Network pharmacology and in vivo and in vitro experiments to determine the mechanism behind the effects of Jiawei Yanghe decoction via TLR4/Myd88/NF-κB against mastitis

In the Qing dynasty, Yanghe decoction was as a therapeutic soup for effectively treating chronic inflammatory disorders. It was used as a therapeutic soup for effectively treating chronic inflammatory disorders. In the clinical use of Yanghe decoction, the adjustment of the medication for a variety of inflammatory diseases have therapeutic effect, including mastitis. Therefore, Jiawei Yanghe decoction (JWYHD) may be used to treat inflammatory breast diseases. First, LM- and JWYHD-related components were retrieved from the database and analysis platform. Next, protein–protein interaction networks were constructed to screen the key targets, and gene ontology and Kyoto encyclopedia of gene and genome enrichment analyses were performed to predict the potential biological functions and mechanisms of JWYHD. Simultaneously, the JWYHD samples were collected and analyzed by UPLC–HRMS. Finally, in vivo and in vitro experiments were conducted to construct animal and cellular inflammation models of mastitis with LPS. Pathological changes in the mammary tissues were detected. Enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and Western blotting was performed to determine the mRNA and protein levels of inflammatory cytokines and toll-like receptor 4/myeloid differentiation primary response 88/nuclear factor kappa B signaling pathway in the breast tissues to elucidate the potential underlying mechanisms of anti-mastitis effects of JWYHD from different aspects. In total, 103 compounds were detected in JWYHD by UPLC–HRMS. 691 active ingredients of JWYHD were screened by network pharmacology, and 47 LM-related targets were identified. The PPI network analysis of the targets revealed the 5 core targets. The KEGG enrichment results established the NF-κB signaling pathways as the core. After JWYHD intervention, low inflammatory enrichment and mild inflammatory damage in breast tissues were observed. Furthermore, JWYHD treatment affected mammary gland inflammatory cytokines and the TLR4/Myd88/NF-κB signaling pathway by considerably reducing the respective protein levels and gene expression; thus, JWYHD alleviated LM symptoms. We hypothesized and demonstrated the anti-inflammatory effects of JWYHD by cytokine regulation via the TLR4/Myd88/NF-κB signaling pathway. In conclusion, JWYHD showed its potential in LM treatment and in treating other acute and chronic inflammatory diseases.