The Mechanism Of The High Expression Of HSP70 Through NO System In Preeclampsia Placental Tissue Involved In The Fetal Intrauterine Development Of Patient With Preeclampsia Complicated With FGR

Background:Preeclampsia is a pregnancy-specific disease with a variety of serious clinical symptoms,serious threat to the health of the mother and fetus.Patient's fetal nutrition is limited and result in fetal growth restriction and the damage of maternal multiple organs.The incidence of FGR on preeclampsia women is 2-3 times higher than normal pregnant women.The earlier happened,the more serious on the ischemia of placental and the hypoxia state of intrauterine fetal.The greater risk of FGR,the poorer prognosis of fetal.Clinical studies showed that the circulating HSP70 level,which was positively correlated with the gestational age and negatively correlated with the age of pregnant women,in the peripheral blood of pregnant women was decreased during normal pregnancy and.In pregnancy asthma,premature birth,preeclampsia,FGR,and other pathological pregnancy,HSP70 was increased in maternal peripheral blood circulation,these suggested that HSP70 may be related to fetal development and pregnancy-related diseases.However,it is not clear how the HSP70 expressed in the circulation induces FGR.The researchers found that HSP70 could stimulate endogenous nitric oxide pathway,inhibit the transcription and synthesis of iNOS,reduce the activity of iNOS,and reduce the intracellular NO synthesis.Therefore,it is unknown whether the overexpression of HSP70 in the placenta and peripheral blood affets the expression of iNOS and/or eNOS,then causes the reduction of NO synthesis of related blood vessels,and final leads to fetal growth retardation.Therefore,we carried out the following three parts:(1)analyze the relationship between the levels of HSP70 and NO in peripheral blood and the disease of preeclampsia combined with FGR;(2)establish JEG-3 and HUVEC co-culture system and explore the influence of JEG-3 cells with different expression levels of HSP70 on HUVEC cells;(3)The effect of HSP70 protein,which was overexpressed in placenta and peripheral blood,on embryonic rat development.Through the research of the above three parts,we made sure that the over expression of HSP70 in placenta and peripheral blood associated with preeclampsia combined with FGR.It may be by influencing the expression of iNOS in the vasculature of fetal circulation,then inhibiting the synthesis of NO in endothelial cells and resulting in inadequate blood supply for feta.In this study,we analyzed the relationship between circulation overexpressed HSP70 and preeclampsia combined with FGR in vivo and cells experiment.All these provide experimental basis for the prevention and treatment of FGR in preeclampsia.Objective:(1)Analyze the relationship between the levels of HSP70 and NO in peripheral blood and the disease of preeclampsia combined with FGR;(2)Establish JEG-3 and HUVEC co-culture system and explore the influence of JEG-3 cells with different expression levels of HSP70 on HUVEC cells;(3)Build animal models through cold stimulation,then artificially induced or interfere with the expression of circulation HSP70 in pregnant rat to explore the effect of the changes of HSP70 protein expression on embryonic rat developmen.Methods:1.30 cases of preeclampsia,25 cases of preeclampsia complicated with FGR and 50 cases of normal pregnant women were collected.The levels of HSP70 and NO in peripheral blood were measured.We compared the hemodynamic changes and the content of HSP70 in placenta of pregnant women in each group,and explored the relationship between the expression of HSP70 protein and preeclampsia complicated with FGR.2.Human choriocarcinoma cell line JEG-3 and human umbilical vein endothelial cell line HUVEC were co-cultured in a 6-well Transwell co-culture system,and then investigate the effect of different expression levels of HSP70 in JEG-3 on HUVEC to provide experimental theoretical basis for the pathogenesis of preeclampsia complicated with FGR.3.The animal model of preeclampsia was established by cold stimulation to simulate the pathophysiological characteristics of preeclampsia pregnant women.At the 15 th day of pregnancy,lentivirus was injected through caudal vein.At the end of pregnancy,we observed the development of fetus and placenta.To investigate the effect of HSP70 expression on preeclampsia infants and to provide experimental evidence for early prevention and treatment of preeclampsia.Results:1.The first part of the study results: The HSP70 expression in peripheral blood of preeclampsia combined with FGR group are significantly increased than preeclampsia group and normal pregnancy group(P<0.05),The NO content in peripheral blood of preeclampsia combined with FGR group preeclampsia(31.47±3.65 ?mol/L)was significantly lower than the normal control group(57.65 ±5.73 ? mol/L)(P<0.05).The results showed that the level of NO released into peripheral blood of pregnant women with preeclampsia combined with FGR are significantly decreased.Western blotting results showed that the expression of HSP70 in the placental tissues of preeclampsia combined with FGR was significantly increased than normal control.2.The second part of the study results: After HSP70 lentivirus infection,JEG-3 were collected for Western blot.The results show that the expression of HSP70 in HSP70 targeted interference lentivirus infected JEG-3 was significantly reduced than negative control group(P<0.05).When use Flag antibody test,only HSP70 overexpressed lentivirus infected cells have protein bands.Meanwhile,when used HSP70 antibody,we found that overexpressed group appeared the endogenous and exogenous dual band,both proved that the expression of HSP70 was significantly higher in HSP70 overexpression lentivirus infected JEG-3(P<0.05).These