| Objective:Investigate the expression of osteopontin(OPN)in decidua stromal cells(DSC)in preterm labor,and analyze its role in decidua immune inflammation to explore the role and mechanism of OPN in preterm labor.Methods:Single-cell transcriptome sequencing was used to the screen pathways of significant changes in DSC before and after labor,and the significantly up-regulated molecular OPN and inflammatory cytokines IL-1,IL-6,IL-8,CCL2 and CCL5 were also screened out in DSC.Then,immunofluorescence,immunohistochemistry and Western Blot were used to verify the decidua of preterm delivery(PTL group),term delivery(TL group)and preterm delivery(PTNL group).Meanwhile,phosphorylated p65 levels in decidua were detected,and the expression levels of OPN,IL-1,IL-6,IL-8,CCL2 and CCL5 in decidua tissues were detected by QRT-PCR.The primary DSC of term pregnant women was isolated and cultured,then treated with LPS,,transfected with OPN small interfering RNA,and co-cultured with CD14~+monocytes.The concentrations of IL-1,IL-6,TNFα,IL-8,CCL2,CCL4,CCL5 and CCL7 in the supernatant of DSC were detected by ELISA,and the levels of phosphorylated p65 and phosphorylated IκBαin DSC were compared,the proportion of p65 into the nucleus was also calculated.Transwell assay was used to detect the migration ability of monocytes.Further,rh CCL5 and CCL5 receptor antagonist Maraviroc were added into the co-culture system respectively or simultaneously for 24 hours to observe the recovery of the migration ability of monocytes.Results:(1)The results of human decidua single cell transcriptome showed that OPN was up-regulated by 2.5 times in DSC after term labor compared with non-labor group(P<0.001),and the cytokine/cytokine receptor interaction pathway and chemokine signal pathway was significantly enriched.(2)OPN was mainly located in DSC,and the OPN protein level in DSC was 7.96±0.22 in PTL group;5.68±0.34 in TL group;2.90±0.34 in PTNL group.PTL group has higher OPN protein expression than TL group and PTNL group(P<0.01).The level of OPN protein in decidua was2.26±0.09 in PTL group;1.13±0.07 in TL group;0.85±0.03 in PTNL group,and PTL group was higher than TL group and PTNL group(P<0.01).(3)Compared with TL group and PTNL group,phosphorylation of p65 in decidua of PTL group was up-regulated(P<0.05),the m RNA levels of OPN and IL-1,IL-6,IL-8,CCL2 and CCL5 in decidua of PTL group were also up-regulated compared with two control groups(P<0.05).(4)After LPS treatment,the m RNA and protein levels of OPN in DSC increased in a dose and time dependent manner(P<0.05),the levels of phosphorylated p65 and IκBαand the proportion of phosphorylated P65 in DSC increased(P<0.05);and the expressions of inflammatory factors IL-1,IL-6,TNFαand IL-8 in the supernatant of DSC increased(P<0.05),the expression levels of chemokines CCL2,CCL3,CCL4,CCL5 and CCL7were also increased(P<0.05);while OPN knockdown significantly OPN knockdown significantly down-regulated the levels of phosphorylated p65and IκBαand the proportion of P-P65 in DSC(P<0.01);and inhibited the LPS-induced secretion levels of these indexes(P<0.001),in which the expression of monocyte chemokine CCL5 was most significantly down-regulated(P<0.001).(5)The migration ability of monocytes in the co-culture system after LPS treatment was enhanced compared with control group(P<0.05),while OPN knockdown inhibited the migration of monocytes induced by LPS(P<0.05).The addition of rh CCL5 in co-culture system partially restored the reduced migration ability of monocytes caused by OPN knockdown(P<0.05),while the CCL5 receptor(CCR5 inhibitor)Maraviroc antagonized the recovery effect of rh CCL5(P<0.05).Conclusion:The expression of OPN in decidua stromal cells is increased during preterm labor,and the activation of NF-ΚB pathway in decidua stromal cells promotes the secretion of inflammation and chemokines,which counted for mediating the infiltration of peripheral blood monocytes in the decidua,contributing to the occurrence of preterm labor.Figure 12,table 14,references 83... |
PDF Full Text Request