| BackgroundRecurrent spontaneous abortion(RSA)is defined as two or more consecutive spontaneous miscarriages occurring before 28 weeks of gestation.Established risk factors are genetic abnormalities,uterine malformations,abnormal endocrine functions,immune imbalance,prethrombotic state and infection.In addition,there are still more than 50%of RSA patients with unclear etiology,known as unexplained recurrent spontaneous abortion.RSA greatly affects the physical and mental health of women of childbearing age.It is of great significance to study its pathogenesis and find effective prevention and treatment methods.Successful pregnancy depends on the maintenance of immune tolerance at the maternalfetal interface.Immune imbalance can lead to a variety of adverse pregnancy states including pregnancy loss.Decidual immune cells are the basis of maternal-fetal immune tolerance,and macrophages are crucial immune cells during pregnancy,participating in the regulation of implantation,vascular remodeling,placenta formation,fetal development,delivery and a series of events.The functions of macrophages include regulating inflammation,removing cell debris and protecting cells from pathogens.After pregnancy,the activity of macrophages in the endometrium decreases and remains at a low level,at which time the activity of macrophages digesting and processing antigens is weakened and the antigen processing ability is reduced,which is conducive to reducing the immunity level of the uterus and protecting the blastocyst from maternal rejection.Overactivated macrophages increase motility and phagocytosis,and produce a large number of pro-inflammatory cytokines and other inflammatory mediators,which further enhance and expand the immune response and inflammatory response,which is not conducive to the maintenance of pregnancy.Autophagy is a catabolic process that relies on lysosomes to participate,degrading senescent organelles,damaged cellular structures,and other biological macromolecules in cells,which is a cellular degradation and circulatory system that plays an important role in maintaining cellular homeostasis.Autophagy plays an important role in macrophage polarization,chronic inflammation,and organ fibrosis.Recent studies have begun to elucidate the intrinsic link between autophagy and macrophage function.The level of autophagy affects the activation,classification,and death of macrophages,as well as the activation of inflammasome and the release of inflammatory cytokines.Gene associated with retinoid-IFN-induced mortality 19(GRIM-19)is a gene that regulates apoptosis and mitochondrial function.GRIM-19 is a fundamental subunit of mitochondrial respiratory chain complex I(RC-1),which is necessary for the assembly of complex Ⅰ-Ⅳ.GRIM-19 deficiency can lead to abnormal mitochondrial structure,function,and intracellular distribution.GRIM-19 participates in the occurrence and development of tumors by regulating tumor cell proliferation,apoptosis,invasion,energy metabolism rearrangement,epithelial-mesenchymal transition,etc.Recent studies have found GRIM-19’s important function in regulating immune response and inflammation.However,at present,the regulatory effect of GRIM-19 on macrophage function has not been reported.ObjectiveThe purpose of this study is to investigate the correlation between abnormal GRIM-19 expression and abortion,and to study the regulation of GRIM-19 on the inflammatory cytokine secretion and phagocytosis of macrophage from the perspective of macrophage autophagy,and to reveal the pathogenesis of RSA.Part Ⅰ Correlation between GRIM-19 expression and the occurrence of abortionMethods1.The Grim-19+/-knockout mouse model was constructed,and the female Grim-19+/mice mated with wild-type male mice to observe the embryo resorption rate,and the correlation between the abnormal expression of GRIM-19 and the occurrence of abortion was studied in the mouse model.2.The decidua tissues of RSA patients and control group were collected,CD14+decidual macrophages were isolated,and GRIM-19 expression was detected by western blot.Clinical specimens were used to determine the correlation between GRIM-19 expression and the occurrence of RSA.Results1.Female Grim-19+/-mice were mated with wild-type male mice,and it was found that the embryo resorption rate of female Grim-19+/-mice was significantly higher than that of wild-type mice,suggesting that the decreased expression of GRIM-19 was related to abortion.2.Western blot results showed that the expression of GRIM-19 in CD14+decidual macrophages of RSA patients was significantly lower than that of control group,suggesting that the decreased expression of GRIM-19 in decidual macrophages was related to RSA.ConclusionsWe confirmed that decreased expression of GRIM-19 was associated with the occurrence of abortion through Grim-19+/-mouse model.The expression of GRIM-19 in decidual macrophages of RSA patients was decreased.Part Ⅱ Regulation of GRIM-19 on autophagy of macrophagesMethods1.Grim-19+/-pregnant mouse uterine mononuclear cells were isolated,and the expressions of autophagy related proteins Beclinl,LC3B Ⅱ/Ⅰ and BNIP3 were detected by western blot.The proportion of CD45+CD11b+F4/80+LC3B+cells in Grim-19+/-mouse uterine mononuclear cells was detected by flow cytometry to study the regulation