A multi-dimensional validation strategy of pharmacological effects of Radix Isatidis Mixtures against the co-infection of Mycoplasma gallisepticum and Escherichia coli in poultry.

Natural drugs possess exceptional pharmacological properties, yet their development is often hindered by a lack of clarity regarding the mechanisms of their pharmacological actions. Building on our previous research, we employed a co-infection model with Mycoplasma gallisepticum (MG) and Escherichia coli (E. coli) to investigate the pharmacological action of Radix Isatidis Mixtures (RIM). To further demonstrate the various mechanisms underlying the pharmacological effects of RIM, we conducted a validation study focusing on gene expression, protein interactions, metabolic pathways, and molecular docking. Through a multi-omics joint analysis network, we identified key targets and metabolites associated with co-infection and conducted targeted verification experiments with RIM aqueous extracts. The experimental results indicated that, compared to the co-infection group, the RIM treatment group significantly modulated the expression of select genes and proteins, particularly MMP2 and TLR4, with a high level of statistical significance (p < 0.01). At the metabolic level, the treatment group exhibited significantly reduced expression levels of Dopamine and γ-Aminobutyric acid. Notably, the molecular docking results highlighted compounds with the most favorable binding affinities: Salvianolic acid A (-10.1 kcal/mol), Licorice (-9.3 kcal/mol), and Isoglycyrrhiza (-8.7 kcal/mol). In conclusion, our multi-level experiments demonstrated that RIM possesses the characteristics of multi-pathway and multi-target treatment for co-infection.