The Protective Effects of Carrageenan Oligosaccharides on Intestinal Oxidative Stress Damage of Female Drosophila melanogaster

Abstract

Carrageenan oligosaccharides (COS) have been reported to possess excellent antioxidant activities, but the underlying mechanism remains poorly understood. In this study, H₂O₂ was applied to trigger oxidative stress. The results showed that the addition of COS could effectively extend the lifespan of female Drosophila, which was associated with improvements by COS on the antioxidant defense system, including a decrease in MDA, the enhanced activities of SOD and CAT, the reduction of ROS in intestinal epithelial cells, and the up-regulation of antioxidant-relevant genes (GCL, GSTs, Nrf2, SOD). Meanwhile, the axenic female Drosophila fed with COS showed almost no improvement in the above measurements after H₂O₂ treatment, which highlighted the antioxidant mechanism of COS was closely related to intestinal microorganisms. Then, 16S rDNA high-throughput sequencing was applied and the result showed that the addition of COS in diets contributed to the diversity and abundance of intestinal flora in H₂O₂ induced female Drosophila. Moreover, COS significantly inhibited the expression of gene mTOR, elevated its downstream gene 4E-BP, and further inhibited autophagy-relevant genes (AMPKα, Atg1, Atg5, Atg8a) in H₂O₂ induced female Drosophila. The inhibition of the mTOR pathway and the activation of autophagy was probably mediated by the antioxidant effects of COS. These results provide potential evidence for further understanding of COS as an intestinal antioxidant.

Keywords: Drosophila melanogaster; carrageenan oligosaccharides; gut flora; intestinal oxidative stress.

Figure 1

The survival curve of H₂O₂-induced female Drosophila with different feeding: (A) normal fruit flies, (B) axenic fruit flies.

Figure 2

Effect of COS on the average lifespan (A,D), half time to death (B,E) and maximum lifespan (C,F) of H₂O₂ treated Drosophila. Charts (A–C) present the results of normal fruit flies, and (D–F) present the results of axenic fruit flies. Differences among treatments were obtained with one-way analysis of variance followed by Tukey’s multiple comparisons test. When compared with the H₂O₂ treated group, * and ** represent p < 0.05 and p < 0.01 respectively.

Figure 3

Effects of COS on SOD (A,D), CAT (B,E) and MDA (C,F) levels in female Drosophila. Charts (A–C) present the results of normal fruit flies, and (D–F) present the results of axenic fruit flies. Differences among treatments were obtained with one-way analysis of variance followed by Tukey’s multiple comparisons test. When compared with the H₂O₂ treated group, * and ** represent p < 0.05 and p < 0.01 respectively.

Figure 4

The effect of COS on gut integrity in female Drosophila. The dye of FD & C Blue No.1 is displayed in female Drosophila (A) and axenic female Drosophila (B) with different treatments. The percentage of “Smurf” flies in normal and axenic types are shown in (C) and (D) respectively. Differences among treatments were obtained with one-way analysis of variance followed by Tukey’s multiple comparisons test. When compared with the H₂O₂ treated group, ** represent p < 0.01 respectively.

Figure 5

The length and width of guts in female fruit flies (A,B) and axenic fruit flies (C,D). Differences among treatments were obtained with one-way analysis of variance followed by Tukey’s multiple comparisons test. When compared with the H₂O₂ treated group, * and ** represent p < 0.05 and p < 0.01 respectively.

Figure 6

Gut dissected from female Drosophila (A,C) or axenic female Drosophila (B,D) with different treatments were stained with 7-AAD (red) and DHE (red) respectively. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Differences among treatments were obtained with one-way analysis of variance followed by Tukey’s multiple comparisons test, * and ** represent p < 0.05 and p < 0.01 respectively.

Figure 7

Analysis of alpha diversity and beta diversity of the intestinal microbiota: (A) Shannon index, (B) Simpson index, (C) principal co-ordinate analysis (PCoA), and (D) nonmetric multidimensional scaling (NMDS) analysis.

Figure 8

The relative abundance of the gut flora of Z-H₂O₂, Z-SUC, and COS-M groups at the phylum level (A), and genus level (B). The relative abundance of the gut flora of Z-H₂O₂, Z-SUC, and COS-M groups with significant differences at the phylum level (C), and genus level (D). Differences among treatments were obtained with one-way analysis of variance followed by Tukey’s multiple comparisons test, * and ** represent p < 0.05 and p < 0.01 respectively.

Figure 9

Carrageenan oligosaccharides alter the gut microbiota and the relevant pathways of H₂O₂-induced female Drosophila. (A) The cladogram of the gut microbiota community of female Drosophila in Z-H₂O₂, Z-SUC and COS-M groups. (B) Prediction of the relative abundance of stress-tolerant bacteria of the gut microbiota of female Drosophila in Z-H₂O₂, Z-SUC and COS-M groups using BugBase. (C) KEGG pathways predicted using PICRUSt 2, and the t-test was used to perform a significance analysis.

Figure 10

The relative expression levels of target genes in Z-SUC, Z-H₂O₂ and COS-M groups. (A) Genes associated with antioxidant activities. (B) Genes relevant to autophagy. (C) Immune-related genes. Differences among treatments were obtained with one-way analysis of variance followed by Tukey’s multiple comparisons test. * and ** represent p < 0.05 and p < 0.01 respectively.