Guben Antai Decoction Inhibits M1 Macrophage Polarization via the NF-κB/HIF-1α Pathway: A Mechanistic Study on the Treatment of Recurrent Spontaneous Abortion

Technical Summary: Guben Antai Decoction Inhibits M1 Macrophage Polarization via the NF-κB/HIF-1α Pathway in Recurrent Spontaneous Abortion

Problem Statement
Recurrent spontaneous abortion (RSA) is a complex condition often linked to immune abnormalities at the maternal-fetal interface. A critical factor in RSA pathogenesis is the imbalance in decidual macrophage polarization. While normal pregnancy is characterized by a predominance of anti-inflammatory, reparative M2 macrophages, RSA patients exhibit a shift toward pro-inflammatory M1 macrophages. This M1 dominance leads to the excessive secretion of inflammatory cytokines (e.g., IL-1β, TNF-α, IL-6), disrupting immune tolerance and impairing trophoblast function, ultimately resulting in pregnancy loss. Although the Guben Antai Decoction (GBAT), a Traditional Chinese Medicine (TCM) formula, has demonstrated clinical efficacy in treating RSA, the specific molecular mechanisms by which it regulates macrophage polarization and restores immune homeostasis remain unclear.

Methodology
This study employed a multi-faceted approach combining clinical sample analysis, single-cell RNA sequencing (scRNA-seq), and in vitro mechanistic experiments:

1. Clinical and Bioinformatics Analysis:

   • Decidual tissue samples were collected from 15 normal pregnancy (NP) patients and 15 RSA patients.
   • scRNA-seq was performed on decidual tissues using the BGI DNBelab C4 platform to profile immune cell composition and identify differentially expressed pathways.
   • RT-qPCR and Western Blotting were used to quantify the expression of macrophage markers (M1: IL-1β, IL-6, iNOS, TNF-α; M2: CD206, IL-10, Arg1) and signaling proteins (p-IκBα, p-NF-κB, HIF-1α) in clinical tissues.
   • Serum Analysis: Peripheral blood samples from 12 RSA patients were analyzed for IL-1β and TNF-α levels before and after GBAT treatment.

2. In Vitro Models:

   • Cell Lines: Human THP-1 monocytes were differentiated into M0 macrophages using PMA, then polarized to M1 (via LPS + IFN-γ) or M2 (via IL-4) phenotypes. The human trophoblast cell line HTR-8/SVneo was used for co-culture assays.
   • GBAT Preparation: GBAT-containing serum was prepared by administering the decoction to SD rats, collecting serum, and heat-inactivating it.
   • Intervention: M1 macrophages were treated with varying concentrations of GBAT-containing serum.
   • Pathway Validation: An NF-κB activator was added to M1 macrophages treated with GBAT to verify the specificity of the NF-κB/HIF-1α pathway.
   • Co-culture System: A Transwell co-culture system was established where treated macrophages (lower chamber) interacted with HTR-8/SVneo cells (upper chamber) to assess trophoblast migration and invasion.

3. Assays:

   • Molecular: RT-qPCR and Western blotting for gene and protein expression.
   • Functional: Flow cytometry and luminescence assays for cytokine levels (IL-1β, TNF-α); Wound healing, Transwell migration, and invasion assays for trophoblast capabilities.

Key Contributions and Results

• Identification of M1 Enrichment and Pathway: scRNA-seq analysis revealed a significantly higher proportion of M1 macrophages in RSA decidual tissue compared to normal pregnancy. KEGG pathway enrichment identified the NF-κB/HIF-1α signaling pathway as a key regulator of this M1 polarization.
• Clinical Correlation: In RSA patients, decidual tissue showed upregulated expression of M1 markers (IL-1β, IL-6, iNOS, TNF-α) and pathway proteins (p-IκBα, p-NF-κB, HIF-1α), while M2 markers were downregulated. Post-treatment with GBAT, serum levels of IL-1β and TNF-α in RSA patients significantly decreased.
• GBAT Inhibits M1 Polarization: In vitro experiments demonstrated that GBAT-containing serum significantly suppressed the expression of M1 markers (IL-1β, IL-6, iNOS, TNF-α) and downregulated the protein levels of p-IκBα, p-NF-κB, and HIF-1α in LPS-induced M1 macrophages.
• Mechanistic Validation: The inhibitory effect of GBAT on M1 polarization was reversed when an NF-κB activator was introduced, confirming that GBAT acts specifically through the suppression of the NF-κB/HIF-1α axis.
• Restoration of Trophoblast Function: In co-culture systems, M1 macrophages inhibited the migration and invasion of HTR-8/SVneo trophoblast cells. GBAT treatment of M1 macrophages reversed this inhibition, restoring trophoblast migratory and invasive capabilities. This protective effect was attenuated by the NF-κB activator, linking the restoration of trophoblast function directly to the modulation of the NF-κB/HIF-1α pathway in macrophages.

Significance and Claims
The authors conclude that Guben Antai Decoction exerts its therapeutic effect on recurrent spontaneous abortion by inhibiting the polarization of macrophages toward the pro-inflammatory M1 phenotype. This inhibition is mediated through the suppression of the NF-κB/HIF-1α signaling pathway.

By downregulating this pathway, GBAT reduces the secretion of inflammatory cytokines (IL-1β, TNF-α), thereby restoring the immune microenvironment at the maternal-fetal interface. Consequently, the decoction alleviates the suppression of trophoblast migration and invasion, which are critical for successful placental formation and pregnancy maintenance. This study provides experimental evidence supporting the use of GBAT for RSA and offers a mechanistic basis for TCM's ability to regulate immune cell polarization, suggesting that targeting the NF-κB/HIF-1α axis is a viable strategy for treating immune-mediated pregnancy loss.

The authors note limitations, including a moderate clinical sample size and the need for further investigation into whether GBAT utilizes other signaling pathways (e.g., PPAR-γ, STAT, MAPK) in addition to NF-κB/HIF-1α. However, the current findings establish a clear link between GBAT, macrophage polarization, and the NF-κB/HIF-1α pathway in the context of RSA.