-based research, to explore mechanism of AOPP induced trophoblastic apoptosis.
[Method] According to Witko-Sarsat methods described to prepare AOPP. The effect of AOPPs on in vitro trophoblast cell function was also examined. Specifically, we exposed trophoblastic cells to AOPPs and measured the production of human chorionic gonadotropin (hCG) as well as their invasion capacity using an in vitro Transwell invasion assay. We also investigated the effect of AOPPs on trophoblastic apoptosis and whether this effect could be mediated through interference in NADPH oxidase signaling. ROS anionic fluorescent probes to detect the impact and role of reactive oxygen molecules AOPP. Repeat the test results of three or more representatives of all the data, expressed as mean±standard deviation, all statistics by the statistical software SPSS13.0completed. Multiple samples were compared using One-Way ANOVA, variance Aceh pairwise comparisons using LSD, using Dunnett's T3law when heterogeneity of variance, P<0.05was considered statistically significant.
[Results] Preparation can be realized without AOPP of endotoxin. AOPPs also affected trophoblastic cell function in vitro by significantly reducing β-HCG (MSA group:F=0.913,P=0.477;AOPP group:F=20.603,P=0.000) production and inhibiting trophoblastic cell invasive capacity(F=197.593;P=0.000). Exposure to AOPPs significantly increased apoptosis in trophoblastic cells(concentration dependent, F=37.813, P=0.000;time dependent, F=24.318, P=0.000), which was mediated through the NADPH oxidase pathway(NOX2:F=21.318, P=0.000; NOX4:F=73.643, P=0.000; p47phox:F=126.678, P=0.000; p22phox:F=4.323, P=0.000; p53:F=40.979, P=0.00; Bax:F=274.420, P=0.00; Bcl-2:F=38.881, P=0.00. AOPP stimulate ROS production.(F=38.277,P=0.00).
[Conclusions] AOPPs expression is increased in PE placentas and exposure to AOPPs adversely affects trophoblastic cell function, which may contribute to the shallow trophoblastic invasion that characterizes this disorder. Additional studies are needed to investigate further the role of AOPPs in the pathogenesis of PE, and to determine whether AOPPs can be used as a biomarker for the diagnosis and/or prediction of PE.
PART3The inhibition and mechanisms of GLP-1of trophoblastic cells apoptosis induced by AOPP
[Objective] Base on the second research part of this study, we confirmed the preliminary AOPP-induced apoptosis in gestational trophoblastic cells. AOPP intervention by GLP-1induced apoptosis in gestational trophoblastic and examine its effect on apoptosis-related molecules and PI3K/Akt pathway. Explore the inhibition and mechanisms of GLP-1of trophoblastic cells apoptosis induced by AOPP.
[Method] CCK-8(cell counting kit-8) colorimetric determines cell viability and value-added capabilities. Detecting fluorescence microscopy active oxygen species (ROS) expression. ELISA was used to detect cell apoptosis protease caspase-9,-3activity. Western-blot to detect the expression of p-Akt, Bcl-2, Bax, Cyto-C and other pathway proteins. Repeat the test results of three or more representatives of all the data, experimental data measured data were expressed as mean±standard deviation, all statistics by the statistical software SPSS13.0completed. Multiple samples were compared using One-Way ANOVA, variance Aceh pairwise comparisons using LSD, using Dunnett's T3law when heterogeneity of variance, P<0.05was considered statistically significant.
[Results] within a certain concentration range (50-150nmol/L), GLP-1-induced pregnancy after treatment AOPP trophoblastic cells, with increasing concentration; the degree of apoptosis is inhibited. GLP-1inhibits gestational trophoblastic intracellular ROS generation (F=86.222P=0.000), which is induced by AOPP. GLP-1may be partially mitigated AOPP caspase-9,-3induced increase in the activity.(caspase-9:F=10.321P=0.004; caspase-3:F=20.989,P=0.000) As the PI3K inhibitor LY294002, the following effects can be antagonized对AOPP of GLP-1:GLP-1 AOPP-induced inhibition of cell viability(F=71.07,P=0.000), GLP-1cells induced by inhibition of AOPP low levels of p-Akt expression(concentration dependent:F=10.679,P=0.004;time dependent:F=45.886P=0.000), GLP-1induced inhibition expression of Bax(F=33.017P=0.00), cyto-c(F=49.483,P=0.00) and stimulate Bcl-2expression (F=45.258P=0.00)
[Conclusion] This study found that GLP-1within a certain range of concentrations antagonistic AOPP inhibition effect of gestational trophoblastic activity, and reduce AOPP-induced cell damage. AOPP effect on pregnancy trophoblast cells, ROS levels increased apoptosis protease caspase-9,-3increased activity, GLP-1can be induced partially reduce these effects AOPP. So presumably oxidative stress, caspase-9,-3GLP-1may be involved in the antagonism AOPP-induced cell apoptosis. GLP-1is at least partially through PI3k/Akt signal transduction pathway plays an anti-apoptotic effect, reducing AOPP-induced cell apoptosis. GLP-1can regulate AOPP induced gestational trophoblastic cells Bcl-2, Bax, cyto-c expression levels, and this regulation and the PI3K/Akt signaling pathway.
