Integrated multi-omics elucidates the dual anti-inflammatory and neuroendocrine mechanisms of novel TCM plasters against primary dysmenorrhea.

Background: Primary dysmenorrhea (PD) is a prevalent gynecological condition primarily driven by uterine inflammation and hormonal imbalances. While non-steroidal anti-inflammatory drugs (NSAIDs) are the first-line treatment, their side effects and failure rate necessitate alternative therapies. The Nuangong Zhitong Plaster (NGZT) and its graphene-modified variant (SMX) are clinically used traditional Chinese medicine (TCM) formulations for PD, but their comprehensive mechanisms of action remain unclear. Methods: A rat PD model was established via estradiol benzoate and oxytocin injection. Rats were treated with NGZT, SMX, ibuprofen, or a loxoprofen patch. Therapeutic effects were assessed through pain behavior scoring, uterine coefficient measurement, and enzyme-linked immunosorbent assay (ELISA) for prostaglandins and β-endorphin. An integrated approach combining network pharmacology, transcriptomics, proteomics, and metabolomics was employed to uncover the mechanisms, followed by experimental validation using Western blot, reverse transcription quantitative polymerase chain reaction (RT-qPCR), and coagulation function tests. Results: Both NGZT and SMX significantly alleviated pain behaviors, reduced uterine swelling, and normalized levels of pain mediators. Network pharmacology and molecular docking predicted multi-target binding against core proteins such as prostaglandin-endoperoxide synthase 2 (PTGS2/COX-2), estrogen receptor 1 (ESR1), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1). Multi-omics analyses revealed that the plasters reversed PD-associated alterations by co-regulating arachidonic acid metabolism and estrogen signaling pathways. Experimental validation confirmed that the plasters suppressed uterine expression of COX-2, interleukin-6 (IL-6), and interleukin-1β (IL-1β), corrected systemic hypercoagulability, and restored sex hormone balance. This was achieved through modulation of key components within the hypothalamic-pituitary-ovarian (HPO) axis-specifically gonadotropin-releasing hormone (GnRH) and its receptor (GnRH-R)-as well as uterine hormone receptors including the estrogen receptor (ER), progesterone receptor (PR), and oxytocin receptor (OTR). SMX demonstrated superior efficacy in modulating certain inflammatory and hormonal parameters, likely attributable to enhanced transdermal delivery facilitated by graphene. Conclusion: This study provides the comprehensive evidence that NGZT and SMX exert their anti-dysmenorrhea effects through a synergistic, multi-target mechanism that concurrently dampens uterine inflammation and rebalances the neuroendocrine system. The integration of multi-omics offers a robust systems-level framework for deciphering the mechanism of complex TCM formulations, solidifying the scientific basis for their clinical application in pain management.