Inhibition of HMGB1 Ameliorates the Maternal-Fetal Interface Destruction in Unexplained Recurrent Spontaneous Abortion by Suppressing Pyroptosis Activation

Abstract

Recurrent spontaneous abortion (RSA) is a common complication of pregnancy that affects the physical and mental health of pregnant women, and approximately 50% of the mechanisms are unclear. Our previous studies have found that high mobility group box 1 (HMGB1) molecules are highly expressed at the maternal-fetal interface of unexplained recurrent spontaneous abortion (URSA) patients. The purpose of this study was to further detect the expression of HMGB1 and pyroptosis in decidual tissue of URSA patients, and explore the potential mechanism of the protective role of HMGB1 in URSA patients and mouse model. The decidua tissues of 75 URSA patients and 75 women who actively terminated pregnancy were collected, and URSA mouse models were established and treated with HMGB1 inhibitor-aspirin. The expression of HMGB1, and their receptors (RAGE, TLR2, TLR4), pyroptosis-associated proteins (NLRP-3, caspase-1, GSDMD) and NF-κB was examined at the maternal-fetal interface of human and mouse. Our study found that HMGB1, NLRP-3, Caspase-1, GSDMD, RAGE, TLR2 and TLR4 were highly expressed and NF-κB signaling pathway were activated in the decidua tissue of URSA group. Moreover, immune cell disorder and co-localization of HMGB1 and macrophages were found at the maternal-fetal interface of URSA mice. However, HMGB1, TLR2, TLR4, NF-κB, and pyroptosis-associated proteins can be down-regulated by administering low-dose aspirin. These data may indicate that highly expressed HMGB1 was actively secreted by macrophages and then activated pyroptosis through the TLR2/TLR4-NF-κB pathway to cause aseptic inflammation, leading to the occurrence and development of URSA. Moreover, low-dose aspirin can reduce HMGB1 protein levels of serum and decidual in URSA.

Keywords: URSA; high mobility group B-1; macrophage; maternal-fetal Interface; pyroptosis.

Figure 1

The expression of HMGB1 and pyroptosis-associated proteins GSDMD, NLRP-3, and Caspase-1 in decidual tissue of URSA patients. (A) HMGB1, GSDMD, NLRP-3, and Caspase-1 protein were detected using Western blotting. (B) Quantitative data analysis for HMGB1. (C) Quantitative data analysis for GSDMD. (D) Quantitative data analysis for NLRP-3. (E) Quantitative analysis for Caspase-1. All the results were representatives of three independent experiments and data were expressed as mean ± SEM. (n=20/group). *P < 0.05, **P < 0.01 compared to the control.

Figure 2

Uterine anatomy and pathological changes of decidua in mice. (A) Left and right uterine horns, fetus, and placenta from normal pregnant mice, aborted mice, and drug-treated mice. (B) The number of fetuses. (C) Decidua of the control group, URSA group, and URSA+ASPL group were stained by H&E staining. (D) Quantitative assessment of infiltrating cells in decidua by counting the number of cell nuclei in H&E staining. The results were representatives of three independent experiments and data were expressed as mean ± SEM. ***P < 0.001. Scale bar represents 50 μm and 20 μm. (n = 6/group).

Figure 3

The expression of HMGB1 in mouse decidual tissue. (A) The expression of HMGB1 in mouse decidual tissue was investigated by immunohistochemistry staining. Scale bar represents 50 μm and 20 μm. (n = 6/group). (B) Quantitative assessment of the HMGB1 positive staining area in decidua using ImageJ software. (C) The protein levels of HMGB1 were identified by Western blot analysis. (D) The quantification data analysis of HMGB1 in the mouse decidual tissues. Data were expressed as mean ± SEM. (n = 6/group). (E) The serum HMGB1 levels of mice among control group, URSA group, and URSA+ASPL group were analyzed by ELISA. All the results were representatives of three independent experiments and data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Chart D n = 11 in control group; n = 14 in URSA group; n = 11 in URSA+ASPL group).

Figure 4

The colocalization between macrophages (F4/80⁺ or CD68⁺) and HMGB1 at the maternal-fetal interface. Double immunofluorescent staining of F4/80 and HMGB1 in human decidua (A) and CD68 and HMGB1 in mouse decidua (B). Quantitative assessment of the HMGB1 (C) and F4/80 (D) positive staining area in human decidua using ImageJ software. Quantitative assessment of the HMGB1 (E) and CD68 (F) positive staining area in mouse decidua using ImageJ software. HMGB1 was labeled with red fluorescent reagent. F4/80 and CD68 were labeled with green fluorescent reagent. The nucleus was stained blue with Hoechst. Scale bar represents 50 μm (human, n = 20/group) and 20 μm (mouse, n = 6/group). All the results were representatives of three independent experiments, and data were expressed as mean ± SEM. *P < 0.05, **P < 0.01.

Figure 5

The analysis of TLR2, TLR4, RAGE, and NF-kB p65 in mouse decidua. (A) The protein levels of TLR2, TLR4, and RAGE were identified by Western blot analysis. (B) Quantitative data analysis for TLR2. (C) Quantitative data analysis for TLR4. (D) Quantitative data analysis for RAGE. (n = 6/group). (E) The expression of NF-κB p65 was detected by immunohistochemistry in decidua among control group, URSA group, and URSA+ASPL group. Scale bar represents 20 μm. (n = 6/group). All the results were representatives of three independent experiments and data were expressed as mean ± SEM. *P < 0.05, ****P < 0.0001.

Figure 6

The analysis of GSDMD, NLRP-3, and Caspase-1 in mouse decidua. (A) The protein levels of GSDMD, NLRP-3, and Caspase-1 were identified by Western blot analysis. (B) Quantitative data analysis for GSDMD. (C) Quantitative data analysis for NLRP-3. (D) Quantitative data analysis for Caspase-1. All the results were representatives of three independent experiments and data were expressed as mean ± SEM. **P < 0.01, ***P < 0.01. (n = 6/group).

Figure 7

Model of aseptic inflammation caused by HMGB1-mediated pyrolysis in decidua tissue of unexplained recurrent spontaneous abortion. At the maternal-fetal interface, macrophages actively secrete HMGB1 and activate the NF-κB signaling pathway through its receptor. Then NLRP-3 inflammasome will be assembled, activate the caspase-1 protein, and release inflammatory factors such as HMGB1, causing aseptic inflammation at the maternal-fetal interface and leading to the development of URSA. Meanwhile, HMGB1 inhibitor could ameliorates the maternal-fetal interface destruction in unexplained recurrent spontaneous abortion.