In this study, in addition to chemical induction with DSS, we used transplantation of stool to induce inflammation in mice. DSS-induced IBD in mice leads to the destruction of intestinal mucus and causes inflammation by inhibiting the repair of intestinal epithelial cells [37]. In our FMT group, bacteria are involved in the development of intestinal inflammation. Human microbiota-associated (HMA) mice are the best available model to study the role of gut microbiota in diseases [38], and in this study, we used the stool of IBD patient to produce an HMA model. Because germ-free mice have biological limitations and the gut microbiota is essential for the maturation of the gut and host immune system [39–41], we used PEG to cleanse the gut microbiota of conventional mice in preparation for transplantation of human stool. Studies have shown that PEG eliminates nearly 99% of intestinal bacteria in mice [10].
The results of the FMT group showed that after transferring human stool to mice, the intestinal microbiota of the mice was altered on day 4. Most of the changes on day 4 involved the phyla of Actinobacteria and Firmicutes, and the species of Clostridium clostridioforme and E. faecalis. Following a decline in the Firmicutes and Actinobacteria population, the abundance of Lactobacillus, Bifidobacterium, Faecalibacterium, and Roseburia was significantly reduced on days 21 and 28 of the study. In contrast, the population of γ-Proteobacteria, especially Enterobacteriaceae, increased. On the other hand, the population of Bacterioidetes increased significantly on day 21, but decreased on day 28 and had a significant decrease compared to the control group.
In general, the microbiota population of the FMT group was similar to that of the DSS group on day 28 of the study, and the increased abundance of γ-Proteobacteria, especially Enterobacteriaceae, in the FMT and DSS groups provided the basis for intestinal inflammation in vivo. The overall results suggest a parallel relationship between gut microbiota and IBD, implying that changes in the population of gut microbiota can cause IBD, just as IBD can alter the population of gut microbiota. Our observations suggest that alterations in the gut microbiota may play a critical role in inflammatory symptoms in the gut and in diseases such as IBD, irritable bowel syndrome, and others [42].
Probiotics as commensals in the gut could modulate the gut microbiota and prevent substantial changes in the microbiome population. In this regard, our results showed that in the FMT + LacBif and DSS + LacBif groups, with an increasing abundance of Lactobacillus and Bifidobacteria, the population of Faecalibacterium and Roseburia also increased, while the population of Enterobacteriaceae and Bactroidetes decreased. The role of Lactobacillus spp. has been shown in inhibiting pro-inflammatory cytokines and maintaining the balance of the gut microbiota [43].
Alteration of the gut microbiota population, characterized by an increase in potentially pathogenic bacteria and a decrease in beneficial bacteria, disrupts normal microbe-host interactions and impairs intestinal homeostasis [44]. Intestinal epithelial cells diagnose microorganisms by specific molecular regions called pathogen-associated molecular patterns (PAMPs) [45]. One of the important pattern recognition receptors (PRRs) for modulating the cellular immune response to PAMPs is the Toll-like receptor (TLR) [44]. TLR activates intracellular signaling via two major pathways, the myeloid differentiation factor 88 (MyD88)-dependent pathway and the MyD88-independent pathway, after pathogen recognition. In the cell, TRAF6, IRAK1, and IRAK4 proteins are activated, and finally, the activation of MAPK increases the production of IL -1, IL -6, and TNF-α [46]. The evaluation of inflammatory genes in our study showed that in the FMT and DSS groups, the expression of inflammatory genes increased compared with the control group. In the FMT group, the expression of MyD88 genes was higher than in the DSS group, so the MyD88-dependent pathway was more activated in the FMT group. Also, the expression of the IRAK4 gene, which is one of the components of the MyD88 signaling pathway, was more expressed in this group than in the DSS group.
In the FMT + LacBif and DSS + LacBif groups, probiotics were able to inhibit the increase of inflammatory factors, and compared to the DSS and FMT groups, the activity of signaling pathways decreased. Probiotics are able to regulate the host immune response by interacting with immune cells and intestinal epithelial cells [47]. The PAMPs of probiotics are recognized by the TLRs of dendritic cells, and unlike pathogenic bacteria that induce inflammation by stimulating TLR, probiotics reduce inflammation by reducing NF-κB [48]. One study has shown that Lactobacillus are able to degrade the chemokine IP-10 by producing a protease called lactocepin [49]. Another study that examined the effect of probiotics on the expression of TLR4 and TLR5 in mice with colitis showed, similar to our results, that probiotics reduced the expression of TLR4 and TLR5 [50].
The intestinal mucosa plays a key role in the development of inflammatory bowel disease. The normal mucosa of the colon has tight junction proteins, and their functional integrity can protect the colon from bacterial invasion [51]. The expression of tight junction proteins was decreased in the DSS and FMT groups compared with the control group. One study reported that the expression of claudin-1 and claudin-2 in the rat model of IBD reached the highest level on day 14 and then gradually decreased [52]. Another study showed that TNF upregulated the expression of claudin-2 [53]. The results of our study also showed a decrease in serum TNF level and claudin-2 expression. Accordingly, cytokines are involved in the expression and distribution of tight junction proteins in the inflammatory state.
However, the expression of claudin-1 and ZO -1 was lower in the FMT group than in the DSS group, which was probably due to the effect of the bacteria in the stool sample transferred to the mice. Thus, with the colonization of the gastrointestinal tract with pathogenic bacteria, the intestinal immune system is activated and inflammatory cytokines and chemokines are produced and cellular junctions are weakened. However, in the groups treated with probiotics, the expression of tight junction proteins was higher than in the FMT and DSS groups. Probiotics are able to decrease gut permeability caused by pathogens and increase tight junction protein expression [54]. The most effective probiotics are Lactobacillus spp. and Bifidobacterium spp. strains, and the effect of combined probiotics, such as the use of multiple bacterial cocktails, is more effective [55].