| Objective:The maternal-fetal interface is formed by decidualized endometrium and extraembryonic tissue during pregnancy.Any factor that leads to abnormal changes of this interface may lead to adverse pregnancy outcomes,among which spontaneous abortion(SA)is one of the most common outcomes.SA refers to the loss of pregnancy(including biochemical pregnancy)when the pregnancy is less than 28 weeks and the fetal weight is less than 1 kilogram,accounting for about 10%to 15% of clinical pregnancies.The etiology and pathogenesis of SA are complicated.At present,the etiology and mechanism of nearly half of the SA cases are unknown,which is called unexplained abortion.Recurrent spontaneous abortion(RSA)refers to 2 or more consecutive SA with the same partner and occurs in about1% to 5% of pregnancies.RSA is more difficult to treat clinically,while no clear therapeutic strategies are available for unexplained RSA(URSA)so far.In recent years,many studies have found that the imbalance of immune-related cytokines at the maternal-fetal interface may play a vital role in the pathogenesis of unexplained abortion.From the point of view of immunology,it can be considered that the embryo is a kind of semi-allograft.The fact that the fetus carries half of the paternal genes and half of the maternal genes in direct contact with the maternal immune system without being rejected is the result of the normal establishment and maintenance of maternal-fetal immune tolerance,that is,the congenital and acquired immunity of mother and fetus reaches a dynamic balance through complex and fine regulatory mechanisms.Maternal-fetal immune tolerance occurs through the maternal-fetal interface,and decidua is a critical component of this interface and contains a large number of pregnancy-related immune cells,including macrophages,natural killer(NK)cells,T lymphocytes,and dendritic cells(DCs),which collectively referred to as decidual immune cells(DICs).DICs can secrete a variety of cytokines,such as interleukin-1β(IL-1β),IL-4,IL-10,IL-17,interferon-γ(IFN-γ),tumor necrosis factor α(TNF-α),and so on;meanwhile,DICs are regulated by these cytokines.When the decidual cytokine balance is disturbed by various factors,the fetus can be immune attacked by the mother,and the initiation of delivery will be triggered.If it is not stopped and repaired in time,pregnancy failure may occur.Therefore,the decidual cytokine balance and its regulatory mechanism is an important starting point for elucidating the pathogenesis of unexplained abortion and exploring therapeutic targets.micro RNA(miR)can result in degradation or translation inhibition by combining with the 3’-untranslated region(3’-UTR)of its target messenger RNA(m RNA).miRs regulate more than 1/3 of gene expression in the human genome at the post-transcriptional level and participates in various physiological and pathological processes.miR-146a-5p is the first miR found to have an immunomodulatory effect.It has been reported in recent years that miR-146a-5p expresses in many kinds of immune cells,such as macrophages,DCs,T lymphocytes,NK cells,and so on.Combined with the cellular composition of DICs,it is speculated that miR-146a-5p may be involved in the pathogenesis of unexplained abortion by regulating the DICs-mediated decidual cytokine balance mediated.According to the literature at home and abroad,it has been found in recent years that miR-146a-5p expressed in decidual tissue of URSA,and its level is lower than that of normal pregnancy.Still,the related mechanism has not been further reported.Therefore,the purpose of this study is to systematically explore the role and related mechanism of miR-146a-5p in the regulation of decidual cytokine balance in unexplained abortion,which will enrich the pathogenesis of unexplained abortion and provide new ideas for subsequent research on prevention and therapeutic targets and strategies.Materials and Methods:1.Clinical study objects and specimen collection.Screening patients with unexplained abortion scheduled to undergo uterine curettage(unexplained abortion group)and normal pregnant women who planned to undergo an