Fetal Lung-derived Exosomes In Term Labor Amniotic Fluid Induce Amniotic Membrane Senescence In Primary Amniotic Epithelial Cells In The Mechanism Of Delivery

Objective:lots of researches show that parturition is associated with amniotic membrane senescence,while animal experiments suggest that pulmonary surfactant accumulate gradually with the extension of pregnancy,which can induce labor.Therefore,our study aimed to analyze the fetal lung-derived exosomes in normal term labor amniotic fluid whether trigger amniotic membrane aging or not.Study design:collecting amniotic membrane characterized by spontaneous term labor,and the membrane was aging identified by senescence-assocaiated β-galactosidase(SA-β-Gal)staining.Exosomes from term labor amniotic fluid(TL-exo)and term not in labor amniotic fluid(TNIL-exo)were isolated by ultracentrifugation.Isolating and culturing primary human amniotic epithelial cells(hAECs),TL-exo and TNIL-exo were used to intervene hAECs respectively,senescence-associated P38 MAPK and P53 signal pathways and damage-associated molecular patterns(DAMPs)expressed by aging and injured cells were detected by western blot.Cellular senescence and apoptosis were examined by senescence-associated β-galactosidase staining and flow cytometry.senescence-associated secretory phenotypes(SASPs)released by apoptotic and damaged cells were detected by ELISA.The human alveolar adenocarcinoma basal epithelial cells(A549)producing pulmonary surfactant was regarded as the alveolar type Ⅱ epithelial cell,and the markers(EpCAM,SP-C)of alveolar type Ⅱ epithelial cell can be expressed by A549 cell.We collected the supernatant of A549 cell,and isolated the exosome from the supernatant(A549-exo)by the same way.After co-culturing with hAECs,western blot was used to detect the level of senescence-associated P38 MAPK and P53 signal pathways and the expression of DAMPs released by aging and injured cells.Senescence-associated β-galactosidase staining and flow cytometry were applied to check cellular senescence and apoptosis.ELISA was used to detect the concentration SASPs released by apoptotic and damaged cells.All these results were compared with the exosome free group which is also called blank control group.Lastly,A549-exos were injected into the amniotic cavity of pregnant mice to observe the rate of vaginal bleeding and premature delivery,compared with the control group injected with 1xPBS.Results:compared with TNIL-exo,TL-exo can activate P38 MAPK and P53 signal pathways regulating cellular senescence and apoptosis,induce amniotic membrane aging,and senescent amniotic epithelial cells release higher level of DAMPs and SASPs.Meanwhile,after stimulating by A549-exo,P38 MAPK and P53 signal pathways of hAECs were activated,and cellular senescence,apoptosis rate,the level of DAMPs and concentration of SASPs were also higher,compared to the blank group.Finally,animal experiment suggested that the rate of vaginal bleeding and premature delivery with pregnant mice were more obvious in A549-exo group.Conclusion:our experiment identified indirectly that the fetal lung-derived exosomes in the term labor amniotic fluid can activate the senescence-related P38 MAPK and P53 signaling pathways,cause higher rate of cellular senescence and apoptosis,and aging and injured amniotic epithelial cell can release more DAMPs and SASPs.Animal experiment showed the fetal lung-derived exosomes induced a higher rate of vaginal bleeding and preterm labor among pregnant mice.In summary,the fetal lung-derived exosome in the term labor amniotic fluid can cause amniotic membrane senescent,and labor occurs.Our study results may provide a new sight for researchers exploring the mechanism of human parturition.