Detection And Analysis Of The Effect Of Low-dose Aspirin On Fatty Acid Metabolism And Differentiation Of Trophoblast Cells

The placenta is an important hub for nutrition metabolism between the mother and the fetus.syncytiotrophoblast(STB)is the most important functional cell on the placenta,and its normal function is closely related to the fetal weight.At present,taking low-dose aspirin(ASA)during pregnancy can improve low birth weight.It has been included in many countries and organizations as a guide for the prevention and treatment of pregnancy-related diseases and related complications.Whether ASA can improve placental function by promoting fatty acid metabolism and proliferation and differentiation of placental trophoblasts remains unclear.This study intends to use human placental tissue,primary trophoblast cells and BeWo cell lines as materials,and use related molecular biology techniques to explore the following issues: low-dose ASA trophoblast cell fatty acid metabolism and gene expression changes in the process of integration,analysis of differences The biological processes influenced by genes;the effect of low-dose ASA on fatty acid transport and synthesis of trophoblast cells;the mechanism of low-dose ASA on trophoblast syncytialization.1.Differentially expressed genes in trophoblast cells before and after treatment with low-dose ASATo clarify the biological role of ASA on cytotrophoblast cell line(BeWo).First,screen the appropriate concentration and treatment time of ASA.This study used 0.01 mM,0.1 mM,0.5 mM,and 1 mM ASA to treat BeWo cells for different periods of time.The results of CCK8 showed that its toxic effect increased with the increase of ASA concentration;and 1mM ASA treated cells for 1h,5h,10 h,24h and 48 h,it was found that its toxic effect also increased with time.Therefore,the final determination of 1mM culture 24 h as working concentration and time.Second,BeWo cells were treated with 1mM ASA for 24 h,and transcriptome sequencing revealed differential expression of key proteins related to fatty acid synthesis,oxidation,and transport,and the expression of trophoblast integration protein was upregulated.GO and KEGG enrichment analysis of the differential genes showed that the differential genes were mainly enriched on the surface of the plasma membrane,mainly involved in the metabolism of nutrients such as fatty acid metabolism of cells;and genes related to trophoblast integration and involved cAMP signaling pathway.2.The effect of low-dose ASA on fatty acid metabolism in trophoblastic cellsAfter treatment of BeWo cells with 1mM ASA,quantitative analysis of genes related to fatty acid metabolism was performed by RT-qPCR.The results showed that fatty acid binding proteins(FABPs),fatty acid transporters(FATPs),fatty acid synthase(FASN),acetyl-CoA carboxylase(ACC)carnitine palmitoyl transferase 1A(CPT1A),fatty acid metabolism related transcription factors(PPAR?,PPAR?)mRNA level was up-regulated,indicating that low-dose ASA affects fatty acid transport,synthesis and oxidation processes.Using confocal microscopy live cell imaging technology,it was observed that low-dose ASA significantly increased the efficiency of BeWo cells taking up free fatty acids;theseresults suggest that low-dose ASA increases the fatty acid metabolism function of trophoblast cells.3.The effect of low-dose ASA on the process of trophoblast integrationIn the development of the human placenta,STB is continuously differentiated and fused from cytotrophoblasts(Cytotrophoblast,CTB).Twenty-four hours after treatment of BeWo cells with 1mM ASA,transcriptome analysis showed that differential genes were enriched in the cAMP signaling pathway,and the expression of the integration marker gene was up-regulated;while the cell fusion-related factor THBS1 and its receptor CD36 were down-regulated,consistent with qRT-RCR quantitative results.Using primary trophoblastic spontaneous integration and establishing a BeWo cell integration model,and through Western blot,RT-qPCR,CO-IP and other technologies,it was found that the expression of THBS1 and CD36 was down-regulated during the integration process and the interaction between the two;and THBS1 Overexpression inhibits the activation of the cAMP signaling pathway and reduces the efficiency of integration.The above results indicate that the down-regulation of THBS1 expression caused by low-dose ASA is beneficial to trophoblast integration.In summary,this study initially explored the effect of low-dose ASA on fatty acid metabolism and cell integration in placental trophoblast cells.By analyzing the differentially expressed genes of BeWo cells before and after ASA treatment by transcriptome sequencing,to explore the changes of genes related to fatty acid metabolism and integration of ASA on trophoblast cells,in the future,it may provide new clues to further explore the function and regulatory mechanism of ASA to improve placental trophoblast cells And provide new ideas for the development and application of other new drugs in the prevention and treatment ofpregnancy-related diseases.