Dual-pathway mechanism of vanadium-induced hepatotoxicity in ducks: Synergistic crosstalk between glucose homeostasis disruption and NADH/FSP1/COQ10 axis-driven ferroptosis

Abstract

In intensive duck production systems, vanadium (V) is widely used as a growth-promoting additive, but excessive supplementation poses health risks to ducks. Previous research indicated that V could cause damage to organs by disrupting the structure and function of mitochondria and the endoplasmic reticulum. However, the precise mechanism of mitochondrial-associated endoplasmic reticulum membranes (MAMs) in V-induced hepatotoxicity remains unclear. To fill this gap, this study employed network toxicology to analyze the hepatotoxicity of V, and further validated the pivotal roles of glucose homeostasis and ferroptosis in this process through targeted MAMs proteomics. The results indicated that V exposure increased liver dysfunction markers, disrupted hepatic cord structure, and widened ER-mitochondria gaps. Besides, V exposure up-regulated the levels of the IP3R-Grp75-VDAC1 complex in MAMs while promoting its dissociation. Moreover, the sequencing results of MAMs demonstrated that V primarily induced hepatotoxicity by disturbing the glycolysis/gluconeogenesis pathway. Notably, V exposure exacerbated lipid peroxides and Fe²⁺ accumulation while inhibiting the NADH/FSP1/CoQ₁₀ axis, down-regulating the expression levels of ferroptosis-related factors in livers. These findings demonstrated that dietary V overexposure impaired hepatic MAMs integrity, disrupted glucose homeostasis, and suppressed the NADH/FSP1/CoQ₁₀ axis, which ultimately induced ferroptosis-mediated liver injury in ducks.

Keywords: MAMs proteomics; NADH/FSP1/CoQ10 axis; ferroptosis; glucose homeostasis; vanadium.

Figure 1

V network toxicology analysis results. (A) Venn diagram for screening V-induced liver damage related genes. (B) KEGG enrichment results of V-induced liver damage related genes. (C) KEGG enrichment circle diagram of V-induced liver damage related genes. (D) PPI network diagram of V-induced liver damage related genes. (E) Level 2 GO terms of V-induced liver damage related genes.

Figure 2

V exposure induced liver injury in ducks. (A) Schematic of the experimental strategy. (B) ALT level. (C) AST level. (D) GGT level. (E) TBIL level. (F) Histopathological observation. The yellow arrows indicated hepatic sinusoids hemorrhage, the bule arrows indicated inflammatory cells infiltration in the livers and the green arrows indicated hepatocyte vacuolation, Scale bar: 100 μm. (G) Ultrastructure observation. The white arrows indicated the swelling of the endoplasmic reticulum, and the red line segments indicated an increase in the distance between the ER and the mitochondria, N: nucleus, Mito: mitochondria, ER: endoplasmic reticulum, Scale bar: 1 μm.

Figure 3

The interconnectivity in the IP3R/Grp75/VDAC1 complex was enhanced under V exposure in duck livers. (A) Schematic diagram of dissociation of the IP3R/Grp75/VDAC1 complex. (B) The relative mRNA levels of IP3R, Grp75, VDAC1, Mfn2, and PACS2. (C)The immunofluorescence colocalization images between VDAC1 and Grp75, scale bar is 50 μm. (D) The immunofluorescence colocalization images between IP3R and Grp75, scale bar is 50 μm. (E) The immunofluorescence colocalization images between VDAC1 and IP3R, scale bar is 50 μm. (F) The images of MAMs associated protein levels (IP3R, Grp75, VDAC1 and β-actin). (G, H) Gray value analysis. “*” indicated P < 0.05, “**” indicated P < 0.01 and “***” indicated P < 0.001 vs. Control group. The same scheme also applies to the remaining figures.

Figure 4

Protein identification and GO annotation of MAMs in duck livers. (A) Flow chart of quantitative proteomic analysis approach utilizing TMT labeling. (B) DEPs venn diagram. (C) Bar chart of DEPs. (D) Volcano Plot for differential protein expression in MAMs. (E) GSEA analysis plot of the top 10 GO-enriched pathways. (F) Circle of enrichment plot of the top 11 GO-enriched pathways. (G) Categorization of differentially expressed proteins based on their functional roles.

Figure 5

Differences in the proteomic profiles of MAMs induced by excessive V in duck livers. (A) Protein identification and KEGG annotation. (B) GSEA analysis plot of the top 10 KEGG-enriched pathways. (C) Circle of enrichment plot of the top 10 KEGG-enriched pathways. (D) KEGG enriched bar chart. (E) KEGG enrichment pathway network diagram. (F) Functional annotation of biological pathways was performed utilizing the KEGG database for in-depth analysis. (G) Differential proteins heatmap analysis. (H) Exploring PPIs among DEPs involved in glycolysis/gluconeogenesis-related pathway and key proteins of MAMs.

Figure 6

V induced the imbalance of glucose metabolism in duck livers. (A) Glycolysis model diagram. (B) The relative mRNA levels of AKR1A1, ALDH1A3 and PGK1. (C) The relative mRNA levels of ENO1, ENO2 and TPIS. (D) The images of glycolysis associated protein levels (AKR1A1, ALDH1A3, TPIS, ENO1, and β-actin). (E) Gray value analysis of AKR1A1, ALDH1A3 and TPIS proteins. (F) Gray value analysis of ENO1 protein. (G) Gluconeogenesis model diagram. (H)The relative mRNA levels of G6pase and FPB1. (I) The relative mRNA levels of PCK1 and PCK2. (J) The images of gluconeogenesis associated protein levels (PCK1 and G6pase, FPB1 and β-actin). (K) Gray value analysis of PCK1 and G6pase proteins. (L) Gray value analysis of FPB1 protein. (M) Representative images of PAS staining. Scale bar is 50 μm. (N) GLU level.

Figure 7

V induced ferroptosis via NADH/FSP1/CoQ₁₀ axis in duck lives. (A) The schematic diagram of molecular mechanism of V induced ferroptosis through interference with glycolysis. (B) Exploring PPIs among proteins involved in glycolysis-related pathways and key proteins of ferroptosis. (C) The immunohistochemical staining of 4-HNE. Scale bar is 50 μm. (D) DAB enhanced Prussian blue staining. Scale bar is 50 μm. (E) C_OQ₁₀H₂ content. (F) C_OQ₁₀ content. (G) The ratio of C_OQ₁₀H₂ content to C_OQ₁₀ content. (H) NADH content. (I) NAD+ content. (J) The ratio of NADH content to NAD+ content. (K) The immunohistochemical staining of FSP1. Scale bar is 50 μm. (L) The immunohistochemical staining of GPX4. Scale bar is 50 μm. (M) Gray value analysis of PTGS2, FSP1 and GPX4 proteins. (N) The images of ferroptosis associated protein levels (PTGS2, FSP1, GPX4 and β-actin). (O) The mRNA level of GPX4, ACSL4, PTGS2, FSP1.