Fig 2: Schematic illustration of the potential mechanisms associated with FU-mediated attenuation of Pb-induced liver injury. Pb exposure disrupts gut microbiota homeostasis, induces microbial dysbiosis, and increases systemic Pb burden, thereby contributing to hepatic oxidative stress, inflammatory responses, and liver injury. FU treatment decreased Pb levels in serum and liver and increased fecal Pb content. In the intestine, FU treatment was associated with remodeling of Pb-disrupted gut microbiota, including increased relative abundance of beneficial bacteria such as Akkermansia muciniphila and Bifidobacterium pseudolongum, as well as alterations in fecal tryptophan metabolism, particularly tryptophol (TOL). Through the gut–liver axis, these FU-associated intestinal changes may contribute to the alleviation of Pb-induced hepatic injury. In the liver, FU protection was associated with IGFBP1-related redox modulation, increased antioxidant capacity, including SOD activity and GSH content, reduced lipid peroxidation, as reflected by decreased MDA levels and suppressed inflammatory responses, including TNF-α, IL-1β, and IL-6. These coordinated changes were accompanied by reduced serum ALT, AST, and ALP levels, alleviated hepatic pathological lesions, and improved hepatic homeostasis.

Fig 3: IGFBP1 is associated with FU-mediated protection against Pb-induced liver injury. (A) Schematic illustration of the experimental design for IGFBP1 rescue experiments; (B) serum IGFBP1 levels in the Model, FU-H, and IGFBP1+FU-H groups; (C) body weight of mice at the end of the experiment; (D) liver organ weight index; (E) representative H&E-stained liver sections from the Model, FU-H, and IGFBP1+FU-H groups. The upper panels show 100×-magnification images (scale bar = 200 μm), and the lower panels show enlarged views of the dashed areas (scale bar = 100 μm). Red arrows indicate inflammatory cell infiltration, and green arrows indicate fatty vacuolation; (F) Histology scores of liver sections. (G–I) Serum levels of hepatic injury biomarkers, including ALT (G), AST (H), and ALP (I); (J–L) serum oxidative stress-related indicators, including SOD activity (J), GSH level (K), and MDA level (L); (M–O) hepatic oxidative stress-related indicators, including SOD activity (M), GSH level (N), and MDA level (O). (P–R) Serum levels of pro-inflammatory cytokines, including IL-6 (P), TNF-α (Q), and IL-1β (R); Data are presented as mean ± SD, n = 6 per group. Statistical significance was determined by one-way ANOVA. Compared with the FU-H group: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Fig 4: Integrated transcriptomic and network toxicology analyses identify potential molecular targets of FU against Pb-induced liver injury. (A) MA plots showing differentially expressed genes in the comparisons of Model versus Control and FU-H versus Model; (B) Venn diagram showing the overlap of differentially expressed genes between the Model versus Control and FU-H versus Model comparisons. A total of 111 common differentially expressed genes were identified; (C) KEGG pathway enrichment analysis of the overlapping differentially expressed genes. Bubble size represents the number of enriched genes, and bubble color represents the adjusted p value; (D) Venn diagram showing the intersection between Pb-related targets and liver injury-associated targets identified by network toxicology analysis; (E) GO enrichment circular plot of the intersecting targets, including biological process, cellular component, and molecular function categories; (F) GO Biological Process network illustrating the relationships between enriched biological processes and their associated genes; (G) KEGG pathway enrichment analysis of the intersecting targets obtained from network toxicology analysis; (H) heatmap showing the expression profiles of MAPK signaling pathway-related differentially expressed genes among the Control, Model, and FU-H groups. IGFBP1 was identified as the most prominently altered candidate gene associated with FU-mediated protection. Differentially expressed genes were screened using the criteria of |log2 fold change| ≥ 1 and adjusted p < 0.05. Enrichment analyses were performed based on significantly enriched GO terms and KEGG pathways.