Iron overload and oxidative stress have been reported to contribute to ferroptosis in endometriotic lesions. However, the possible roles of iron overload on macrophages in endometriosis (EMs) remain unknown. Based on recent reports by single-cell sequencing data of endometriosis, here we found significant upregulations of ferroptosis-associated genes in the macrophage of the endometriotic lesion. Additionally, there was an elevated expression of HMOX1, FTH1, and FTL in macrophages of peritoneal fluid in EMs, as well as iron accumulation in the endometriotic lesions. Notably, cyst fluid significantly up-regulated levels of intracellular iron and ferroptosis in Phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cells. Additionally, high iron-induced ferroptosis obviously reduced PMA-stimulated THP-1 cells’ phagocytosis and increased the expression of angiogenic cytokines, such as vascular endothelial growth factor A (VEGFA) and interleukin 8 (IL8). Baicalein, a potential anti-ferroptosis compound, increased GPX4 expression, significantly inhibited ferroptosis, and restored phagocytosis of THP-1 cells in vitro. Collectively, our study reveals that ferroptosis triggered by high iron in cyst fluid promotes the development of EMs by impairing macrophage phagocytosis and producing more angiogenic cytokines (e.g., IL8 and VEGFA). Baicalein displays the potential for the treatment of EMs, especially in patients with high ferroptosis and low phagocytosis of macrophages.

PurposeSanjie Zhentong Capsule (SZC) is gradually becoming widely used in the treatment of endometriosis (EMs) and has demonstrated an excellent curative effect in the clinic. However, the active components and mechanisms of Sanjie Zhentong Capsule (SZC) in the treatment of endometriosis (EMs) remain unclear, and further research is needed to explore the effects of Sanjie Zhentong Capsule (SZC).Materials and methodsFirst, a drug target database of Sanjie Zhentong capsule (SZC) was established by consulting the TCMSP database and related literature. An endometriosis (EMs) disease target database was then established by consulting the GeneCards, OMIM and Drug Bank databases. The overlapping genes of SZC and EMs were determined, and protein-protein interactions (PPIs), gene ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) analyses were performed to predict the potential therapeutic mechanisms. Molecular docking was used to observe whether the key active ingredients and targets predicted by network pharmacology had good binding energy. Finally, in vitro experiments such as CCK-8, flow cytometry and RT-PCR assays were carried out to preliminarily verify the potential mechanisms.ResultsThrough the construction of a pharmacological network, we identified a total of 28 active components in SZC and 52 potential therapeutic targets. According to GO and KEGG enrichment analyses, the effects of SZC treatment may be related to oxidative stress, steroid metabolism, apoptosis and proliferation. We also experimentally confirmed that SZC can regulate the expression of steroid hormone biosynthesis-related genes, inhibit ectopic endometrial stromal cell (EESC) proliferation and oxidative stress, and promote apoptosis.ConclusionThis study explored the potential mechanism of SZC in the treatment of EMs through network pharmacology and experiments, providing a basis for further future research on SZC in the treatment of EMs.