| Preeclampsia(PE) belongs to the family of hypertensive disorders in pregnant and is a pregnant-specific disease that is characterized mainly by new onset of hypertension and proteinuria after 20 gestational weeks.PE is widely considered to be one of the leading causes of maternal and perinatal morbidity and mortality.The incidence of PE worldwide is 2%-8%,more than 50000 pregnant women are death,and 10.4% in China.however,but its pathogenesis has not been fully elucidated.Preeclampsia is considered to be a complex disorder which is the result of comprehensive action by a variety of factors(nerve, hormone, placenta, genetic,environmental, immune factors, etc.).At present, "two stage" theory to the pathogenesis of preeclampsia is more recognized.The first stage, the potential pathological changes in the first trimester caused by the changes of placental perfusion state:uterine spiral artery trophoblast recasting obstacle and placental perfusion decreasing lead to placental ischemia and anoxia and releasing a variety of placenta factors.The second stage,involvement of maternal multiple organ in the second and third trimester and appearance of maternal clinical symptoms:a variety of placenta factors entering into the maternal blood circulation,which can activate the systemic inflammation and endothelial damage,thus leading to various clinical symptoms of preeclampsia and eclampsia.Due to PE appears along with the formationof the placenta, improves or disappears along with delivery of the placenta, so that the developing of PE may be closely related to placental dysfunction.Studies have shown that oxidative stress and vascular endothelial damage plays an important role in the development of PE.Nrf2-ARE pathway is the key factor of regulating expression of many antioxidants in cells and has kinds of biological activity,including antioxidant, anti-inflammatory, inhibiting apoptosis,and so on.NQO1 is a downstream regulator of Nrf2- ARE pathways, its expression level is related to the developing of a variety of cancers(such as cervical cancer, lung cancer, ovarian cancer,pancreatic cancer, etc).The increasing of NQO1 expression in cancerous tissue maybe protect cells against oxidative stress injury, so as to promote cell proliferation and lead to the occurrence and development of cancers,but it has not yet been reported in PE.The current study aimed to detect the expression of Nrf2 and NQO1 in the human placenta of normal pregnancy and severe PE patients, in order to preliminarily evaluate the relationship between the expression levels of Nrf2 and NQO1 and the pathogenesis of PE.ObjectiveThe current study aimed to detect the expression level of Nrf2 and NQO1 m RNA and protein in the human placenta of normal pregnancy and severe PE patients, locate their position in the placenta tissue, in order to evaluate the relationship between the expression levels of Nrf2 and NQO1 and the pathogenesis of PE.Materials and Methods1 Materials30 severe PE patients(the s PE group)and 30 parturients(the control group)were involved in the study and all of pregnant women underwent caesarean section from January 2014 to March 2015 at the Third Affiliated Hospital of Zhengzhou University, the diagnostic criteria of severe PE refered to the eighth edition of“obstetrics and gynecology” published by the People’s Medical Publishing House.Real-Time PCR and immunohistochemistry were employed to detect the expression of Nrf2 and NQO1 in placenta.All subjects were single live births, and those with premature rupture of membranes, placental abruption, placenta previa,hypertension disease, renal disease, diabetes mellitus, polycystic ovary syndrome,and other pregnancy complications were excluded. This study gained the approval of the ethics committee in our hospital and got the informed consent of all the research objects.2 Methodswithin 15 min after delivery, 5 pieces of full-thickness placenta,2 ×2 × 1 cm in size,were taken,one form the central portion and others respectively from each quadrant(3,6,9,12 o’clock of the placenta).Areas with the edge,calcification,hemorrhage and necrosis were avoided when collecting the samples. Blood in the tissues was wiped by sterile filter paper again and again.Freeze a portion of the placental tissues immediately in liquid nitrogen and keep them in a refrigerator at-80℃,and fix the rest in 10% formalin for immunohistochemistry(IHC).Real-Time PCR was employed to detect the expression of Nrf2 and NQO1 m RNA in the placenta