DSS is widely used to construct the IBD animal model. It has been assumed in most former researches that DSS induces epithelial damage mainly in the colon and pathological change in the small intestine is ignored. In this research, however, it was observed that DSS induced damage in both the small intestine and the colon when it was given orally in drinking water. Local mucosa of the small intestine and the colon was damaged by ulcer and hemorrhagic necrosis, along with inflammatory cell infiltration. This result was in agreement with the report from Elsheikh et al.31 and suggested that the DSS induced IBD was more similar to CD in the pathological features. In comparison with the colon, the small intestine is longer, with larger mucosa surface, which is favourable for nutrition adsorption. The damage of small intestine mucosa would lead to innutrition, which could cause various mobidness including weight loss and the decrease of immunological defence. Thus, the protection of the small intestinal structure and function is more important than protection of the colon. Based on the pathological characteristics of IBD, the prevention and curation of IBD should focus on the inhibition of inflammatory reaction and the protection of the mucosa both in the small intestine and in the colon from inflammatory injury. Because the CD, especially small-bowel Crohn's disease, possessed the higher disease severity and more extensive injury in the intestinal mucosa (including small intestine and colon) than UC,37–41 the protection of small intestine from inflammatory damage and promotion of the mucosal healing in the small intestine of IBD are more effective for prevention and curation of this disease especially for small-bowel Crohn's disease. Meanwhile, the promotion of epithelial stem cell proliferation and differentiation, and the reconstruction of the integrate mucosa barrier are essential for recovery from IBD. Besides the traditional therapy of anti-inflammatory drugs, the application of regulators for the gut flora is a novel therapeutic strategy in preventing recurrence and ameliorating refractoriness of IBD.
This study focused on the protective and repairing effects of the B. subtilis fermented milk on both small intestinal and colonic mucosa in the DSS-induced IBD mouse model, and aimed at exploring the action mechanisms of the B. subtilis fermented milk. The results indicated that oral intake of DSS could induce extensive injury of the small intestinal mucosa and the colonic mucosa. In the active phase of the DSS-induced IBD, the major pathological characteristics included necrosis of the epithelium and the ulcers in the mucosa. The damage in the colon was more severe than in the small intestine. In the recovery stage, the injury in the small intestine could partially recover automatically, while the ulcers of the colonic mucosa were replaced by inflammatory scars. For the small intestine, in the active phase (DSS inducing phase), oral intake of the B. subtilis fermented milk could prevent the inflammatory injury; And in the recovery phase (after DSS inducing), the B. subtilis fermented milk could promote the repairing of the injury and then completely reconstruct the microstructure of the mucosa. For the colon, in the active phase, oral intake of the B. subtilis fermented milk could lessen the ulcer of the mucosa; And in the recovery phase, oral intake of the B. subtilis fermented milk could inhibit the formation of the scars in the colonic mucosa, and promote the regeneration of the epithelium. These results suggested that B. subtilis fermented milk possessed the double-function of both prevention and curation for DSS-induced IBD. Meanwhile, the results also showed that oral intake of the B. subtilis fermented milk could induce the goblet cells to secret more mucus which was stained blue via alcian blue. The mucus secreted by the goblet cells was important for construction of an integrate mucus barrier, which can protect the deep area of the mucosa from the infiltration of the pathogenic bacteria, and thus inhibit the local inflammatory reaction.42,43
In this research, the action mechanisms of the B. subtilis fermented milk for treatment of DSS-induced IBD were explored. The B. subtilis fermented milk could inhibit the MPO+ neutrophil infiltration and the expression of pro-inflammatory cytokine TNF, and promote the expression of the anti-inflammatory cytokine IL-10 in the intestinal mucosa, which reduced local inflammatory injury of the intestinal mucosa. It has been reported that TNF was over-expressed in the intestinal mucosa and could lead apoptosis of the intestinal epithelial cells (IECs) in IBD.44–46 However, anti-inflammatory cytokine IL-10 exerted essential functions to maintain tissue homeostasis during infection and inflammation through restriction of excessive inflammatory responses and promotion of tissue repairing mechanisms.47,48 IL-10 could secreted by both immune cells and IECs.49,50 IL-10 binds to a specific receptor on IECs and may regulate the contribution of epithelial cells to the inflammatory and immune response in the digestive tract via auto-secretion pathway.51–54 Many experimental results indicated that IL-10-deficient mouse has susceptibility to IBD, and over-expression of IL-10 was related to good therapeutic effect of drugs for IBD.55–57 In this study, the results of immunohistochemical staining and western-blotting indicated that oral intake of the B. subtilis fermented milk could inhibit expression of TNF and promote over-expression of IL-10 in the IECs, which might be an important action mechanism of the B. subtilis in treatment of DSS-induced IBD.