妊娠期高血压疾病是妊娠期特有的全身性疾病,我国发病率为9.4%-10.4%,国外报道发病率为7%-12%。该疾病不但严重影响了母婴健康,至今为止仍然是母婴发病率及死亡率较高的主要原因。子痫前期(preeclampsia)是该疾病最常见的类型。流行病学调查结果显示,子痫前期孕产妇的死亡率约为5%-10%,且围产儿死亡率在子痫前期及子痫患者更高达33.9%-68.6%。子痫前期以妊娠20周以后首次出现高血压、蛋白尿、水肿为主要特征,并可伴有全身多脏器的损害;病情严重者可能因抽搐、脑出血、肺水肿、心功能衰竭、弥漫性血管内出血而导致死亡。临床上处理子痫前期的患者,主要以对症治疗和终止妊娠为主。由于缺乏明确的预测手段和防治对策,使子痫前期成为导致孕产妇死亡的第二大原因,也是导致围产儿发病及死亡的主要原因。其发病机制是多因素的,且目前尚不十分明确。现多数学者认为子痫前期的发生、发展的原因和过程可能与以下几种因素和学说相关：免疫失衡机制,胎盘浅着床,血管内皮细胞损伤和胎盘滋养细胞缺血,胰岛素抵抗,钙平衡失调,遗传因素、营养缺乏等。其中细胞功能损伤及凋亡均与氧化应激有着密切的关系。
现有部分研究表明氧化应激学说可能是子痫前期的重要机制之一。子痫前期患者体内氧化系统和抗氧化系统的平衡遭到了破坏；活性氧族(Reactive Oxygen Species, ROS)与抗氧化体系之间失去平衡。其中氧化作用明显加强,而抗氧化作用则相对减弱,总平衡向氧化方向增强。其实在正常妊娠孕妇体内也存在着不等量的自由基及其产物,但是由于妊娠期孕妇体内抗氧化能力相应提高,二者之间仍处于平衡状态,正常的生理性妊娠不致于形成氧化应激。据文献报道,蛋白、多肽、氨基酸对各种自由基及其相关氧化物相当敏感。在子痫前期患者体内,氧化产物增多(脂质过氧化物、蛋白氧化产物、核酸)而抗氧化能力降低,导致子痫前期患者体内呈现氧化应激状态。氧化应激在许多与细胞功能受损相关的疾病中发挥着重要作用。由于子痫前期患者的缺血、缺氧可以导致大量的自由基生成、脂质、蛋白质过氧化物产生、内皮细胞损伤等；这种过程在子痫前期疾病发生发展的过程中呈正反馈且加重了氧化应激,导致了疾病进一步恶化发展。目前研究表明,蛋白质也是氧化应激攻击的主要靶分子之一,主要表现为氨基酸残基氧化、肽链断裂或者蛋白质分子的交联聚合、蛋白质的疏水性与对水解酶的抵抗力增强,从而使蛋白质丧失生物活性并易于沉积。细胞的损伤及功能失调是子痫前期疾病特征性病理变化之一,它也是子痫前期疾病多器官功能损伤的病理生理学基础。许多子痫前期患者在临床症状出现以前,胎盘的形态与功能方面的改变就已经出现,并且临床上大部分子痫前期患者在胎盘娩出后其临床症状和体征很快就随之消失；这均表明了胎盘在子痫前期疾病的发生发展过程中可能起到了关键作用。基础研究表明,子痫前期的发病机制可归纳为胎盘浅着床和全身血管内皮细胞的激活两个阶段；第一阶段的胎盘血液灌注减少,产生过多的活性氧,造成氧化应激,导致孕妇血管内皮细胞损伤,从而引发第二阶段全身血管内皮细胞的病理性损伤。全身血管内皮损伤和系统性炎症反应又加重了机体的氧化应激失衡,最终导致了子痫前期的一系列临床症状和体征。根据目前的临床和基础研究,我们有理由相信子痫前期是一种胎盘源性疾病,胎盘的氧化-抗氧化的失衡是其发病的不可或缺的环节。
晚期氧化蛋白产物(advanced oxidation protein products, AOPP)是一种新型的蛋白类氧化应激标志物,是氧化应激引起体内各种蛋白质氧化所形成的终末产物的总称。它于1996年由Witko-Sarsat首次报道,于尿毒症患者血浆中首次发现,在慢性肾功能衰竭患者的血浆中分离出来。它是血浆白蛋白被自由基和反应性氧系氧化后形成的双酪氨酸蛋白交连产物,其主要成分来自于：血清白蛋白被自由基或反应性氧系(主要为中性粒细胞髓过氧酶产生的氯化氧化物)氧化而成。AOPP相对于既往普遍关注的脂质过氧化物标志而言,它形成早,存在时间长,更加稳定,是反映蛋白氧化应激损伤的可信指标；同时也是氧化应激时重要的促炎症因子,炎症介质是参与细胞损伤的重要物质。体外研究已经证实,AOPP可通过不同途径参与诱导肾脏足细胞凋亡、血管内皮细胞激活、胰岛素抵抗等一系列病理生理过程。目前已经证实在高龄、慢性肾脏疾病、冠状动脉疾病、糖尿病、代谢综合征、多囊卵巢综合征等等疾病患者中均存在慢性AOPP蓄积。该类人群在妊娠过程中与正常人群相比更容易出现早产、子痫前期、胎儿宫内生长受限、胎盘早剥、重复的胎儿丢失、宫内死胎等等妊娠并发症。进一步而言,对于AOPP为何沉积于上述疾病患者的体内,其机制的研究也日渐深入和明晰。Hideaki Kaneda等的研究表明慢性冠状动脉疾病与尿毒症中一样,血浆白蛋白发生了氧化,该类疾病患者的血浆中AOPP水平和尿毒症患者血浆中同样增高,并且AOPP水平与慢性冠状动脉疾病的发生以及该类疾病的严重程度相关；该实验证明了氧化应激可能与慢性冠状动脉疾病的病理生理过程密切相关。Agnieszka Piwoman等学者研究发现,如妊娠期高血压疾病、妊娠期糖尿病等均属于复杂性代谢紊乱性疾病,其发病机制与活性氧的产生及抗氧化系统功能失衡有关,在糖尿病患者中TRAP(抗酒石酸酸性磷酸酶)及SH(蚕丝水解物)水平均有不同程度的降低,而CO2、NH2及AOPP水平增高；AOPP的表达水平在Ⅱ型糖尿病患者、子痫前期患者血浆中明显增高(本课题组前期实验结果),可推测AOPP可能是一种评估糖尿病病人氧化应激介导蛋白损伤的有用指标。AOPP不仅仅是氧化应激的产物,它还可以引起单核细胞(中性粒细胞)的呼吸爆发,从而诱导单核细胞产生更多的活性氧,正反馈诱导或加重氧化应激反应,使得机体呈现出一种持续氧化应激状态。
胰高血糖素样肽链-1(Glucagon-like peptide-1,GLP-1)主要是由肠道内分泌L细胞分泌的多肽,作为一种安全、有效的促胰岛素分泌剂越来越受到关注,它本是治疗Ⅱ型糖尿病的一种新途径。GLP-1除了广为人知的促进胰岛β细胞增值、抑制β细胞凋亡、促进胰岛素分泌等等作用外；近些年的研究显示其在抑制氧化应激中也承担着重要的角色。它可以降低TNF-a等诱导的炎症水平,以减少高凝状态和炎症反应对于细胞的损伤；此外,GLP-1可抑制单核细胞粘附于人的主动脉内皮细胞,减轻血管炎性损伤,组织冠状动脉粥样硬化及其相关疾病的进展。大量的临床及实验性研究资料表明,GLP-1保护细胞生理功能可能与抑制氧化应激有关,但是其机制尚未十分明确。研究表明,在胰岛β细胞、心肌细胞、胆囊细胞中,GLP-1均表现出了明显的抗氧化应激作用。那么在妊娠滋养细胞中,GLP-1是否具有抑制AOPP抗氧化应激的作用,其作用机制是否与PI13k/Akt信号传导通路有关。这些问题将在本实验中得到初步的探索性答案。
