Effect And Mechanism Of Prostaglandin E2 In Endometrial Stromal Cells On The Defect Of Decidual Angiogenesis Caused By Obesity

Object:In recent years,the increased number of obese people attracted great concentration around the world.The glucose intolerance and insulin resistance caused by obesity are important factors leading to various diseases.More than half of adverse pregnancy outcomes like infertility,abortion,and perinatal complications are attributed to the obesity women’s embryo implantation failure.Embryo implantation is the first step of a successful pregnancy,and involves a series of complex physiological processes including blastocyst implantation and decidualization after implantation.Decidualization is the proliferation and differentiation of endometrial stromal cells into decidual cells,along with angiogenesis,providing a suitable uterine microenvironment for blastocysts development.Our previous study found a damaged decidualization in high-fat diet mice,but whether obesity could impair angiogenesis during decidualization is still unknown.In the process of decidualization,endometrial stromal cells and vascular endothelial cells are both activated by the implantation signal.The prostaglandins secreted from endometrial stromal cells play a role during decidualization by promoting angiogenesis.However,it is not known the role of prostaglandins secreted from endometrial stromal cells during decidualization in high-fat diet mice.Our aim is to investigate the effect of prostaglandins secreted from endometrial stromal cells on angiogenesis during decidualization in high-fat diet mice,to further explore the mechanism of decidualization damage caused by obesity.Method:1.To establish high-fat diet mice modelFour-week-old female C57BL/6J mice were fed with high-fat diet(HFD,60%kcal)or control diet(CTD,10%kcal)for 12 weeks,the weight was recorded every week.The trend of mice weight growth,the glucose tolerance test,insulin tolerance test,serum level of triglyceride,and total cholesterol were analyzed to confirm the model was successfully established.2.To investigate whether obesity induced by high-fat diet impairs decidualization(1)The HFD/CTD female mice were mated with C57BL/6J male mice to construct normal pregnancy mice model.Morphological observation and HE staining were used to analyze the development of decidual on days 6-7 of pregnancy.The expression of decidualization markers(HOXA10,BMP2,MMP2,MMP9)were tested by western blot and immunohistochemical.(2)The mouse endometrial stromal cells(mESCs)decidualization was induced into decidual endometrial stromal cells(mDSCs)by estrogen(E2)and progestin(P4),to construct mESCs induced decidualization model in vitro.Oleic acid(OA)and palmitic acid(PA)were used to simulate the high-fat environment.The expression of decidualization marker HOXA10 was tested by western blot,and the cytoskeleton of mDSCs was tested by F-actin staining.3.To investigate the angiogenesis during decidualization in high-fat diet miceFirstly,the uterine blood flow on day 7 of pregnancy was tested by Doppler ultrasound in HFD/CTD mice.Secondly,The expression and location of vascular marker CD34 and vascular signaling markers VEGFA、VEGFR1、p-VEGFR2、ANG1、ANG2,and TIE2 were tested by immunofluorescence and western blot on days 6-7 of pregnancy in HFD/CTD mice.Further,the co-location of VEGFR1 and vascular endothelial cells marker vWF was tested by immunofluorescence to investigate the expression of VEGFR1 in the vascular endothelial cells of HFD/CTD mice.4.To investigate the prostaglandins synthesis of endometrial stromal cells during decidualization in high-fat diet mice(1)The high fat treated endometrial stromal cells(mESCs)were induced into decidual endometrial stromal cells(mDSCs)in vitro.The prostaglandins(PGI2,PGE2,PGD2,PGF2a,and TXA2)content was tested by gas chromatography-mass spectrometry(GC-MS)of the mDSCs supernatant.The expression of cyclooxygenase 2(COX2),Prostaglandin E2 synthase(m-PGES-1),and Prostaglandin 12 synthase(PTGIS)were tested by western bolt.(2)In the normal pregnancy mice model,the arachidonic acid and prostaglandins were tested by the GC-MS in HFD/CTD mice endometrium on day 6 of pregnancy.Further,the expression and location of COX2,mPGES-1,and PTGIS were tested by western bolt and immunohistochemistry.5.To investigate the effect of disordered prostaglandins of endometrial stromal cells on angiogenesis in high-fat diet mice during decidualization(1)To investigate whether prostaglandins of endometrial stromal cells regulate VEGFA in high-fat diet mice during decidualizationThe expression of COX2 was knocked down by siRNA and m-PGES1 was overexpressed in the mESCs induced decidualization model.After high-fat treatment,the prostaglandins content of mDSCs supernatant was tested by GC-MS,and the expression of VEGFA in mDSCs was tested by RT-PCR.