The therapeutic mechanism of Fuzheng-Qushi formula on lung injury in mice revealed by microbiomics, metabolomics and transcriptomics
Aim of the study: The objective of this study was to elucidate the protective effects of Fuzheng-Qushi formula (FZQS) against lung injury in mice, and to uncover its potential targets and key biological pathways for the treatment of lung injury in combination with transcriptomics, microbiomics, and untargeted metabolomics.Materials and methods: LPS was used to construct a mouse lung injury model by nasal inhalation. The ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was used to analyze all the chemical constituents contained in FZQS. HE staining was used to analyze the histopathological changes in the lungs of mice. Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect cytokines in mouse lung tissues. Macrophage polarization and neutrophil activation were detected by flow cytometry. RNA sequencing (RNA-seq) was deployed to screen for differentially expressed genes (DEGs) in lung tissue. Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot were utilized to validate key DEGs and target proteins in lung tissue. 16S rRNA sequencing was employed to characterize the gut microbiota of mice. Metabolites in the gut were analyzed using untargeted metabolomics.Results: FZQS treatment significantly ameliorated lung histopathological damage, decreased pro-inflammatory cytokine levels and increased anti-inflammatory cytokine levels. The level of M1 macrophages in the peripheral blood of lung-injured mice decreased, the level of M2 macrophages increased, and activated neutrophils were inhibited. Importantly, transcriptomic analysis showed that FZQS downregulated macrophage and neutrophil activation, migration and adhesion pathways by reversing 51 DEGs, which was further confirmed by RT-qPCR and Western blot. In addition, FZQS modulated the dysbiosis of the gut microbiota by reversing the abundance of Corynebacterium, Facklamia, Staphylococcus, Paenalcaligenes, Lachnoclostridium, norank__f__Muribaculaceae, and unclassified__f__ Lachnospiraceae. Meanwhile, metabolomics analysis revealed that FZQS significantly regulated tryptophan metabolism by reducing the levels of 3-Indoleacetonitrile and 5-Hydroxykynurenine.Conclusions: The present study demonstrated that FZQS ameliorated LPS-induced lung injury mainly by inhibiting the activation, migration and adhesion of immune cells, as well as modulating the disruption of the gut microbiota and its metabolites.