The Relationship Between The Expression Of Inflammatory Factors TNF-? And IL-6 In The Placenta And Preeclampsia

BackgroundPreeclampsia is characterized by new-onset hypertension and proteinuria after 20 weeks of gestation.In the absence of proteinuria,diagnosis need the presence of hypertension together with evidence of other organ damage such as acute renal and liver function impairment,thrombocytopenia,nervous system complications,pulmonary oedema,uterine and placental dysfunction,etc.Preeclampsia is a serious complication during pregnancy,which affects about 3-8%of all pregnancies.It is the leading cause of maternal and perinatal morbidity and mortality wordwide especially in the developing countries,causing about 70,000 maternal deaths and more than 50,000 perinatal deaths every year.Preeclampsia is of special relevance in the developing countries due to the lack of assessment and monitoring of the health status of pregnant women and subsequently missing timely intervening measures at early stages.The incidence of preeclampsia is high and often deteriorates rapidly without any signs and seriously affects the health of mother and fetus.For pregnant woman with preeclampsia can occur heart failure,kidney and liver injury or failure,disseminated intravascular coagulation(DIC),adult respiratory distress syndrome,central nervous system(CNS)damage,stroke,pulmonary and cerebral edema and hemorrhage,HELLP(hemolysis,elevated liver enzymes,low platelets),and even death.Preeclampsia is deleterious effect not only on the mother,but also on the fetus.To the fetus preeclampsia may cause intrauterine growth restriction(IUGR),placental abruption,preterm birth and associated complications including neonatal respiratory distress syndrome,cerebral palsy,cerebral hemorrhage necrotizing enterocolitis,retinopathy and even perinatal death.The risk of stillbirth and neonatal death with preeclampsia increased significantly.In recent years,the proportion of preterm births in preeclampsia is significantly higher than in the past.In addition to immediate effects,preeclampsia can also have long-term effects on mothers and offspring.Women with a history of preeclampsia are more likely to develop hypertension,cardiovascular disease,stroke,and type 2 diabetes mellitus later in life and have a higher risk for cognitive impairment in later life.Preeclampsia as an independent factor increases the risk of maternal death jfrom cardiovascular disease.Preeclampsia also has an overall adverse effect on cardiovascular,immune and neurological health of the offspring.It has been confirmed that there is most prominent and reliable associations between gestational hypertension and higher blood pressure of offsprings.Preeclampsia also reduce the cognitive function of the offspring.Preeclampsia is still a great challenge for obstetricians at present.There are no effective treatments or preventive measures at present.Delivery is still the only treatment for severe preeclampsia.Despite the breakthroughs in the understanding of preeclampsia etiopathogenesis,the physiopathology is still not completely understood.However,it is clear that the development of preeclampsia is related to the placenta,the clinical syndrome will not occur if the placenta is not present and it will disappear soon after placental delivery.Preeclampsia is considered to evolve in two stages.In the first stage,inadequate remodeling of spiral arterioles leads to shallow placentation.This abnormal shallow placentation reduces uteroplacental blood perfusion and consequently leads to local placental hypoxia.This oxidative stress further aggravates the vascular function of the placenta,which consequently cause insufficient blood perfusion,structural damage,inflammation,apoptosis etc.In the second stage,factors released into the maternal circulation from the poorly perfused placenta activate inflammatory reaction and cause the endothelial dysfunction that leads to the typical symptoms of preeclampsia:hypertension and proteinuria.In the above processes,cytokines became the intersection of immune and inflammatory reaction disorders,placental shallow implantation,and vascular endothelial injury in the etiology of preeclampsia.Pregnancy itself is considered to be a moderate inflammatory response,but over inflammatory response plays an important role in the pathophysiological process of preeclampsia.In the past few decades,evidence supporting this idea has been published by many groups suggesting the importance of the maternal pro-inflammatory cytokines in the pathogenesis of preeclampsia.An increase in pro-inflammatory cytokines can cause the damage of uteroplacental blood perfusion throughout pregnancy,which in turn aggravated clinical symptoms.This suggests that inflammatory factors play an important role in the occurrence and development of preeclampsia and to some extent can reflect the severity of the disease.The inflammatory cytokines tumor necrosis factor-alpha(TNF-?)and interleukin-6(IL-6)as important media of the maternal immune system play important roles in the development of preeclampsia.In studies on pregnant women,it has been proven that both TNF-? and IL-6 induce