artificial abortion because of no fertility plan(normal pregnancy group).After obtaining the written informed consent,the decidual tissues were collected and stored separately for subsequent cell separation,gene and protein detection,histopathological analysis,and so on.2.Detection of miR-146a-5p and cytokine in decidual tissue.The level of the miR-146a-5p gene was detected by polymerase chain reaction(PCR),and the level of cytokines was detected by enzyme-linked immunosorbent assay(ELISA).The differences between the two groups were analyzed,and the correlation between the levels of miR-146a-5p and cytokines was analyzed.3.Study on the regulation of miR-146a-5p on the secretion of cytokines in DICs.DICs were separated from normal pregnancy-derived and unexplained abortion-derived decidual tissues by enzymatic digestion.Flow cytometry(FCM)was used to detect the positive percentage of CD45.After transfection with miR control,miR-146a-5p mimic,or inhibitor,the levels of miR-146a-5p and cytokines were measured,and the differences between groups were analyzed.Bioinformatics methods were used to predict human miR-146a-5p target genes,and the key target molecules(hereafter called "target molecules")that regulate the balance of cytokines and their enriched signaling pathways were screened.The decidual tissues from normal pregnancy and unexplained abortion were taken to detect the gene and protein expression of target molecules by PCR and immunohistochemistry(IHC),and the differences between groups were analyzed.DICs were separated from unexplained abortion-derived decidua.After transfection with miR control,miR-146a-5p mimic,or inhibitor,the gene and protein expression of the target molecules were detected by PCR and western blotting(WB),and the differences between groups were analyzed.4.Regulation and mechanistic verification of miR-146a-5p on M1/M2 polarization of decidual macrophages.(1)The expressions of M1/M2 macrophage polarization markers CD86,inducible nitric oxide synthase(i NOS),CD206,and arginase 1(ARG1)in nine normal pregnancy-derived and nine unexplained abortion-derived decidual tissues were detected by WB.The expression of miR-146a-5p in decidual macrophages and M1/M2 macrophages was detected by immunofluorescence co-staining with CD68,CD86,or CD206 and miR-146a-5p,respectively,and the differences between groups were analyzed.(2)THP-1 cells were cultured,and M0 macrophages were induced by 100ng/m L phorbol12-myristate 13-acetate(PMA),and the positive percentage of CD14 and CD11 b were detected by FCM.(3)M0 macrophages were cultured,M1 macrophages were induced by 100ng/ml lipopolysaccharide(LPS)+ 20ng/ml IFN-γ,and M2 macrophages were induced by 20ng/ml IL-4.The positive percentage/quantity of CD86 and CD206 was detected by FCM and WB.(4)miR negative control,miR-146a-5p mimic,or inhibitor was pre-transfected into M0 cells,and then M1 and M2 macrophages were induced.The level of miR-146a-5p was detected by PCR,and the protein expression of macrophage polarization markers,cytokines,and miR-146a-5p target molecules was detected by WB.The differences between groups were analyzed.5.In vivo intervention of miR-146a-5p on the abortion-prone(AP)pregnant mice model.The mouse miR-146a-5p target genes were predicted by bioinformatics methods,and the key target molecules with the same prediction results as human miR-146a-5p were screened.In the normal pregnant mouse model group,CBA/J female mice(8-10 weeks old)and BALB/c male mice(8-10 weeks old)were mated in cages at a 2:1 ratio.The next morning,the vaginal suppository was examined to determine whether the mating was successful,and the day it was found was set as0.5 days of pregnancy.Normal saline(NS)was injected through the tail vein on days 0.5,3.5,6.5,9.5 of pregnancy.In the AP pregnant mouse model group,female CBA/J mice(8-10 weeks old)and male DBA/2 mice(8-10 weeks old)were mated in cages at a 2:1 ratio,the vaginal suppository was considered as successful mating and was set as 0.5 days of pregnancy,On days 0.5,3.5,6.5,9.5 of pregnancy,NS,miR negative control,miR-146a-5p antagonist,or agonist were injected intravenously.The