Radix Pseudostellaria polysaccharides alleviate sepsis-induced liver injury by modulating the gut microbiota via the TLR4/NF-κB pathway

Abstract

Background: Sepsis-induced liver injury (SLI) is a life-threatening complication with limited therapeutic options. Radix Pseudostellariae polysaccharides (RPPS), a component of traditional Chinese medicine, exert immunomodulatory, anti-inflammatory, and antioxidant properties. Herein, we investigated the therapeutic effects and mechanisms of RPPS on SLI.

Methods: A murine sepsis model was established using cecal ligation and puncture. Mice were pretreated with RPPS or saline for 14 days. Subsequently, multi-omics integration-including metagenomics, proteomics, and network pharmacology-was employed to elucidate the mechanisms of RPPS. Liver injury was assessed via serum biomarkers, histopathology, and transmission electron microscopy, while intestinal barrier integrity was evaluated through histopathological analysis. Gut microbiota composition and functional pathways were examined using metagenomic sequencing. Furthermore, Kyoto Encyclopedia of Genes and Genomes enrichment analyses of gut microbiota, liver proteomics, and network pharmacology data were integrated to predict key target pathways, which were experimentally validated in mice.

Results: RPPS pretreatment significantly improved survival, reduced liver injury markers, attenuated hepatic necrosis and inflammation, and restored intestinal barrier integrity. RPPS also modulated the gut microbiota by enriching beneficial taxa and suppressing pathogens. Multi-omics integration identified the toll-like receptor 4 (TLR4)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway as the core mechanism, and experimental validation confirmed that RPPS inhibited TLR4 membrane expression, MyD88/IKKα/β activation, NF-κB p65 phosphorylation, and nuclear translocation. In conclusion, RPPS alleviates SLI by protecting the intestinal barrier, modulating gut microbiota, and suppressing the TLR4/NF-κB signaling pathway.

Conclusion: This study provides a scientific foundation for RPPS as a potential therapeutic candidate in sepsis treatment.

Keywords: Radix Pseudostellariae polysaccharides; TLR4/NF-κB; gut-liver axis; multi-omics; sepsis.

FIGURE 1

Radix Pseudostellariae polysaccharides (RPPS) attenuate CLP-induced liver inflammation and injury in mice. (A) The experimental design and procedures for RPPS administration. (B) Survival rate. (C) MSS in the NS + CLP and RP + CLP groups. (D) Liver morphology. (E) Histological representation of liver sections using hematoxylin and eosin (H,E) staining (×100 and ×200 magnification, n = 10). (F) Immunohistochemical staining of CD68 (×100 and ×200 magnification, n = 6). (G) Representative transmission electron microscopy images of hepatocytes. Bar = 1 μm (n = 6–8). (H–J) Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) levels. (K) Histopathological score from H&E sections. (L) The rate of CD68-positive cell density in the liver. (M–O) Levels of TNF-α, IL-1β, and IL-6 in liver tissue. Data are presented as mean ± standard error of mean. A one-way analysis of variance with Tukey’s analysis was used for multiple comparisons. Significance levels are represented as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, with “ns” indicating “not significant.”

FIGURE 2

Radix Pseudostellariae polysaccharides (RPPS) restore intestinal morphology and intestinal barrier function in cecal ligation and puncture (CLP) sepsis-induced mice. (A) Colon morphology. (B,C) Histological representation of colonic sections using hematoxylin and eosin (H&E) and Periodic acid-Schiff (PAS) staining. (×40 and ×200 magnification, n = 6–10) (D) Immunofluorescence staining of occludin in colonic sections. (E) Histopathological scoring from H&E sections. (F) Colon crypt depth of each group from H&E sections (n = 6). (G) The number of goblet cells in the colon from PAS sections (n = 6). (H) The occludin expression in colon tissues; the scale bar represents 625 μm (n = 6). Data are presented as mean ± standard error of mean. A one-way analysis of variance with Tukey’s analysis was used for multiple comparisons. Significance levels are represented as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, with “ns” indicating “not significant.”

FIGURE 3

Radix Pseudostellariae polysaccharides (RPPS) modulate the composition of gut microbiota. (A) Alpha diversity of gut microbiota. (B) Beta diversity difference analysis. (C) Principal Coordinate Analysis (PCoA) analysis at the species level. (D) Bar plots of the relative abundance of intestinal microbiota at the species level. (E–H) The relative abundance of Lactobacillus johnsonii, Muribaculum intestinale, Duncaniella spp. increased, and that of Escherichia coli decreased across different groups. (I,J) Linear discriminant analysis Effect Size (LEfSe) Hierarchical Clustering Tree Plot and the histogram of linear discriminant analysis (LDA) between the NS + CLP and RP + CLP groups. Data are expressed as the mean ± SD. An LDA score of >4.0 was considered statistically significant. Significance levels are represented as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, with “ns” indicating “not significant.” Kruskal–Wallis rank-sum test (Dunn’s test) in (A), Wilcoxon rank-sum test in (B), Kruskal–Wallis H-test at a species level in (E–H), Spearman’s rank-correlation coefficients were calculated for correlation analysis, p < 0.05; n = 6–8.

FIGURE 4

Gut microbiota is correlated with liver function and hepatic inflammatory factors. (A) Correlation heatmap of intestinal microbiota and hepatic inflammatory cytokines. (B) Correlation heatmap of intestinal microbiota and liver function. (C) KEGG enrichment assay between the NS + CLP and RP + CLP groups.

FIGURE 5

Proteomics reveals the critical role of NF-κB signaling pathway in the phenotype of sepsis-induced liver injury (SLI) following Radix Pseudostellariae polysaccharides (RPPS) treatment. (A) Principal component analysis (PCA) of liver proteomes. (B) Gene set enrichment analysis (GSEA) comparing RP + CLP and NS + CLP groups, highlighting significant enrichment of the NF-κB signaling pathway in the RP + CLP group. (C) Detailed GSEA results for the MMU04064 gene set showing differential expression of NF-κB pathway proteins between the RP + CLP and NS + CLP groups.

FIGURE 6

Prediction of targets of Radix Pseudostellariae polysaccharides (RPPS) treatment in sepsis-induced liver injury (SLI). (A) The common target of RPPS in the treatment of SLI. (B) protein–protein interaction (PPI) network of common targets. (C) KEGG enrichment analysis of core targets. (D) Potentially active ingredients that intersect with sepsis targets. (E) The chemical structures of the screened small molecules. (F) Molecular docking between the TLR4 and screened small molecules.

FIGURE 7

The beneficial effects of Radix Pseudostellariae polysaccharides (RPPS) on sepsis progression are associated with the TLR4/NF-κB pathway. (A) Immunofluorescent staining of Toll-like receptor 4 (TLR4) in liver tissues. (×200 magnification, n = 3). (B) Quantification of the percentage of TLR4-positive cells. (C) Immunofluorescent staining of p-p65 in liver tissues. (D) Quantification of the percentage of p-p65-positive cells. (Bar = 50 μm, n = 3). (E) Representative Western blot images of the NF-κB pathway. (F–J) Expression and quantification of MyD88, IKK α/β, NF-κB (p65), and pNF-κB (pp65) proteins using Western blot (n = 3). (K) Immunohistochemical staining for IL-1β and TNFα (×100 and ×200 magnification). (L,M) Percentage of IL-1β and TNFα positive area in liver tissue (n = 6). Data are expressed as the mean ± standard error of mean, and the significance of the differences between groups was assessed using one-way analysis of variance (Tukey’s test). *p < 0.05, **p < 0.05.