dysfunction of endothelial cells which was associated with many forms of hypertension and was at least partially mediated by increased levels of proinflammatory cytokines in human studies.The levels of TNF-? and IL-6 significantly increase when preeclampsia happened and can mediate some clinical manifestations of preeclampsia.According to some studies anti-inflammatory targeting drugs may play a role in the control of preeclampsia.TNF-? plays important role in both immune regulation and inflammatory response.It also has a toxic effect on vascular endothelium and promotes the expression of endothelial cell adhesion factor.At the same time,TNF-? can also induce antibody dependent cytotoxic effects which causing endothelial cell damage,increasing vascular permeability,promoting the exudation of body fluids and proteins from blood vessels,and activating the body's coagulation system.In addition,TNF-?also affects the synthesis and release of vascular active substances,causing extensive spasm of the systemic arterioles,which ultimately elevated blood pressure.Change of TNF-? level is associated with pathological pregnancy.The increase of maternal TNF-? is related to premature delivery,hemorrhage necrosis of placenta and fetal intrauterine growth retardation.At the same time,the up-regulated expression of TNF-? is also associated with the occurrence and development of preeclampsia.Both the expression of TNF-? and the apoptosis of placental trophoblasts increased when preeclampsia occurred.IL-6 is an important immune regulatory factor in the body,released by various cells such as endothelial,smooth muscle cells,macrophages.Studies have shown that there is a close relationship between elevated levels of IL-6 and preeclampsia.Studies have shown that serum levels of IL-6 in preeclampsia are significantly higher than those in the control group,and there is a correlation between IL-6 levels and TNF.The level of oxygen free radicals and TNF were significantly up-regulated in the presence of preeclampsia,both of which stimulated immune-active cells and vascular endothelial cells,thereby promoting the production of IL-6 in both cells.This augmented expression of IL-6 may play roles in the pathogenesis of preeclampsia by serving as a source of a key circulating factor that promotes systemic maternal endothelial cell dysfunction and subsequent vascular dysfunction;indirectly impeding cytotrophoblast invasion by contributing to excess macrophage infiltration of the decidua.Therefore,TNF-? and IL-6 are associated with preeclampsia and are promising as a molecular marker for predicting the disease.Due to the ethical problems of studying the molecular mechanism of human preeclampsia,and the possible impact on maternal and infant,animal models have been widely used in preeclampsia studies.An ideal animal model of preeclampsia would be one characterized by the development of pregnancy specific maternal hypertension,proteinuria,endothelial dysfunction and an imbalance of angiogenic factors.Furthermore,these changes would resolve following delivery of the placenta.It has also been demonstrated that hypoxia is correlated with hallmark features of preeclampsia,inducing trophoblast cell death,the release of proinflammatory cytokines,and oxidative stress.Some scholars exposed pregnant rats to hypoxic stress that was hypothesized to induce conditions similar to those of preeclampsia.Rats showed clinical manifestations such as elevated blood pressure,proteinuria,and fetal growth limitations,and successfully established a rat model of preeclampsia.ObjectiveWe hypothesized that gestational hypoxia induces preeclampsia-like symptoms via heightened TNF-? and IL-6 in the placenta.We presume that the heightened expression of TNF-? and IL-6 in placenta may be related to the clinical symptoms of preeclampsia.In this study,we induced hypoxic stress in rats during pregnancy to reproduce physiological conditions associated with preeclampsia and detected the expression change of TNF-? and IL-6 in placenta.Then the relationship between the expression change of TNF-? and IL-6 in placenta and blood pressure,urine protein,liver and kidney function damage,fetal weight are explored.The aim of this study is to find effective biomarkes which can predict preeclampsia and monitor the severity of the disease.As a result,we can prevent the occurrence of serious complications,and then improve the prognosis of pregnant women and perinatal infants.It is also expected to provide a breakthrough for the treatment of preeclampsia.MethodsSpragne-Dawley rats were bred in the specific-pathogen-free(SPF)environment in which sufficient water and fodder were guaranteed.The purchased adult rats were bred in a cage at a ratio of female:male=4:5.Vaginal secretions of female rats were scraped at 9:00 every morning,which were then smeared on glass slides to be observed under a microscope.If sperms were found,the day would be recorded as day 0 of pregnancy,indicating that all the females were pregnant.These rats were normally bred until day 15 of pregnancy,and then 60 pregnant rats were randomly divided into the normoxia with normal blood pressure group rats,(control