Anwei decoction alleviates chronic atrophic gastritis by modulating the gut microbiota-metabolite axis and NLRP3 inflammasome activity

Figure 1 Experimental design: After 7 days of acclimatization, 10 rats were randomly selected as the normal control group. The remaining rats underwent chronic atrophic gastritis model induction. Upon successful modeling, rats were randomly divided into the model group receiving saline gavage, the Anwei decoction (AWD) intervention group receiving AWD (10.71 g/kg) and the Weifuchun capsules (WFC) intervention group (receiving WFC at 0.43 g/kg) daily for 8 weeks. The normal control group was provided regular feeding and drinking water free. CAG: Chronic atrophic gastritis; MNNG: N-methyl-N’-nitro-N-nitrosoguanidine; HE: Hematoxylin-eosin; IL: Interleukin; TNF: Tumor necrosis factor; LPS: Lipopolysaccharide; ELISA: Enzyme-linked immunosorbent assay; qRT-PCR: Quantitative real-time polymerase chain reaction; mRNA: Messenger RNA.

Figure 2 Total ion chromatogram for natural product identification in samples. A: Total ion chromatogram (TIC) of blank serum (positive ion mode); B: TIC of blank serum (negative ion mode); C: TIC of serum containing Anwei decoction (AWD) (positive ion mode); D: TIC of serum containing AWD (negative ion mode); E: TIC of AWD (positive ion mode); F: TIC of AWD (negative ion mode).

Figure 3 Hematoxylin-eosin-stained histopathological images of gastric mucosa from the normal control group, model group, Anwei decoction group, and Weifuchun capsules intervention groups. Detachment of gastric mucosal epithelial cells (yellow arrow); dilation of gastric glands (blue arrow); atrophy or reduction of gastric glands (black arrow); hyperplasia of fibrous connective tissue (orange arrow); inflammatory cell infiltration (purple arrow). The black square indicates the location of the magnified field. NC: Normal control; MD: Model; AWD: Anwei decoction; WFC: Weifuchun capsules.

Figure 4 NLRP3 messenger RNA expression levels in gastric mucosa. Compared with the normal control group and compared with the model group. ^cP < 0.001. mRNA: Messenger RNA; NC: Normal control; MD: Model; AWD: Anwei decoction; WFC: Weifuchun capsules.

Figure 5 Western blotting analysis. A: Western blot detection results of NLRP3 inflammasome-related protein expression levels in each group of cells; B: Protein expression levels of caspase-1; C: Protein expression levels of interleukin-1β; D: Protein expression levels of NLRP3. Compared with the normal control group and compared with the model group. ^cP < 0.001. IL: Interleukin; NC: Normal control; MD: Model; AWD: Anwei decoction; WFC: Weifuchun capsules.

Figure 6 Analysis of intestinal microbial diversity. A: Venn diagram; B: Heatmap integrated with Metastats significance markers, displaying differences in microbial composition determined by 16S rRNA amplicon sequencing; C: Non-metric multidimensional scaling analysis results. NC: Normal control; MD: Model; AWD: Anwei decoction; NMDS: Non-metric multidimensional scaling.

Figure 7 Structure and changes in gut microbes. A: Relative abundance of microbial taxa at the phylum level; B: Relative abundance of microbial taxa at the genus level; C: Linear discriminant analysis score distribution indicating microbial taxa significantly differing among groups; D: Taxonomic cladogram illustrating taxa with significant differences among groups. NC: Normal control; MD: Model; AWD: Anwei decoction; LDA: Linear discriminant analysis.

Figure 8 Circular diagrams illustrating metabolite category composition. A: Positive-ion metabolites; B: Negative-ion metabolites. FA: Fatty acids; GL: Glycerolipids; GP: Glycerophospholipids; SP: Sphingolipids; ST: Steroid.

Figure 9 Volcano plots of differential metabolites. A: Model group vs normal control group; B: Anwei decoction group vs model group. Each dot represents a metabolite, with green dots indicating downregulated metabolites, red dots indicating upregulated metabolites, and gray dots indicating metabolites with no significant difference. VIP: Variable importance in projection.

Figure 10  Kyoto Encyclopedia of Genes and Genomes analysis. A: Kyoto Encyclopedia of Genes and Genomes (KEGG) classification of differential metabolites (model group vs normal control group); B: KEGG enrichment analysis (model group vs normal control group); C: KEGG classification of differential metabolites (Anwei decoction group vs model group); D: KEGG enrichment analysis (Anwei decoction group vs model group). KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 11  Correlation analysis. A: Correlation heatmap of top 20 differential metabolites and microorganisms (model group vs normal control group); B: Correlation heatmap of top 20 differential metabolites and microorganisms (Anwei decoction group vs model group). In the correlation heatmap, microorganisms are represented by rows, while metabolites correspond to columns. Positive correlations are indicated by red ellipses, whereas negative correlations are shown with blue ellipses. Ellipse narrowness reflects correlation strength, with narrower ellipses denoting stronger associations. Cells lacking ellipses represent correlations that were not statistically significant (P > 0.05).