of GRIM19 on macrophage autophagy.2.Mouse mononuclear macrophages RAW264.7 cells and human leukemia mononuclear cell line THP-1 cells were cultured.After cells transfected with specific siRNA downregulated GRIM-19 expression or transfected with pcDNA3.1-GRIM-19 plasmid up-regulated GRIM-19 expression,the expressions of Beclinl,LC3B Ⅱ/Ⅰ and BNIP3 proteins were detected by western blot.The formation of autophagosomes was observed by transmission electron microscopy after downregulation of GRIM-19 in RAW264.7 cells.3.The decidua tissues of RSA patients and control group were collected,CD14+ decidual macrophages were isolated,and the expressions of autophagy related proteins Beclinl,LC3BⅡ/Ⅰ and BNIP3 were detected by western blot.Results1.Western blot results showed that the expression of autophagy related proteins Beclinl,LC3B Ⅱ/Ⅰ and BNIP3 in uterine mononuclear cells of Grim-19+/-pregnant mice were significantly increased.Flow cytometry showed that the proportion of CD45+CD11b+F4/80+LC3B+ cells in uterine mononuclear cells of Grim-19+/-mice was significantly higher than that of wild-type mice.2.Transfection of specific siRNA could significantly down-regulate GRIM-19 expression in RAW264.7 cells and THP-1 cells,and western blot results showed that expression of autophagy related proteins Beclinl,LC3B Ⅱ/Ⅰ and BNIP3 were significantly increased after down-regulation of GRIM-19.Meanwhile,after transfection with pcDNA3.1GRIM-19 plasmid significantly up-regulated GRIM-19 expression,autophagy related proteins Beclinl,LC3B Ⅱ/Ⅰ and BNIP3 expression were significantly decreased.The results of transmission electron microscopy showed that the number of autophagosomes of RAW264.7 cells increased after GRIM-19 downregulation.3.Western blot results showed that the expression of autophagy related proteins Beclinl,LC3B Ⅱ/Ⅰ and BNIP3 in CD14+decidual macrophages of RSA patients were significantly higher than those in the control group.ConclusionsWe confirmed that GRIM-19 regulates autophagy of decidual macrophages through Grim-19+/-mouse model,in vitro cultured macrophage cell lines and decidual specimens of RSA patients.Part Ⅲ The mechanism of GRIM-19 participating in RSA by regulating autophagy of macrophagesMethods1.Grim-19+/-pregnant mice uterus mononuclear cells were isolated,and the expressions of inflammatory cytokine IL-1β,IL-6 and TNF-α were detected by quantitative real-time PCR to study the regulation of GRIM-19 on the expression of inflammatory cytokine.2.RAW264.7 cells were cultured and transfected with siRNA to down-regulate GRIM19 expression.The cells were treated with autophagy inhibitor 3-MA,and the levels of inflammatory cytokines IL-β,IL-6 and TNF-α in the supernatant were detected by ELISA.3.RAW264.7 cells were cultured and transfected with siRNA to down-regulate GRIM19 expression.The cells were treated with autophagy inhibitor 3-MA,and the phagocytosis ability of the cells was detected by flow cytometry.4.THP-1 cells were cultured,macrophages were induced by PMA,siRNA was transfected to down-regulate GRIM-19 expression,and differentially expressed genes were screened by transcriptomic sequencing,and KEGG and GO analysis were performed.Results1.Quantitative real-time PCR results showed that the expression of IL-1β,IL-6 and TNF-α in uterine mononuclear cells of Grim-19+/-pregnant mice were significantly higher than those of wild-type mice.2.ELISA results showed that the down-regulation of GRIM-19 expression in RAW264.7 cells significantly increased the levels of inflammatory cytokines IL-1β,IL-6 and TNF-α in the supernatant,and the inhibition of autophagy by 3-MA could significantly reduce the levels of inflammatory cytokines.3.Flow cytometry showed that the phagocytosis ability of macrophages was significantly improved after GRIM-19 expression was down-regulated in RAW264.7 cells,and the phagocytosis ability of macrophages was significantly reduced after autophagy was inhibited by 3-MA.4.Transcriptome analysis results showed that 980 genes were downregulated and 914 genes were upregulated.The enrichment and Gene Ontology(GO)analyses showed that the changed genes were enriched in many immune response terms,including regulation of immune response,inflammatory response,immune response,neutrophil,monocyte,leukocyte,and eosinophil chemotaxis.The KEGG analysis of these hub genes was also enriched in many immune and inflammatory pathways,including PI3K-AKT signaling pathway,cytokinecytokine receptor interaction,chemokine signaling pathway,TNF signaling pathway.These results indicate that GRIM-19 mainly modulates immune and inflammatory related pathways,leading to cytokine production,and thus contributing to inflammation.ConclusionsGRIM-19 affects the secretion of inflammatory cytokines and phagocytosis ability of macrophages by regulating autophagy,and participates in the occurrence of RSA.Conclusions1.We confirmed that decreased expression of GRIM-19 was associated with the occurrence of abortion through Grim-19+/-mouse model.The expression of GRIM-19 in decidual macrophages of RSA patients was decreased.2.GRIM-19 affects the secretion of inflammatory cytokines and phagocytosis ability of macrophages by regulating autophagy,and participates in the occurrence of RSA. |
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