| ObjectionHypertensive disorder complicating Pregnancy is the most common obstetric complications,the incidence rate is 9.40%in our country,which is the main reason of maternal perinatal morbidity and mortality.Pre-eclampsia and eclampsia are serious stage of pregnancy-induced hypertension,so far that etiology and pathogenesis is unknown make it difficult to prevent and treat.In recent years,experts scholars at home and abroad have done many studies for its etiology and pathogenesis,and raised a lot of theory.Theory of pathogens that now is commonly accepted is immune,or ischemic in placental trophoblast cells leaf theory,genetics,vasoactive substance doctrine,the theory of calcium imbalance,vascular endothelial injury theory,but none of them can fully satisfactied explain its cause.Scholars now have put forward a new theory, stimulate inflammation may be involved in the pathogenesis of preeclampsia,and there were scholars successfully established an animal model of hypertensive disorder complicating Pregnancyby endotoxin,and anti-inflammatory treatment is effective. This proved that infection can lead to maternal-fetal immune imbalance,immune injury, ischemia and trophoblastic abnormal placenta accreta,increased placental ischemia,the release of plasma toxic factors,arising from systemic vascular endothelial damage, eventually leading to pre-eclampsia and eclampsia.Placenta is an important immune organ,containing a large number of mononuclear cells,which is considered sources of "toxicity factor" in patients of pre-eclampsia in vivo,and the role of placental trophoblast is popular concerned.Some scholars believe that the trophoblast cells during pregnancy is also a kind of immune cells,are natural components of the immune system,impact immune response of maternal-fetal. Trophoblast cells and macrophages have certain common characteristics:phagocytosis, the formation of syncytial cell,tissue infiltration function;the expression of CD4, CD14,IgG receptor;secretion of IL--1,2,6(IL-1,IL-2,IL-6),interferon-γ(INF-γ) and tumor necrosis factorα(TNF-α) and other cytokines,and express their receptors.In recent years,the role of calprotectin(Cal) in hypertensive disorder complicating Pregnancydisease caused a lot of the attention of scholars.Calprotectin are found in recent years as a new calcium-binding protein,which has close relationship with the inflammatory response,belongs to the S100 protein family,heterodimers consisting S100A8 and S100A9,mainly expressed in neutrophils,single nuclear - macrophages, can inhibit cell proliferation and induced apoptosis,immune regulation,inflammatory response,and so on,participation in a variety of biological functions,and in many acute and chronic inflammatory diseases was found increased expression,so calprotectin has raised to a certain extent reflect the severity of inflammatory diseases. Advanced glycation end products receptor(RAGE),a receptor of calprotectin,may be combined calprotectin to start a number of signaling pathways,mediate a variety of inflammation pathological process.Tumor necrosis factor-α(TNF-α) is a proinflammatory cytokine,regulating the local activity of the uterus in the pregnancy tissue through autocrine or paracrine,and then participate in many physiological and pathological processes.At present,the expression of calprotectin at maternal pre-eclampsia was reported only a little in foreign,there are no reports in domestic.So far no study about it in maternal blood,placenta,trophoblast cells have reported at home and abroad.In order to investigate proinflammatory role of calprotectin in trophoblast cells and the pathogenesis of preeclampsia,we intend to detected expression of calprotectin and RAGE in pregnancy-induced hypertension,induction role of calprotectin on trophoblast cells to produce inflammatory mediators and regulation of NF-kB,p38 MAPK,ERK signal transduction pathway in vitro.Building a new way to find a drug inhibiting calprotectin increase,thereby inhibiting the release of inflammatory mediators,and whether or not it is need to combined with antibiotics to treat hypertensive disorder complicating Pregnancydiseases.Methods1.Expression of calprotectin and its receptor,RAGE and TNF-αin pregnancy-induced hypertension. By immunohistochemical streptavidin-biotin-protein - enzyme-linked peroxidase (SP) detection Cal,RAGE and TNF-α's expression in placenta of patients with pregnancy-induced hypertension,by enzyme-linked immunosorbent assay(ELISA) method to detect expression of Cal and TNF-αin pregnancy serum of patients with hypertensive disease.2.Induction of calprotectin to human trophoblast cells of expressing TNF-αCalprotectin treat early primary culture of trophoblast cells,MTT assay the role of calprotectin on proliferation of trophoblastic activity,observed by transmission electron microscope ultrastructural changes of trophoblastic after treated with calprotectin, ELISA,Real Time PCR detect the influence under different times and dose effect of calprotectin to release of TNF-αfor trophoblast cells.3.Human calprotectin-induced trophoblast expression of TNF-αsignal transduction pathwayCultured human trophoblast cells in primary separately treat with the ERK inhibitor PD98059,p38MAPK inhibitor SB203580 and NF-κB inhibitor BAY11-7082, United SB203580 and PD98059,incubated 2h,the medium