目前已发现子痫前期血浆AOPP水平明显高于正常孕妇。因此我们推测孕妇胎盘AOPP蓄积可能是诱导滋养细胞损伤、功能障碍并引起子痫前期发生过程中新的致病物质。本研究从探讨子痫前期孕妇胎盘中AOPP表达水平与子痫前期严重程度的相关性以及子痫前期患者胎盘AOPP高表达可能的临床意义,作为切入点。在体外的细胞水平进行实验探索性研究,旨在证明AOPP通过NADPH通路诱导妊娠滋养细胞凋亡；以及GLP-1抑制AOPP诱导的凋亡及其机制。为进一步寻找子痫前期的发病原因及机制提供理论基础。
第一部分AOPP在子痫前期与正常妊娠胎盘组织中的表达
【目的】通过测定AOPP在子痫前期患者和正常孕妇胎盘组织中的表达水平及其差异；探讨AOPP与子痫前期发病机制的相关性；为进一步研究氧化应激在子痫前期发病机制中的信号转导通路提供线索及切入点。
【方法】随机选取2012年9月-2013年9月南方医科大学南方医院收治并以剖宫产方式分娩的子痫前期患者共50例,其中子痫前期轻度(mildpreeclampsia, MPE)25例,子痫前期重度(severe preeclampsia, SPE)25例；选取同期足月因社会因素行剖宫产分娩的孕妇50例作为对照组。运用免疫组织化学方法检测子痫前期患者(实验组)与正常妊娠孕妇(对照组)胎盘组织中AOPP的表达水平；测定切片平均灰密度值,半定量胎盘中AOPP的表达量。采用SPSS13.0统计软件,计量资料以均数±标准差表示。组间比较采用ANOVA检验。P<0.05为差异具有统计学意义。
[结果](1)正常孕妇组胎盘组织基本无染色。子痫前期组胎盘组织中均可见棕褐色阳性染色。子痫前期轻度组AOPP主要表达于绒毛合体滋养层,偶见于绒毛细胞滋养细胞。子痫前期重度组AOPP在绒毛合体滋养层细胞以及绒毛细胞滋养细胞中均明显表达。(2)与对照组(30.746±6.99)相比,轻度子痫前期患者(64.056±9.63)胎盘组织中的AOPP表达水平明显增高,且差异具有显著性(F=415.39；P<0.05)。与对照组(30.746±6.99)相比,重度子痫前期患者(97.516±13.72)胎盘组织中的AOPP表达水平亦明显增高,差异具有显著性(F=415.39；P<0.05)。重度子痫前期患者胎盘组织中的AOPP表达水平高于轻度子痫前期患者胎盘组织中的表达量(F=415.39；P<0.05)。
[结论]AOPP在子痫前期患者的胎盘中的主要表达位置为合体滋养层细胞以及绒毛细胞滋养细胞。子痫前期患者胎盘组织中AOPP的表达水平与正常妊娠孕妇胎盘中的表达相比,具有显著性差异。本部分实验结果提示孕妇胎盘中AOPP的表达增高是子痫前期的重要病理变化,追溯其根源可能与子痫前期疾病有关,同时AOPP诱导加重了子痫前期的发生和发展；进一步说明了子痫前期的发病机制是多因素多因子共同参与,相互作用的结果。
第二部分AOPP通过NADPH通路诱导妊娠滋养细胞凋亡
[目的]通过一系列体外细胞水平的实验室基础研究,探讨AOPP诱导妊娠滋养细胞凋亡的机制。
[方法]采用Witko-Sarsat等介绍的方法制备AOPP。用ELISA试剂盒复孔检测β-HCG的含量。用侵袭小室试验检测AOPP刺激后妊娠滋养细胞侵袭能力的变化。用Hoechst33258荧光法检测凋亡。用Western Blotting检测凋亡相关蛋白的表达以及检测尼克酰胺腺嘌呤二核苷酸磷酸(Nicotinamide Adanine Dinucleotide Phosphate, NADPH)氧化酶亚基的表达。用活性氧分子(Reactive Oxygen Species,ROS)阴离子荧光探针,检测AOPP对活性氧分子的影响和作用。所有数据代表三次以上的重复试验结果,以均数±标准差表示,所有统计由统计软件SPSS13.0完成。多个样本均数的比较采用One-Way ANOVA,方差齐时两两比较采用LSD,方差不齐时采用Dunnett's T3法,P<0.05为差异具有统计学意义。
[结果]可实现无内毒素的AOPP的制备。AOPP抑制妊娠滋养细胞β-HCG的分泌(MSA刺激组F=0.913, P=0.477;AOPP刺激组F=20.603,P=0.000)。AOPP抑制妊娠滋养细胞的侵袭能力(F=197.593;P=0.000)。AOPP组妊娠滋养细胞凋亡率明显升高(不同浓度刺激组,F=37.813,P=0.000;不同时间刺激组,F=24.318,P=0.000),apocynin阻断AOPP此效应。AOPP诱导了体外的NADPH氧化酶及其相关亚基的活化(NOX2:F=21.318,P=0.000;NOX4:F=73.643,P=0.000;p47phox: F=126.678, P=0.000;p22phox:F=4.323, P=0.000),NADPH氧化酶的抑制剂apocynin可阻断AOPP此效应。AOPP诱导凋亡相关蛋白的表达(p53:F=40.979,P=0.00;Bax:F=274.420, P=0.00;Bcl-2:F=38.881, P=0.00)。 AOPP在体外刺激了ROS的表达(F=38.277,P=0.00),apocynin可阻断AOPP此效应。
[结论]这一部分的实验研究证实,AOPP抑制P-HCG的分泌和妊娠滋养细胞的侵袭能力可能与胎盘浅着床(子痫前期发病机制中比较受肯定和公认的理论之一)的理论相符合,因此AOPP的高表达是不利于妊娠顺利进行的。AOPP以时间和剂量的方式诱导了妊娠滋养细胞的凋亡,其途径主要是通过NADPH通路。P53、Bax蛋白参与AOPP诱导的妊娠滋养细胞凋亡,P53、Bax蛋白在NAPDH氧化酶下游。NADPH氧化酶抑制剂apocynin干预阻断了AOPP诱导的细胞损伤。因此,AOPP诱导的妊娠滋养细胞NADPH氧化酶的活化以及产生的大量ROS是导致细胞损伤的重要因素之一,这一结论为临床防治子痫前期等妊娠期相关病变提供了一定程度上的理论依据。
第三部分GLP-1抑制AOPP所诱导的凋亡及其机制
[目的]在本课题第二部分初步证实了AOPP诱导妊娠滋养细胞凋亡的研究基础上,通过GLP-1干预AOPP诱导妊娠滋养细胞凋亡的作用,并检验其对凋亡相关分子及PI3K/Akt通路的影响,初步探讨GLP-1对AOPP诱导细胞凋亡的影响及其分子机制。
[方法]CCK-8(cell counting kit-8)比色法测定细胞活力及增值能力。荧光显微镜检测活性氧(ROS)的表达。ELISA法检测细胞凋亡蛋白酶caspase-9,-3的活性。Western-blot检测p-Akt等通路相关蛋白的表达。ELISA法检测凋亡相关蛋白Bcl-2、Bax、Cyto-C的表达。所有数据代表三次以上的重复试验结果,实验数据计量资料以均数±标准差表示,所有统计由统计软件SPSS13.0完成。多个样本均数的比较采用One-Way ANOVA,方差齐时两两比较采用LSD,方差不齐时采用Dunnet's T3法,P<0.05为差异具有统计学意义。