indicated that the expression level of gene HSP70 correspondingly increased and reduced in JEG-3 after infected the lentivirus infection.HUVEC cells were in a spindle shape when cultured single,and arranged into a paving stone appearance.The morphological changes were not observed after co-culture.When HUVEC cells were co-cultured with JEG-3 with reduced expressed levels of HSP70,no significant difference was found in the intracellular NO content compared with the negative control group(P>0.05).However,when HUVEC cells were co-cultured with JEG-3 with overexpressed levels of HSP70,significant difference was found in the incerased intracellular NO content compared with the negative control group(P<0.05).These results indicated that JEG-3 with different HSP70 expression levels could influence the expression of NO in HUVEC cells through certain cytokines when co-cultured with HUVEC.After HUVEC co-cultured with JEG-3,which respectively with low level and high level expression HSP70,we collected HUVEC cells and used the CCK-8 method to dectect cell viability.We found that there was no significant difference in cell appreciation rate between the treatment group and the control group.This results indicate that JEG-3 with different levels of HSP70 did not affect the vitality of HUVEC cells after co-cultuered with HUVEC.After HUVEC was co-cultured with JEG-3 of different HSP70 level,we detected the apoptosis level of each group by using Annexin V-FITC/PI double standard method.Compared with the control group,there was no significant change in the apoptosis level of HUVEC cells after co-culture with JEG-3(P > 0.05).The results showed JEG-3 with different levels of HSP70 did not affect the apoptosis level of HUVEC after co-cultuered with HUVEC.After HUVEC was cultured with JEG-3 of different HSP70 level,we detected the cell cyclel of each group by using PI standard method.Compared with the control group,there was no significant change in cell cycle level of HUVEC cells after co-culture with JEG-3(P > 0.05).The results showed JEG-3 with different levels of HSP70 did not affect the cell cycle level of HUVEC after co-cultuered with HUVEC.Western blotting results showed that the expression levels of iNOS,eNOS and HSP70 protein in HUVEC were not statistically significant difference compared with the control group after co-culture with JEG-3 with low expression level of HSP70(P>0.05).However,when HUVEC co-culture with JEG-3 with high expression level of HSP70,we found that the expression of iNOS was significantly lower than control group(P<0.05),while the expression of eNOS and HSP70 showed no significant difference.3.The third part of the study results: There was no significant difference on pregnant rats' fetal mortality and litter size,while the total number of live births in the interference control group and overexpression group has a tendency to rise,but no statistical differences compared with other groups(P>0.05),which may be related to the individual differences of pregnant rats.There was no statistically significant difference of fetal rats' body length and placental weight in each experimental group(all P>0.05).The fetal rat weight of HSP70 interference expression group was significantly higher than control group and the HSP70 overexpression group,and the difference was statistically significant(P<0.05).The placental weight of HSP70 interference expression group was lower than the control group,and the difference was statistically significant(P<0.05).4.Compared the NO content in peripheral blood with each group: the NO content was significantly reduced in cold stimulate model group than normol group(P<0.05).The NO content was significantly increased in HSP70 interference expression group than negative control group(P<0.05).However,the NO content was increased in HSP70 overexpression group than negative control group but not statistical significance(P>0.05).Compared the expression of HSP70 in placental tissues with each group: Western blotting results shown that the expression of HSP70 was significantly increased in cold stimulate model group than normol group(P<0.05).The expression of HSP70 was significantly reduced in HSP70 interference expression group than negative control group(P<0.05).The expression of HSP70 was significantly increased in HSP70 overexpression group than negative control group(P<0.05).Compared the expression of eNOS and iNOS in funicle tissues with each group: the expression of HSP70 was significantly reduced in cold stimulate model group than normol group(P<0.05),this is accordance with the results of the NO content in peripheral blood.The expression of eNOS and iNOS was significantly increased in HSP70 interference expression group than negative control group(P<0.05).The expression of iNOS but not eNOS was significantly reduced in HSP70 overexpression group than negative control group(P<0.05).The results may be explained by the increased expression of iNOS,which induced by the overexpression of HSP70,resulting in the compensatory increase of the expression eNOS.Conclusion:1.The increased expression of HSP70 in peripheral blood and placental tissues may relate to the development of preeclampsia combined with FGR.2.After co-culture with JEG-3 with high expression level of HSP70,the expression of iNOS in HUVEC cells was signigicantly reduced and then lead to the decreased release of NO.3.Cellular biological function of HUVEC did not change after co-culture with JEG-3 with different level of HSP70.4.The fetal rat weight was significantly decreased in cold stimulation model group than normol group,as well as the significantly increased expression of HSP70 in placental tissues and the significantly decreased NO synthase in funicle tissues.5.It is conducive to the growth of fetal rat by means of the suppressed expression of HSP70 in placenta ltissues.