Semi-Rational Screening of Probiotics from the Fecal Flora of Healthy Adults against DSS-Induced Colitis Mice by Enhancing Anti-Inflammatory Activity and Modulating the Gut Microbiota
1Department of Gastroenterology, Cangzhou Central Hospital, No. 16 Xinhua West Road, Cangzhou, Hebei 061001, P.R. China, 2Tianjin Institute of Acute Abdominal Diseases, Nankai Hospital, Nankai District, Tianjin, 300100, P.R. China, 3Endoscopy Room, Cangzhou Central Hospital, No. 16 Xinhua West Road, Cangzhou, Hebei 061001, P.R. ChinaCorrespondence to:
J. Microbiol. Biotechnol. 2019; 29(9): 1478-1487
Published September 28, 2019
Copyright © The Korean Society for Microbiology and Biotechnology.
Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn’s disease (CD) . Ulcerative colitis (UC), a chronic or long lasting IBD, is difficult to cure due to its rising incidence in recent decades . To our best knowledge, UC causes many sporadic symptoms, including abdominal pain, diarrhea, and bloody mucopurulent stool , which is often accompanied by an increase in intestinal tract disorders  and inflammatory mediators .
Gut microbiota can be divided into three categories: (A) Symbiotic bacteria, including
Inducing remission and preventing recurrence were the goal of UC treatment . Nowadays, the major drugs used in UC management are corticosteroids, 5-aminosalicylates, immunomodulators and biologics. However, the severe adverse side effects are also very obvious, such as nephrotoxicity, fever, rash, drug hypersensitivity, hepatitis, pancreatitis, lymphadenopathy, abdominal pain, nausea, vomiting, diarrhea exacerbation, and myalgia . Due to dissatisfaction with these medications, probiotics, which were only discovered within recent years, are now seen as a potential complementary and alternative therapy for the treatment of UC and related illness.
Probiotics can enhance the intestinal barrier, regulate the immune system and improve health performance . Probiotics have been used in UC remission via barrier function modulation, which involves the competition of nutrients and the production of bacteriocin or antibacterial proteins. It has been reported that probiotics, such as
Fecal microbiota transplantation (FMT), also called fecal bacteriotherapy, has been used for 50 years for treatment of
Materials and Methods
Screening of Strains
Lactic acid bacterial strains were isolated from fecal microbiota of healthy adults. To ensure general health, the criteria for the healthy adult volunteers had to meet the following standards: 1) be free of known metabolic and gastrointestinal diseases, with no history of metabolic or gastrointestinal diseases; 2) avoid taking medications or alcohol that would impact gut function; 3) be willing to complete all necessary study questionnaires and to donate stool specimens as required; 4) to voluntarily sign a written informed consent form before participation in the study. Lactic acid bacteria were isolated on MRS agar (pH 5.6) incubated at 30°C for 72 h. Acid-resistance of lactic acid bacteria colonies isolated from the MRS agar was screened by culturing on MRS agar (pH 3.0). The bile salt tolerance of the colonies was screened by culturing on MRS agar containing 0.30% ox bile salts at 37°C.
C57BL/6J mice (seven weeks old, male) were purchased from Super-B&K Laboratory Animal Corp., Ltd. (China). The mice were kept at 22°C under a 12-h light/dark cycle, supplied with sufficient water and standard rodent diet, and acclimatized to laboratory conditions for 7 days before conducting experiments. Mice were divided into 4 groups (
Analyses of Hemoglobin Concentration in Blood and DAI
Hemoglobin concentration was determined according to the method of HiCN . The disease activity indices (DAI) composed of three parameters (weight loss, stool consistency, and bloody stools) were calculated to assess severity of colitis according to the report of Sang LX . Briefly, the disease activity index consisted of scoring for stool consistency (0–3) body weight loss (0–3), and visual observation of blood in feces (0–4).
