Fermented Astragalus and its metabolites regulate inflammatory status and gut microbiota to repair intestinal barrier damage in dextran sulfate sodium-induced ulcerative colitis

Abstract

Fermentation represents an efficient biotechnological approach to increase the nutritional and functional potential of traditional Chinese medicine. In this study, Lactobacillus plantarum was used to ferment traditional Chinese medicine Astragalus, the differential metabolites in the fermented Astragalus (FA) were identified by ultra-performance liquid chromatography-Q Exactive hybrid quadrupole-Orbitrap mass spectrometry (UPLC-Q-Exactive-MS), and the ameliorating effect of FA on dextran sulfate sodium (DSS)-induced colitis in mice were further explored. The results showed that 11 differential metabolites such as raffinose, progesterone and uridine were identified in FA, which may help improve the ability of FA to alleviate colitis. Prophylactic FA supplementation effectively improved DAI score, colon length and histopathological lesion in DSS-treated mice. The abnormal activation of the intestinal immune barrier in mice was controlled after FA supplementation, the contents of myeloperoxidase (MPO) and IgE were reduced and the contents of IgA were increased. The intestinal pro-inflammatory factors TNF-α, IL-1β, IL-6, and IL-17 were down-regulated and the anti-inflammatory factors IL-10 and TGF-β were up-regulated, suggesting that FA can intervene in inflammatory status by regulating the balance of Th1/Th2/Th17/Treg related cytokines. In addition, FA supplementation modified the structure of the intestinal microbiota and enriched the abundance of Akkermansia and Alistipes, which were positively associated with the production of short-chain fatty acids. These microbes and their metabolites induced by FA also be involved in maintaining the intestinal mucosal barrier integrity by affecting mucosal immunity. We observed that intestinal tight junction protein and mucous secreting protein ZO-1, occludin, and MUC2 genes expression were more pronounced in mice supplemented with FA compared to unfermented Astragalus, along with modulation of intestinal epithelial cells (IECs) apoptosis, verifying the intestinal mucosal barrier repaired by FA. This study is the first to suggest that FA as a potential modulator can more effectively regulate the inflammatory status and gut microbiota to repair the intestinal barrier damage caused by colitis.

Keywords: Astragalus; fermentation; gut microbiota; inflammation; intestinal barrier.

FIGURE 1

FA alleviated the severity of DSS-induced colitis in mice: (A) schematic diagram of the animal experiment; (B) FA ameliorated the body weight loss associated with the dextran sulfate sodium (DSS)-induced colitis in mice; (C) DAI score; (D) representative images of colon tissues; (E) colonic lengths from different groups; (F) representative Hematoxylin and Eosin (H&E)-stained colon sections are shown at 20 × magnification. The same locations are marked with the same symbols. (a) Edema in loose connective tissue (*moderate edema); (b) crypt and goblet cell (*crypt distortion and loss of goblet cells); (c) epithelial layer (*broken epithelial layer); (d) inflammatory infiltration; (G) the histological score of the colons. Data are mean ± SD (n = 6). ^***P ≤ 0.001 and ^****P ≤ 0.0001 vs. the M group; ^##P ≤ 0.01 vs. the A group.

FIGURE 2

FA attenuated the colonic inflammation and modulated the inflammatory cytokines balance in DSS-induced mice: (A) MPO; (B) IgE; (C) IgA; (D) TNF-α; (E) IL-1β; (F) IL-6; (G) IL-10; (H) IL-17; (I) TGF-β in serum or colonic tissue. Data are mean ± SD (n = 5–6). *P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001, and ^****P ≤ 0.0001 vs. the M group; ^#P ≤ 0.05 and ^##P ≤ 0.01 vs. the A group.

FIGURE 3

FA altered the microbial diversity and gut microbiota composition: (A) venn plot that illustrated the observed ASV counts in samples; (B) NMDS analysis based on Jaccard distance; (C) phylum level of the gut microbiota composition; (D) genus level of the gut microbiota composition; (E) relative abundance of the ten most predominant gut microbiota at the genus level (n = 4). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 vs. the M group.

FIGURE 4

Key microbial communities characteristics and function of gut microbiota: (A) analysis of differences in the microbiota shown by LEfSe (linear discriminant analysis effect size); (B) the content of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and caproic acid in feces of DSS-induced mice; (C) correlation between key microbes, short chain fatty acids and parameters of colitis. Data are mean ± SD (n = 4). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 vs. the M group; ^#P ≤ 0.05 and ^##P ≤ 0.01 vs. the A group. The red color denotes a positive correlation, while blue color denotes a negative correlation. The intensity of the color is proportional to the strength of Spearman correlation.

FIGURE 5

FA repaired intestinal mucosal barrier structure and ameliorated cells apoptosis in DSS-induced mice: (A) DAO activity in serum; (B) the expression of ZO-1 in colon tissue; (C) occludin; (D) MUC2; (E) the level of the nucleosome in colon tissue; (F) the expression of Bax in colon tissue; (G) Bcl-2. Data are mean ± SD (n = 3–4). *P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001, and ^****P ≤ 0.0001 vs. the M group; ^#P ≤ 0.05, ^##P ≤ 0.01, and ^###P ≤ 0.001 vs. the A group. ns, no significance.