[结果]在一定浓度范围内(50-150nmol/L),GLP-1处理AOPP诱导的妊娠滋养细胞后进行实验,结果表明GLP-1抑制AOPP诱导的妊娠滋养细胞内ROS的生成(P=86.222P=0.000)。GLP-1可部分减轻AOPP诱导caspase-9、 caspase-3活性的增加(caspase-9:F=10.321P=0.004; caspase-3:F=20.989,P=0.000)。LY294002作为P13K的抑制剂,可拮抗GLP-1对AOPP的以下作用：GLP-1抑制AOPP诱导的细胞活力下降(F=71.07,P=0.000)、GLP-1抑制AOPP诱导细胞p-Akt水平低表达(不同浓度刺激时F=10.679,P=0.004；不同时间刺激时F=45.886P=0.000),GLP-1抑制AOPP诱导细胞Bax(F=33.017P=0.00)、 cyto-c(F=49.483,P=0.00)高表达以及Bcl-2的低表达(F=45.258P=0.00)。
[结论]本实验发现GLP-1在一定的浓度范围内有拮抗AOPP抑制妊娠滋养细胞活力的作用,而且减少AOPP诱导的细胞损伤。AOPP作用于妊娠滋养细胞后,ROS水平升高,凋亡蛋白酶caspase-9、-3的活性增加,GLP-1可部分性减少AOPP诱导的上述效应。因此推测氧化应激、caspase-9、-3可能参与了GLP-1拮抗AOPP诱导的细胞凋亡的作用。GLP-1至少部分通过PI3k/Akt信号转导通路发挥抗凋亡作用,减轻AOPP诱导的细胞凋亡。GLP-1可调节AOPP诱导妊娠滋养细胞内Bcl-2、Bax、cyto-c表达水平,且此调节作用与PI3K/Akt信号通路有关。
Background
Hypertensive disorder complicating pregnancy is the systemic disease, which is idiopathic in the gestational period; the morbidity in China is9.4%-10.4%, while it is7%-12%abroad. The disease is not only a serious impact on maternal and child health. So far it also remains high maternal and child morbidity and mortality of the main reasons. Preeclampsia (PE) is the most common type of the disease. Epidemiological investigations showed that maternal mortality of preeclampsia is about5%to10%. As well as, perinatal mortality rate was as high as33.9%～68.6%in preeclampsia and eclampsia. The main feature of preeclampsia is the first occurrence of hypertension after20weeks gestation, proteinuria and edema. It also can be accompanied by systemic multi-organ damage; severe cases may be dead due to convulsions, cerebral hemorrhage, pulmonary edema, heart failure, and disseminated intravascular hemorrhage. Clinically we treated patients with pre-eclampsia mainly basing on symptomatic treatment and termination of pregnancy. Due to the lack of a clear prediction methods and countermeasures that preeclampsia become the second leading cause of maternal mortality. Furthermore, it is the main reasons of perinatal morbidity and mortality. Its pathogenesis is multifactorial and the pathogenesis is not very clear. Most scholars now believe that the incidence of preeclampsia, the causes and course of development may be related to several factors and theories related to:immune imbalance mechanism, shallow placental implantation, vascular endothelial cell injury and ischemic placental trophoblastic cells, insulin resistance, calcium balance disorders, genetic factors, nutritional deficiencies and so on. Among the factor mentioned above, endothelial dysfunction and oxidative stress in cells is closely related.