The results of IHC and western blotting for the marker of intestinal stem cells (ISCs) Lgr5 and epithelial marker CDX2 indicated that oral intake of the B. subtilis fermented milk could promote epithelial regeneration via protection of the intestinal stem cells (ISCs) from inflammatory injury and induced proliferation of the ISCs. ISCs can proliferate and differentiate into the intestinal epithelial cells and goblet cells.58–62 DSS leaded to the loss of Lgr5 + cells, however, B. subtilis could increase Lgr5 + ISCs and then result regeneration of the epithelium lined the intestinal mucosa injured by DSS induced IBD. In addition, the B. subtilis fermented milk could induce the small intestinal and colonic epithelium to over-expression the CDX2. CDX2 is an intestinal specific transcription factor located in the nuclei of IECs and modulates a diverse set of cellular behaviours, including cell proliferation and differentiation, and cell adhesion and migration. CDX2 is an essential regulator of intestinal epithelium homeostasis.63 TNF-α could impair the functions of CDX2 in IBD leading to the mucosal injury.64 Inducing expression of CDX2 by the B. subtilis fermented milk suggested that the B. subtilis could maintain intestinal epithelium homeostasis via its inhibition against the TNF-α or via direct action to stimulate expression of CDX2. Meanwhile, oral intake of B. subtilis fermented milk could promote expression of Mucin2 in the goblet cells and expression of Villin in the epithelial cells. Mucins are the main components of mucus, which is secreted by goblet cells and forms a protective homeostatic barrier between the resident microbiota and the underlying immune cells in the colon.65,66 It has been reported that 5 weeks after birth, MUC2 knockout animals develop spontaneous colitis and display increased susceptibility to experimental DSS colitis.67 Plaisancié et al. reported that a novel bioactive peptide produced from bovine β-casein in yoghurts could induce expression of the gel-forming MUC2 mucin in the human intestinal mucus-producing cells (HT29-MTX) and enhanced the number of goblet cells and Paneth cells along the small intestine.68 In this study, over-expression of Mucin2 by oral intake of B. subtilis fermented milk suggested that B. subtilis or/and some bioactive peptide produced from bovine β-casein in the fermented milk might play a role in treatment of DSS-induced IBD via the protective function of Mucin2 as well. Villin is an actin regulatory protein expressed in the intestinal epithelium and possesses the epithelial cell-specific anti-apoptotic function.69 Absence of Villin predisposes mice to DSS-induced colitis by inducing apoptosis of the IECs.70 Inducing expression of Villin suggested that B. subtilis fermented milk could play the anti-apoptotic role in treatment of DSS-induced IBD. More importantly, oral intake of the B. subtilis fermented milk could promote expression of ZO-1 which showed dotted line like localization on the membrane of the IECs. The proteins of tight junction, ZO-1 and ZO-2 can bind directly to F-actin and other cytoskeletal proteins, and these proteins are relevant both to cellular organization and epithelial morphogenesis.71,72 It has been reported that the expression of ZO-1 was significantly higher in the patients with quiescent UC with mucosa healing compared with those without mucosal healing, and the loss of ZO-1 could increase permeability of the intestinal epithelium and promoted the development of significant intestinal inflammation in animals with DSS colitis.73,74 Peng et al. reported that the probiotic B. subtilis CW14 could reduce disruption of the epithelial barrier and toxicity of ochratoxin A to Caco-2 cells via improving ZO-1 protein expression.75 In this study, the high expression level and the dotted-line like distribution of ZO-1 along the surface of the IECs in the normal group and DSS + B. subtilis fermented milk group suggested that ZO-1 participated in the construction of the tight junction between the intestinal epithelial cells. These results suggested that B. subtilis could promote reconstruction of the epithelium barrier which prevented pathogenic bacterial invasion and protected the intestinal mucosa from inflammatory injury in DSS-induced IBD.
