The Interaction between Oxidative Stress Biomarkers and Gut Microbiota in the Antioxidant Effects of Extracts from Sonchus brachyotus DC. in Oxazolone-Induced Intestinal Oxidative Stress in Adult Zebrafish

Abstract

Oxidative stress is a phenomenon caused by an imbalance between the production and accumulation of reactive oxygen species in cells and tissues that eventually leads to the production of various diseases. Here, we investigated the antioxidant effects of the extract from Sonchus brachyotus DC. (SBE) based on the 0.2% oxazolone-induced intestinal oxidative stress model of zebrafish. Compared to the model group, the treatment group alleviated oxazolone-induced intestinal tissue damage and reduced the contents of malondialdehyde, reactive oxygen species, IL-1β, and TNF-α and then increased the contents of superoxide dismutase, glutathione peroxidase, and IL-10. The 16s rDNA gene sequencing findings demonstrated that SBE could increase the relative abundance of Fusobacteriota, Actinobacteriota, and Firmicutes and decrease the relative abundance of Proteobacteria. Based on the correlation analysis between the oxidative stress biomarkers and intestinal flora, we found that the trends of oxidative stress biomarkers were significantly correlated with intestinal microorganisms, especially at the genus level. The correlations of MDA, IL-1β, and TNF-α were significantly negative with Shewanella, while SOD, GSH-Px, and IL-10 were significantly positive with Cetobacterium, Gemmobacter, and Flavobacterium. Consequently, we concluded that the antioxidant effect of SBE was realized through the interaction between oxidative stress biomarkers and gut microbiota.

Keywords: Sonchus brachyotus DC.; extract; gut microbiota; oxidative stress; zebrafish.

Figure 1

Changes in ROS content and MDA content caused by oxazolone injection time. The values are expressed as the mean ± SD. (n = 6 per group). Statistical differences between groups during the indicated time course were obtained according to repeated-measurement ANOVA (compared with the CON group, ^# p < 0.05, ^## p < 0.01).

Figure 2

Effects of SBE on the intestinal tissue of zebrafish. Representative hematoxylin and eosin (H&E)-stained intestinal sections and semiquantitative scoring showed tissue damage. The values are expressed as the mean ± SD. (n = 3). The thin black arrows represent degeneration, necrosis, and detachment of mucosal epithelial cells. The hollow arrows represent the exposed muscle layer. The folded arrow represents neutrophil infiltration. Thick black arrows represent villi breakage. Groups with different letters statistically differ (compared with the CON group, ^# p < 0.05, ^## p < 0.01).

Figure 3

Effects of SBE on the oxidative stress biomarkers. Zebrafish intestines were collected, and the ROS relative intensity of fluorescence (A), the MDA contents (B), and the enzyme activities of SOD (C), GSH-Px (D), and CAT (E) are shown. Fold changes are expressed as means ± SD (n = 30 per group). Groups with different letters statistically differ (compared with the CON group, ^# p < 0.05, ^## p < 0.01; compared with the Oxa group, * p < 0.05, ** p < 0.01).

Figure 4

Effects of SBE on the inflammatory factors. Zebrafish intestines were collected, and quantified real-time PCR and mRNA expressions of TNF-α (A), IL-1β (B), and IL-10 (C) are shown. Fold changes are expressed as means ± SD (n = 30 per group). Groups with different letters statistically differ (compared with the CON group, ^## p < 0.01; compared with the Oxa group, ** p < 0.01).

Figure 5

Effects of SBE on intestinal microbiota in zebrafish. A Venn diagram shows the overlap of the OTUs identified in the intestinal microbiota among CON, Oxa, and SBE groups (A). Analysis of alpha diversity (Shannon index) was used to detect differences between the groups (B). Plots of unweighted UniFrac-based PCoA (C). The top 15 bacteria, with a maximum abundance of gut bacteria at the phylum level (D). The top 15 bacteria, with a maximum abundance of gut bacteria at the genus level (E).

Figure 6

The correlation analysis between the contents of oxidative stress biomarkers and intestinal flora. The variation of oxidative stress biomarkers with relation to the intestinal microbiota. Correlation between biomarkers and intestinal microbiota at the phylum level (A). Histogram about the abundance of microbe that correlated with biomarkers contents at the phylum level (B–D). Correlation between biomarkers and intestinal microbiota at the genus level (E). Histogram about the abundance of microbe that correlated with biomarkers contents at the genus level (F–I). Mechanistic diagram of SBE regulating oxidative stress biomarkers via intestinal flora in zebrafish (J). A redder color indicates a positive correlation, while a bluer color indicates a negative correlation (* p < 0.05, ** p < 0.01. n ≥ 3/per group).

Figure 7

The correlation analysis between intestinal inflammatory factors and intestinal flora. Correlation between the mRNA expressions of TNF-α, IL-1β, and IL-10 and intestinal microbiota at the phylum level (A). Histogram about the abundance of microbe that significantly correlated with the expression of IL-10 at the phylum level (B,C). Correlation between the mRNA expressions of TNF-α, IL-1β, and IL-10 and intestinal microbiota at the genus level (D). Histogram about the abundance of microbe that significantly correlated with the expression of TNF-α, IL-1β, and IL-10 at the genus level (E–H). Mechanistic diagram of SBE regulating inflammatory factors via intestinal flora in zebrafish (I). A redder color indicates a positive correlation, while a bluer color indicates a negative correlation (* p < 0.05, ** p < 0.01. n ≥ 3/per group).