High Level Insulin Infulence Angiogenesis In The Endometrium OG Early Pregnant Mice

Objective: In the past,the mechanism of insulin was mainly focused on the terms of metabolism.In some diseases such as obesity,diabetes and polycystic ovary syndrome,which is related to high insulin hematic,and could lead to impaired endometrial receptivity and embryo implantation abnormality.The endometrium plays an important role in the development of embryo.Previous studies have shown that embryonic development is abnormal under the direct action of insulin,but the effect of insulin deficiency on the endometrium is little known about.Our previous study found that high insulin result in impaired endometrial receptivity and abnormal decidualization.Angiogenesis is an important process of endometrial decidualization.Decidual angiogenesis forms a new vascular network that serves as the first exchange apparatus between the maternal circulation and the developing embryo and thus is a crucial and fundamental process for embryonic survival and a successful pregnancy.Studies have shown that high levels of insulin have a very important role of angiogenesis on other tissues and organs.So,we hypothesized that high level insulin may also have an effect on the formation of endometrial decidual angiogenesis.This study aims to construct animal and cell models of high level insulin,to investigate the effect and the mechanism of insulin on decidual angiogenesis.Method:1.The establishment of high level insulin model.(1)Mice were randomly divided into two groups.High insulin group was taken of subcutaneous injection of insulin,and the control group was injected with 0.9% normal saline at the same time.(2)Normal pregnancy model: adult female mice were mated with fertile males of the same strain to induce pregnancy.The appearance of the vaginal plug was considered to indicate Day1 of pregnancy.Uterine endometrial tissue on D6,D7,and D8 was collected on ice and quickly stored at-80 °C for further analyses.(3)Artificial decidualization model: Adult female mice were mated with vasectomized males of the same strain to induce pseudopregnancy.Artificial decidualization was induced by intraluminal infusion of 25 ul of sesame oil into one uterine horn on the morning of Day 4 of pseudopregnancy,whereas the contralateral uninject uterine horn served as a control.The uterin were collected on Day 8 of pseudo-pregnancy.Parts of the uteri horns were fixed in formalin and embedded in paraffin,and the rest of the tissues were stored stored at-80 °C for further analyses.2.Detection of decidual marker gene: Real-time PCR and Western-blot analysis the expression of BMP2 and PRL in the decidual tissue of pregnant mice on D6 ? D7 ? and D8.Real-time PCR and Western-blot analysis the expression of BMP2 and PRL in endometrium of artificially decidualization model.Immunohistochemistry analysis the expression of BMP2 in endometrium of pregnant mice on D8.3.Detection of angiogenesis and tube formation: Immunohistochemistry analysis the expression of CD34 in endometrium of pregnant mice on D7 and D8.Immunohistochemistry analysis the expression of CD34 on endometrium of artificially decidualization model(induced side).Tube formation of HUVECS was analyzed between high insulin group and control group.4.Detection of angiogenesis marker gene: Real-time PCR analysis the expression of ANG-1? TIE2?VEGFA?VEGFR2 in endometrium of pregnant mice on D6?D7?and D8.Western-blot analysis the expression of ANG-1?TIE2?VEGFA in endometrium of pregnant mice in endometrium of pregnant mice on D6?D7?and D8.The expression of ANG-1?TIE2? VEGFA?and VEGFR2 were analyzed in artificially decidualization model.Real-time PCR analysis the expression of ANG-1? TIE2?VEGFA?VEGFR2 in endometrium of artificially decidualization model.Western-blot analysis the expression of ANG-1?TIE2?VEGFA in endometrium of artificially decidualization model.Western-blot analysis the expression of ANG-1?TIE2?VEGFA in HUVECS.Immunofluorescence staining with VEGFA in HUVECS cells was analyzed between high insulin group and control group.5.Detection of autophgy marker gene: Western-blot was used to detect the autophagy markers: LC3? P62? Beclin1? Atg5? ULK1? p-ULK1 in endometrium of pregnant mice on D6?D7?and D8.Immunofluorescence staining with LC3 in HUVECS cells was analyzed between high insulin group and control group.Tube formation of HUVECS was analyzed among CON?IN?and IN+3-MA group.Immunofluorescence staining with VEGFA in HUVECS was analyzed among CON?IN?and IN+3-MA group.Result:1.Serum insulin level were significantly increased in the high-insulin group(p<0.001)and blood glucose levels were significantly increased in the high-insulin group(p< 0.05).The high insulin model was successfully established.2.Real-time PCR results showed that PRL and BMP2 m RNA was significantly decreased following high insulin treatment in endometrium of pregnant mice on D6?D7?and D8.Moreover,the expression of BMP2 and PRL proteins was markedly reduced in high insulin group from D6 to D8,as revealed using Western-blot analysis.Immunohistochemistry studies showed that BMP2 proteins are widely decreased in high insulin group on D8.The m RNA and protein levels of BMP2 and PRL in artificially decidualization model were also significantly decreased under high insulin treatment.3.The large-sized vascular sinus foldings in the central region were markedly samller on D7 and D8 of high insulin group as evidenced with CD34.The size of vascular sinus foldings were also significantly smaller in high insulin group compared with control group in the artificially decidualization model.Furthermore,high level insulin negative affect the tube formation of HUVECS cells.4.The m RNA expression of ANG-1 and TIE2 in the high insulin mice was significantly increased compared with the normal mice from D6 to D8,while the VEGFA and VEGFR2 m RNA expression in the high insulin mice was decreased from D6 to D8.The protein expression levels of ANG-1?TIE2? and VEGFA analyzed by Western-blot were correlated with the expression level of m RNA.Immunohistochemistry studies showed that ANG-1?VEGFA proteins are widely distributed in the high insulin group,the number of VEGFA positive cells was significantly decreased in high insulin group,but the expression of ANG-1 in high insulin group was markedly increased.The expression of ANG-1?TIE2? VEGFA?and VEGFR2 were analyzed in artificially decidualization model.The m RNA expression of VEGFA and VEGFR2 was significantly decreased in high insulin group,but the m RNA expression of ANG-1 and TIE2 in high insulin group was markedly increased.The protein expression of VEGFA in the high insulin group was significantly decreased compared with the normal group,while the ANG-1 and TIE2 protein expression in the high insulin mice was increased.Furthermore,High level insulin negative affect the expression of VEGFA in HUVECS.5.High levels of insulin significantly increased the expression of LC3-?,Atg5,Beclin1 and p-ULK1 in endometrium of pregnant mice on D6?D7?and D8,and significantly reduced the expression of P62.The expression of LC3 was significantly increased in HUVECS following high insulin treatment.The tube formation and the expression of pro-angiogenic factor VEGFA was reversed by the inhibition of autophay.Conclusion:In conclusion,endometrial angiogenesis is a complex process that is regulated by many factors.High level of insulin leads to abnormal decidual angiogenesis,resulting in the inhibition of decidual angiogenesis.Autophagy may be involved in the abnormal decidual angiogenesis induced by high insulin,but the specific mechanism remains to be further studied.