uterus and embryo growth of normal pregnant mice and AP pregnant mice were observed on day 11.5 of pregnancy.The embryo absorption rate was calculated,and the differences between groups were analyzed.The expression of miR-146a-5p in decidual tissue was detected by fluorescence staining,and the levels of macrophage polarization markers and miR-146a-5p target molecules were detected by IHC,and the differences between groups were analyzed.Results:1.Abnormal expression of miR-146a-5p and cytokines in decidual tissue of unexplained abortion(significantly different from that of normal pregnancy group).Thirty-eight cases of unexplained abortion decidual tissue and 40 cases of normal pregnancy decidual tissue were collected.There was no significant difference in age,gestational days,and family history of SA between the two groups(p>0.05).The levels of miR-146a-5p and anti-inflammatory cytokines IL-4 and IL-10 in the unexplained abortion group were significantly lower than those in the normal pregnancy group(p<0.05),while the levels of proinflammatory cytokines IL-1β,IL17,and IFN-γ in the unexplained abortion group were significantly higher than those in the normal pregnancy group(p<0.05).The level of miR-146a-5p was positively correlated with the levels of anti-inflammatory cytokines IL-4 and IL-10(p<0.05)but negatively correlated with the levels of proinflammatory cytokines IL-1β,IL17,and IFN-γ(p<0.05).2.miR-146a-5p improved the imbalance of decidual cytokines of unexplained abortion.The DICs were successfully isolated from normal pregnancy and unexplained abortion decidual tissue,and the positive percentage of CD45 was97.28 ±0.78% and 96.72 ±1.06%,respectively.In DICs of both groups,the levels of miR-146a-5p and IL-10 in the miR-146a-5p inhibitor group were significantly lower than those in the miR control group(p<0.05).In comparison,the levels of IL-1β and IL-17/IFN-γ were significantly higher in the miR-146a-5p inhibitor group than in the miR control group(p<0.05).In unexplained abortion DICs,the levels of miR-146a-5p,IL4,and IL-10 in the miR-146a-5p mimic group were significantly higher than those in the control group(p<0.05).A total of 104 human miR-146a-5p target genes were predicted by bioinformatics,of which TRAF6,IRAK1,CCL5,and CD80 were enriched in the Toll-like receptor(TLR)signaling pathway and involved in regulating the pro-inflammatory process mediated by many cytokines such as IL-1β and IL-6.The gene levels of TRAF6,IRAK1,CCL5,and CD80,as well as the positive percentage,in decidual tissue of unexplained abortion were significantly higher than those in normal pregnancy(p<0.05),and the positive percentage of these four molecules was negatively correlated with the level of miR-146a-5p(p<0.05).In unexplained abortion DICs,compared with the miR control group,CCL5 and CD80 gene levels and TRAF6,IRAK1,CCL5,and CD80 protein levels were significantly decreased in the miR-146a-5p mimic group(p<0.05).In contrast,CCL5 gene level and CD80 protein level were significantly increased in the miR-146a-5p inhibitor group(p<0.05).3.Abnormal polarization of macrophages in decidual tissue of unexplained abortion.In human decidual tissue,the protein levels of CD86 and i NOS were significantly higher.In comparison,the levels of CD206 and ARG1 protein were significantly lower in the unexplained abortion group than in the normal pregnancy group(p<0.05).miR-146a-5p expressed in decidual macrophages,M1 macrophages,and M2 macrophages in the two groups,and the expression of miR-146a-5p in decidual macrophages and their subtypes in the unexplained abortion group was lower than that in the normal pregnancy group.4.miR-146a-5p regulated macrophage polarization through the TLR signaling pathway.M0,M1,and M2 macrophages were successfully induced from THP-1cells.The positive percentage of CD14 and CD11 b in M0 macrophages was 55.31±2.79% and 95.20 ±0.46%,respectively;the positive percentage and protein level of CD86 in M1 macrophages were significantly higher than that in M2 macrophages(p<0.05),while CD206 was significantly lower than that in M2 macrophages(p<0.05).miR-146a-5p and its target molecules