Investigation of Gynura segetum root extract (GSrE) induced hepatotoxicity based on metabolomic signatures and microbial community profiling in rats

Abstract

In recent years, many reports focus on the hepatotoxicity of Gynura segetum root extract (GSrE), but the interaction between GSrE and the gut microbiota is still unclear. This study investigated the mechanism of GSrE-induced hepatotoxicity of different doses and exposure durations by combining metabolomics and gut microbiota analysis. SD rats were divided into 3 groups: blank, low-dose (7.5 g/kg), and high-dose (15 g/kg) groups. Urine and feces samples were collected on day 0, day 10, and day 21. Metabolomics based on gas chromatography-mass spectrometry (GC-MS) was carried out to identify metabolites and metabolic pathways. 16S rDNA gene sequencing was applied to investigate the composition of gut microbiota before and after GSrE-induced hepatotoxicity. Finally, a correlation analysis of metabolites and gut microbiota was performed. Differential metabolites in urine and feces involved amino acids, carbohydrates, lipids, organic acids, and short chain fatty acids. Among them, L-valine, L-proline, DL-arabinose, pentanoic acid, D-allose, and D-glucose in urine and D-lactic acid and glycerol in fecal metabolites depended on the exposure of time and dose. In addition, 16S rDNA sequencing analysis revealed that GSrE-induced hepatotoxicity significantly altered the composition of gut microbiota, namely, f_Muribaculaceae_Unclassified, Lactobacillus, Bacteroides, Lachnospiraceae_NK4A136_group, f_Ruminococcaceae_Unclassified, Prevotellaceae_Ga6A1_group, and Escherichia-Shigella. The correlation analysis between gut microbiota and differential metabolites showed the crosstalk between the gut microbiota and metabolism in host involving energy, lipid, and amino acid metabolisms. In summary, our findings revealed that peripheral metabolism and gut microbiota disorders were time- and dose-related and the correlation between gut microbiota and metabolites in GSrE-induced hepatotoxicity.

Keywords: Gynura segetum (Lour.) Merr.; correlation analysis; gut microbiota; hepatotoxicity; metabolomics.

Figure 1

The Principal Component Analysis (PCA) between different groups of urine (A–D) and fecal (E–H) samples. (A,E) Different doses of 10 day-groups; (B,F) different doses of 21 day-groups; (C,G) 0, 10, 21 days of low-dose groups; (D,H), 0, 10, 21 days of high-dose groups. (n = 4–8).

Figure 2

The orthogonal partial least squares-discriminant analysis (OPLS-DA) between different groups of urine (A–H) and fecal (I–P) samples. (A,I) blank group vs. low-dose group on the 10th day; (B,J) blank group vs. high-dose group on the 10th day; (C,K) blank group vs. low-dose group on the 21st day; (D,L) blank group vs. high-dose group on the 21st day; (E,M) 0-day vs. 10-day of low-dose groups; (F,N) 0-day vs. 21-day of low-dose groups; (G,O) 0-day vs. 10-day of high-dose groups; (H,P) 0-day vs. 21-day of high-dose groups. (n = 4–8).

Figure 3

The Venn diagram of differential metabolites in urine (A) and fecal (B) samples.

Figure 4

Metabolic pathway analysis of urine samples with different groups. (A) pathways of low-dose and high-dose groups on the 10th day. (B) pathways of low-dose and high-dose groups on the 21th day. (C) pathways on the 10th day and 21st day of low-dose groups. (D) pathways on the 10th day and 21st day of high-dose groups.

Figure 5

Metabolic pathway analysis of fecal samples with different groups. (A) Pathways of low-dose and high-dose groups on the 10th day. (B) Pathways of low-dose and high-dose groups on the 21th day. (C) Pathways on the 10th day and 21st day of low-dose groups. (D) Pathways on the 10th day and 21st day of high-dose groups.

Figure 6

Gut microbiota analysis of each group. (A) Alpha diversity analysis. (B–E) PCA analysis on the 10th day groups (B), 21st day groups (C), low-dose groups (D), and high-dose groups (E). (F) Relative abundance of significantly altered taxa at the phylum level. (G) Relative abundance of significantly altered taxa at the genus level. * P < 0.05, compared with the blank groups; ** P < 0.01, compared with the blank groups; # P < 0.05, compared with the low dose groups, n = 4–8.

Figure 7

Species with significantly relatively high abundance and significant differences at genus level. (A) f_Muribaculaceae_Unclassified; (B) Lactobacillus; (C) Bacteroides; (D) Lachnospiraceae_NK4A136_group; (E) f_Ruminococcaceae_Unclassified; (F) Prevotellaceae_Ga6A1_group; (G) Escherichia-Shigella. * P < 0.05, ** P < 0.01, compared with the blank groups; # P < 0.05, ## P < 0.01, compared with the low dose groups; & P < 0.05, && P < 0.01, comparison between groups at the same dose, n = 4–8.

Figure 8

Heatmaps of correlation analysis between top 10 abundance species at genus level and urine (A) or fecal (B) metabolites. * P < 0.05; ** P < 0.01.