Vitamin B6 Metabolic Pathway is Involved in the Pathogenesis of Liver Diseases via Multi-Omics Analysis

Abstract

Purpose: To clarify the underlying regulatory mechanisms of progression from liver cirrhosis to hepatocellular carcinoma (HCC), we analyzed the microbiomics, metabolomics, and proteomics in plasma and tissues from patients with HCC or decompensated liver cirrhosis (DC).

Patients and methods: Tissues and plasma from 44 HCC patients and 28 patients with DC were collected for metabolomic analysis. 16S rRNA sequencing was performed in nine HCC tissues (HCCT), four distal noncancerous tissues (HCCN), and 11 DC tissues (DCT). Five HCC tissues had liver cirrhosis (HCCT-LC). Five hepatocellular carcinoma tissues without liver cirrhosis (HCCT-NLC) and five DCT were selected for proteomic sequencing. After combining proteomic and metabolomic analysis, we constructed a mouse model of chronic liver injury using carbon tetrachloride (CCl4) and treated them with vitamin B6 (VB6).

Results: 16s rRNA sequence results showed that HCC tissues had higher alpha diversity. The highest LDA scores were detected for Elizabethkingia in HCCT, Subsaxibacter in DCT, and Stenotrophomon in HCCN. Metabolomics results demonstrated some metabolites, including capric acid, L-threonate, choline, alpha-D-Glucose, D-ribose, betaine, 2E-eicosenoic acid, linoleic acid, L-palmitoylcarnitine, taurodeoxycholic acid, L-pyroglutamic acid, androsterone sulfate, and phthalic acid mono-2-ethylhexyl ester (MEHP), had better diagnostic efficacy than AFP (AUC: 0.852; 95% CI: 0.749, 0.954). In a combined analysis of metabolomics and proteomics, we found that HCCT-LC had more obvious disorders of VB6 metabolism and pentose and glucuronate interconversions than DCT, and kynurenine metabolism disorder was more significant in HCCT-LC than in HCCT-NLC. In the CCl4-induced chronic liver injury model, after VB6 supplementation, inflammatory cell infiltration, hepatocyte edema, and degeneration were significantly improved.

Conclusion: We found significant differences in the flora distribution between HCCT and DC; MEHP was a new diagnostic biomarker of HCC, and VB6 ameliorated the inflammatory cell infiltration, hepatocyte edema, and degeneration in chronic liver injury.

Keywords: cirrhosis; hepatocellular carcinoma; metabolomics; microbiomics; proteomics; vitamin B6.

Figure 1

The flow chart of our study. Plasma isolated from peripheral blood was used for metabolomics analysis. Hepatocellular carcinoma tissues, normal adjacent tissues and decompensated liver cirrhosis tissues were used for metabolomics analysis, proteomics analysis, and 16S rRNA sequencing.

Figure 2

Tissue microbiota composition of HCCT, HCCN and DCT. (A) Stacked graph depicting composition of bacterial genera with relative abundance >10⁻⁵ in the cohort. (B) Diversity analysis of liver tissue microbiota of the whole cohort. ****P < 0.001; ***P < 0.01; ns, P ≥ 0.05; by one-way analysis of variance (ANOVA). (C) Characteristics of liver tissue microbiota in the cohort were evaluated with LEfSe analysis. Bacterial populations with LDA score >2 are displayed. (D) Phylogenetic classification of bacterial populations with LDA score >2. (E) Frequencies of the tissue bacterial genera according to LDA score. Mean±SE is displayed. (F) Heat map depicting the weight distribution of the 54 genera with LDA score >2. HCCT: hepatocellular carcinoma tissue.

Figure 3

Common metabolites in the blood between groups HCCB vs NCB and DCB vs NCB. (A) Venn diagram of the overlapping metabolites in negative ion mode. (B) Heatmap of the overlapping 19 metabolites in negative ion mode. (C) Heatmap of the overlapping 19 metabolites in negative ion mode. (D) Venn diagram of the overlapping metabolites in positive ion mode. (E) Heatmap of the overlapping 23 metabolites in positive ion mode. (F) Heatmap of the overlapping 23 metabolites in positive ion mode.

