IBS is a functional gastrointestinal disorder characterized by visceral hypersensitivity, intestinal immune activation and gut microbiota dysfunction [32]. Recently, a growing body of evidence has suggested that gut the microbiome plays a pivotal role in colonic inflammation [33]. As a probiotic mixture, Bifico is supplied for the treatment of microbiota disorders or alleviating the inflammatory reaction [34, 35]. However, how Bifico treatment functions in IBS is still unclear. Here we analyzed the relationship between the gut microbiota and the inflammatory cytokines in IBS after treatment with Bifico, which might provide a theoretical basis for the clinical use of Bifico. Our studies showed that Bifico may relieve the symptoms of IBS by reducing the protein expression level of IL-6 and TNF-α, altering fecal metabolites and gut microbiota. Further studies revealed that Bifidobacterium genera may play important roles in treatment.
Previous studies have demonstrated that Bifidobacterium and Lactobacillus could specifically relieve the symptoms of IBS [36]. Moreover, multispecies probiotics containing strains of more than one genus show enhanced effects in treating antibiotic-associated diarrhea in children, which suggests that probiotic mixtures may more be effective than a single-strain [37]. Bifico is a type of multispecies probiotic. Lactobacillus acidophilus could produce bifidogenic growth factors to stimulate the growth of Bifidobacterium longum in pure culture [38]. Enterococcus spp. has been used as a probiotic to defend against gut infection and prevent the colonization of more pathogenic bacteria [39]. In addition, Enterococcus faecalis could create anaerobic conditions, which might be of benefit for survival of Bifidobacterium [40].
WRS is an accepted method of creating an acute stress-induced IBS model [24]. In this study, we observed that treatment of Bifico significantly alleviated intestinal visceral hypersensitivity and reduced weight loss. These results were consistent with most of the previous studies that effective treatment could reduce the AWR score of IBS [41–43]. Since IBS often involves gut microbiome dysbiosis, we collected fecal samples to analyze the gut microbiota. Community richness was measured by Chao1 and Observed_otus, while community diversity was measured by Simpson Evenness and Shannon diversity. Results showed that Chao1 and Observed_otus of the IBS group had no significant difference compared to the control group, but Simpson Evenness and Shannon diversity was lower in the IBS group than in the control group, were similar to previous findings by Fukui H. et al [44]. Despite treatment, Bifico did not alter alpha diversity of IBS. In beta diversity, the bacterial composition of the IBS + Bifico group was closer to the control group through treatment of Bifico. To further analyze the gut microbiota and their relative abundance according to LEfSe. In the phylum level, the relative abundance of Proteobacteria and Actinobacteria were markedly lower in the IBS group than in the control group and the IBS + Bifico group. In the genera level, Parabacteroides and Sutterella were increased in the IBS group compared to the control group and the IBS + Bifico group. Parabacteroides and Parabacteroides merdae (belongs to Parabacteroides genera) were considered as potentially pathogenic bacterium that were reported to be frequently enriched in the hypertensive gut microbiome [45, 46]. Moreover, in infectious diseases, Parabacteroides merdae is generally considered an opportunistic pathogen, which is able to develop antimicrobial drug resistance [47]. Sutterella is a controversial bacterium. From a previous review, Sutterella was related to better outcomes in patients with IBD [48]. Berer K et al. held the opinion that Sutterella had anti-inflammatory functions in vitro [49]. But some studies have suspected Sutterella plays a role in the disease progression of IBD [50]. Furthermore, in clinical studies, no difference was observed in the prevalence of Sutterella spp. between the IBD patients and the healthy subjects [51, 52]. Surprisingly, Lactobacillus expressed higher levels in the IBS group than in the control group and the IBS + Bifico group. Similarly, clinical research from Japan also observed that IBS patients had significantly higher counts of Lactobacillus [53]. Another clinical study considered that Lactobacillus may have no effect on IBS patients [54]. Compared to the control group and the IBS + Bifico group, the IBS group showed a significant decrease in the abundance of Muribaculum and Bifidobacterium. Yuan Y et al. noted that reduction of Muribaculum could result in inflammation, dyslipidemia and glucose intolerance [55]. As for Bifidobacterium, which belongs to Actinobacteria phylum, numerous studies have shown that it has benefits in improving epithelial barrier function in mice, act as an anti-inflammatory agent and a source of SCFAs [56, 57].
SCFAs describe acetic acid, propionic acid, butyric acid, valeric acid and caproic acid, which are the main byproducts of gut metabolites [58]. In the colon, the proportion of acetate, propionate, and butyrate can reach 90–95% of SCFAs [59]. It has been widely reported that SCFAs play pivotal roles in anti-inflammatory and maintenance of intestinal health such as locomotion recovery [60–62]. What’s more, SCFAs are involved in lipid metabolism and glucose metabolism [63]. According to the altered gut bacteria, we speculate that colonic metabolites may have changed. Through 1H NMR spectroscopy, we found that the abundance of acetate, propionate, butyrate and valerate were decreased in the IBS group, which supported the view of suggesting that patients with IBS had lower levels of SCFA [64]. Treatment of Bifico could increase their abundance. From Spearman's correlation analysis, we found that Actinobacteria phylum and Bifidobacterium genera were positively correlated with propionate, butyrate and valerate, Proteobacteria phylum and Muribaculum genera were positively correlated with valerate. The Sutterella genera were negatively correlated with propionate, butyrate and valerate.
Changes in the gut microbiota can induce or aggravate inflammation [65]. Recent research regards TNF-α as a vital inflammatory cytokine in IBS [66]. In addition, IL-6 has reproducibly been detected to be elevated in IBS patients and rats [67–69]. In our studies, the protein level in colonic IL-6 and TNF-α were higher in the IBS group than in the control group, further confirmed by the previous results. Zhao HM et al. found that in the colon of mice with colitis, the level of TNF-α could be significantly reduced by Bifico [70]. Our results supported the that Bifico could reduce both the protein level of IL-6 and TNF-α in the colon, alleviating the inflammation to a certain extent. In further correlation analysis, Actinobacteria phylum and Bifidobacterium genera were negatively correlated with IL-6 and TNF-α, verifying the anti-inflammatory function reported by Chichlowski M et al. [71]. Interestingly, Sutterella genera were positively correlated with IL-6 and TNF-α. This result stood suggested that the Sutterella genera had a pro-inflammatory capacity in the human gastrointestinal tract by Hiippala K et al. [72].
Taken together, using a widely developed IBS mice model, we found that Bifico regulated the gut microbiota and elevated SCFAs to reduce inflammation and relieve visceral hypersensitivity of IBS mice. These results hint at the potential clinical utility of treatment with Bifico.