The Role Of HMGB1 In Regulating The Pyroptosis Of Macrophages At The Maternal-fetal Interface In Unexplained Recurrent Spontaneous Abortion

Objective:Recurrent spontaneous abortion（RSA）is a common pregnancy complication that seriously affects the physical and mental health of pregnant women,and the pathogenesis of approximately 50%is still unclear.Previous studies by our group have found that high mobility group box 1（HMGB1）molecules are highly expressed at the maternal-fetal interface in patients with unexplained recurrent spontaneous abortion（URSA）,but the specific mechanism of action is still unclear.In this study,we further examined the expression of HMGB1 and pyroptosis in the decidual tissue of URSA patients,while downregulating HMGB1 levels in URSA mice,to investigate the specific mechanism of action and therapeutic significance of HMGB1 in URSA patients and mouse models.Methods:In this study,we established an animal model of URSA mice and used aspirin,a commonly used drug for clinical treatment of abortion,to down-regulate HMGB1 and investigate the effects of blocking HMGB1 on the prevention,treatment,and reversal of URSA.The URSA mouse model was divided into the normal group,the URSA group,and intervention group.Low-dose aspirin was given before and after matting.On the14th day of pregnancy,the mice were sacrificed by cervical dislocation,and serum and decidual tissues were obtained.Seventy-five cases of aborted decidual tissues from patients with URSA early pregnancy and 75 cases from patients with voluntary termination of early pregnancy were also collected to verify the role of HMGB1 in URSA.HE staining,immunohistochemistry,Western Blot,immunofluorescence,and ELISA were used to detect the expression of HMGB1 and its receptors（RAGE,TLR2,TLR4）,pyroptosis-associated proteins（NLRP-3,Caspase-1,GSDMD）,and NF-κB at the maternal-fetal interface of humans and mice.Results:（1）A mouse model of unexplained recurrent spontaneous abortion was successfully established,and the embryos in the control group were the same in size and more in number;the embryos in the URSA model group were smaller in number,different in size,and some fetuses were smaller in size and a necrotic state;aspirin intervention group blocking HMGB1 could restore the number of embryos and reduce aborted embryos.（2）The results of HE staining showed that compared with the control group,the number of decidual tissue cells was increased,the cells were crowded and disorganized,nuclear rupture or nuclear pyknosis occurred,and a large number of immune cells were infiltrated into the tissue in the URSA model group,while aspirin intervention could significantly improve the inflammatory response at the maternal-fetal interface in URSA mice.（3）Immunohistochemical results showed that in the control group,HMGB1protein was stained brown and mainly expressed in the nucleus,while HMGB1 protein was not only expressed in the nucleus but also stained a large number of positive cells in the cytoplasm and intercellular space in the decidual tissue of URSA mice.In the control group,a small amount of NF-κB was concentrated in the cytoplasm and nucleus,and the decidual cytoplasm and extracellular space of mice in the URSA group were filled with positive staining and translocated into the nucleus.After aspirin intervention,HMGB1 in the decidual tissue returned to be mainly expressed in the nucleus,and NF-κB positive cells were reduced.（4）Western blot results showed that the expression of HMGB1 protein and its related receptors（RAGE,TLR2,TLR4）in the decidua of the maternal-fetal interface in the URSA group was higher than that in the control group,meanwhile,the expression of local pyroptosis-associated proteins（NLRP-3,Caspase-1,GSDMD）was also higher in the URSA group.However,after aspirin intervention,the expression of HMGB1 and its related receptors and pyroptosis-associated proteins were down-regulated.（5）Immunofluorescence results showed that HMGB1 was expressed in the nucleus in the decidua of control mice,and the mean fluorescence intensity of CD68（macrophages）and HMGB1 was weak,the number of positive cells was small and the overlap was small,indicating that there were fewer macrophages in the maternal-fetal interface of pregnant mice.HMGB1 was expressed in the nucleus and extranuclear space in the decidua of URSA mice,and HMGB1 and CD68（macrophages）double-positive cells were abundantly expressed,with high mean fluorescence intensity,and most of the two could overlap,indicating that HMGB1 in the maternal-fetal interface of URSA was mainly produced by CD68⁺macrophages.However,aspirin significantly reduced the number of HMGB1⁺,CD68⁺,and double-positive cells in decidual tissues after the intervention,indicating that blocking HMGB1 may reduce HMGB1 levels by reducing the number of macrophages,or may directly prevent macrophages to produce HMGB1.（6）The decidual tissues from abortions in the population were collected.The expression levels of HMGB1 and pyroptosis-associated proteins（NLRP-3,Caspase-1,GSDMD）in decidual tissues of URSA patients were increased by Western Blot.Immunofluorescence results showed that HMGB1 highly expressed in the decidua of URSA patients was derived from active secretion by decidual macrophages at the maternal-fetal interface.Conclusion:（1）HMGB1 is involved in the development of URSA.The main mechanism may be that macrophages at the maternal-fetal interface actively secrete HMGB1,which enters the cells extracellularly through its receptor and induces pyroptosis after activating the NF-κB signaling pathway,thereby inducing aseptic inflammation,ultimately leading to the destruction of the maternal-fetal interface and the occurrence of URSA.（2）It has preventive and therapeutic effects on unexplained recurrent spontaneous abortion by administering low-dose aspirin as a blocker of HMGB1.