Role Of Leptin In The Regulation Of Endometrial Epithelial Cell Function And MAPK Signaling Pathway In Sheep

Xinjiang is a large sheep breeding area,and the number of herds ranks second in the country all the year round.However,the low breeding efficiency of local breeds is a bottleneck problem restricting the development of mutton sheep breeding.How to improve the sheep breeding efficiency in this region needs to carry out systematic research.Leptin is a key factor that regulates the success or failure of embryo implantation in sheep.It is of great significance to study the related mechanism of leptin in regulating embryo implantation to improve sheep fecundity.Objective: In order to clarify the localization characteristics of leptin in pregnant and non-pregnant sheep endometrium tissues,and to explore its functional verification in sheep endometrial epithelial cells,the study of leptin-induced MAPK signaling pathway was carried out.Methods:(1)The endometrial epithelial tissue of adult healthy sheep was collected during the estrus period,and the localization of leptin and receptors in the sheep uterus was confirmed by immunohistochemical method,cultured by tissue block method,and purified by trypsin digestion Primary endometrial epithelial cells,characterized by immunohistochemical identification.Taking advantage of the feature that keratin is a specific expression protein of epithelial cells,the immunofluorescence method was used to detect whether the sheep endometrial epithelial cells that can be normally passaged,frozen and thawed,and grown in vitro were successfully established.(2)Functional verification of leptin at the cellular level.Add(0,100,200,250,500 μg/m L)leptin to treat cells respectively.The effect of leptin on the proliferation of sheep endometrial epithelial cells was detected by cck-8 method,and the key genes of apoptosis of endometrial epithelial cells treated with leptin were detected by Western blotting and RT-q PCR after screening the optimal concentration The cell scratch test was used to detect the migration of endometrial epithelial cells in each group,and the effect of leptin on the expression of embryo implantation-related factors was detected by RT-q PCR.(3)To study the effect of the MAPK pathway on the function of leptin,the MAPK signaling pathway blockers SB203580,U0126-Eto H,and SP600125 were screened for drug concentration by the cck8 method,and added to the leptin-treated sheep endometrial epithelial cells In the experiment,kits were used to detect the effect of cell antioxidant capacity;Western blotting was used to detect the expression of key MAPK genes;signal pathway blockers were used as the control group,and RT-q PCR was used to detect the effect of leptin on the expression of embryo implantation-related factors.Results:(1)Leptin protein was mainly expressed in the stroma and epithelial tissue of the endometrium of non-pregnant sheep,while it was mainly expressed in the endometrial epithelial tissue of pregnant sheep,and there was no significant change in the expression of leptin receptor;tissue block method The primary sheep endometrial epithelial cells with typical epithelioid cell characteristics and expressing keratin CK18 can be successfully obtained;(2)250 μg/m L leptin promoted the proliferation and migration of sheep endometrial epithelial cells,significantly up-regulated sheep embryo implantation-related factors and apoptosis inhibition-related genes Bcl-2,and down-regulated the transcription of BAX and Casepase 3 genes and the transcription of BAX and Expression of Casepase 9 protein.It suggested that leptin may regulate the process of sheep embryo implantation by inhibiting the apoptosis of sheep endometrial epithelial cells,promoting cell proliferation and migration.(3)After screening the drug concentrations of MAPK signaling pathway blockers SB203580,U0126-Eto H,and SP600125,add them to leptin-treated endometrial epithelial cells;leptin down-regulates the concentration of MDA in cells,up-regulates the expression of SOD m RNA,and increases the expression of GSH-PX and CAT enzyme activity,thereby enhancing the ability of cells to resist oxidative damage;leptin significantly up-regulated the key genes of the MAPK pathway,and compared with MAPK signaling pathway blockers,the positive regulation ability of leptin on embryo implantation-related factors was weakened.It is suggested that leptin may enhance the antioxidant capacity of cells through the MAPK signaling pathway,and affect the regulation of the process of sheep embryo implantation.Conclusion: In this study,primary sheep endometrial epithelial cells were successfully isolated and purified by trypsin + tissue block culture method combined with trypsin differential digestion method;leptin was specifically expressed in the endometrium of pregnant sheep,and leptin It can significantly promote cell proliferation and migration,inhibit cell apoptosis,and affect the expression changes of key embryo genes,thereby regulating the basic functions of endometrial epithelial cells and promoting the process of sheep embryo implantation.Leptin-mediated MAPK pathway significantly enhanced cell antioxidant capacity and MAPK key genes,and affected the expression changes of key genes in sheep embryo implantation,thus playing a positive regulatory role in sheep embryo implantation.