Nigella A ameliorates experimental colitis via interacting with gut microbiota on dextran sulphate sodium (DSS)-induced C57BL/6 mice

Nigella A, also named Sieboldianoside A, has been extracted from many kinds of Traditional Chinese Medicine (TCM), such as Nigella glandulifera, Stauntonia chinensis DC., and the leaves of Acanthopanax sieboldianus. Nigella A exhibited potential analgesic, anti-inammatory, anti-tumor, and antioxidant activities. However, whether Nigella A could treat ulcerative colitis (UC) is still unknown. As saponins always be regarded as the kinds of ingredients that could regulate immunity and intestinal ora. This research aimed to investigate the therapeutic effect of Nigella A on UC and explore its effect on intestinal ora. We noted that Nigella A and SASP could signicantly improve the signs and symptoms, alleviate colonic pathological injury in DSS-induced mice. The changing of many specic bacterial genus such as Lactobacillus, Porphyromonadaceae, Bacteroides and Escherichia might closely related to the recovery of intestinal inammatory response. This study initially conrmed the therapeutic effect of Nigella A and SASP on DSS-induced colitis by improving the diversity of intestinal microbial composition. Nigella A has the potential to be developed for the treatment of UC and other disorders related to the imbalance of intestinal ora.


Introduction
In ammatory bowel disease (IBD) is an idiopathic disease that affects the ileum, rectum and colon, including ulcerative colitis (UC) and crohn's disease (CD). UC is a kind of serious disease of digestive system with high incidence worldwide (Ng et al. 2018). In ammation, intestinal ora, and immunity were strongly related to the occurrence and recovery of UC (Huang and Chen, 2016). The interaction between the intestinal microbial community and the mucosal immune system has been identi ed as the key to chronic in ammation, and the changes of The seeds of Nigella glandulifera Freyn et Sint (N. glandulifera), which were known as black cumin (black seeds) and were widely used as a traditional medicine for the treatment of numerous in ammatory diseases ; Hu et al. 2020). N. glandulifera seed powder, extracts (ethanolic, volatile oil, total avonoids, and total saponins, etc.), and some active ingredients showed signi cant anti-in ammatory effects and might be effective against many kinds of in ammatory diseases (Boubertakh et al. 2013). For example, the total saponins from N. glandulifera (TSN) exhibited potential analgesic, anti-in ammatory, anti-tumor, and antioxidant activities. The content of Nigella A was 60.36 ± 1.25 g/100 g existed in TSN (Zhao et al. 2013). Our previous results revealed that saponins were the principal active components of N. glandulifera with the highest content (64.5%) and Nigella A (also named Sieboldianoside A) is a major oleanane triterpenoid saponin isolated from N. glandulifera ; Zheng et al. 2020). We speculated that it could be used to treat a variety of in ammation-related diseases by regulating ora and immune.
Although the anti-in ammatory activity of N. glandulifera and its main constitutes have gained a great deal of attention in recent years, no research has been conducted on its anti-colitis activity. In our previous study, a large amount of Nigella A was extracted and puri ed from the seeds of N. glandulifera (Hu et al. 2014;Hu et al. 2020). Due to these healthcare functions, it is worthwhile to explore the therapeutical effect of Nigella A, a main active triterpene saponin extracted from N. glandulifera, on UC. Meanwhile, the mechanisms of action remain unclear and saponins always be regarded as the kinds of ingredients that could regulate immunity and intestinal ora in vivo. Based on our previous achievements, this study aimed to investigate the effects of Nigella A on dextran sodium sulfate (DSS) induced UC mice and the regulating effect of intestinal ora using modern of pharmacological techniques.

Material And Methods
Drugs and reagents Similar to our previous research , DSS was bought from MP Biomedicals (MP Biomedicals, USA). Nigella A was isolated and puri ed from N. glandulifera in our lab with the purity of 98.4% (Hu et al. 2014), and its structure was con rmed by MS, 1H and 13C NMR spectra.

The design of experiments
As we previously reported without any changes , thirty male C57BL/6 SPF mice were obtained from the Experimental Animal Center of Zhejiang Province (Hangzhou, China). All mice were adaptability raised in 18 to 23℃ temperature, light/dark cycle environment after randomly divided into ve groups with six mice each.
Mice of the control group were free to drink pure water, other groups were allowed to drink freely of 4%DSS solution for 7 days (every 2 days replace new con guration DSS solution) to induce in ammatory bowel disease. The corresponding drugs were given to the stomach twice a day at the same time according to the dosing volume of 0.1 mL/10g for 7 days. Based on the reported results and our preliminary experimental results, the optimal dosages of Nigella A (50, 100 mg/kg) and Salicylazosulfapyridine (SASP, 200 mg/kg) were chosen in animal experiments. The control and DSS groups were given of normal saline, and all mice were sacri ced for cervical dislocation on the 8th day for anatomical sampling.
The evaluation of anti-UC effects Disease activity index (DAI) score is a common index to evaluate the colon injury model of experimental animals. It can be used to evaluate the mice's body weight loss, diarrhea, blood in the stool and other uncomfortable symptoms after DSS treatment. Therefore, as we reported before, the body weight, activity status, fecal characteristics and blood in the stool of mice were evaluated in a blind manner every day according to the DAI scoring system ). On the 8th day of the experiment, after the colon was dissected, pathological changes such as edema, adhesion, ulcer and necrosis were observed. The length of the colon was measured with a ruler and photographed.
According to the above two indicators, the e cacy of the drug on UC was preliminarily determined.

