| Background:Preterm labor,the delivery of more than 28 weeks but less than 37 weeks of gestation,is the most common pathological pregnancy and the leading cause of perinatal mortality and morbidity.There is currently a lack of effective preventive measures for preterm labor due to the regulatory mechanisms that underlie the initiation of labor have not been fully elucidated.To clarify the mechanism of human labor initiation and effectively prevent and treat premature birth is an urgent need for China’s major national policy of "improving maternal and infant health and improving population quality".Labor initiation is a complex process with multicellular involvement at the maternal-fetal interface and is precisely regulated by signaling molecules produced locally at the maternal-fetal interface.Our group’s previous finding that male mice with double gene knockout of the steroid receptor coactivators SRC-1/2(SRC-1-/-,SRC-2+/-)mated to wild-type females resulted in significantly expired births suggests the important role of fetal original factors in the initiation of labor.However,there is still a lack of relevant research about wheather placenta,as a special fetal organ in pregnancy,could produce fetal original factors that influence the course of parturition in this overdue delivery mouse model.Spermidine belongs to polyamines,there are several pathways that spermidine induces cellular autophagy.However,the induction effect of spermidine on autophagy in placental cells and its role in labor initiation have not been reported.It is worth noting that spermidine can be used to synthesize unconventional amino acid-hypusine,which currently has only one known target of post-translational modification,namely eukaryotic translation initiation factor 5A(EIF5A).It has been reported that spermidine mediated hypusination of EIF5A can induce autophagy,which can be regulated by GC-7,the specific inhibitor of EIF5A hypusination.Therefore,the present study aims to investigate the autophagy inducing effect of placental spermidine on trophoblast cell and its role in the initiation of labor.Methods:The placental tissues of SRC-1/2 deficient mice were collected,and the differentially expressed genes and related metabolic pathways in the placentas of expired mice and wild-type mice were screened by high-throughput transcriptome sequencing combined with targeted metabonomics technology,and verified in normal placenta and preterm placenta samples of mice and human,respectively.Human placental trophoblast cell line JEG-3 were cultured.Spermidine or difluoromethylornithine monohydrate(DFMO)which inhibits the formation of endogenous spermidine or GC-7 or EIF5A siRNA was added to explore whether spermidine could increase the autophagy level of placental trophoblast cells through EIF5A and its hypusination.The changes of placental endocrine function under different treatments were detected by synchronously collecting the cell supernatant.The mouse model of trophoblast specific knockdown of spermidine oxidase Aoc1 was constructed by adeno-associated virus(AAV).The delivery phenotype of AAV-shAoc1 mice was observed,and the placental tissues of the mice were collected.Premature mouse model was established to study the effect of spermidine on the delivery phenotype of premature mice by intraperitoneal injection.Human uterine smooth muscle cell line was treated with spermidine to observe the direct effect of spermidine on labor initiation.ChIP-qPCR was used to investigate the regulation of diamine oxidase Aoc1.Results:The expression of SRC-1 and SRC-2 in the placentas of SRC-1/2 dKO mice was significantly decreased in the overdue delivery mouse model caused by the deletion of SRC1/2.In SRC-1/2 dKO placentas,the poly amine metabolic pathway was significantly enriched,and the expression of diamine oxidase AOC1 was significantly decreased.The substrate spermidine was significantly accumulated,and spermidine metabolism was abnormal.The expression level of AOC1 protein in the placentas of wild-type pregnant mice during late gestation from 15.5dpc(days post-coitum)to labor was increased with the progress of pregnancy.The expression of AOC1 was significantly increased in preterm mouse placenta and human preterm placenta.The increase of endogenous AOC1 was accompanied by the decrease of spermidine production and the endogenous knockdown of Aoc1 resulted in the increase of spermidine production.At the overall animal level,placental specific knockdown of Aoc1 delayed the initiation of labor in mice,and exogenous spermidine could significantly reverse the premature delivery phenotype of mice.Further mechanism research found that,spermidine could increase autophagy of placental trophoblast cell through EIF5A hypusination pathway,thereby reducing the production of placental estrogen and prostaglandins and spermidine also has a direct relaxation effect on the contraction of uterine smooth muscle.Estrogen and its receptor a increased the transcriptional activity and expression of Aoc1 in an SRC-1/SRC-2 dependent manner,thus a dual positive feedback regulation is formed between estrogen and spermidine through AOC1.Conclusion:The expression of diamine oxidase AOC1 is developmentally upregulated in placentas during late gestation and is significantly decreased in placentas of SRC-1/2 dKO mice,accompanied with an increase in the AOC1 substrate spermidine.The accumulated spermidine exerts dual intrauterine effects.Spermidine induces autophagy of placental trophoblast cells by hypusination modification of EIF5A and leads to the decrease secretion of estrogen and prostaglandin.On the other hand,spermidine decreases the expression of contractionassociated proteins in uterine smooth muscle cells.Comprehensively,we speculate that spermidine inhibits the contraction of uterine smooth muscle through the direct and indirect effects mentioned above,thus conducive to the maintenance of uterine resting state during pregnancy;In late pregnancy,with the increase of placental AOC1 expression,the content of spermidine decreases,which contributes to the initiation of labor. |
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