Some studies indicate that oxidative stress existing doctrine may be one of the main mechanisms of preeclampsia. Oxidative balance system and antioxidant systems in preeclampsia have been destroyed and balance lose between reactive oxygen species (ROS) and antioxidant system. Oxidation strengthened and the relative weakening of antioxidant enhance the overall balance of the oxidation direction. In normal pregnant women also have the amount of free radicals and their products, but because of pregnancy corresponding increase in vivo antioxidant capacity, it is still in the balance between the two and prevented the formation of oxidative stress. According to the literature, proteins, peptides and amino acids are quite sensitive to a variety of related oxide radicals. In preeclampsia, increased oxidation products (lipid peroxidation, protein oxidation products, nucleic acids) and reduced antioxidant capacity, resulted in patients showing oxidative stress. Because ischemia and hypoxia of preeclampsia patients, that can cause a lot of free radical production, lipid, protein peroxides, endothelial cell injury. This process occurs in the process of developing preeclampsia will increase oxidative stress CKS feedback, resulting in a further development of the disease. The present study showed also the major target of oxidative stress proteins attack, mainly for the oxidation of an amino acid residue, a peptide or protein molecule strand cross-linking polymerization of hydrophobic protein with increased resistance to enzymatic hydrolysis increased, so that it will resulted in the loss of biological activity and protein tend to accumulate. Injury and dysfunction of endothelial cells is one of the pathological changes characteristic of preeclampsia disease, it is also pathophysiology foundation of multiple organ dysfunction in preeclampsia. Many patients before clinical symptoms of preeclampsia, placental morphological and functional aspects of the change have occurred, and after the placenta delivery, clinical symptoms and clinical signs soon disappeared. This indicates that the placenta may play a critical role in the development of preeclampsia disease. Basic research showed that the pathogenesis of preeclampsia can be summarized as the formation of the placenta and systemic adverse vascular endothelial cells activated in two stages;first stage reduced placental perfusion, excessive ROS cause oxidative stress, resulting in pregnant women vascular endothelial cell injury, causing damage to the second phase of pathological systemic vascular endothelial cells. Systemic vascular endothelial injury and systemic inflammation and oxidative stress increased the imbalance in the body, eventually leading to a series of pre-eclampsia clinical signs and symptoms. Based on current clinical and basic research, we have reason to believe that pre-eclampsia is a placental-borne disease, placental oxidation-antioxidant imbalance is an indispensable part of its incidence.