The evaluation of pathological features of colon tissues
The colon tissues were isolated, measured and photographed as quickly as possible, and a part of the colon tissue was xed with 4% paraformaldehyde for two days. Then, the colon fragments were embedded, sectioned, stained and observed under the microscope. The histological score was evaluated according to the scoring criteria as we previously reported ). The sum of each score was used to evaluate colon histopathological improvement. . Paired-end reads was assigned, truncated and merged using FLASH. Chimeric sequences were ltered by using Vsearch software (v2.3.4). Sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTUs) by Vsearch (v2.3.4). Representative sequences were chosen for each OTU, and taxonomic data were then assigned to each representative sequence using the RDP (Ribosomal Database Project) classi er. The differences of the dominant species in different groups, multiple sequence alignment were conducted using the mafft software (V 7.310) to investigate the phylogenetic relationship of different OTUs. All of these indices in our samples were calculated with QIIME (Version 1.8.0).

Statistical analysis
For intestinal ora analysis, FLASH 1.2.8 software was used to splicing sequences, Vsearch 2.3.4 software was used to lter chimeras and OTU clustering, QIIME 1.8.0 software was used to analyze the diversity of micro ora, and R 3.4.4 (R Core team) language mapping software was used. Other experimental data were revealed as Mean ± SD of three experiments. The statistical signi cance was assessed by one-way ANOVA followed by post hoc Tukey's test or Student's t-test when appropriate by using GraphPad 7.0 software.

Results
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 8 th 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 signi cantly 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 in ltration of immune cells, etc. were observed in submucosa and muscle layer of the colon, and the histopathologic score was signi cantly increased than that of the control group ( Fig. 2b). The in ammatory in ltration 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 signi cantly decreased. The e cacy of Nigella A is better than SASP, especially the high dosage of Nigella A.
Microbial diversity analysis -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 signi cantly 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 ora was signi cantly decreased in mice induced by DSS, and the diversity of intestinal ora 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 su cient 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 signi cantly improve the microbial diversity in DSS-treated mice.

β-Diversity analysis
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 ora to normal. The results of non-metric multi-dimensional scaling (NMDS) suggested that model group and control group showed signi cant 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 signi cantly inclined to the control group.

Cluster analysis
Metastatistical analysis showed that, at the phylum level, there were three phyla, including Firmicutes, Bacteria_unclassi ed and Proteobacteria, that displayed signi cant differences in the relative abundance between the model group and the control group (Table 2). These three phyla in the model group exhibited signi cant 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 pro ling showed a signi cant lower proportion of Lactobacillus, Porphyromonadaceae_unclassi ed, Alistipes, Acetivibrio, Barnesiella, Bacteria_unclassi ed and an a signi cant 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 signi cantly reversed the levels of these changes. In addition, our results showed that there were signi cant 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 signi cantly regulate the changes of a variety of bacterins and then affect the intestinal ora 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_unclassi ed. However, the abundances of these main bacterial genus were signi cantly reversed by Nig_A_100 and SASP (Table   S1).