of recombinant human separately add calprotectin,incubated 6h.ELISA detect TNF-αcontent in the culture supernatant,Real Time PCR detect of mRNA expression of TNF-α.Results1.Expression of calprotectin and its receptor,RAGE and TNF-αin pregnancy-induced hypertension.The calprotectin and TNF-αlevels of serum in hypertensive disorder complicating Pregnancywas significantly higher than control group,the two groups has statistical significance difference(P＜0.01);patients with severe pre-eclampsia in patients was significantly increased compared with less severe pre-eclampsia(P＜0.01),while patients with mild preeclampsia was significantly higher than that in patients with hypertensive disorder complicating Pregnancy(P＜0.05),hypertensive disorder complicating Pregnancywas higher than control group,but the difference has not statistically significant(P＞0.05). In two groups of pregnant women,villous stromal macrophagocyte and neutrophils was both positive expression of calprotectin,mainly located in the cell membrane and cytoplasm.Hypertensive group was higher than control group,the difference has statistical significance(P＜0.01);light,severe pre-eclampsia patients was significantly higher than control group(P＜0.05,P＜0.01),and the difference between mild and severe pre-eclampsia patients has statistically significant(P＜0.05);there was no significant difference between hypertensive disorder complicating Pregnancypatients and control group(P＞0.05).Two groups of pregnant women placental of villi syncytiotrophoblast cells and decidual cells have RAGE,TNF-αexpression,located mainly in the cell membrane and cytoplasm.Hypertensive group pregnancy placenta RAGE,TNF-αexpression was significantly higher than control group,the difference has statistical significance in two groups(P＜0.01);pregnancy-induced hypertension, light,severe pre-eclampsia patients was gradually increasing,and the difference between mild and severe pre-eclampsia patients has statistically significant(P＜0.05).The calprotectin serum levels of hypertensive disorder complicating Pregnancyhad positively correlated with that of TNF-α(r=0.359,P＜0.05);control group had no(r= 0.184,P＞0.05).Calprotectin pregnancy placenta levels of hypertensive group was positively correlated with thai of TNF-α(r=0.383,P＜0.01);control group had not.(r =0.130,P＞0.05).The calprotectin serum and placenta level of Pregnancy-induced hypertension group and control group were positively correlated(r=0.365,P＜0.01;r =0.675,P＜0.01).2.Induction of calprotectin to human trophoblast cells of expressing TNF-αAfter treated with calprotectin for 24 hours,the drug concentration was a negative correlation with the proliferative activity of cytotrophoblasts,namely,as calprotectin increase,cell growth was inhibited.Especially at 800 ng/ml and the more higher the more obvious(P＜0.01).However,at low dose the concentration of drug(＜0.39 ng/ml) stimulated,calprotectin can stimulate cytotrophoblast proliferation,especially in the concentration of 0.20 ng/ml more evident Department(P＜0.01).Time for 48 hours, proliferation and activity of these relations between calprotectin and cytotrophoblast cell becomes more apparent.By transmission electron microscope,after calprotectin-treated cytotrophoblast nuclear can be seen small nuclear ambiguity,nuclear heterochromatin margination,was like a half months,showing apoptosis changes,mitochondria's cristae reduced in cytoplasm,some vacuolar degeneration,membrane dissolved.Add calprotectin to to the primary cultured trophoblast cells during early culture medium significantly stimulated the secretion of TNF-α.At the scope of 0～500ng/ml, ELISA,Real Time PCR detected that calprotectin-induced trophoblast secretion of TNF-αwere increased,showing a dose-dependent relationship,in1000ng/ml induction of calprotectin to TNF-αsecretion reduced.Calprotectin Group trophoblastic secretion of TNF-αat 3～48h showed a bimodal shape,in 6h and 24h the secretion of TNF-αenhanced.3.Human calprotectin-induced trophoblast expression of TNF-αsignal transduction pathwayELISA assay calprotectin induced human trophoblast TNF-αincreased,PD98059, SB203580 and BAY11-7082 can be significantly reduced TNF-αinduced by calprotectin in human trophoblast cells(P＜0.05),while the use of PD98059 and SB203580 could enhance the inhibition of the secretion of TNF-αlevels(P＜0.05).Real Time PCR detect mRNA xpression of TNF-αincreased by calprotectin-induced,PD98059,SB203580 or BAY11-7082 can be significantly reduced TNF-αmRNA(P＜0.05),PD98059 and SB203580 has a synergistic inhibitory effect(P＜0.05).Conclusion1.The expression of calprotectin,RAGE,TNF-αprotein in hypertensive disorder complicating Pregnancyin progress is increased as the disease gradually severe,which would be expected to be the assessment target of the severity of pre-eclampsia,disease prognosis and prediction of the effective.2.High doses of calprotectin in vitro has a clear anti-proliferation and induction of apoptosis to cytotrophoblast early pregnancy,calprotectin can induce trophoblastic secretion of TNF-α,it was confirmed that calprotectin play a role in the pathological process of pre-eclampsia.3.Signaling molecules ERK,p38MAPK and NF-κB are involved in expression of TNF-αby calprotectin-induced early pregnancy cytotrophoblast.α...
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