The Study Of Relationship Between Autophagy Mediated By HIF-1?/BNIP3 And Missed Abortion

Missed abortion is a special form of abortion in which there is embryonic or fetal demise without timely discharged,also called overdue abortion.At present,the rate of the occurrence of missed abortion is as high as 13.4% in our country.The incidence rate is increasing year by year with the release of the national two child policy and the increasing of maternal age,brings heavy burden to the society and family.The missed abortion is not easy to peel because of placental tissue is organized and adhere to the uterine wall.If the missed time is too long,the degeneration of placenta thromboplastin will release into the maternal circulation,leading to severe coagulopathy and even DIC,which will harm the life of the pregnant woman,so the missed abortion is a type of tricky clinical abortion.Recent studies showed that 50% of missed abortion closely relate to chromosome abnormalities,immune factors,endocrine factors,genital malformation,environmental factors,the other reasons are unknown etiology,the pathogenesis of missed abortion is still unclear.Pregnancy is a complex physiological process,the maternal fetal interface is the place of transporting nutrients and gas and removing waste parts between maternal and fetal,and the trophoblast cells on the interface is important to undertake this function.The study has found that the trophoblast cells is controlling proliferation and apoptosis,invasion and vascular remodeling and other biological characteristics under the combined action of various mechanisms,to ensure normal pregnancy.Trophoblast cell dysfunction may lead to a series of placental dysfunction diseases,such as abortion,stillbirth,fetal growth restriction,pre eclampsia and so on.The domestic and foreign researches shows that in early pregnancy,before the establishment of maternal and fetal circulation,embryo and placenta survive under physiological hypoxia,namely the trophoblast cells are in physiological hypoxia environment.At the same time,in the tumor,hypoxic-ischemic encephalopathy and other diseases,a large number of studies have found that hypoxia can induce autophagy,and mainly through the HIF-1 alpha /BNIP3 signal pathway and autophagy induced by hypoxia has important relations with cell apoptosis?invasion and so on.So that autophagy induced by hypoxia may have important relationship with placental abnormalities,and play a key role in the occurrence and development of missed abortion.Physiological hypoxia in early pregnancy is a key factor in the process of placental invasion and development,Rodesch found that the interstitial oxygen concentration is less than 20 mm Hg(2.67 Kpa)in the 8-week of pregnancy,and reaches the peak at 11-12 weeks of gestation.Studies have shown that,as an important regulator of oxygen balance,hypoxia inducible factor-1(HIF-1)in trophoblast cells increased in early pregnancy,and reached the peak at 11-12 weeks,and then began to decline,playing an important role to tolerance the hypoxia environment.Studies have found that HIF-1?is expressed in the trophoblast cells of early normal pregnancy,and the expression in missed abortion reduced significantly.HIF-1 is a heterodimer,consisting of two subunit of ? and ?,in which HIF-1? is a structural subunit,expressing stably in cells,HIF-1? is a functional subunit,its expression is regulated by oxygen content,accumulating under hypoxia in the cells,and dimerize with HIF-1? after transfer to the nucleus to regulate a series of downstream genes.BNIP3(BCL2/adenovirus E1 B 19k Da interacting protein 3),is an important downstream target of HIF-1?,which promoter region contains a hypoxic response element,can be combined with HIF-1? and increase the expression in hypoxia.There is a BH3 domain in the N terminal of BNIP3,this domain is related to mitochondrial autophagy,and can compete with Beclin1 to destroy Beclin1-Bcl2 or Beclin1-Bcl-XL complex,which causes the Beclin1 to be free to induce autophagy.LC3(Light chain 3)is an important marker of autophagy,there are two types,when autophagy is becoming,LC3 I can be transformed into LC3 II,the latter is always located in the autophagic membrane,so LC3 II / I can represent the level of autophagy.Autophagy,also known as type II programmed cell death,is a highly conserved cell degradation process,it isolate some of the cytoplasm and organelles in the vesicles of the bilayer membrane,and then transport to the organelle degradation(vacuole / lysosome)to decompose,and finally recycle macromolecules produced by the decomposition.It is ubiquitous in all eukaryotic cells and appears as a stress response when hypoxia?undernutrition and injury occur.This process can alleviate the adverse effects of various stress factors on cells and plays a crucial role in cell development and differentiation.In addition,autophagy involves a variety of diseases,including cancer,neurodegenerative diseases(Alzheimer's disease ? Huntington's disease ? Parkinson),diabetes,organ transplantation and aging,and the related research of pregnancy disease is rarely reported.ObjectiveIn this study,we detect the key protein of HIF-1 alpha /BNIP3 signaling pathway in the villi of missed abortion to observe the expression of the key protein of HIF-1 alpha /BNIP3 signaling pathway and investigate the relationship between autophagy and missed abortion and provide further theoretical basis for the prevention and treatment of missed abortion.Materials and Methods 1 Materials 30 cases of missed abortion patients,diagnosed by B ultrasound and ?-h CG dynamic monitoring and combined with clinical confirmed in obstetrics clinic,were collected in the Third Affiliated Hospital of Zhengzhou University from October2015-April 2016.The diagnostic criteria of missed abortion is according to?obstetrics and Gynecology?