Nigella A relieved the symptoms of DSS indued UC mice
During continuous administration, mice of the control group grew well and slowly gained weight, while mice of DSS group and each drug intervention group lost weight, among which the DSS group had the highest degree of decline at the 8th day. During the administration period (2-8 days), mice that were given Nigella A showed a rapid weight loss from day 2 to day 6 when compared with the DSS group. The rapid weight loss of DSS group occurred on the day 5 to day 7. The weight loss of mice and the shorten of colon length were both improved. Colon length is one of main parameters to evaluate the severity of colitis. The reduction of colon length was approximately 23.8%, 13.1%, and 15.6% in colon length in DSS group, Nigella A groups and SASP group when compared with the normal group (Fig. 1a-b). Furthermore, the DAI scores were significantly increased in mice treated with Nigella A and SASP (Fig. 1c).
Nigella A alleviated the colonic injury of DSS-induced colitis mice
The colon structure of mice in the normal group was intact, and the distribution between mucosa, submucosa, muscularis and outer membrane was clear and complete (Fig. 2a). In the model group, colonic injury, such as the erosion of epithelial monolayer, crypt loss and infiltration of immune cells, etc. were observed in submucosa and muscle layer of the colon, and the histopathologic score was significantly increased than that of the control group (Fig. 2b). The inflammatory infiltration in SASP (200 mg/kg) and Nigella A (50 and 100 mg/kg) group was slightly improved, but goblet cell loss and epithelial cell proliferation were observed. Only a few gland pits and goblet cells were observed. Compared with the DSS group, the histopathological score of SASP and Nigella A groups were significantly decreased. The efficacy of Nigella A is better than SASP, especially the high dosage of Nigella A.
Microbial diversity analysis
As shown in the Venn diagram of Fig. 3a, 1234 OTUs appeared in all groups, while 25 OTUs overlapped in the control and model groups, suggesting that DSS could significantly decrease the diversity of bacteria. 69 OTUs overlapped in the control and SASP groups, suggesting that the positive drug SASP could evaluate the diversity of bacteria to some extent. 64 OTUs overlapped in the control group and Nigella A (100 mg/kg) group (Nig_A_100), suggesting that Nig_A_100 showed similar improvement of bacteria diversity to SASP. The OTUs of control group, model group, SASP group and Nig_A_100 were 100, 14, 21 and 52, respectively. These results suggested that the diversity of intestinal flora was significantly decreased in mice induced by DSS, and the diversity of intestinal flora in SASP group and Nig_A_100 group were improved to some extent.
The rarefaction curves tended to reach the saturated plateau, which indicated that the sequencing coverage is sufficient for further data analysis. As is shown in Fig. 3b and Table 1, DSS administration repressed the microbial richness (Chao1) when compared with healthy mice. The results from the observed species index showed there was changes in alpha diversity between the control group and the model group (Fig. 3c). Meanwhile, it was largely reversed in alpha diversity after the treatment of SASP and Nig_A_100, even though the Shannon and Simpson indices of these four groups were similar (Fig. 3d-e). These results showed that SASP and Nig_A_100 could significantly improve the microbial diversity in DSS-treated mice.
The results of principal component analysis (Fig. 4a), principal coordinate analysis (PCoA) of weighted (Fig. 4b) and unweighted Unifrac distance (Fig. 4c) suggested that no overlap between the control group and model group, indicating that the number of differential OTUs was high. However, SASP and Nig_A_100 groups were more inclined to the control group, suggesting that SASP group and Nig_A_100 groups had the function of regulating the tendency of flora to normal. The results of non-metric multi-dimensional scaling (NMDS) suggested that model group and control group showed significant isolated clusters, indicating a variation tendency of the altered main microbial composition structures (Fig. 4d-e). However, the community structure of SASP and Nig_A_100 groups were significantly inclined to the control group.
Metastatistical analysis showed that, at the phylum level, there were three phyla, including Firmicutes, Bacteria_unclassified and Proteobacteria, that displayed significant differences in the relative abundance between the model group and the control group (Table 2). These three phyla in the model group exhibited significant differences of the relative abundance with SASP and Nig_A_100 group (Fig. 5a). At the genus level, the changes of model group were observed in many bacterial genus (Fig. 5b). The taxonomic profiling showed a significant lower proportion of Lactobacillus, Porphyromonadaceae_unclassified, Alistipes, Acetivibrio, Barnesiella, Bacteria_unclassified and an a significant increase of Parabacteroides, Desulfovibrio, Romboutsia, Bacteroides, Escherichia, Clostridium_XlVb, Meniscus in the model when compared with the control group (Table 3). However, Nig_A_100 and SASP significantly reversed the levels of these changes. In addition, our results showed that there were significant changes in up to 61 genera after DSS treatment, and most of the changes could be reversed by SASP and Nig_A_100 (Table S1 and Fig. S1). These results showed that at least at the level of genus, Nig_A_100 and SASP could significantly regulate the changes of a variety of bacterins and then affect the intestinal flora in the model of ulcerative colitis mice.
Overall structure modulation of gut microbiota after Nig_A_100 treatment
Consistent with other results, the cladogram, generated from the linear discriminant analysis effect size (LEfSe) analysis, showed distinct gut microbiota compositions among mice from all groups (Fig. 5c-d). Great changes have happened of dominant bacterial taxa after the intervention of DSS. The comparison of dominant bacterial taxa at genus level demonstrated that DSS increased the relative abundance of p__Bacteroidetes/c__Bacteroidia/o__Bacteroidales/f__Bacteroidaceae/g__Bacteroides and p__Proteobacteria/c__Gammaproteobacteria/o__Enterobacteriales/f__Enterobacteriaceae/g__Escherichia. Meanwhile, DSS decreased the relative abundance of p__Firmicutes/c__Bacilli/o__Lactobacillales/f__Lactobacillaceae/g__Lactobacillus and p__Bacteroidetes/c__Bacteroidia/o__Bacteroidales/f__Porphyromonadaceae/g__Porphyromonadaceae_unclassified. However, the abundances of these main bacterial genus were significantly reversed by Nig_A_100 and SASP (Table S1).