Ulcerative colitis is one common chronic diseases of digestive system. At present in clinical, doctors use anti-inflammatory drugs or immunosuppressive drugs to treat UC. However, these drugs usually cause some side effects. UC has a long course of disease, which is easy to recur, and cannot maintain long-term clinical remission. The need of new treatment methods is urgent.
SFN is a kind of natural isothiocyanate, which exists in cruciferous plants, such as cabbage and broccoli. SFN can activate the nuclear factor E2 related factor 2 (Nrf2) pathway, and induce the body to produce type II detoxification enzymes, which have antioxidant, anti-cancer activity and immune regulation functions (Sun et al. 2015). In recent years, SFN has also been found to have good anti-inflammatory and antibacterial effects. Wagner (Wagner et al. 2013) showed that SFN pretreatment had a protection on DSS-induced colitis by reducing the expression of inflammatory biomarkers in intestinal mucosa and increasing the expression of Nrf2 dependent genes. Morever, Hubbard (Hubbard et al. 2017) suggested that the broccoli riched in SFN affect the structure of intestinal microbial community and attenuate colitis by alylhydrogen receptor (AHR) dependent mode in Ahrb/b mice. At present, there is no study on the effect of SFN on the intestinal flora of UC mice. Therefore, the purpose of this study is to find out the effect of SFN on the intestinal microbial community of UC mice.
DSS-induced UC in mice is one of the most mature methods at present. The influencing factors of DSS modeling include: molecular weight and concentration of DSS, research environment, mouse species, administration time, etc. (Kitajima et al. 2000). After 7 days of DSS intervention, the mice in the DSS group lost weight, shortened the length of blood stool and colon, and significantly increased the DAI score. The DAI score, colon length and histopathological score in the SFN group were all improved (P < 0.05) than that in the DSS group. The MPO activity of the SFN group was significantly lower than that of the DSS group (P < 0.05), which indirectly proved that SFN reduced the inflammatory degree of acute ulcerative colitis in mice.
Dysbacteriosis of intestinal flora is intimately correlated to the pathogenesis of DSS-induced UC (Chen et al. 2016). The results showed that, compared with the blank group, after 7 days of DSS intervention, the Shannon index of intestinal microflora in other two groups descended, that is, the diversity of microflora decreased. PCoA analysis showed that the distance between the blank group and the DSS group was far from each other, indicating that there was a great difference in the composition of the flora between the two groups, which was same with the previous conclusion of Yang et al. (2017). The DSS intervention could change the β diversity of the intestinal microbial community in mice, that is, change the species composition.
According to the results of phylum level, the three major phyla were Firmicutes, Proteobacteria and Bacteroidetes in the three groups. In comparision to the blank group, the proportion of Firmicutes and Proteobacteria in the DSS group grown. Meanwhile, the proportion of Bacteroidetes and Verrucomibia dropped, and the ratio of F/B grown (P < 0.05). In comparison to the DSS group, SFN group had lower Firmicutes, higher Bacteroides and lower F/B (P < 0.05). There was no obviously difference in F/B between SFN group and blank group. Wu et al. (2016) showed that during AOM/DSS induced colitis related colon cancer in mice, the level of Bacteroides decreased and that of Firmicutes increased, which was consistent with the results of this experiment. However, Yeom et al. (2016) showed that after DSS intervention, the proportion of Bacteroides in mouse intestinal microflora increased, while that of Firmicutes decreased. Some studies have suggested that the increase of F/B ratio, namely, the increase of Firmicutes, and the decrease of Bacteroides have a protective effect on inflammatory bowel disease. There are two reasons for the difference: First, the intestinal flora of mice can be affected by many factors such as feed, drinking water, environment and so on. At the same time, the structure of intestinal flora is intimately correlated to the disease factors such as the course of ulcerative colitis, the location of lesions, the degree of inflammation and so on. Secondly, in this study, the number of mice in each group is less, and the individual differences are larger. Therefore, the detection of intestinal flora is also biased.
