The Regulation Of High Insulin Levels On Uterine Apoptosis In Early Pregnant Mice

Objective:Metabolic syndrome has become a health problem worldwide.Metabolic syndrome and its comorbidities,characterized by hyperglycemia,obesity,insulin resistance and hypertension,are a serious threat to the health of the population.Insulin resistance and the associated metabolic syndrome are associated with reproductive problems.Uterinedecidualization is a critical event during mammalian pregnancy.Aberrant decidualization has been linked to numerous pregnancy complications,including infertility,recurrent miscarriage,preeclampsia,intrauterine growth restriction,and preterm delivery.Our previous indicated that high level of insulin has effect on uterine decidualization in early pregnant mice,but the mechanism remains elusive.Studies show that insulin could regulates apoptosis through a series of signaling pathways,and the balance of apoptosis of endometrial cells is essential to normal decidualization.Here,we studied the role of high insulin levels on uterine endometrial cells apoptosis during early pregnancy,and further explored whether the the possible mechanism.Methods:1.Establishment of animal model and sample collection: 8-week-old KUNMING mice obtained from the Laboratory Animal Centre of Chongqing Medical University.The high insulin-exposed group and control group were established.Adult female mice from two groups were mated with fertile(or vasectomized)males to induce pregnancy(or pseudopregnancy).The day in which a vaginal plug was found after mating was considered the first day of pregnancy(D1/PD1).Serum and uterus tissues on D6/PD6,D7 and D8/PD8 were collected on ice for further analyses.On PD4,one side of the uterine horn was infused with corn oil to artificially induce decidualization,and the other side of the uterine horn was served as the control.The mice were sacrificed on PD8,serum and uterus tissues were collected.2.Uterine endometrial cells apoptosis is impaired in early pregnancy mice with high insulin treatment: Flow cytometry used to detect apoptosis of the uterine endometrial cells in D6,D7,D8 and ID.TEM was used to observe the apoptotic bodies in D8.Immunohistochemistry was used to detect the protein expression of Bax in D7 and D8.RT-PCR was used to detect the expression of Bax.Western blot used to detect the expression of apoptosis related proteins in D6,D7,D8,PD6,PD8 and ID.3.PI3K/AKT signalling pathway is involved in apoptosis inhibited by high insulin levels: Western blot was used to detect the expression of PI3K/AKT signalling pathway and apoptosis related proteins in uterine endometrial cells in mice.To further survey the role of the PI3K/AKT pathway on the alteration in apoptosis in the high insulin-exposed mice,LY294002,a specific inhibitor of the PI3k/Akt pathway,was injected into the lumen of one uterine horn adjacent to the ovary in mice.Western blot used to detect the expression of PI3K/AKT signalling pathway and apoptosis related proteins in uterine endometrial cells in mice.4.Mouse uterine endometrial stromal cells culture and induced decidualization in vitro: Isolate and culture stomal cells from mouse endometrium(normal media or high levels of insulin media)and identify purity by immunofluorescence of Vimentin.Add E2 and P4 to mice endometrial stomal cells to induce decidualization in vitro,detect the expression of dtprp by RT-PCR and Western blot.5.Mice uterine decidual cells apoptosis are impaired with high insulin treatment: Flow cytometry and TUNEL were used to detect apoptosis of the decidual cells.JC-1 detection was used to observe the release of cytochrome c.Western blot used to detect the expression of apoptosis and PI3K/Akt pathway related protein.PI3K/Akt pathway specific inhibitor LY294002 was added to two groups and further analysis of the effect of high levels of insulin on apoptosis and decidualization of endometrial cells.Results:1.The model of high levels of insulin in mice was established successfully: The blood glucose of tail vein was measured before the mice were killed.The results showed that the blood glucose was significantly increased in the high insulin-exposed group.In addition,the results showed that serum insulin level was significantly increased by insulin exposure.2.Uterine endometrial cells apoptosis is impaired in early pregnancy mice with high insulin treatment: Flow cytometry detection showed that high insulin-exposed group had less apoptosis.TEM was observed that cells of mice in the high insulin-exposed group had less apoptotic bodies.Western blot indicated that high insulin-exposed group expressed less Bax,cleaved-Casepase3,cleaved PARP while expression of Bcl2 was increased.RT-PCR and immunohistochemistry results of Bax and Bcl2 were consistant with western blot.3.PI3K/AKT signalling pathway is involved in apoptosis inhibited by high insulin levels: Western blot showed that p-Akt was obviously activated in the high insulin-exposed group compared with the control group.However,LY294002 eliminated the effect of the high insulin level on the upregulation of Akt phosphorylation in the high insulin-exposed group.4.Mice uterine decidual cells apoptosis and decidualization are impaired with high insulin treatment: Flow cytometry,TUNEL,JC-1 and western blot detection showed that high insulin-exposed group had less apoptosis.RT-PCR and Western blot results showed that mouse related decidualization marker genes dtprp of control group were up-regulated in vitro decidualization,whereas decidualization were obviously impaired in high insulin-exposed group.Western blot results showed that decidualization related proteins were obviously impaired in high insulin-exposed group.However,LY294002 eliminated the effect of the high insulin level on the downregulation of apoptosis and decidualization in the mouse and human endometrial cells.Conclusion:The apoptosis of endometrial cells was inhibited with high levels of insulin treatment during the process of decidualization.Meanwhile,expression of related proteins in PI3K/Akt signaling pathway from the insulin group have significantly differences.All of these results showed that endometrial cells apoptosis is imbalanced under the role of insulin and then impaired the process of decidualization,the PI3K/AKT signalling pathway might participate in this process.