tissue of severe PE and normal pregnancy group,and IHC was applied to evaluated the localization and expression of Nrf2 and NQO1 protein in two groups of placenta tissue.The 2-△△CTmethod was exploited for analyzing the relative expression of Nrf2 and NQO1 m RNA in the placental tissues of severe PE patients and normal controls pregnancies from Real-Time PCR data.PBS was used to instead of the first antibody as a negative control in IHC,the placenta tissue structure clear,buffy or brown staining in cytoplasm or nuclei,and dyeing significantly higher than background microscopically was regarded as the positive staining. semi-quantitative integral method was used to analyze the expression of Nrf2 and NQO1 protein in placenta tissues of two groups.3 Statistics analysisSPSS 21.0 software was used for statistical analysis.Quantitative data werepresented as mean ± standard deviation,the comparison between the two groups used two independent samples t test when the data of two groups were adequately normally distributed or the Mann- Whitney U test if the data distribution of any group was skew; enumeration data were compared using the2c test;comparison of two groups of ordered categorical data used Mann-Whitney U test.P-value <0.05 was thought that difference between two groups was statistically significant.Result1 Clinical data between severe PE patients and normal controls pregnancy womenAverage age of s PE group and normal control group pregnancy women were respectively 28.87±4.94 years and 29.97±3.89 years,BMI at delivery were respectively29.89±3.12 kg/m2 and 28.48±3.05 kg/m2,gestational age at delivery were respectively35.07±2.92 weeks and 38.23±1.28 weeks,the birth weight were respectively 2288.33±691.16 g and 3381.67±486.63 g,Systolic blood pressure were respectively 164.53±12.64 mm Hg and 116.67±9.41 mm Hg, diastolic blood pressure were respectively108.43±9.73 mm Hg and 74.87±7.14 mm Hg.Comparison of average age,BMI at delivery between s PE group and normal control group pregnancy women were not significant differences(P > 0.05).gestational age at delivery and the birth weight in s PE group were significantly lower than those of normal control group(P <0.05).Systolic and diastolic blood pressure and urine protein qualitative case in s PE group were significantly higher than in the normal control group(P < 0.05).In addition, maternal urine protein of s PE group was regarded as "+" in 5 cases, as "+ +" in 19 cases, as "+ + +" in 6 cases while normal control group were all negative, and the difference between s PE group and the control group was statistically significant(P< 0.05).2 Nrf2 m RNA expression in the placenta tissues of s PE group and the control group pregnancy womenCompared with the control group,the expression levels of Nrf2 m RNA in thes PE group was lower(the relative expression of s PE group and the control group were respectively 3.840±0.426,4.183±0.604),and the difference between between s PE group and the control group was statistically significant(P < 0.05).3 NQO1 m RNA expression in the placenta tissues of s PE group and the control group pregnancy womenCompared with the control group,the expression levels of NQO1 m RNA in the s PE group was lower(the relative expression of s PE group and the control group were respectively 4.608±0.776,5.358±0.811), and the difference between s PE group and the control group was statistically significant(P < 0.05).4 The location and expression of Nrf2 protein in the placenta tissues of s PE group and the control group pregnancy womenNrf2 was mainly expressed in the the cytoplasm of trophoblastic cells,while rarely in the nuclei.The expression of Nrf2 protein in the s PE group was significantly lower than that in control group(the positive rates of Nrf2 in the trophoblastic cells of s PE group and the control group were respectively46.7%,76.7%,P < 0.05).5 The location and expression of NQO1 protein in the placenta tissues of s PE group and the control group pregnancy womenNQO1 was expressed in the cytoplasm of trophoblastic cells.The expression of NQO1 protein in the s PE group was significantly lower than that in control group(the positive rates of Nrf2 in the trophoblastic cells of s PE group and the control group were respectively43.3%,70.0%,P < 0.05).ConclusionThe decreased expression of Nrf2 and NQO1 in the placental of s PE patients might be involved in the process of pathogenesis of PE by weakening antioxidant stress of the placenta. |
PDF Full Text Request