According to the results of family level, after DSS modeling, in comparision to the blank group, the bacterial of DSS-induced mice changed dramatically. However, the intestinal flora of SFN group was closer to that of blank group. In comparison to the DSS group, Erysipelototrichaeae and Campylobacteraceae was found significantly decreased in the blank and SFN group. While the relative abundance of Bacteroidales_S24-7, Rikenellaceae and Prevotellaceae increased (P < 0.05). According to relevant research reports, Bacteroidales_S24-7 is a kind of intestinal probiotics, which is negatively related to intestinal inflammation. Rikenellaceae is a hydrogen producing bacterium, which can selectively neutralize cytotoxic reactive oxygen species (ROS) and protect cells from oxidative stress, thus improving the symptoms of inflammatory bowel disease (Rooks et al. 2014). In general, Bacteroidales_S24-7 and Rikenellaceae are more likely to have beneficial bacteria and protect the intestine. The role of Prevotellaceae is unclear. On the one hand, it has been reported that Prevotellaceae is prominent in IBD patients. Wright et al. (2000) suggested that Prevetelaceae may damage the function of intestinal mucosal barrier by producing sulfatase which can degrade mucopolysaccharide, which is increased in IBD patients’ intestinal biopsies. Prevotellaceae may also lead to the deterioration of chronic intestinal inflammation in IBD mice. On the other hand, Monk et al. (2016) showed that cranberry bean rich diet can reduce the symptoms of ulcerative colitis in mice, and beans can increase the abundance of Prevetelaceae and Bacteroidales_S24-7 in mice intestine. De Cruz et al. (2015) proved that Prevetellaceae is a kind of intestinal microorganism which has relation to the remission of inflammatory bowel disease. Therefore, the role of Prevotellaceae needs further study in the future.
Currently, in antiretroviral treatment of patients infected with human immunodeficiency virus, it has been proved that Erysipelototrichaeae is associated with elevated TNF levels and chronic intestinal inflammation (Dinh et al. 2015). Hubbard et al. (2017) study found that eating broccoli can degrade the relative abundance of Erysipelototrichaeae in the intestine of colitis mice and alleviated colitis. Campylobacteraceae belongs to Proteobacteria. Many Campylobacter species may be relative to the pathogenesis of IBD. A meta-analysis by Natalia (Castano-Rodriguez et al. 2017) showed that Campylobacter (especially C. showae and C. concisus) raise the chance of IBD. The results of Lefse analysis of mouse intestinal flora showed that Erysipelototrichaeae and Campylobacteraceae is also a biomarker in the DSS group. The intervention of SFN can reduce the relative abundance of the two bacteria.
In order to further analyze the specific strains, seven specific strains were found out by comparing the intestinal flora of mice in the blank group and the DSS group on the 14th day of the experiment with the metastats method, the specific species were Ruminiclostridium, Alloprevotella, Turicibacter, Butyricicoccus, Lachnospiraceae_UCG-006, Ruminococcaceae_UCG-013 and Family_XIII_UCG-001(P < 0.05). The relative abundance of Butyricicoccus in the DSS group was the lowest among the three groups, while the relative abundance of Turicabaracter was the highest. Turiribcharacter belongs to Erysipelototrichaeae. Previous studies have shown that the abundance of Tricibacter in the gastrointestinal tract of colitis mice (DSS induced mice and IL-22 deficient mice) is decreased (Zenewicz et al. 2013; Collins et al. 2014). More recently, it has been introduced that in AOM/DSS induced colitis related colon cancer, the relative abundance of Turicabaracter increased (Wu et al. 2016). In the pathogenesis of human ileal pouch inflammation, which previously suffered from UC, Turicabaracter is closely related to the well-known pathogenic bacterium Clostridium perfringens (Falk et al. 2007). Therefore, it is believed that Turibacharacter has two factors affect with the pathogenesis of mammalian immune system and IBD.
Butyricococcus is a Clostridial cluster IV that produces butyrate, and its number is reduced in feces of patients with ulcerative colitis (Falk et al. 2007). Butyrate, a short chain fatty acid, was generated in the fermentation of dietary fiber in colon. Butyrate is not only the main energy source of colonic cells, but also maintains colonic homeostasis by regulating various cell functions, including proliferation, differentiation, apoptosis and control of intestinal epithelial permeability (Hamer et al. 2008). In addition, butyrate is also an effective anti-inflammatory medium, which can promote the function of epithelial barrier, induce the ability of colon regulatory T cell differentiation, and inhibit the expression of cytokines (Hamer et al. 2008). In this study, RT-PCR was used to present quantitative analysis. The number of Butyricicoccus in the SFN group was more than that in the DSS group, but there was no statistical difference between the two groups. But there was a statistical difference of the number of Butyricoccus in the blank group, which was much higher than that in the other two groups. According to the results, the relative abundance of microbial species altered by SFN treatment showed the difference of gut bacterial compositions compared to the DSS group. SFN has the protective effect on intestine by Butyricococcus. There are many bacteria can produce Butyrate, such as Clostridium cluster IV and XIVa bacteria. In this study, only one of them was detected, so the effect of other butyrate producing bacteria on intestinal protection could not be completely denied. In the future experiments, the alleviative effect of SFN on colitis in mice can be further explored.