Nuciferine Regulates Immune Function and Gut Microbiota in DSS-Induced Ulcerative Colitis

Abstract

Nuciferine, a major aporphine alkaloid obtained from the leaves of Nelumbo nucifera, exhibits anti-cancer and anti-inflammatory properties; however, its protective effects against inflammatory bowel diseases (IBD) has never been explored. In this study, an ulcerative colitis (UC) model was established in BALb/c mice by the continuous administration of 5% dextran sulfate sodium (DSS) in drinking water for 1 week. From day 8 to day 14, the DSS-treated mice were divided into a high-dose and a low-dose nuciferine treatment group and were intraperitoneally injected with the corresponding dose of the drug. Body weight loss, disease activity index (DAI), and colon length were measured. Histological changes were observed using hematoxylin and eosin staining. T lymphocyte proliferation was assessed by MTT assay. The ratio of CD3+, CD4+, CD8+, Th1, Th2, Th17, and Treg cells were estimated by flow cytometry. Finally, 16S rRNA sequencing was performed to compare the composition and relative abundance of the gut microbiota among the different treatment groups. The results showed that nuciferine treatment led to a significant improvement in symptoms, such as histological injury and colon shortening in mice with DSS-induced UC. Nuciferine treatment improved the Th1/Th2 and Treg/Th17 balance in the DSS-induced IBD model, as well as the composition of the intestinal microflora. At the phylum level, compared with the control group, the abundance of Firmicutes and Actinobacteriota was decreased in the model group, whereas that of Bacteroidetes increased. Meanwhile, at the genus level, compared with the control group, the numbers of the genera Lachnospiraceae_Clostridium, Bilophila and Halomonas reduced in the model group, while those of Bacteroides, Parabacteroides, and Paraprevotella increased. Notably, nuciferine administration reversed this DSS-induced gut dysbiosis. These results indicated that nuciferine modulates gut microbiota homeostasis and immune function in mice with DSS-induced UC.

Keywords: T cells; Th1/Th2 cells; Treg/Th17 cells; gut microbiota; immune function; nuciferine; ulcerative colitis.

Figure 1

Nuciferine reduced clinical signs in DSS-activated colitis (n = 6). (A) The illustrative images of colon samples (B) Average body weight (C) Average colon length and (D) Disease activity index score. (E) H&E staining of the distal colon tissues The dataare expressed as the mean ± S.D. Significant differences were considered at P < 0.05. ^a,b Bars without the same superscripts differ significantly (P < 0.05).

Figure 2

Effects of nuciferine on B cell, (A) The percentage of B220 cells in the PP nodes. (B) The percentage of IgA cells in the PP nodes (n = 6). The dataare expressed as the mean ± S.D. Significant differences were considered at P < 0.05. ^a−c Bars without the same superscripts differ significantly (P < 0.05).

Figure 3

Nuciferine regulated T cell proliferation and differentiation (n = 6). Effects of nuciferine on T cell proliferation in spleen (A) and mesenteric lymph nodes (B). (C) The percentage of CD3 cells in the mesenteric lymph nodes. (D) The percentage of CD4 cells in the mesenteric lymph nodes. (E) The percentage of CD8 cells in the mesenteric lymph nodes. (F) Comparison on the ratio of CD4/ CD8 between groups, The dataare expressed as the mean ± S.D. Significant differences were considered at P < 0.05. ^a−c Bars without the same superscripts differ significantly (P < 0.05).

Figure 4

Nuciferine restored the balance of Th1/Th2 Th17/Treg cells (n = 6). (A) The percentage of IL-4 cells in the spleens. (B) The percentage of IFN cells in the spleens. (C) The Th1/Th2 ratio in the spleens. (D) Flow cytometry of IL-4 and IFN in the spleens. (E) The percentage of Tregs cells in the spleens. (F) The percentage of Th17 cells in the spleens. (G) Flow cytometry of Tregs in the spleens. (H) Flow cytometry of Th17 in the spleens. The data are expressed as the mean ± S.D. Significant differences were considered at P < 0.05. ^a−d Bars without the same superscripts differ significantly (P < 0.05).

Figure 5

Species annotation and evaluation (n = 4). (A) pecies accumulation curve of samples. (B) Operational taxonomic units (OTU) relative abundance in individual samples of the control, DSS, Nuc, L-Nuc DSS and H-Nuc DSS groups. (C) Statistical chart of microbial groups at each classification level. The abscissa is arranged according to the sample name, and the abscissa is the number of microbial groups contained in each of the six classification levels of phylum, class, order, family, genus, and species. (D) Chao1 rarefaction curve. (E) Shannon rarefaction curve.

Figure 6

Change of gut microbiota composition in DSS-induced UC mice model under different drug interventions. (A) Gut microbial compositions at phylum level between groups, (B,C) Comparison on relative abundances of Bacteroidetes between groups, (C) Comparison on relative abundances of Firmicutes between groups (D), Comparison on relative abundances of Actinobacteria between groups, (E) Gut microbial compositions at genus level, (F) Comparison on relative abundances of Bacteroides between groups (G) Comparison on relative abundances of Halomonas between groups, (H) Comparison on relative abundances of Parabacteroides between groups, (I) Comparison on relative abundances of BacteroideParaprevotellas between groups, (J) Comparison on relative abundances of Bilophila between groups. (K) Comparison on relative abundances of Lachnospiraceae_Clostridium between groups The data are expressed as the mean ± S.D (n = 4). Significant differences were considered at P < 0.05. ^a−d Bars without the same superscripts differ significantly (P < 0.05).

Figure 7

Change of gut microbiota structure in DSS-induced UC mice model under different drug interventions (n = 4). (A) Principal components analysis (PCA) for DSS- induced UC mice model with different treatments. (B) Principal coordinates analysis (PCoA) for DSS-induced UC mice model with different treatments. (C) Orthogonal Partial Least Squares Discriminant Analysis, and (D) Venn diagram analysis.

Figure 8

Nuciferine repaired intestinal mucosal damage. (A) Representative immunohisto chemical (IHC) staining of ZO-1. (B) IHC staining of occludin. (C) IHC analysis of claudin-1 in colon tissues. (D) Immunohistochemical analysis of ZO-1. (E) Immunohistochemical analysis of occludin. (F) Immunohistochemical analysis of claudin-1. The dataare expressed as the mean ± S.D (n = 6). Significant differences were considered at P < 0.05. ^a−c Bars without the same superscripts differ significantly (P < 0.05).