group,n=20),the 10%hypoxia-induced preeclampsia group(preeclampsia group,n=20)and the group in which caudal veins were consecutively administered with an anti-hypertensive drug,Treprostinil for one week after 10%hypoxia-induced preeclampsia(treatment group,n=20).The caudal vein blood pressure of the three groups of pregnant rats was tested on day 15,18 and 21 of pregnancy with a rat caudal artery non-invasive blood pressure monitor,respectively.Followed by collection of 24 h urine proteins on day 15,18 and 21 of pregnancy and the urine proteins level was detected by BCA.On day 21 of pregnancy,chloral hydrate was used for anesthesia,and serum was collected to detect liver function(ALT,AST)and renal function(BUN,Cr).As fllowed pregnant rats were sacrificed via cervical dislocation and placental tissue,liver tissue,kidney tissues were collected and fetal weight were measured.The abnormal changes of rat liver and kidney tissues were observed,and histopathological examination was performed.The placenta was taken to measure the expression of IL-6 and TNF-? by real-time PCR,western blot,and immunohistochemistry.Correlation analyses of IL-6 and TNF-? with blood pressure,urine proteins,ALT,AST,BUN,Cr,fetal weight were conducted using Pearson's correlation analysis.Results1.Comparisons of the average blood pressure and urine protein in the three groups.Compared with the control group,blood pressure and urine proteins in the preeclampsia group were significantly increased at different time points(P<0.05).However,there were no statistically significant differences in blood pressure and urine proteins at different time points between the control group and the treatment group(P>0.05).2.Comparisons of liver function(ALT,AST)and renal function(BUN?Cr)in the three groups.Compared with the control group,the levels of ALT,AST,BUN,Cr in preeclampsia group were significantly higher(P<0.05);However,there were no statistically significant differences between the treatment group and the control group(P>0.05).3.Comparison of fetal weight in the three groups.Compared with the control group the average weight was significantly decreased in preeclampsia group(P<0.05),However,there were no statistically significant differences between the treatment group and the control group(P>0.05).4.Histopathological changes of liver,kidney in three groups of rats.In the preeclampsia group,glomerulosclerosis was found in the kidney,accompanied extensive endothelial swelling,narrowing,and occlusion of capillary lumen.The hepatocyte cords were disorganized,hepatocyte degeneration,fibrin thrombus with focal necrosis were found.The control group and the treatment group did not change significantly.5.mRNA levels of IL-6 and TNF-? in placenta tissues of pregnant rats.Results showed that mRNA levels of IL-6 and TNF-? in the preeclampsia group were higher than those in the control group(P<0.05),while there were no statistically significant differences in mRNAlevels of IL-6 and TNF-? between the treatment group and the control group(P>0.05).6.Protein levels of IL-6 and TNF-? in placenta tissues by western blot analysis.Results showed that the average protein levels of IL-6 and TNF-? in the preeclamsia group were higher than those in the cont:rol group(P<0.05),while there were no statistically significant differences in protein levels of IL-6 and TNF-? between the treatment and control groups(P>0.05),suggesting that the expression levels of IL-6 and TNF-? are positively correlated with preeclampsia.7.Detection of IL-6 and TNF-? in placenta tissues by immunohistochemistry.IL-6 and TNF-? were positively expressed in three groups with yellow or brownish-yellow granular stain.IL-6 were mainly expressed in the nucleus of trophoblastic cells and also were seen in villous vascular endothelial cells.TNF-?were mainly located in the nucleus and cytoplasm of trophoblastic cells.8.Correlation analyses of IL-6 level in placenta tissues with blood pressure,urine proteins,ALT,AST,BUN,Cr and fetal weight.Pearson's correlation analysis results showed that the IL-6 protein level was positively correlated with blood pressure,urine proteins,ALT,AST,BUN and Cr,but negatively correlated with fetal weight.9.Correlation analysis of TNF-? level in placenta tissues with blood pressure,urine proteins,ALT,AST,BUN,Cr and fetal weight.Pearson's correlation analysis results showed that the TNF-? protein level was positively correlated with blood pressure,urine proteins,ALT,AST,BUN and Cr,but negatively correlated with fetal weight.Conclusion1.Exposure rats to 10%O2 during day 15 through day 21 of gestation causes the development of preeclamptic symptoms,including hypertension,proteinuria,impairment of liver and kidney function,fetal growth restriction,successfully established a model of preeclampsia.2.The expression of IL-6 and TNF-? in placenta of preeclampsia rats was significantly up-regulated;3.IL-6 and TNF-? were positively correlated with blood pressure,urine proteins,ALT,AST,BUN and Cr,but negatively correlated with fetal weight.4.IL-6 and TNF-? are involved in the occurrence of preeclampsia and their concentration are closely related to the severity of the disease.