Effect Of PPARγ-Mediated Lipid Metabolic Pathway On The Biological Function Of Trophoblast Cells In Sheep

Object:Under the regulation of maternal hormones,embryonic implantation gradually establishes close connections between embryonic trophoblast cells and maternal endometrial cells（including endometrial stromal cells and epithelial cells）,thus leading to the process of structural and functional remodeling.The destruction of embryo implantation will lead to poor pregnancy outcomes.Trophoblast cells,as the embryonic ectoderm in contact with the maternal uterine epithelium,show a variety of basic and important functions in the process of embryo attachment.Peroxisome proliferators-activated receptorsγ（PPARγ）is a nuclear receptor highly expressed in the trophoblast during implantation.As a key gene in the regulation of lipid metabolism in cells,it plays an important role in physiological processes including embryo implantation and pregnancy maintenance.On this basis,this study used sheep trophoblast cells as the research object to explore the regulatory effect of PPARγon lipid metabolism and biological function of trophoblast cells.Methods:（1）Uterine tissue of normal pregnancy and abortion was collected,and trophoblast tissue was isolated.Immunohistochemistry,Real-time quantitative PCR（RT-qPCR）and Western blot were used to detect the expression level of PPARγand the expression levels of genes related to lipid metabolism and prostaglandin metabolism in tissues.The content of triglyceride in trophoblast tissue was determined by colorimetry and the level of lipid oxidation in trophoblast tissue was determined by MDA.（2）Subcellular localization of PPARγprotein in sheep trophoblast cells（STCs）was performed by immunofluorescence method.The appropriate concentrations of rosiglitazone and GW9662 were screened and exogenous treated with sheep trophoblast cells to activate and inhibit PPARγactivity.mRNA expression levels of lipid metabolism-related genes and prostaglandin metabolism-related genes were detected by RT-qPCR,and lipid drops were fluorescently stained.The secretion levels of PGE2 and PGF2were detected by ELISA.The proliferative ability,mobility and apoptosis rate of trophoblast cells were detected by CCK-8 assay,scratch assay and TUNEL method.（3）Sheep trophoblast cells were treated with estradiol,progesterone and IFN-τ.The expression of PPARγprotein and mTOR pathway-related proteins were detected by Western blot,and the transcription levels of PPARγand ISG15 were detected by RT-qPCR.The secretion of PGE₂and PGF_2αwas detected by ELISA,and the ratio of PGE₂to PGF_2αwas calculated.After treatment with exogenous drugs,under the condition of hormone stimulation,the levels of PGE₂and PGF_2αin trophoblast cells were detected by ELISA,and the expression levels of p70S6K,p-p70S6K,LC3B-Ⅱ,LC3B-Ⅰ and BAX proteins were detected by Western blot.（4）Based on the correlation between PPARγ,mTOR/autophagy and lipid metabolism.In trophoblast cells,PPARγactivity was activated and inhibited in vitro,autophagy was promoted and inhibited by rapamycin and 3-MA,and PPARγactivity was activated by rapamycin treatment and 3-MA pretreatment after inhibiting PPARγactivity.Western blot,triglyceride content detection,MDA detection and lipid droplets（BODIPY 493/503）and lysosomes（Lyso-Tracker Red）co-localization fluorescence staining were used to detect the expression of autophagy-related proteins and lipid metabolism in trophoblast cells.Results:（1）In the trophoblast tissue of abortion,the expression level and activity of PPAR were significantly decreased.The transcription levels of lipid metabolism genes PLIN2,FABP3,SCD,ACBP and prostaglandin metabolism-related genes PLA2G4A and PTGS2 decreased to varying degrees in the trophoblast tissues of abortion,while the triglyceride content decreased and the lipid oxidation level increased.（2）PPARγprotein was observed to be localized in the nucleus and cytoplasm of trophoblast cells under an inverted fluorescence microscope.The relative expression of PPARγprotein in trophoblast cells was the highest under 5μM rosiglitazone treatment,while the expression level was the lowest under 20μM GW9662 treatment.After activating PPARγactivity in trophoblast cells,the expression levels of lipid metabolism-related genes FABP4,PLIN2,and prostaglandin-related genes PTGS2,PLA2G4A and PTGES were significantly increased,which were represented by lipid droplet accumulation and increased PGE₂/PGF_2αratio.However,the expression of FABP3 was significantly reduced,with no significant changes in SLC27A1,SCD,ACBP,and SLCO2A1.Correspondingly,after inhibiting PPARγactivity,the expression of FABP4,PLIN2 and PTGS2 in trophoblast cells were significantly decreased,while the expression of FABP3,SLC27A1,SCD,ACBP and SLCO2A1 increased significantly,and the expression of PLA2G4A did not change significantly.Activation of PPARγactivity in trophoblast cells promotes cell proliferation,increases cell migration rate,and inhibits apoptosis.（3）Hormone treatment promoted the expression of PPARγ,inhibited the autophagy induced by mTOR pathway,and increased the ratio of PGE₂/PGF_2α.Inhibition of PPARγactivity reduced the release of prostaglandins in trophoblastic cells,and blocking mTOR pathway could restore the functional release of PGs in trophoblastic cells after inhibition of PPARγactivity.The ratio of PGE₂ to PGF_2αincreased.（4）Rosiglitazone treatment inhibited the mTOR pathway of trophoblast cells,induced the increase of autophagy and lysosome number,promoted the accumulation of lipid droplets and the increase of triglyceride content,while GW9662 treatment showed the opposite trend,and the content of lipid oxidation MDA increased.The results of rapamycin pretreatment were similar to those of rosiglitazone treatment.3-MA pretreatment blocked autophagy,inhibited lysosome number and lipid droplet accumulation,reduced triglyceride content,and increased lipid oxidation MDA content.Inhibition of mTOR pathway induced autophagy and promoted lipid accumulation capacity of trophoblast cells under the premise of inhibition of PPARγactivity.In contrast,activation of PPARγactivity after inhibition of autophagy was able to inhibit the lipid accumulation capacity of trophoblast cells compared to trophoblast cells under activation of PPARγactivity only.Conclusion:Finally,this study discovered that PPARγ-mediated lipid and prostaglandin related metabolism is involved in the regulation of trophoblast cell function,and it partially revealed the molecular regulation mechanism of PPARγ,the mTOR pathway,and autophagy on on lipid metabolism of sheep trophoblast cells.This study’s findings give a theoretical foundation for further research into the functional regulatory network of trophoblast cells during pregnancy in sheep.