Quantitative Real-Time Polymerase Chain Reaction (qrtPCR)
A fresh colon sample was used for total RNA extraction from with TRIZOL reagent (Invitrogen Life Technologies Co Ltd., USA) following the manufacturer’s protocol. The relative mRNA expression of cytokines was determined by quantitative real-time polymerase chain reaction (qrt-PCR) with optimal concentrations of primers and probes. Mouse TGF-β primer (forward 5’-ACGAC ATGATAGTCACTGACAACA and reverse 5’-TTGGGGTCA TGGCAAACTGTCTC); TNF-α primer (forward 5’-AGATGTGGA ACTGGCAGAGG; antisense 5’-CACGAGCAGGAATGAGAAGAG), IL-10 primer pair (forward 5’-ACTGCTATGTTGCCTGCTCTT; antisense 5’-GGTCTGGCTGACTGGGAAGT) and IL-6 primer pair (sense 5’-TAGTCCTTCCTACCCCAATTTCC; antisense 5’- TTGGTC CTTAGCCACTCCTTC) were used as the primers or probes. Mouse GAPDH primers (forward: 5’-GTATGACTCCACTCACGGCAAA and reverse, 5’-GGTCTCGCTCCTGGAAGATG) were used as internal controls. The thermal cycler conditions were 30 cycles of 95°C for 15 sec, 58°C for 20 sec, and 72°C for 30 sec. All samples were run in duplicate. Relative quantity or fold change in gene expression of target genes relative to the house keeping gene 2-ΔΔCt, where ΔΔCt = ΔCt control – ΔCt treated.
Analysis of Intestinal Bacteria
The composition of the bacteria present in colonic contents of the mice was detected by quantitative PCR according to the method of Long T
Measurement of the Colonic Mucosal Permeability In Vitro
The colonic mucosal permeability was examined by Evans Blue (EB) assay . A small bowel was infused with 0.3 ml EB solution. After washing with normal saline, the small bowel was dried for 24 h, and incubated with 1 ml of formamide for 24 h. After centrifuging, the supernatant was collected and examined under an ultraviolet spectrophotometer at a wavelength of 620 nm.
FMT has been used for treatment of
Fig. 1. Strains screened from the fecal flora of healthy adults (
A). Survival rate % (PBS, pH=3.0)= N4/N0 ×100 %; Survival rate % (0.3 % bile salt)= N4b/N0b ×100 %. N4, Number of lactic acid bacteria (log CFU/ml) after incubation for 4 h at 37°C; N0, Number of lactic acid bacteria (log CFU/ml) after incubation for 0 h at 37°C in MRS broth (pH=3.0); N4b, Number of lactic acid bacteria (log CFU/ml) after incubation for 4 h at 37°C in MRS broth containing 0.30% bile salts (pH=6.0); N0b, Number of lactic acid bacteria (log CFU/ml) after incubation for 0 h at 37°C in MRS broth containing 0.30% bile salts (pH=6.0). The concentrations of the candidates were 1 × 106 CFU/ml. The concentrations of IL-10 were measured by ELISA. Screening of the functional Bifidobacteriumstrains on inhibition of E. coliisolated from bloody stools of DSS-induced colitis mice ( C). The E. coli(1 × 108 CFU/ml) were cocultured with Bifidobacteriumfor three hours at 37°C. ( D) Disease activity index, a composite measure of weight loss, stool consistency and blood in stool. Data presented indicate the mean ± SD. ## p< 0.01 vs C group, * p< 0.05 and ** p< 0.01 vs DSS group.
Fig. 4. The abundances of microbial phyla in mice fecal samples (
A). In DSS-induced colitis mice and other experimental groups, pooled fecal samples were provided separately. The Bacteriodetes/ Firmicutes(B/F) ratio of different groups ( B). Alteration of microbiota composition in C57BL/6J mice ( C, D). Relative quantity of four representative bacteria was measured by qPCR. Values were expressed as the mean ± SD, * p< 0.05, ** p< 0.01 as conducted.
Effects of Probiotic PB on DSS-Induced Colitis Mice
The function of probiotic PB in DSS-induced colitis mice was examined in vivo. C57BL/6J mice were pre-treated with probiotics P or PB for four days and then administered DSS in drinking water for another four days to induce colitis. Treatment with probiotic PB was continued after DSS administration. The result was shown in Fig. 2. Comparing to the control group, the colon length of DSS group mice was significantly decreased and colon weight/colon length were increased (Fig. 2A). The DSS group had a significantly higher colon weight/length ratio (63 ± 0.92 mg/cm) compared to the other groups. Moreover, it was observed that the colon weight/length ratio of the DSS-induced mice could be reduced by probiotic PB. Also, the role of probiotic P was enhanced by
Fig. 2. PB ameliorated colonic injury in dextran sodium sulfate (DSS)-induced chronic colitis mice. (
A) Colon length. ( B) Colon weight/Colon length (mg/cm). Abbreviation: C, Control group; DSS, DSS group; DSS+P, DSS + Lactobacillus sakei 07(L07); (4) DSS + PB, DSS + Lactobacillus sakei 07(L07) + Bifidobacterium bifidumB10. Values were expressed as the mean ± SD, * p< 0.05, ** p< 0.01 as conducted.