are expressed in both M1 and M2 macrophages induced by THP-1 cells.The relative expression of miR-146a-5p and its target molecules in M2 macrophages was significantly higher than in M1macrophages(p<0.05).In contrast,the protein levels of TRAF6,IRAK1,and CD80 in M2 macrophages were significantly lower than those in M1 macrophages(p<0.05).In M1 macrophages,the gene levels of miR-146a-5p and the protein levels of CD206,IL-10,and ARG1 in the miR-146a-5p mimic group were significantly higher than those in the control group(p<0.05);in contrast,the protein levels of CD86,IL-1β,IL-17,i NOS,and TRAF6 were significantly lower than those in the control group(p<0.05).On the contrary,miR-146a-5p gene level and IL-4protein level decreased significantly(p<0.05),while IL-1β,i NOS,IRAK1,and CD80 protein levels increased significantly in the miR-146a-5p inhibitor group(p<0.05).In M2 macrophages,compared with the control group,the levels of miR-146a-5p gene and CD206 protein were significantly increased(p<0.05),while the protein levels of CD86,IL-17,i NOS,TRAF6,and IRAK1 were significantly decreased in the miR-146a-5p mimic group(p<0.05).On the contrary,the gene level of miR-146a-5p and the protein levels of IL-4,IL-10,and ARG1 were significantly decreased(p<0.05).In contrast,IL-1β,IL-17,i NOS,CCL5,and CD80 protein levels were significantly increased in the miR-146a-5p inhibitor group than in the control group(p<0.05).5.miR-146a-5p treatment can effectively reduce the abortion rate of AP pregnant mice.A total of 79 mouse miR-146a-5p target genes were predicted by bioinformatics,and TRAF6 and IRAK1 were the same as those of human miR-146a-5p.Comparing the two pregnant mouse models injected with NS,the embryo absorption rate and the percentage of CD86-,TRAF6-and IRAK1-positive cells in decidual tissue of AP pregnant mice were significantly higher than those of normal pregnant mice(p<0.05).In contrast,the expression of miR-146a-5p in decidual tissues of AP pregnant mice was lower than that of normal pregnant mice.The percentage of CD206-positive cells in decidual tissue of AP pregnant mice was also significantly lower than that of normal pregnant mice(p<0.05).In AP pregnant mice,the embryo absorption rate and the percentage of CD86-,TRAF6-and IRAK1-positive cells in decidual tissue in the miR-146a-5p agonist group were significantly lower than those in the control group(p<0.05).In contrast,the expression of miR-146a-5p in decidual tissue was more than that in the control group,and the percentage of CD206-positive cells in decidual tissue in the miR-146a-5p inhibitor group was significantly higher than that in the control group(p<0.05).Conclusions:1.The decreased expression of miR-146a-5p is closely related to the decidual cytokine imbalance in unexplained abortion and a significant inducement of abnormal secretion of cytokines in normal pregnancy-derived DICs.2.miR-146a-5p can improve the DICs-mediated disorder of cytokine balance to some extent,and the mechanism may be related to the targeted inhibition of the key molecules of the TLR signaling pathway TRAF6,IRAK1,CCL5,and CD80.3.Abnormal M1/M2 polarization is found in decidual macrophages of unexplained abortion,showing a shift to M1.The expression of miR-146a-5p in unexplained abortion decidual macrophages,M1,and M2 macrophages is less than that in human normal pregnancy decidual macrophages and subtypes.In THP-1-induced macrophages,upregulation of miR-146a-5p enhances IL-4-induced M2 polarization and promotes the secretion of anti-inflammatory cytokines while weakens LPS+IFN-γ-induced M1 polarization and inhibits the expression of proinflammatory cytokines and the key molecules of the TLR pathway.4.The decidual macrophages of AP pregnant mice also show abnormal polarization of M1/M2,a shift to M1.Upregulating the level of decidual miR-146a-5p can effectively reduce the abortion rate in AP pregnant mice.The mechanism is related to the fact that miR-146a-5p promotes the migration of decidual macrophages to M2 and inhibits TRAF6 and IRAK1,the key molecules of the TLR signaling pathway. |
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