Figure 4

Common metabolites in the tissue between groups HCCT vs HCCN and DCT vs HCCN. (A) Venn diagram of the overlapping metabolites in negative ion mode. (B) Heatmap of the overlapping 47 metabolites in negative ion mode. (C) Heatmap of the overlapping 47 metabolites in negative ion mode. (D) Venn diagram of the overlapping metabolites in positive ion mode. (E) Heatmap of the overlapping 44 metabolites in positive ion mode. (F) Heatmap of the overlapping 44 metabolites in positive ion mode. HCCT: hepatocellular carcinoma tissue.

Figure 5

Characteristics of the biomarker signature from HCC VS DC (A) Heatmap of 16 differential metabolites among four groups: HCCB vs NCB, DCB vs NCB, HCCT vs HCCN and DCB vs HCCN. (B) ROC curve of AFP and 16 differential metabolites.

Figure 6

Proteomic analysis between HCCT-LC vs DC and HCCT-LC vs HCCT-NLC (A) Flow chart of proteomics. (B) The histogram of the differential proteins between the two groups of HCCT-LC vs DC and HCCT-LC vs HCCT-NLC. (C) Heatmap of up-regulated and down-regulated differential proteins in HCCT-LC vs DC group. (D) Log2 differential proteins between HCCT-LC and DC group is plotted on the x-axis, and the –log10 p-value is plotted on the y-axis. Probes that are identified to be significantly different between two groups are colored in red (up-regulation) and blue (down-regulation). (E) Domain enrichment shows that top 20 domain of HCCT-LC vs DC group. (F) KEGG plot indicates that enrichment pathways of HCCT-LC vs DC group. (G) Heatmap of up-regulated and down-regulated differential proteins in HCCT-LC vs HCCT-NLC group. (H) Log2 differential proteins between HCCT-LC and HCCT-NLC group is plotted on the X-axis, and the –log10 p-value is plotted on the Y-axis. Probes that are identified as significantly different between two groups are colored in red (up-regulation) and blue (down-regulation). (I) Domain enrichment shows that top 20 domain of HCCT-LC vs HCCT-NLC group. (J) KEGG plot indicates that enrichment pathways of HCCT-LC vs HCCT-NLC group.

Figure 7

Combined analysis of metabolomics and proteomics. (A) The Venn diagram of the pathways involved in the differential metabolites and proteins between HCCT-LC group and DC group, and the overlap represents the shared pathways. (B) The top 10 pathways in quantity of metabolites and proteins co-participation between HCCT-LC group and DC group. (C) The KEGG pathway enrichment based on the differential metabolites and proteins between HCCT-LC group and DC group. (D) The Venn diagram of the pathways involved in the differential metabolites and proteins between HCCT-LC and HCCT-NLC group, and the overlap represents the shared pathways. (E) The top 10 pathways in quantity of metabolites and proteins co-participation between HCCT-LC and HCCT-NLC group. (F) The KEGG pathway enrichment based on the differential metabolites and proteins of HCCT-LC and HCCT-NLC group. Different colors represent different omics, blue represents proteomics, and Orange represents metabolomics.

Figure 8

Schematic diagram of the proteins and metabolites pathways between HCCT-LC vs DCT and HCCT-LC vs HCCT-NLC. (A) Vitamin B6 metabolism. (B) Pentose and glucuronate interconversions metabolism. (C) African trypanosomiasis. Green means that the expression of proteins and metabolites is down-regulated and red means up regulated, the FDR-adjusted p-values were less than 0.001.

Figure 9

VB6 has protective effect on CCl4-induced chronic liver injury model. (A) Flow chart of experimental design of VB6 intervention on liver cirrhosis in mice. (B) AST, ALT and TBIL in mice after the intervention with CCl4 and/or VB6. (C) Liver pathological changes in HE staining and Masson staining after intervention with CCl4 and/or VB6. (D) Ishak score after Masson staining of liver. (E) Collagen volume fraction calculated by ImageJ after Masson staining of liver. ns means P ≥ 0.05.