Modified Shaofu Zhuyu Tang, based on the Wnt3a/β-catenin/TCF4/GPX4 signaling pathway, mediates iron death in ectopic endometrial tissues of rats with endometriosis

The study aimed to investigate the potential molecular mechanism by which Modified Shaofu Zhuyu Tang (MSZT) mediates ferroptosis in ectopic endometrial tissue through the wingless type MMTV integration site family member 3A (Wnt3a)/β-catenin/T-cell transcription factor 4 (TCF4)/glutathione peroxidase 4 (GPX4) signaling pathway.

Eighty-four female SPF grade, SD rats were divided into blank (n = 12), sham operation (n = 12), and modeling groups (n = 60), according to the random number table method. The blank group received no intervention, the sham operation group underwent laparotomy only, and the modeling group was established with an endometriosis (EMs) rat model of cold coagulation and blood stasis syndrome via autologous transplantation combined with ice-water bath. After successful modeling, the modeling group was further subdivided into model, gestrinone (0. 25 mg/[kg·3 d]), and low-, medium-, and high-dose MSZT groups (7. 5, 15. 0, and 30. 0 g/[kg·d], respectively). After four weeks of administration, traditional Chinese medicine syndrome scores and writhing responses were evaluated, and the volume of ectopic endometrial tissue was recorded upon sample collection. Pathological changes in ectopic tissues were assessed using HE staining, Prussian blue staining, and transmission electron microscopy. Serum transforming growth factor-β1 (TGF-β1) levels were measured via enzyme-linked immunosorbent assay, whereas reduced glutathione (GSH) and malondialdehyde (MDA) levels in ectopic tissues were determined using microplate assays. The relative protein and mRNA expression levels of Wnt3a, β-catenin, Cyclin D1, TCF4, and GPX4 in ectopic tissues were analyzed via Western blotting and real-time quantitative PCR.

The model group exhibited higher cold coagulation and blood stasis syndrome score than the blank group (P<0. 05). The ectopic endometrial tissue in the model group resembled eutopic endometrium, with reduced iron deposition and increased mitochondrial numbers. Serum TGF-β1 (P<0. 05) and ectopic tissue GSH levels (P<0. 05) were elevated. Protein and mRNA expression levels of Wnt3a, β-catenin, Cyclin D1, TCF4, and GPX4 were upregulated in ectopic tissues (P<0. 05). Compared with the model group, the cold coagulation and blood stasis syndrome scores of medium-and high-dose MSZT groups decreased (P<0. 05). The gestrinone and medium-to high-dose MSZT groups exhibited prolonged writhing latency and reduced writhing times (P<0. 05), decreased ectopic endometrial tissue volume (P<0. 05); pathological results indicated increased iron deposition, epithelial and glandular cell shrinkage, and detachment, alongside mitochondrial cristae blurring, vacuolization, and outer membrane rupture. Serum TGF-β1 (P<0. 05) and GSH or MDA levels of ectopic tissue (P<0. 05) declined, whereas Wnt3a, β-catenin, Cyclin D1, TCF4, and GPX4 protein and mRNA expressions were downregulated (P<0. 05) in the gestrinone and MSZT groups.

MSZT inhibits the proliferative capacity of ectopic endometrial tissue and induces ferroptosis in ectopic endometrial tissue by affecting key proteins in the Wnt3a/β-catenin/TCF4/GPX4 signaling pathway, thereby alleviating the symptoms of cold-congestion and blood stasis syndrome in EMs.