(Eighth Edition).30 cases normal early pregnancy womenwith live fetus of unwanted pregnancy confirmed by B-mode ultrasound were selected in the same period.Two groups of pregnant women were 20-45 years old,with gestational age of 7-9w;singleton,without history of hypertension,diabetes and hyperthyroidism and other systemic diseases;No smoking,long term medication history;Anti nuclear antibodies,anticardiolipin antibody,antisperm antibody,anti endometrial antibody and anti ovary antibody examination are normal;Rubella virus Ig M,cytomegalovirus Ig M,herpes simplex virus Ig M,Toxoplasma gondii Ig M,genital mycoplasma,Chlamydia examination is normal;No abnormal genital anatomy;Chorionic chromosome examination without abnormality;No acute or chronic infectious diseases during pregnancy.All the subjects had informed consent to the study and had passed the approval of the ethics committee.2 Methods 2.1 specimen collection During the 3 min after abortion,the villi were collected quickly and washed with sterile saline.The villi are divided into 4 parts,the first part was enclosed in 10% formalin solution,dehydrated and embed by paraffin conventionally and characterized after fixed 24 h.The other two parts are placed in the high-pressure sterilization frozen storage tube,and quickly put into the liquid nitrogen tank,finally placed in-80? refrigerator to be preserved for testing after overnight;The last part of the villi was about 1 mm3 and stored at 2.5% glutaraldehyde in 4? refrigerator to be preserved for electron microscopy.2.2 RT-PCR The total RNA of two groups of villus tissues were extracted to reverse transcriptase after detecting the total RNA integrity?concentration and purity,with the aim of detecting the m RNA expression of HIF-1 and BNIP3 using the 2-??Ct method.2.3 Immunohistochemistry The two groups of villi were made paraffin sections to detect the expression of HIF-1?,BNIP3 and LC3 protein by SP.Regaring the PBS as the negativecontrol,when it appeared brown yellow or brown significantly higher than that of the background under the microscope,we take it as positive expression.2.4 Western blot The total protein was extracted to detect the expression of HIF-1?,BNIP3 and LC3 protein in the villi.2.5 Transmission electron microscope The two groups were treated with ultrathin sections,and the formation of autophagy in the villi of the two groups was observed under transmission electron microscope.3 Statistical analysis SPSS 19 software was used for statistical analysis of data processing,all data was expressed as (?)±s,Comparison between two groups was analysed by independent sample t test,Correlation analysis was performed by Pearson method.A value of ?=0.05 was considered as a test standard.Result 1 General situation comparison The average age of missed abortion patients was 28.04 ± 3.496 y,mean gestational age was 58.70 + 8.636d;The average age of normal early pregnancy group was 30.09 ±5.206 y,with an average gestational age of 62.87±12.593 d,the difference between the two groups was not statistically significant(P>0.05).2 The the expression location of HIF-1?,BNIP3 and LC3 in villi of two groups HIF-1??BNIP3 and LC3 could be found by immunochemical staining in villous tissues from the two groups,HIF-1? was mainly localized in the nucleus and cytoplasm of trophoblast cells,BNIP3 and LC3 were mainly located in the cytoplasm of trophoblast cells 3 The the expression of HIF-1?m RNA and protein in the villi of two groups 3.1 The relative expression level of HIF-1?m RNA in the villi of two groups The relative expression level of HIF-1?m RNA in missed abortion group was1.11 ± 0.42,and the normal pregnancy group was 1.00 ± 0.39.The level of HIF-1?m RNA between the two groups had no obvious difference,the difference was not statistically significant(P>0.05).3.2 The relative expression level of HIF-1? protein in the villi of two groups The relative expression level of HIF-1? protein in missed abortion group was 0.226±0.053,and the normal pregnancy group was 0.432±0.055.The level of HIF-1? protein in missed abortion group was significantly lower than the normal pregnancy group(P<0.05).4 The the expression of BNIP3 m RNA and protein in the villi of two groups 4.1 The relative expression level of BNIP3 m RNA in the villi of two groups The relative expression level of BNIP3 m RNA in missed abortion group was 0.75 ± 0.27,and the normal pregnancy group was 0.99± 0.37.The level of BNIP3 m RNA in missed abortion group was significantly lower than the normal pregnancy group(P<0.05).4.2 The relative expression level of BNIP3 protein in the villi of two groups The relative expression level of BNIP3 protein in missed abortion group was 0.524±0.340,and the normal pregnancy group was 1.461±0.487.The level of BNIP3 protein in missed abortion group was significantly lower than the normal pregnancy group(P<0.05).5 The the expression of LC3?/?protein in the villi of two groups The relative expression level of LC3?/?protein in missed abortion group was 3.227±2.601,and the normal pregnancy group was 16.592±6.248.The level of LC3?/? protein in missed abortion group was significantly lower than the normal pregnancy group(P<0.05).6 The Correlation analysis of HIF-1? and BNIP3 protein,BNIP3 and LC3 II / I ?HIF-1? and LC3 II / I protein expression The protein expression of BNIP3 was positively affected by HIF-1? in the villous tissues(r=0.837,P <0.01),The protein expression of LC3 II / I was positively affected by BNIP3(r=0.876,P<0.01),The protein expression of LC3 II / I was positively affected by HIF-1?(r=0.853,P<0.01).7 Observing the autophagy with transmission electron microscopy We can observe double membrane autophagosomes in the normal control group and the group of missed abortion villi,and it decreases significantly in missed abortion group,and the structure is abnormal.Conclusion Autophagy mediated by HIF-1?/BNIP3 in the villous trophoblast cells has an important relationship with the occurrence and development of missed abortion.