| BackgroundHypertensive 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 differenceWhen 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. |
PDF Full Text Request