Saireito, a Japanese herbal medicine, alleviates leaky gut associated with antibiotic-induced dysbiosis in mice

Abstract

Antibiotics disrupt normal gut microbiota and cause dysbiosis, leading to a reduction in intestinal epithelial barrier function. Disruption of the intestinal epithelial barrier, which is known as "leaky gut", results in increased intestinal permeability and contributes to the development or exacerbation of gastrointestinal diseases such as inflammatory bowel disease and irritable bowel syndrome. We have previously reported on a murine model of intestinal epithelial barrier dysfunction associated with dysbiosis induced by the administration of ampicillin and vancomycin. Saireito, a traditional Japanese herbal medicine, is often used to treat autoimmune disorders including ulcerative colitis; the possible mechanism of action and its efficacy, however, remains unclear. In this study, we examined the efficacy of Saireito in our animal model for leaky gut associated with dysbiosis. C57BL/6 mice were fed a Saireito diet for the entirety of the protocol (day1-28). To induce colitis, ampicillin and vancomycin were administered in drinking water for the last seven consecutive days (day22-28). As previously demonstrated, treatment with antibiotics caused fecal occult bleeding, cecum enlargement with black discoloration, colon inflammation with epithelial cell apoptosis, and upregulation of pro-inflammatory cytokines. Oral administration of Saireito significantly improved antibiotics-induced fecal occult bleeding and cecum enlargement by suppressing inflammation in the colon. Furthermore, Saireito treatment ensured the integrity of the intestinal epithelial barrier by suppressing apoptosis and inducing cell adhesion proteins including ZO-1, occludin, and E-cadherin in intestinal epithelial cells, which in turn decreased intestinal epithelial permeability. Moreover, the reduced microbial diversity seen in the gut of mice treated with antibiotics was remarkably improved with the administration of Saireito. In addition, Saireito altered the composition of gut microbiota in these mice. These results suggest that Saireito alleviates leaky gut caused by antibiotic-induced dysbiosis. Our findings provide a potentially new therapeutic strategy for antibiotic-related gastrointestinal disorders.

Fig 1. Effect of Saireito on antibiotic-induced fecal occult bleeding and cecum enlargement.

Mice in the normal group were given a normal diet and normal drinking water; ABT group mice were given a normal diet and A + V solution in drinking water for the last 7 days; ABT + SRT group mice were fed a 1.5% Saireito diet until the last day and given A + V solution in drinking water for the last 7 days. (A) Images of fecal occult bleeding test (FOBT) on day 28. If FOBT is positive, it will turn blue. (B) Graph shows the images of large intestines. (C) Bar graphs show the weight of cecums. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s multiple comparisons test. Data are shown as mean ± SEM (n = 4, each group). ****P < 0.0001.

Fig 2. Effect of Saireito on antibiotics-induced colon inflammation.

Mice in normal group were given a normal diet and normal drinking water; ABT group mice were given a normal diet and A + V solution in drinking water for the last 7 days; ABT + SRT group mice were fed a 1.5% Saireito diet until the last day and given A + V solution in drinking water for the last 7 days. (A) The colon was excised on day 28, sectioned, and stained with H&E (×200). (B) Graph shows the histological score. (C) mRNA expression level of pro-inflammatory cytokines was determined by quantitative RT-PCR. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s multiple comparisons test. Results are expressed as the mean ± SEM (n = 4, each group). * P < 0.05, ** P < 0.01, *** P < 0.0001, **** P < 0.0001, and NS: not significant.

Fig 3. Effect of Saireito on antibiotic-induced colonic epithelial barrier dysfunction and cell apoptosis in the colon.

Normal group mice were given a normal diet and normal drinking water; ABT group mice were given a normal diet and A + V solution in drinking water for the last 7 days; ABT + SRT group mice were fed a 1.5% Saireito diet until the last day and given A + V solution in drinking water for the last 7 days. (A) Graph shows FITC-dextran levels in the serum of each mouse. One representative of two independent experiments (n = 5, normal group. n = 11, ABT group and n = 5, ABT + SRT group). (B) Graph shows TUNEL staining of the colon (× 400). (C) Graph shows the number of TUNEL positive apoptotic cells in each section (n = 4, each group). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test and student t test. Data are shown as mean ± SEM. * P < 0.05.

Fig 4. Effect of Saireito on expression of cell adhesion proteins.

Normal group mice were given a normal diet and normal drinking water; ABT group mice were given a normal diet and A + V solution in drinking water for the last 7 days; ABT + SRT group mice were fed a 1.5% Saireito diet until the last day and given A + V solution in drinking water for the last 7 days. (A) mRNA analysis of cell adhesion proteins. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s multiple comparisons test. Data are shown as mean ± SEM (n = 4, each group). * P < 0.05, **P < 0.01. (B) The figure shows immunohistochemistry for ZO-1, occludin, and E-cadherin (×200).

Fig 5. Effect of Saireito co-treatment on the induction of cell adhesion protein in vitro.

(A) mRNA analysis of cell adhesion proteins in vitro. The concentrations of Saireito were 1 μg/ml or 3 μg/ml. Statistical analysis was carried out using Dunnett’s test. Data are shown as mean ± SEM (n = 3, each group). * P < 0.05, **P < 0.01 vs Control.

Fig 6. Effect of Saireito on gut microbiota of mice.

(A) Visualization of principal coordinates analysis (PCoA) of unweighted Unifrac distances to show differences in bacterial composition. Each point represents the bacterial fecal microbiota in a single sample. (B) α-diversity was measured by the Shannon diversity index of microbiota in fecal samples from different groups (n = 4, normal group. n = 5, ABT and ABT + SRT group). Mann-Whitney U test was used to determine statistical significance, * P < 0.05, **P < 0.01. (C) DNA in cecum sample was extracted and copy number of rRNA gene was quantified by qPCR. Mann-Whitney U test was used to determine statistical significance, * P < 0.05. (D) Fecal samples were subjected to 16S ribosomal RNA sequencing to evaluate the composition of gut microbiota. The relative bacterial abundance is shown at the genus level. Each bar shows relative bacterial abundance in individual mice. (E) The relative bacterial abundance is shown at the species level. Data are shown as mean ± SEM (n = 4, normal group. n = 5, ABT and ABT + SRT group) **** P < 0.0001.

Fig 7. A model for the effect of Saireito on leaky gut associated with antibiotic-induced dysbiosis.

The figure summarizes the results of this study. (A) Antibiotic therapy disturbs the normal gut microbiota. Pseudomonas spp., which is mostly occupied in the colon of mice treated with antibiotic, disrupts the intestinal epithelial barrier by decreasing the expression of TJPs and increasing epithelial cell apoptosis. (B) Saireito may suppress the growth of Pseudomonas spp., enhance the expression of TJPs and inhibit intestinal epithelial apoptosis, which contributes to alleviates leaky gut in antibiotic-treated mice.