Regulatory role of MRTF-A in macrophage polarization and the therapeutic mechanism of Bushen Wenyang Huayu decoction in endometriosis.

[ETHNOPHARMACOLOGICAL RELEVANCE] Traditional Chinese Medicine (TCM) offers multiple advantages in the treatment of endometriosis (EMS). Bushen Wenyang Huayu Decoction (BWHD) is widely used in clinical practice for managing EMS. It can significantly alleviate patients' pain symptoms, regulate disordered reproductive hormone levels, improve the hypoxic state of lesions, and thereby enhance patients' quality of life. However, the precise mechanisms by which BWHD inhibits the progression of EMS remain incompletely elucidated.

[AIM OF THE STUDY] To investigate the regulatory role of MRTF-A/SRF in macrophage polarization during EMS progression and to assess the therapeutic effect of BWHD and its potential molecular targets.

[METHODS] Fifty rats were randomly divided into five groups: sham-operated, model, high-dose BWHD, low-dose BWHD, and Western medicine groups. Following EMS induction, treatments were administered via gavage for 3 weeks. Flow cytometry was performed to detect macrophage polarization in peritoneal fluid, in situ endometrium, and ectopic lesions. Western blot and RT-PCR were used to measure protein and mRNA expression of myocardin-related transcription factor-A (MRTF-A), serum response factor (SRF), brahma-related gene 1 (BRG1), and M2 macrophage markers. Immunohistochemistry (IHC) and immunofluorescence (IF) assays were conducted to determine the localization of these proteins. THP-1 cells were induced into M2 macrophages and co-cultured with 12Z cells and HUVECs, respectively. MRTF-A and BRG1 were knocked down or overexpressed, and cells were treated with BWHD serum. Protein and mRNA levels of MRTF-A, SRF, BRG1, and M2 macrophage markers were measured by WB and RT-PCR. Mixed lineage leukemia protein 1 (MLL1) and histone 3 lysine 4 trimethylation (H3K4me3) mRNA levels were detected by RT-PCR. Cell migration, proliferation, and invasion were evaluated using wound healing, CCK-8, and Transwell assays, respectively. Angiogenic ability of HUVECs was assessed by tube formation assay. Co-immunoprecipitation (Co-IP) was conducted to examine the interaction between MRTF-A and SRF, and macrophage polarization was assessed by flow cytometry.

[RESULTS] In animal experiments, the model group exhibited increased M2 macrophage polarization, elevated expression of MRTF-A/SRF, BRG1, and M2 macrophage markers, and enhanced migration, invasion, and angiogenesis compared to the sham-operated group. BWHD at high and low doses, as well as Western medicine, significantly reversed these changes. In cell experiments, overexpression of MRTF-A enhanced migration, proliferation, and invasion of 12Z cells in co-culture, whereas MRTF-A knockdown and BWHD treatment markedly reduced these abilities. Similarly, overexpression of MRTF-A significantly promoted angiogenesis in HUVECs, while MRTF-A knockdown and BWHD treatment reduced this effect. MRTF-A overexpression in THP-1 cells increased M2 macrophage polarization, MRTF-A/SRF interaction, BRG1 activation, and H3K4me3 methylation levels. Conversely, MRTF-A knockdown and BWHD treatment significantly attenuated these effects.

[CONCLUSION] MRTF-A/SRF promotes histone H3K4me3 methylation by regulating BRG1, thereby inducing macrophage polarization toward the M2 phenotype and enhancing migratory, invasive, and angiogenic abilities of ectopic endometrial cells, ultimately contributing to EMS progression. BWHD exerts therapeutic effects in EMS by downregulating MRTF-A, attenuating MRTF-A-mediated M2 macrophage polarization, and restoring macrophage immune homeostasis.