Bushen Guchong decoction facilitates M2 polarization through JAK2/STAT3 for URSA treatment

Unexplained recurrent spontaneous abortion (URSA) poses a significant threat to women's reproductive health. While macrophage polarization imbalance has been implicated in URSA pathogenesis, the therapeutic potential of traditional Chinese medicine (TCM) formulations targeting this process remains underexplored. Bushen Guchong Decoction (BSGCD) has been extensively utilized in the treatment of URSA; however, the bioactive constituents and the underlying mechanisms remain insufficiently characterized.

This study aims to conduct comprehensive investigation of molecular mechanisms underlying the therapeutic effects of BSGCD in the management of URSA to bridge TCM and reproductive immunology.

RNA sequencing (RNA-seq) was conducted to investigate transcriptomic aberrations in decidua tissues of URSA. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed to analyze the natural ingredients as well as the bioactive compounds derived from both aqueous extract and blood samples of BSGCD. Subsequently, network pharmacology was applied to delineate the potential mechanism through which BSGCD exerts its therapeutic effects on URSA. Furthermore, a murine model of URSA was established to assess the impact of BSGCD on pregnancy outcomes in mice. Flow cytometry, qRT-PCR, Western blot, ELISA, molecular docking, CETSA and SPR were utilized to validate the therapeutic mechanisms of BSGCD.

RNA-seq analysis elucidated that the aberrant polarization of macrophage significantly contributed to the pathogenesis of URSA. A total of thirty-four bioactive compounds from BSGCD were identified as being absorbed in vivo. Network pharmacology analysis revealed that BSGCD may exert therapeutic effects on URSA by modulating macrophage polarization through targeted regulation of the JAK2/STAT3 signaling pathway. In addition, in vivo assays confirmed that BSGCD markedly reduced embryonic resorption in the murine model of URSA and upregulated the expression of M2 macrophage marker genes, the corresponding proteins, and the regulatory cytokines. Moreover, BSGCD was found to enhance the phosphorylation of STAT3 and JAK2 in the decidual cells of URSA mice. Our findings indicate that the principal bioactive constituents of BSGCD, including baicalin, salicylic acid, wogonin, oroxylin A, calycosin, glycyrrhizin, daidzein, and hyperin, may underlie its therapeutic effects.

Our work represents a comprehensive demonstration that BSGCD functions as a novel TCM formula capable of restoring maternal-fetal tolerance through immuno-regulation of JAK2/STAT3-mediated macrophage polarization, thereby establishing a new paradigm for TCM modernization.