Modified Pulsatilla decoction alleviates 5-fluorouracil-induced intestinal mucositis by modulating the TLR4/MyD88/NF-κB pathway and gut microbiota

Figure 1  Experimental timeline and procedures.

Figure 2 The total negative and positive ion chromatograms of modified Pulsatilla decoction. A: Total negative ion chromatograms; B: Total positive ion chromatograms.

Figure 3 Modified Pulsatilla decoction compounds ligand dock to the TLR4/MyD88/NF-κB pathway. A-E: Docking of glycyrrhizic acid with lipopolysaccharide lipopolysaccharide (LPS; A), TLR4 (B), MyD88 (C), TRAF6 (D), and NF-κB (E); F-J: Limonin with LPS (F), TLR4 (G), MyD88 (H), TRAF6 (I), and NF-κB (J).

Figure 4 Modified Pulsatilla decoction alleviates symptoms in intestinal mucositis mice. A: Percentage of initial body weight (%); B: Diarrhea scores; C: Perianal condition; D: Intestinal length; E: H&E staining (60 × and 200 × magnification); F: Length of the small intestine; G: Length of the colon; H: Histopathological scores; I: MPO expression level. Data are shown as mean ± SD (n = 6-8). ^aP < 0.01 vs control group; ^bP < 0.05, ^cP < 0.01 vs 5-fluorouracil group. 5-FU: 5-fluorouracil; LO: Loperamide; PD: Pulsatilla decoction.

Figure 5 Modified Pulsatilla decoction inhibits reactive oxygen species production in intestinal mucositis mice. A: Reactive oxygen species (ROS) staining images of frozen ileum tissue sections; B: Average fluorescence intensity of ROS, normalized to the control group (set as 1). Data are shown as mean ± SD (n = 3). ^aP < 0.01 vs control group; ^bP < 0.01 vs 5-fluorouracil group. 5-FU: 5-fluorouracil; LO: Loperamide; PD: Pulsatilla decoction.

Figure 6 Modified Pulsatilla decoction exerts inhibitory effect on TLR4/MyD88/NF-κB pathway. A and B: Western blotting images of TLR4, MyD88, TRAF6, β-actin (A), and NF-κB P65, Histone3 (B); C-F: Ratios of TLR4/β-actin (C), MyD88/β-actin (D), TRAF6/β-actin (E), and P65/Histone3 (F); G-N: Immunofluorescent images for TLR4 (G), MyD88 (I), TRAF6 (K), and NF-κB (M) with corresponding fluorescence intensities (H, J, L and N), normalized to the control group (set as 1). Data are shown as mean ± SD (n = 3). ^aP < 0.01 vs control group; ^bP < 0.01 vs 5-fluorouracil group. 5-FU: 5-fluorouracil; LO: Loperamide; PD: Pulsatilla decoction.

Figure 7 Modified Pulsatilla decoction promotes inflammatory response balance. A-H: Levels of inflammatory markers include lipopolysaccharide (A), TNF-α(B), IFN-γ (C), IL-1β(D), IL-6 (E), IL-8 (F), IL-10 (G), IL-17 (H). Data are presented as mean ± SD (n = 6). ^aP < 0.01 vs control group; ^bP < 0.01 vs 5-fluorouracil group. 5-FU: 5-fluorouracil; LO: Loperamide; LPS: Lipopolysaccharide.

Figure 8 Modified Pulsatilla decoction enhances intestinal barrier function. A-F: Expression levels and average fluorescence intensity of Occludin-1 (A and B), Claudin-1 (C and D), ZO-1 (E and F); G and H: Immunohistochemical staining and scoring of Mucin-2. Data are presented as mean ± SD (n = 3). ^aP < 0.01 vs control group; ^bP < 0.01 vs 5-fluorouracil group. 5-FU: 5-fluorouracil; LO: Loperamide; PD: Pulsatilla decoction.

Figure 9 Modified Pulsatilla decoction ameliorates gut microbiota dysbiosis. A and B: Amplicon sequence variant (ASV) Venn diagram (A) and counts (B); C and D: Stacked bar charts of relative species abundance at genus (C), and species (D) levels; E-J: Alpha diversity indices including observe ASV (E), ACE (F), Chao1 (G), Shannon (H), Simpson (1-D; I), and Pulsatilla decoction whole tree (J); K-P: Beta diversity analysis of Unweighted PCoA (K), Unweighted PCoA with UPGMA (L), Weighted PCoA (M), Weighted PCoA with UPGMA (N), Bray-Curtis PCoA (O) and NMDS (P). ^aP < 0.01 vs control group; ^bP < 0.05, ^cP < 0.01 vs 5-fluorouracil group. ASV: Amplicon sequence variant; LO: Loperamide; PD: Pulsatilla decoction.

Figure 10  The relationships between biological markers of the gut microbiota. A and B: LDA value distribution bar chart (A) and cladogram (B) from LEfSe analysis; C: The correlation between these biomarkers and the relative abundance of gut microbiota at genus and species levels, with positive correlations shown in red and negative correlations in blue and green, respectively. Significance levels are marked as ^aP < 0.05, ^bP < 0.01).

Figure 11  Gut microbiota functional prediction. A and B: Heatmaps generated by the Tax4Fun2 tool illustrate predicted metabolic pathways across groups at levels 2 (A) and 3 (B). LO: Loperamide; PD: Pulsatilla decoction.

Figure 12  The mechanism of modified Pulsatilla decoction in intestinal mucositis mitigation. LPS: Lipopolysaccharide; ROS: Reactive oxygen species.