Revealing active ingredients, potential targets, and action mechanism of Ermiao fang for treating endometritis based on network pharmacology strategy

Ethnopharmacological relevance: Ermiao fang (EMF) is a traditional Chinese medicinal herbal formula from ancient times and recorded in the pharmacopeia of the People's Republic of China. It is composed of two typical Chinese herbal medicines, Cortex Phellodendri (Huangbai), the bark of Phellodendron chinensis Schneid. (Rutaceae), and Rhizoma Atractylodis (Cangzhu), the rhizome of Atractylodes lancea (Thunb.) DC. (Compositae). EMF has been clinically used for the treatment of endometritis for many years in China. Aim of the study: This study was aimed to identify the active ingredients, potential targets, and mechanism of action of EMF for the treatment of endometritis. Materials and methods: In this research, the pharmacological effects of EMF on endometritis were first evaluated by establishing a rat model of endometritis. A network pharmacology-based analytical strategy was then used to predict its targets and signaling pathways. An endometritis-related protein target and compound database was built for EMF. The compounds in EMF and those absorbed into the blood were identified by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS). High-throughput virtual screening and molecule docking methods were used to predict the protein targets of EMF. The surface plasmon resonance analysis (SPR) method was used to validate the affinity between the compound and proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to predict the related pathways. Western blotting analysis was used to evaluate the expression of key proteins in the related pathways. Results: The animal study showed that EMF could reduce uterine inflammation in rats with endometritis. Then, an ingredient database including 187 compounds and a protein target database including 836 proteins were constructed. Twenty-four compounds in EMF were identified by UHPLC-Q-TOF/MS, among which eight compounds were present in rat plasma after an oral administration of EMF. Afterward, 39 potential target proteins were predicted by the high-throughput screening method, and 20 of them were selected after further screening using molecular docking. Subsequently, an ingredient-target network was constructed, and the target proteins were classified into the NF-kappa B and MAPK signal pathways by KEGG pathway enrichment analysis. Finally, the affinity between the active ingredients and the target proteins was verified by SPR. The Western blotting analysis showed that EMF significantly inhibited the elevated NF-kappa B and MAPK pathway proteins in rats with endometritis. Conclusions: EMF exhibited a significant pharmacological effect on rats with endometritis. Network pharmacology analysis revealed that eight compounds were absorbed into the blood after oral administration and interacted with 20 targets. Western blotting analysis indicated that EMF exerted anti-inflammatory effects by inhibiting the NF-kappa B and MAPK signaling pathway proteins in the treatment of endometritis.