Furthermore, the effect of probiotic PB on mRNA expression of cytokines in DSS-induced colitis mice has been investigated. The mRNA expression of IL-10 and TGF-β was down-regulated in DSS-induced mice as compared to control group. The TGF-β and IL-10 expression of the DSS group was up-regulated by administration of probiotic P or PB, however, the difference between probiotic P and probiotic PB is not significant (Figs. 3A and 3B). The results demonstrated that mRNA levels of IL-6 and TNF-α were elevated in DSS group compared with the control group. Colonic IL-6 and TNF-α expression of DSS-induced mice was significantly reduced after administration of probiotic P or PB (Figs. 3C and 3D).
Fig. 3. Effects of probiotics on inflammation marker expression. Relative expression level of TGF-β (
A); Relative expression level of IL-10 ( B); Relative expression level of IL-6 ( C); Relative expression level of TNF-α ( D). Abbreviation: C, control; DSS: DSStreated group; DSS+P: Probiotic P-treated group; DSS+PB: Probiotic combination-treated group. Values were expressed as the mean ± SD, * p< 0.05, ** p< 0.01 as conducted.
Effect of Probiotic PB on the Bacterial Communities of DSS-Induced Colitis Mice
The relative abundances of different phyla in feces samples were shown in Fig. 4. The high-abundant (>1% of community) were investigated, including
In addition, the
Furthermore, the relative representative four bacteria (
Effect of Probiotic PB on LPS and Homocysteine Levels of DSS-Induced Colitis Mice
In order to further evaluate the effect of probiotic PB on the intestinal tract, the concentrations of fecal and plasma endotoxin were investigated. The result showed that the fecal LPS and plasma LPS levels of DSS group were higher than the control group. What’s worse, the plasma LPS levels of DSS group were three times that of control group (Fig. 5A). The
Fig. 5. Effect of probiotic PB on the fecal LPS and plasma LPS levels (
A). LAL assay was used to measure the fecal and plasma endotoxin concentrations. All values are indicated as the mean ± SD. Effect of probiotic PB on the PLPS/ FLPS rate ( B). Abbreviation: PLPS, plasma LPS levels; FLPS, fecal LPS levels. Values were expressed as the mean ± SD, * p< 0.05, ** p< 0.01 as conducted.
Furthermore, several studies have found that the endothelial cell barrier can be destroyed by homocysteine (Hcy), resulting in increased endothelial cell permeability and promoted inflammation [14, 22]. Therefore, the levels of homocysteine in plasma and colon have been investigated. The result showed that the levels of homocysteine in plasma and colon were increased in DSS-induced colitis mice, comparing to the healthy mice (Fig. 6A). As assessed by the Evans blue permeability test, we detected a significant increase in intestinal permeability of DSS-induced colitis mice compared with the healthy mice. Upon administration of PB, there was an approximately 2.01-fold decrease in intestinal permeability of DSS-induced colitis mice (Fig. 6B).
Fig. 6. The levels of homocysteine (Hcy) in plasma and colon mucosa in mice with DSS-induced colitis (
A). Abbreviation: C, control; DSS: DSS-treated group; DSS+PB: Probiotic combination-treated group. Effect of probiotic PB on the colonic mucosal permeability of DSS-induced colitis ( B). Values were expressed as the mean ± SD, ## p< 0.01 vs C group, ** p< 0.01 vs DSS group.
FMT, also called fecal bacteriotherapy with a resolution rate of 83% -92%, has been used for 50 years for treatment of
During the last decade, abundant evidence has shown that colonic inflammation is associated with inflammation and gut microbiota . In present study, we found that
Lipopolysaccharide (LPS; endotoxin) produced by gram-negative bacteria increases the permeability of the gut mucosal barrier, increasing LPS translocation into the circulation, augmenting endotoxemia with consequent systemic inflammation . It was found that the plasma endotoxin concentrations were decreased by probiotic PB treatment in this study. These results indicate that there are a lot of endotoxins absorbed and accumulated by the intestine rather than excreted out of the body in DSS-induced colitis mice, comparing to the healthy mice. Consequently, the
Conflicts of Interest
The authors have no financial conflicts of interest to declare.
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