AOPP is a new class of protein markers of oxidative stress; oxidative stress is caused by a variety of proteins in vivo oxidation of AGEs formed in general. It was first reported in1996by the Witko-Sarsat, in uremic patients plasma and it is separated from the plasma in patients with chronic renal failure. It is a double cross-linked product of tyrosine protein is serum albumin and the reactive oxygen radicals formed by oxidation of the system, the main component of serum albumin or reactive oxygen free radical system (mainly neutrophils myeloperoxidase IDO chloride produced oxide). AOPP general concern with respect to previous mark of lipid peroxides, which formed early, existence of long time, more stable, is a reflection of the credibility of protein oxidative stress indicators. Simultaneously, it also important pro-inflammatory cytokines, inflammatory mediators when oxidative stress happened, which is an important substance involved in endothelial injury. In vitro studies have demonstrated that, AOPP through different pathways involved in a series of pathophysiological processes, such as the induction of apoptosis in renal podocytes, endothelial cell activation, insulin resistance, etc. Now it has been confirmed in elderly patients with chronic kidney disease, coronary artery disease, diabetes, metabolic syndrome, polycystic ovary syndrome, etc. are present in chronic AOPP accumulation. Such people are more prone to preterm labor, preeclampsia, intrauterine growth restriction, placental abruption, repeated fetal loss, intrauterine fetal death, and so pregnancy complications compared to the normal population during pregnancy. Further, why AOPP easy the deposition to these diseases the patient's body, to study of the mechanism increasing depth and clarity. Hideaki Kaneda and other studies have shown that chronic coronary artery disease, the same as uremia, plasma albumin oxidation occurs. In patients with such diseases, AOPP levels in plasma and plasma uremic patients also increased. AOPP levels and the incidence of chronic coronary artery disease, as well as, severity of these diseases have close relationship. The experiment proved that oxidative stress might be associated with the pathophysiology of chronic coronary artery disease. Agnieszka Piwoman scholars found that diabetes is a complex metabolic disorders, which produces and antioxidant system function in the pathogenesis and reactive oxygen species imbalance, in diabetic patients TRAP (tartrate-resistant acid phosphatase) and SH (silk hydrolyzate) lower levels of varying degrees, while the CO2, NH2and AOPP levels increased. AOPP expression levels in patients with type II diabetes was significantly increased, AOPP presumably may be a useful indicator for diabetes oxidative stress-mediated protein damage. AOPP is not just a product of oxidative stress, it can cause monocytes neutrophil respiratory burst, thereby inducing monocytes to produce more reactive oxygen species, a positive feedback to induce or aggravate oxidative stress, making body showing a sustained oxidative stress. Glucagon-like peptide-1(Glucagon-like peptide-1, GLP-1) is mainly secreted by the intestinal endocrine L-cells of a polypeptide. As a safe and effective insulin secretion agent more and more attention, it is a new way to treat this type II diabetes. In addition to promoting cell proliferation of islet cell β, inhibit cell β apoptosis, insulin secretion promoting, recent studies show it also plays an important role in the inhibition of micro vascular lesions. It can reduce the induction of TNF-a and other inflammatory molecule level. In order to reduce hypercoagulable state and inflammation damage to endothelial cells. Besides, GLP-1inhibits monocyte adhesion to human aortic endothelial cells, reducing vascular inflammatory injury, the progress of atherosclerosis and its related organizations coronary artery disease.A large number of clinical and experimental research data shows, GLP-1protects endothelial cells by inhibiting oxidative stress, but its mechanism is not very clear. GLP-1receptor agonist exendin-4activation PKA-PI13k/Akt-eNOS signaling pathways to stimulate endothelial cell proliferation, in order to facilitate the rapid repair of vascular injury. Research shows that in pancreatic β cells, cardiomyocytes, gallbladder cells, GLP-1exhibited significant anti-oxidative stress. So in gestational trophoblastic cells, whether GLP-1inhibition effect of AOPP or not, what its mechanism may be related to, these issues will have a preliminary exploratory answers in this experiment.
Preeclampsia has been found in plasma AOPP levels were significantly higher than normal pregnant women. Therefore, we hypothesized that placental trophoblastic AOPP, as a new pathogenic substances, its accumulation could be induced cell damage and dysfunction caused by in the process of preeclampsia occurs. This study investigates AOPP expression and severity of preeclampsia correlation in the placenta from pregnant women with preeclampsia. It also investigates AOPP high expression of possible clinical significance in preeclampsia placenta. That is the stating point of the assay. Experimental exploratory research at the cellular level in vitro is to study AOPP induced by NADPH gestational trophoblastic apoptosis pathway. Beside, GLP-1inhibited AOPP-induced apoptosis and its mechanism. The purpose of this study is providing a theoretical basis for further search and mechanisms of pathogenesis of preeclampsia.
