Integrated in silico transdermal prediction, serum pharmacochemistry, and experimental validation reveal the active constituents and therapeutic mechanism of Aifu Nuangong plaster against primary dysmenorrhea.

BACKGROUND

Primary dysmenorrhea (PD) is a prevalent gynecological disease that significantly impairs women's quality of life. Although Aifu Nuangong Wan (AFNGW) has demonstrated therapeutic efficacy on PD, its conventional oral administration is hindered by the first-pass effect and gastrointestinal adverse reactions. This study developed a novel traditional Chinese medicine transdermal plaster, Aifu Nuangong Plaster (AFNGP), based on Aifu Nuangong Wan (AFNGW). Nevertheless, the molecular mechanisms by which AFNGP alleviates PD have not yet been fully clarified.

AIM OF THE STUDY: To reveal the bioactive constituents and potential mechanisms of AFNGP in treating PD.

METHODS

UPLC-Q-Exactive Orbitrap-MS was employed to identify the chemical components in AFNGP extract and drug-containing serum, while the Deep-PK model was utilized to predict the transdermal properties of components in AFNGP extract. Network pharmacology and molecular docking were performed based on the analysis of blood-absorbed prototype components. A PD rat model was established using estradiol benzoate and oxytocin to evaluate the therapeutic effects of AFNGP. Furthermore, the functional mechanism of AFNGP on PD was predicted using network pharmacology and transcriptomics, followed by further validation using Western blotting.

RESULTS

Pharmacodynamic assessment demonstrated that AFNGP improved symptoms in PD rats, as demonstrated by reduced writhing responses, alleviated uterine histopathological damage, and decreased levels of PGF₂α and IL-6 in serum and uterine tissues in a dose-dependent manner. A total of 82 compounds in AFNGP extract and 19 prototype components in serum. Integrated network pharmacology and RNA-seq analysis indicated that AFNGP exerted therapeutic effects on PD through the PI3K-AKT signaling pathway. Moreover, Western blotting revealed that AFNGP inhibited the expression of key proteins in the PI3K-AKT signaling pathway.

CONCLUSION

AFNGP protected against PD by targeting the PI3K-AKT signaling pathway, thereby providing novel evidence for its potential application in PD.