Wuda granules target estrogen receptors and modulate gut microbiota to alleviate postoperative ileus: A multi-omics perspective

Figure 1 Wuda granules improved gastrointestinal motility in postoperative ileus mice. A: Body-weight change; B: Gastric emptying rate; C and D: Statistical results and representative images of charcoal powder propulsion distance in the small intestine; E-G: Serum levels of cholecystokinin, gastrin, and motilin. Data are presented as mean ± SD (n = 6). ^aP < 0.05 vs postoperative ileus, ^bP < 0.01 vs postoperative ileus, and ^cP < 0.001 vs postoperative ileus. POI: Postoperative ileus; WDG: Wuda granules; MTL: Motilin; GAS: Gastrin; CCK: Cholecystokinin.

Figure 2 Effects of Wuda granules on histopathological changes and inflammatory cytokine levels in the colon and small intestine of postoperative ileus mice. A and B: Hematoxylin and eosin-stained images of the small intestine and colon tissues (magnification: 200 ×); C-F: Enzyme-linked immunosorbent assay analysis of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and myeloperoxidase expression in colon tissue; G-J: Enzyme-linked immunosorbent assay analysis of IL-6, IL-1β, tumor necrosis factor-α, and myeloperoxidase expression in small intestine tissue. ^aP < 0.05 vs postoperative ileus, ^bP < 0.01 vs postoperative ileus, and ^cP < 0.001 vs postoperative ileus. POI: Postoperative ileus; WDG: Wuda granules; IL: Interleukin; TNF-α: Tumor necrosis factor-α; MPO: Myeloperoxidase.

Figure 3 Wuda granules improve intestinal barrier structure and function in postoperative ileus mice. A and B: Alcian blue-periodic acid Schiff staining of the small intestine and colon (magnification: 200 ×); C: Serum D-lactic acid levels; D and E: Relative mRNA expression of tight-junction protein-1 and occludin in small-intestinal tissue, as determined by reverse transcription-quantitative polymerase chain reaction. Data are presented as mean ± SD (n = 6). ^aP < 0.05 vs postoperative ileus, and ^cP < 0.001 vs postoperative ileus. HE: Hematoxylin and eosin; POI: Postoperative ileus; WDG: Wuda granules; TJP-1: Tight-junction protein-1.

Figure 4 Proteomics analysis of Wuda granules in colon tissues of postoperative ileus mice. A: Venn diagram showing differentially expressed proteins shared between the postoperative ileus model and Wuda granules-treated groups; B: Volcano plot indicating significantly upregulated and downregulated proteins (red dots) with |log₂ fold changes| > 1 and P < 0.05; C: Heatmap illustrating the expression profiles of differentially expressed proteins across samples, with red to blue indicating high to low abundance; D: Gene Ontology enrichment analysis of differential proteins categorized by cellular component, molecular function, and biological process; E: Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially expressed proteins. WDG: Wuda granules.

Figure 5 Effects of Wuda granules on the expression of the enteric pacemaker-hormonal-neuronal regulation axis in the small intestine of postoperative ileus mice. A: Representative immunohistochemical staining of tyrosine-protein kinase kit, neuronal nitric oxide synthase, estrogen receptor (ER), and anoctamin-1 in small-intestinal tissues from different groups (magnification: 200 ×); B: Enzyme-linked immunosorbent assay analysis of ERα and ERβ protein levels in the small intestine (n = 6); C: Western blot analysis of tyrosine-protein kinase kit, neuronal nitric oxide synthase, and ER protein levels in intestinal tissues; D: Quantification of protein expression levels normalized to 3-phosphate dehydrogenase (n = 4). Data are presented as mean ± SD. ^aP < 0.05 vs postoperative ileus, and ^cP < 0.001 vs postoperative ileus. POI: Postoperative ileus; WDG: Wuda granules; c-Kit: Tyrosine-protein kinase kit; nNOS: Neuronal nitric oxide synthase; ER: Estrogen receptor; ANO1: Anoctamin-1; GAPDH: 3-phosphate dehydrogenase.

Figure 6 Molecular docking of representative chemical constituents from Wuda granules with estrogen receptors. A: Binding modes of linderane, quercetin, and quercitrin with estrogen receptor α (PDB ID: 1a52); B: Binding modes of linderane and quercetin with estrogen receptor β (PDB ID: 5toa). Electrostatic surface representations (left), binding pocket interactions (middle), and two-dimensional interaction diagrams (right) are shown. The interaction types are indicated by different colors: Hydrogen bonds, yellow; hydrophobic interactions, blue; and salt bridges, orange. ER: Estrogen receptor.

Figure 7 Effects of Wuda granules on the intestinal flora in postoperative ileus mice. A-C: Α-diversity indices: Chao1, Shannon, and Simpson; D: Principal coordinates analysis plot based on the Bray-Curtis distance showing β-diversity differences among groups; E: Bray-Curtis Anosim test validating community structure differences; F: Linear discriminant analysis revealing significantly different taxa between postoperative ileus and Wuda granules groups (linear discriminant analysis score > 3); G and H: Gut microbiota composition at the genus and species levels; I and J: Relative abundance of key differential genera (Parabacteroides, Bacteroides) and species (Parabacteroides goldsteinii, Bacteroides acidifaciens). ^aP < 0.05 vs postoperative ileus. POI: Postoperative ileus; WDG: Wuda granules; PCoA: Principal coordinates analysis; P. goldsteinii: Parabacteroides goldsteinii; B. acidifaciens: Bacteroides acidifaciens.

Figure 8 Effects of Wuda granules on fecal short-chain fatty acid levels in postoperative ileus mice. A-G: Concentrations of various short-chain fatty acids: Acetic acid, butyric acid, caproic acid, isobutyric acid, isovaleric acid, propionic acid and valeric acid; H: Spearman correlation of the top 10 gut microbial genera and short-chain fatty acids. ^aP < 0.05 vs postoperative ileus; ^dP < 0.05, ^eP < 0.01, and ^fP < 0.001, gut microbial genera vs short-chain fatty acids. AA: Acetic acid; BA: Butyric acid; CA: Caproic acid; IBA: Isobutyric acid; IVA: Isovaleric acid; PA: Propionic acid; VA: Valeric acid; SCFA: Short-chain fatty acid.