MS-based molecular networking and network pharmacology analysis of XFBD. (a) Base peak mass spectrum of XFBD with positive and negative modes. (b) The main clusters of molecular networking of XFBD. Nodes with different colors represent different compound classifications. Yellow: flavonoid; red: terpenes; green: carboxylic acids; blue: glycosides.

XFBD inhibits LPS-induced acute inflammation in mice. (a) Scheme of XFBD administration and LPS injection. (b–d) Effects of XFBD (high: 4.32 g·kg^–1; low: 2.16 g·kg^–1) on inflammatory cytokines secretion in serum induced by LPS (20 mg·kg^–1 for 4 h). At least six animals were examined in each group (*: P < 0.05, **: P < 0.01 vs saline; #: P < 0.05, ##: P < 0.01 vs LPS). (e) H&E staining of the lung tissue of different groups. (f) Quantification of mice lung pathological sections: (size of blank area/size of total area) × 100%. For analysis, an image of one section was taken for each mouse and three mice were included in each group (*: P < 0.05, **: P < 0.01 vs saline). i.p.: intraperitoneal injection; i.g.: intragastrical administration.

Transcriptomic analysis suggested that XFBD regulates gene expression in multiple infection- and inflammation-related pathways. (a) Counts and distribution of differentially expressed genes (DEGs) of LPS-stimulated mice with or without XFBD treatment. Red: upregulated genes; green: downregulated genes; blue: genes without significant changes. Padj: adjust P value. (b) Venn diagram of DEGs in respective groups. (c) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of 357 overlapping DEGs.

XFBD and its active compounds inhibited macrophage activation in vitro. (a) Scheme of macrophage activation and analysis. (b) XFBD extracts of 6.25–100 μg·mL^–1 inhibited IL-6 release on the macrophage-activation model in a dose-dependent manner after 24 h of treatment (n = 3, ***: P < 0.001 vs P2P). (c) Protein level of IL-6 in macrophage cells with treatment with different compounds (n = 3, **: P < 0.01, ***: P < 0.001 vs P2P). DEX was used as the positive control. The detailed concentrations of each treatment are listed in the Section 2.9. (d) The effects on IL-6 release of representative XFBD compounds with different doses in the P2P-stimulated macrophage model. The relative IL-6 concentrations were calculated by comparing the value of each compound-treated group with those of the model group. (e, f) Effect of isoliquiritin (50 μmol·L^–1) on the phosphorylation of JNK (p-JNK), p38 (p-p38), and MEK (p-MEK). Representative western blots are shown in (e) and statistical graphs are shown in (f) (n = 3; *: P < 0.05 vs P2P; #: P < 0.05, ##: P < 0.01 vs control). CTR: control.

XFBD inhibits inflammation-induced macrophage recruitment in zebrafish. (a) Illustration of the timeline of drug protection and tail amputation. Red dotted line represents the amputation site, and the blue box represents the region for the cell counting of recruited inflammatory cells. (b) Toxicity effects of different doses of XFBD (50–800 μg·mL^–1) on fish survival and general development. (c) Representative images and (d) quantification of the accumulation of macrophages at the wounding sites of the control or the XFBD-treated (50–200 μg·mL^–1) embryos. (e) Representative images and (f) quantification of the accumulation of neutrophils at the wounding sites of the control or the XFBD-treated (50–200 μg·mL^–1) embryos. (g) Time-lapse imaging of macrophage movement in the control and the XFBD-treated (200 μg·mL^–1) embryos at indicated times after tail amputation. (h) Quantification of the moving velocity of macrophages. (i) Gene expression of cytokines IL-6 and IL-1β in tail-amputated embryos with or without XFBD (200 μg·mL^–1) treatment. White dotted lines outline the fish embryos and yellow (c, e) or red (g) dashed lines mark the transection site. Yellow and red asterisks mark the moving trajectory of the representative macrophages. dpf: days after fertilization; mpa: minutes after amputation, UC: tail-uncutted embryos; CTR: tail-cutted embryos without treatment; XFBD groups: tail-cutted embryos with XFBD treatment of different dosages. *: P < 0.05; **: P < 0.01.

Screen for active herbs, herbal combinations, and compounds of the XFBD recipe in macrophage mobilization. Quantification of the number of macrophages accumulated at the wounding site of embryos treated with (a) different XFBD herb extracts, (b) herb combinations, and (c) compounds. The dosages of each treatment are listed in the Sections 2.9 and 2.11. **: P < 0.01. Groups labeled without a common letter were significantly different (P < 0.05) in (b). (d) Molecular structure of compounds with significant effects in regulating macrophage recruitment. (e) Model of the regulatory effects of XFBD and its components on macrophage-mediated inflammation response. Red boxes highlight the active herbs identified from the macrophage activation model in P2P-stimulated RAW 264.7 cells; blue boxes highlight active herbs identified from the macrophage mobilization model in caudal fin-wounding zebrafish. CTR: tail-cutted embryos without treatment.