The inflammatory injury in the intestine was reported to be related with the imbalance of the intestinal flora and reduction of the abundance and diversity of the gut microbiota.76,77 As reported by Sjöberg et al.,10 pyrosequencing revealed that the gut microbiota of patients with ulcerative colitis contained fewer Operational Taxonomic Units (OTU) per individual than the controls, and this reduction in richness of the gut microbiota was observed in Firmicutes, Actinobacteria, Collinsella, Lactobacillus, and Bacillus. In this research, oral intake of the B. subtilis fermented milk increased the species diversity of the normal intestinal microbiota. And the decrease in abundance of Alistipes, Rikenella, Barnesiella, Macellibacteroides, and Lactobacillus was observed in the DSS-induced IBD models, while the abundance of Escherichia and Bacteroides increased. According to the LEfSe analysis results, Clostridiales and another genius in Clostridium (Clostridium_IV) were the most significantly increased genius in the DSS group and the DSS + milk group. Some species in the Clostridiium genius were reported to be opportunistic pathogens associated with intestinal infection. Clostrodiium difficile was reported to be related with diarrhea.77 In accordance with our results, it was reported in several researches that the relative abundance of Bacteroides was higher in DSS-induced IBD models.78 On the other hand, the results suggested that the B. subtilis fermented milk could increase the total species and diversity of the gut microbiota which were reduced by DSS-induced IBD. Meanwhile, the fractions of Lactobacillae and Porphyromonadaceae in the DSS + B. subtilis fermented milk group were increased to the similar level of the normal group. Oral intake of the B. subtilis fermented milk significantly increased the abundance of Bacillus, Barnesiella, Alistipes, and Saccharibacteria, and the total OTU detected was largely increased. Another genius in Clostridiaceae (different from the genius detected in other groups) was detected in DSS-B. subtilis fermented milk group, but the LDA score was lower than the Clostridiales genius detected in the DSS group. Meanwhile, the abundance of Escherichia, which was reported to be related with inflammatory reactions, was reduced.79,80 The dramatic increase of Bacillus abundance after oral intake of the B. subtilis fermented milk suggested that B. subtilis was successfully implanted in the intestine. Among the species increased through B. subtilis intake, Barnesiella and Alistipes have been approved beneficial to intestinal health. Alistipes was reported to reduce the inflammatory reaction in the intestine and regulate the lipid metabolism.81 And Barnesiella could facilitate cyclophosphamide, which possesses anti-cancer activity.82 Besides, the abundance of short-chain fatty acid (SCFA)-producing Ruminococcus was significantly higher in the DSS + B. subtilis fermented milk group than in the other groups, and it was reported that the absence of Ruminococcus was related with CD.83,84 According to the results in this study, oral intake of B. subtilis fermented milk could efficiently increase the gut flora diversity of the intestinal microbiota and restore the balance of gut flora which was disturbed by DSS; The B. subtilis in the fermented milk might play the important role in the prevention and curation of IBD, via regulation of the intestinal flora.
In conclusion, this research focused on the prevention and curation effects of the B. subtilis fermented milk on the DSS-induced IBD and exploring the action mechanisms, including inhibition of inflammation, promotion of reconstruction of intestinal mucosal barrier, and regulation of the intestinal flora. It was demonstrated that oral intake of the B. subtilis fermented milk could reduce the inflammatory injury of both the small intestinal and colonic mucosa, and promote the epithelial regeneration and reconstruction of the intestinal mucosa barrier. Oral supplement of the B. subtilis could increase the total species and diversity of the bowel microbiota and regulate the gut flora balance which was disturbed by DSS-induced IBD. The results indicated that oral intake of the B. subtilis fermented milk could prevent and cure the DSS-induced IBD in mice. The B. subtilis fermented milk would be a potential novel functional food for the application in the therapy of IBD. However, it remains to be further explored what bioactive ingredients (the probiotic B. subtilis, its metabolites or these cooperative factors) in the B. subtilis fermented milk possessed the functions of prevention and curation of IBD. In addition, the molecular signaling pathway related to the function of B. subtilis in treatment of IBD is worthy of further study.