PART1AOPP expression in normal pregnancy and preeclampsia placenta
[Objective] by measuring the expression levels and their differences of AOPP in placental tissue form patients with preeclampsia and normal pregnant women, explore the correlation between AOPP and pathogenesis of preeclampsia. This can provide clues and the starting point for further study of oxidative stress in the pathogenesis of preeclampsia in the signal transduction pathway.
[Method] This study was performed in50cases of pregnant women with preeclampsia in the obstetrical department of NanFang Hospital from September2012to September2013, in which25cases were mild preeclampsia (MPE) and25 cases were severe preeclampsia (SPE).50cases the same period in term pregnant women delivered by cesarean section due to social factors as a control group. Immunohistochemistry was used to detect preeclampsia and normal pregnant women in AOPP (control group) placental tissue expression levels. The average ash density values measured, semi-quantitative expression of the placenta AOPP. Using SPSS13.0statistical software, data were expressed as mean±standard. Groups comparing using ANOVA test. P<0.05was considered statistically significant.
[Results](1) Normal pregnant group placentas almost no staining. Preeclampsia placentas were visible brown staining. Mild preeclampsia group AOPP will mainly expressed in the villous syncytiotrophoblastic, occasionally in villous trophoblastic cells. Severe preeclampsia group AOPP in villous syncytiotrophoblastic and villous trophoblastic cells were significantly in expression.(2) Compared with the control group (30.746±6.99),mild preeclampsia (64.056±9.63) placenta AOPP expression levels were significantly higher, and the difference was significant (F=415.39;P <0.05). Compared with the control group (30.746±6.99), patients with severe preeclampsia placenta (97.516±13.72) AOPP expression level was also significantly increased, the difference was significant (F=415.39;P<0.05). The patient with severe preeclampsia will have higher AOPP placenta expression levels compare with mild preeclampsia placenta tissue (F=415.39; P<0.05).
[Conclusion] The mainly expressed position of AOPP in the placenta from preeclampsia is syncytiotrophoblastic and villous trophoblastic cells. There is a significant difference
When we mentioned AOPP expression levels between preeclampsia patients and normal pregnant women in placenta, the results of this part showed, expression in the placenta of pregnant women increased AOPP is an important pre-eclampsia pathological changes. Trace its roots, that may be associated with pre-eclampsia, while AOPP induced increased incidence of preeclampsia and development; further illustrate the pathogenesis of preeclampsia is multifactorial joint participation and the results of the interaction.
PART2AOPP-induced apoptosis in gestational trophoblastic pathway through NADPH
[Objective] Through a series of in vitro cell-based research, to explore mechanism of AOPP induced trophoblastic apoptosis.
[Method] According to Witko-Sarsat methods described to prepare AOPP. The effect of AOPPs on in vitro trophoblast cell function was also examined. Specifically, we exposed trophoblastic cells to AOPPs and measured the production of human chorionic gonadotropin (hCG) as well as their invasion capacity using an in vitro Transwell invasion assay. We also investigated the effect of AOPPs on trophoblastic apoptosis and whether this effect could be mediated through interference in NADPH oxidase signaling. ROS anionic fluorescent probes to detect the impact and role of reactive oxygen molecules AOPP. Repeat the test results of three or more representatives of all the data, expressed as mean±standard deviation, all statistics by the statistical software SPSS13.0completed. Multiple samples were compared using One-Way ANOVA, variance Aceh pairwise comparisons using LSD, using Dunnett's T3law when heterogeneity of variance, P<0.05was considered statistically significant.
[Results] Preparation can be realized without AOPP of endotoxin. AOPPs also affected trophoblastic cell function in vitro by significantly reducing β-HCG (MSA group:F=0.913,P=0.477;AOPP group:F=20.603,P=0.000) production and inhibiting trophoblastic cell invasive capacity(F=197.593;P=0.000). Exposure to AOPPs significantly increased apoptosis in trophoblastic cells(concentration dependent, F=37.813, P=0.000;time dependent, F=24.318, P=0.000), which was mediated through the NADPH oxidase pathway(NOX2:F=21.318, P=0.000; NOX4:F=73.643, P=0.000; p47phox:F=126.678, P=0.000; p22phox:F=4.323, P=0.000; p53:F=40.979, P=0.00; Bax:F=274.420, P=0.00; Bcl-2:F=38.881, P=0.00. AOPP stimulate ROS production.(F=38.277,P=0.00).
[Conclusions] AOPPs expression is increased in PE placentas and exposure to AOPPs adversely affects trophoblastic cell function, which may contribute to the shallow trophoblastic invasion that characterizes this disorder. Additional studies are needed to investigate further the role of AOPPs in the pathogenesis of PE, and to determine whether AOPPs can be used as a biomarker for the diagnosis and/or prediction of PE.
