Molecular Mechanism Of Low-dose Aspirin Promoting Fatty Acid Transport And Syncytilization In Placental Trophoblast Cells

The placenta is the link between the mother and the fetus during pregnancy,Abnormalities in the placenta’s function is closely related to poor pregnancy outcomes.Fetal growth restriction（FGR）is one of common complications of pregnancy in gynecology and obstetrics,mainly caused by placental insufficiency and fetal nutritional deficiency.Trophoblast cells,as the principal functional cells in placenta,are responsible for the secretion of hormones,nutrition transmission and immune defense.Current studies have found that low-dose aspirin（ASA）in early pregnancy can effectively reduce the occurrence of FGR and low birth weight in twins.However,its specific pharmacological mechanism has not been fully elucidated.In this study,we planned to use in vivo and in vitro experiments,label-free quantitative proteomics experiment and molecular and cellular biology experiments and other experimental techniques,to investigate the intervention effect of low-dose ASA treatment during pregnancy on FGR rat model,the effect of ASA on fatty acid metabolism of Be Wo cells and the change of acetylation level of related genes,and the effect of ASA on Be Wo cell syncytialization.1.Low dose ASA improved the birth weight of the fetus of FGR rats and twins in humans and the acetylation of placental in FGR rat modelL-NAME was used to induce FGR rat model,and low dose ASA was given to intervene.On the 21st day of gestation,after euthanasia death of rats,observation and measurement of various fetal and placental indicators showed that low-dose ASA significantly reduced the incidence of FGR,increased the average fetal weight,and improved placental efficiency.In addition,the mean birth weight of the fetus after ASA intervention in twin pregnancy was significantly higher than that in the non-intervention group.Further,the levels of placental acetylation in control group,FGR model group and ASA intervention group of animal model were detected by Western blot.It was found that continuous intervention of low-dose ASA during pregnancy could significantly increase the level of total placental protein acetylation in the intervention group.2.ASA enhanced the acetylation of FABPpm and promoted the transport of long-chain fatty acidsBe Wo cells were treated with different concentrations of ASA（0.01,0.1,0.5,1,5 m M）and different time（1,5,10,24,48h）,and the effect of ASA on cell viability was detected by MTT cytotoxicity assay and the effect of ASA on cell protein acetylation level was detected by Western blot.The results showed that 1m M ASA treated cells for 24h had no significant effect on cell viability and the level of cell protein acetylation was significantly up-regulated.Therefore,subsequent experiments were conducted under this condition.Bodipy^TMFL C16 and[9,10³H]palmitate were used to detect fatty acid uptake,and cell oil red O assay was used to detect fatty acid storage.The results showed that the uptake and storage capacity of long chain fatty acids of Be Wo cells were significantly improved after ASA treatment.Further acetylated label-free quantitative proteomics analysis also showed that protein acetylation in cell metabolism,especially in fatty acid metabolism-related pathways,was up-regulated after ASA treatment.Among them,the acetylation levels of 3 sites of plasma membrane-associated fatty acid-binding protein（FABPpm）were directly regulated by ASA,and IP experiments also confirmed that ASA could directly regulate the acetylation levels.Taken together,these results suggest that ASA may enhance Be Wo cells’ability to take up long-chain fatty acids by regulating the acetylation level of FABPpm.3.The effects of ASA upregulation of PPBP expression on Be Wo cell syncytializationSyncytiotrophoblast（STB）plays an important role in the transport of nutrients.During the development of placenta,STB is mainly derived from cytotrophoblast（CTB）.The potential regulatory genes of FGR were screened out by analysis of transcriptome sequencing data of FGR placenta.Treatment with 1m M ASA for 24h upregulated key potential regulatory genes of FGR in Be Wo cells.The structure of FGR placenta was evaluated by HE staining and immunofluorescence,and it was found that FGR placenta was impaired in syncytialization.Further,knockout of PPBP,the cell fusion rate detected by immunofluorescence,the markers related to syncytialization detected by RT-q PCR and Western blot all showed that the syncytialization process of Be Wo cells was inhibited.Finally,Western blot was used to detect CREB protein and the phosphorylation level of CREB.After knockout of PPBP,p-CREB was down-regulated and the activation of c AMP signaling pathway was inhibited.These results suggested that ASA up-regulated PPBP was beneficial to Be Wo cell syncytilization.In conclusion,this study combined with FGR animal model of SD rats and cell experiments to preliminarily explore the improvement effect of low-dose ASA intervention during pregnancy on FGR;The effect of ASA treatment on the ability of trophoblast cells to transport long chain unsaturated fat;The acetylated label-free quantitative proteomics was used to analyze the proteins related to fatty acid transport regulated by ASA direct acetylation and the relationship between the acetylation modification and the function of FABPpm.Combined with transcriptome analysis,potential regulatory genes of FGR were found and histomathological analysis revealed that FGR placental syncytilization was impaired.ASA affected the process of trophoblast syncytilization by affecting the expression of PPBP,a potential regulatory factor of FGR.These results will provide a potential reference for ASA to improve the function of placental associated cells and prevent placental diseases during pregnancy.