(2)To investigate the effect of disordered prostaglandins of endometrial stromal cells on angiogenesis in high-fat diet mice during decidualizationFirstly,the supernatant of high fat treated mDSCs/high fat treated mDSCs overexpressed m-PGES-1 were used to culture the human umbilical vein endothelial cell(HUVEC).The cell apoptosis level of HUVEC was tested by flow cytometry.Further,the high fat treated mDSCs/high fat treated mDSCs overexpressed m-PGES-1 were co-cultured with HUVEC.The cell migration,invasion,and tube formation ability of HUVEC were tested by wound healing test,transwell,and tube formation test.In the Matrigel plugs formation experiment,a mixture of 400μl Matrigel and 100μl mESCs supernatant was subcutaneously injected into nude mice.The Matrigel plugs were collected after 15 days for further experiment.The hemoglobin content,expression of vascular marker CD34,and cell number of the vascular endothelial cells were tested by hemoglobin testing kit,immunofluorescence and HE staining.Result:1.The high-fat diet mice model was successfully establishedThe weight of high-fat diet mice was significantly increased compared with control mice.The serum total cholesterol and triglyceride were significantly increased,and Glucose intolerance and insulin tolerance were present in high-fat diet mice.2.High-fat diet impairs the decidualization of endometrial stromal cells during earing pregnancyMorphological observation and HE staining showed the slower development of decidua in high-fat diet mice on days 6-7 of pregnancy.Western blot showed that the expression of decidualization markers HOXA10、BMP2、MMP2 and MMP9 were significantly decreased in high-fat diet mice.Further,the endometrial stromal cells(mESCs)were induced into decidual endometrial stromal cells decidualization(mDSCs)in vitro.After high-fat treatment,western blot showed the decreased expression of decidualization markers HOXA10 and F-actin showed the damaged cytoskeleton of mDSCs.3.High-fat diet impairs the decidual angiogenesis during earing pregnancyDoppler ultrasound showed that the average uterine blood flow velocity,peak systolic velocity,and end diastolic blood flow velocity were significantly decreased,and the resistance index and pulsatile index significantly were significantly increased on day 7 of pregnancy in high-fat fat diet mice.Immunofluorescence and western blot showed that the expression of vascular marker CD34 and vascular signaling markers VEGFA,and VEGFR1 significantly decreased on days 6-7 of pregnancy in high-fat diet mice.Further,the reduced co-location of VEGFR1 and vascular endothelial cells marker vWF showed the significantly decreased expression of VEGFR1 in vascular endothelial cells on day 7 of pregnancy in high-fat diet mice.4.High-fat diet downregulates the PGE2-VEGFA axis during endometrial stromal cells decidualization in earing pregnancy mice(1)High-fat diet disorder the PGE2/PGI2 content during endometrial stromal cells decidualization in earing pregnancy miceGC-MS showed that PGE2 content was significantly reduced,and PGI2 content was significantly increased in the supernatant of high fat treated mDSCs.Further,arachidonic acid content was significantly increased in the endometrium of high-fat diet mice.In addition,PGE2 content was also reduced and PGI2 content was increased in the endometrium of high-fat diet mice.Immunofluorescence and western blot showed that the expression of COX2 and PTGIS were significantly upregulated and m-PGES-1 was significantly down-regulated in the endometrium of high-fat diet mice.(2)High-fat diet down-regulates the PGE2-VEGFA axis of endometrial stromal cells during decidualization in vitroThe high fat treated mESCs was induced into mDSCs in vitro.Western blot showed the expression of COX2 and PTGIS were not changed,but m-PGES-1 and VEGFA were significantly down-regulated.After m-PGES-1 overexpressed in mDSCs,GC-MS showed a significant increase in PGE2,and RT-PCR showed a significant up-regulation in VEGFA.5.Reduced PGE2 of endometrial stromal cell impair uterine angiogenesis during decidualization in high-fat diet miceFirstly,the supernatant of high fat treated mDSCs was used to culture HUVEC.Flow cytometry showed the cell apoptosis of HUVEC was significantly increased.Then,the high fat treated mDSCs were overexpressed m-PGES-1,to collect the supernatant for HUVEC culture,and the cell apoptosis of HUVEC was significantly decreased.Further,HUVEC was co-cultured with the high fat treated mDSCs,wound healing,transwell,and tube formation tests showed the migration,invasion,and tube formation ability of HUVEC were significantly inhibited.After HUVEC was co-cultured with the high fat treated mDSCs overexpressed m-PGES-1,the migration,invasion,and tube formation ability of HUVEC were significantly improved.At last,the Matrigel plugs formation experiment showed that,after the high-fat treatment in mDSCs,the hemoglobin content,expression of vascular marker CD34,and the vascular endothelial cell number were significantly inhibited in the Matrigel plugs.But after the high fat treated mDSCs overexpressed m-PGES-1,the hemoglobin content,expression of vascular marker CD34,and the vascular endothelial cell number were significantly improved.Conclusion:1.The decidual angiogenesis was impaired by the high-fat diet induced obesity during early pregnancy.2.The decrease of PGE2 of uterine endometrial stromal cells downregulated the expression of VEGFA,further promoting cell apoptosis and inhibiting cell migration,invasion,and tube formation ability of the vascular endothelial cell,impaired angiogenesis during decidualization in the high-fat diet mice.