PART3The inhibition and mechanisms of GLP-1of trophoblastic cells apoptosis induced by AOPP
[Objective] Base on the second research part of this study, we confirmed the preliminary AOPP-induced apoptosis in gestational trophoblastic cells. AOPP intervention by GLP-1induced apoptosis in gestational trophoblastic and examine its effect on apoptosis-related molecules and PI3K/Akt pathway. Explore the inhibition and mechanisms of GLP-1of trophoblastic cells apoptosis induced by AOPP.
[Method] CCK-8(cell counting kit-8) colorimetric determines cell viability and value-added capabilities. Detecting fluorescence microscopy active oxygen species (ROS) expression. ELISA was used to detect cell apoptosis protease caspase-9,-3activity. Western-blot to detect the expression of p-Akt, Bcl-2, Bax, Cyto-C and other pathway proteins. Repeat the test results of three or more representatives of all the data, experimental data measured data were expressed as mean±standard deviation, all statistics by the statistical software SPSS13.0completed. Multiple samples were compared using One-Way ANOVA, variance Aceh pairwise comparisons using LSD, using Dunnett's T3law when heterogeneity of variance, P<0.05was considered statistically significant.
[Results] within a certain concentration range (50-150nmol/L), GLP-1-induced pregnancy after treatment AOPP trophoblastic cells, with increasing concentration; the degree of apoptosis is inhibited. GLP-1inhibits gestational trophoblastic intracellular ROS generation (F=86.222P=0.000), which is induced by AOPP. GLP-1may be partially mitigated AOPP caspase-9,-3induced increase in the activity.(caspase-9:F=10.321P=0.004; caspase-3:F=20.989,P=0.000) As the PI3K inhibitor LY294002, the following effects can be antagonized对AOPP of GLP-1:GLP-1 AOPP-induced inhibition of cell viability(F=71.07,P=0.000), GLP-1cells induced by inhibition of AOPP low levels of p-Akt expression(concentration dependent:F=10.679,P=0.004;time dependent:F=45.886P=0.000), GLP-1induced inhibition expression of Bax(F=33.017P=0.00), cyto-c(F=49.483,P=0.00) and stimulate Bcl-2expression (F=45.258P=0.00)
[Conclusion] This study found that GLP-1within a certain range of concentrations antagonistic AOPP inhibition effect of gestational trophoblastic activity, and reduce AOPP-induced cell damage. AOPP effect on pregnancy trophoblast cells, ROS levels increased apoptosis protease caspase-9,-3increased activity, GLP-1can be induced partially reduce these effects AOPP. So presumably oxidative stress, caspase-9,-3GLP-1may be involved in the antagonism AOPP-induced cell apoptosis. GLP-1is at least partially through PI3k/Akt signal transduction pathway plays an anti-apoptotic effect, reducing AOPP-induced cell apoptosis. GLP-1can regulate AOPP induced gestational trophoblastic cells Bcl-2, Bax, cyto-c expression levels, and this regulation and the PI3K/Akt signaling pathway.
引文
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4. Silva, L.M., et al., Low socioeconomic status is a risk factor for preeclampsia: the Generation R Study. J Hypertens,2008.26(6):p.1200-8.
5. Steegers, E.A., et al., Pre-eclampsia. Lancet.376(9741):p.631-44.
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8. Little, R.E. and B.C. Gladen, Levels of lipid peroxides in uncomplicated pregnancy:a review of the literature. Reprod Toxicol,1999.13(5):p.347-52.
9. Uzun, H., et al., Circulating oxidized low-density lipoprotein and paraoxonase activity in preeclampsia. Gynecol Obstet Invest,2005.60(4):p. 195-200.
10. Witko-Sarsat, V., et al., Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int,1996.49(5):p.1304-13.
11. Witko-Sarsat, V., et al., Advanced oxidation protein products as novel mediators of inflammation and monocyte activation in chronic renal failure. J Immunol,1998.161(5):p.2524-32.
12. Kalousova, M., J. Skrha, and T. Zima, Advanced glycation end-products and advanced oxidation protein products in patients with diabetes mellitus. Physiol Res,2002.51(6):p.597-604.
13. Martin-Gallan, P., et al., Biomarkers of diabetes-associated oxidative stress and antioxidant status in young diabetic patients with or without subclinical complications. Free Radic Biol Med,2003.34(12):p.1563-74.
14. Atabek, M.E., et al., Protein oxidation in obesity and insulin resistance. Eur J Pediatr,2006.165(11):p.753-6.
15. Wisdom, S.J., et al., Antioxidant systems in normal pregnancy and in pregnancy-induced hypertension. Am J Obstet Gynecol,1991.165(6 Pt 1):p. 1701-4.
16. Dordevic, N.Z., et al., Oxidative stress and changes in antioxidative defense system in erythrocytes of preeclampsia in women. Reprod Toxicol,2008. 25(2):p.213-8.
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20. Tomas, E. and J.F. Habener, Insulin-like actions of glucagon-like peptide-1:a dual receptor hypothesis. Trends Endocrinol Metab,2010.21(2):p.59-67.
21. Puddu, A., et al., Glucagon-like peptide-1 counteracts the detrimental effects of Advanced Glycation End-Products in the pancreatic beta cell line HIT-T 15. Biochem Biophys Res Commun,2010.398(3):p.462-6.
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