Discussion
N. glandulifera has been widely used in Islamic countries as a kind of traditional medicinal herb and a food additive since ancient times (Zheng et al. 2020). The whole herb of N. glandulifera has been used for a variety of in ammatory disease, such as colds, coughs, and insomnia (Chinese Pharmacopoeia, 2015). N. glandulifera seeds also traditionally were used as a spice or remedy to treat various in ammatory diseases (Boubertakh et al. 2013).
For example, N. glandulifera and TSN were con rmed to exhibit signi cant antioxidant and anti-in ammatory properties (Zhao et al. 2013;Zheng et al. 2020). Triterpene saponins are the dominant group of anti-in ammatory compounds existed in this ethnomedicinal plant (Hu et al. 2014;Hu et al. 2020) and Nigella A was a major ingredient of triterpene saponins extracted from N. glandulifera. Nigella A was 60.36 ± 1.25 g/100 g total saponins of N. glandulifera (Zhao et al. 2013). All of these results supplied su cient theoretical evidence for the anti-UC effect of triterpene saponins and speci cally Nigella A in the therapeutic eld of UC.
It has been reported that saponins always exhibited protective effects against DSS-induced intestinal in ammatory injury fermented with probiotic in animal models (Lu et al. 2020;Jang et al. 2017). Our previous review revealed that many kinds of extractions of N. glandulifera seeds and some active ingredients always exhibited signi cant antiin ammatory effects and might be effective on various in ammatory diseases . For example, TSN could be considered to be a potential analgesic, anti-in ammatory, anti-tumor, and antioxidant agent (Zhao et al. 2013). Our present results suggested that Nigella A, a major component of triterpene saponins extracted from N. glandulifera, could signi cantly attenuate colonic shortening, induced a reduction in body weight, and led to the DAI of mice. These results basically con rmed the e cacy of Nigella A on experimental UC mice. Our results demonstrated that DSS could signi cantly decrease the proportion of Lactobacillus and Porphyromonadaceae and increase the proportion of Bacteroides and Escherichia. However, the maximum variation of these four bacterial genus could signi cantly reversed by SASP and Nigella A. The maximum variation of Lactobacillus, Porphyromonadaceae, Bacteroides and Escherichia levels suggested that these four kinds of bacterial genus may be the main bacterial genus affected by SASP and Nigella A.
In addition, we also detected a signi cantly up-regulated or down-regulated of a large number of other bacterial genus after the intervention of DSS. We detected signi cant changes of 62 genera after the intervention of DSS, and most of the changes could be reversed by SASP and Nig_A_100. As for the regulation of intestinal ora and the therapeutic effect of UC, although current experimental results can't provide accurate correlation results, the regulation of intestinal ora is at least one of the mechanisms of SASP and Nigella A. Taken together, SASP and Nigella A could alleviate microbiota dysbiosis in DSS-induced colitis model by regulating intestinal ora. In addition, there are many similar saponins present in N. glandulifera that show similar pharmacological activities. Nigella A was only one of these representative components, but it exhibits a similar anti-UC effect to SASP. Due to the fact that Nigella A was also found in many other plants and Traditional Chinese Medicine (TCM), other saponins and TCM extractions containing Nigella A may also exhibit anti-UC effects.
It's worth mentioning that SASP has been used in clinic for many years for the therapy of UC, the regulation of intestinal ora is also one of its mechanisms of action (Chen et al. 2005). And once again, our results proved that SASP has the functions of regulating intestinal ora in DSS induced mice (Jia et al. 2020). It is interesting to note that Nigella A (100 mg/kg) exhibited very similar regulatory effects of intestinal ora to SASP (100 mg/kg). Considering the similar anti-UC activities of SASP and Nigella A, as well as the multiple mechanisms of action due to SASP. We speculated that Nigella A may also have other mechanisms of action for the treatment of UC, regulating gut ora is at least one of the mechanisms by which Nigella A works. Nigella A has been widely reported to have signi cant anti-in ammatory and immune-regulating functions ), which may also play a role in the mechanisms of its anti-UC effect. All of these speculations need to be con rmed by further experimental results.
In summary, the oral administration of Nigella A and SASP exhibited therapeutic effects on DSS-induced UC in mice. The therapeutic effects manifested as the improvement of the general symptoms and the remission of in ammatory injure. The underlying mechanism of action of these effects involved the preventing the imbalance of intestinal ora and restoring the relative abundances of vital bacteria. These results revealed that Nigella A had a protective effect on DSS-induced UC by regulating the intestinal ora. Nigella A was also considered to be a novel attractive intestinal microecological improver with great development prospects. YZ, XY and XH provided technical guidance and modi ed the manuscript. QZ, as the corresponding author, provided nancial support and contributed in organizing, writing, editing and revising the manuscript. All authors reviewed and approved the manuscript.   Figure 1 The e ciency of Nigella A in colitis tissues. a Nigella A effectively improved the shorten of the colon tissue. b Quantitative results of colon length in different groups. c Changes in body weight in mice of different groups over the whole course of this experiment. d DAI change in different experimental groups. Each point represented the mean ± SD (n = 6). ###P < 0.001 vs. control, *p < 0.05 and **p < 0.01. Analysis -diversity of the differential microbial community among the control group, model group, SASP and Nig_A_100-treated group. a Venn diagram; b Chao1; c Observed species index; d Shannon; e Simpson.

Figure 5
Structural comparison of fecal ora among the control, model, SASP and Nig_A_100 groups. Bacterial taxonomic pro ling at the phylum lever (a) and genus level (b). LEfSe analysis of fecal ora among the control, model, SASP and Nig_A_100 groups. c LEfSe analysis showed the distribution histogram of different groups in gut microbiota (LDA sore >4). d Cladogram. The size of each node represents the relative abundance of the species. (p, phylum; c, class; o, order; f, family; g, genus; s, species).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TableS1Group.ControlvsModelvsSASPvsNigA100.diff.xlsx