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Method Article
Here, we present a protocol to establish the antibiotic-induced pseudo-germ-free dextran sulfate sodium-induced colitis mouse model to investigate the role of gut microbiota in regulating the positive effects of Bacillus cereus on colitis.
Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in alleviating colitis remain undefined. Most analysis methods rely on limited diversity and abundance of gut microorganisms. Therefore, observational studies cannot establish causation. In this study, we applied antibiotic-induced pseudo-germ-free mice to investigate the role of gut microbiota in regulating the probiotic effects of Bacillus cereus (B. cereus) on dextran sulfate sodium (DSS)-induced colitis in mice. This process allows for evaluating the bidirectional regulating effect of B. cereus supplementation on health and provides stable and reproducible results. Here, the detailed protocols for B. cereus cultivation, gavage operation, stool collection, and antibiotic clearance treatment on colitis mice are provided. The optimization methods are also applicable for other chronic inflammatory-associated disorders. The results showed that B. cereus administration decreased body weight loss, colon length shortening, disease activity index, and histopathological scores. However, treatment with antibiotics suppressed the positive effect of B. cereus on colitis. These results indicate that gut microbiota are needed for the alleviating effects of B. cereus on colitis. Therefore, exploring the beneficial effects of probiotics in this research is a promising approach for developing novel treatment strategies for alleviating the symptoms of chronic inflammatory-associated disorders.
Inflammatory bowel diseases (IBDs) are common chronic gastrointestinal inflammatory disorders, including Crohn's disease and ulcerative colitis (UC)1. The current therapeutic avenues for inflammatory colitis are 5-aminosalicylates, corticosteroids, azathioprine, and antibiotics2. Moreover, these drugs have considerable side effects3. Therefore, we should pay more attention to the effect of probiotics on the treatment of colitis.
Alternative therapeutic approaches include probiotic administration, which has been used in animal models and clinical trials. Previous studies have indicated that different probiotic administration, such as Bacillus cereus or Bifidobacterium infantis, could alleviate colitis4,5. In preclinical studies, the effectiveness and safety of probiotics must be investigated in animal models.
Dextran sulfate sodium (DSS) colitis mouse models can help explore the mechanisms of colitis and evaluate the positive effects of probiotics. The DSS inducement results in erosions of the intestinal mucosa, gut barrier dysfunction, and an increase in intestinal epithelial permeability6. Most mouse models for probiotics have mainly highlighted biological effects. However, the mechanism behind the administration of this probiotic is challenging to explore, given the limitation in further verifying the causal relationship between probiotics and the alleviation of colitis. Thus, there is a need to develop a standardized method for investigating the mechanism of probiotics.
Traditionally, these studies require inoculating certain probiotics into germ-free mice7. However, there are some laboratory limitations in using germ-free mice as receptors for probiotics, such as the low immune capacity and expensive germ-free facilities8. In order to avoid these limitations, an alternative DSS-induced colitis model was established using pseudo-germ-free mice. The pseudo-germ-free mouse model was established by the application of antibiotics as previously described9,10.
In this article, we establish pseudo-germ-free mice with an antibiotic cocktail. We also describe in detail the methodology for establishing and evaluating mouse colitis models and investigate the effects of probiotics on alleviating colitis symptoms. The protocol below also provides the methods of probiotic delivery by gavage.
All procedures were carried out in accordance with the Guidelines for Care and Use of Laboratory Animals of Anhui University, China, and all procedures were approved by the Animal Ethics Committee of Anhui University, China (No. IACUC(AHU)-2020-014).
1. Administration of antibiotics
2. Preparation of DSS drinking water and induction of colitis
3. Quantification of B. cereus
4. Preparation of B. cereus for gavage
5. Application of B. cereus supplements by gavage
6. Determination of the disease activity index
NOTE: The disease activity index (DAI) was calculated daily by the mice's body weight loss, fecal occult blood, and stool consistency.
7. Tissue collection
8. Assessment of histological damage
B. cereus supplementation and colitis
The acute experimental colitis model was induced by the DSS intervention in drinking water. Figure 1A shows the experimental protocol for B. cereus administration to the colitis mouse model. DSS inducement obviously decreased the body weight (Figure 1B) and colon length. The alleviating effect of B. cereus through oral gavage was investigated on the colitis model mice. Compar...
In order to use probiotics in clinical studies, it is necessary to evaluate the efficacy and safety of probiotics in animal models. The provided protocols have been previously optimized for evaluating colitis severity. Mice treated with DSS is a model of intestinal inflammation, which mimics the clinical and histological features characteristic of ulcerative colitis12. Acute colitis model mice are characterized by body weight loss, diarrhea, fecal bleeding, and inflammatory cell infiltration
The authors have nothing to disclose.
This study was supported at least in part by the National Natural Science Foundation of China (K.S., grant No. 32000081), the Natural Science Foundation of Anhui Province (K.S., grant No. 1908085QC120), the Open Project Program of State Key Laboratory of Food Science and Technology, Jiangnan University (K.S., grant No.SKLF-KF-201920), the Natural Science Foundation of Anhui Higher Education Institutions of China (K.S., grant No. KJ2019A0040), the Doctoral Scientific Research Foundation of Anhui University (K.S., grant No. J01003316 ), the National Natural Science Foundation of China (Y.W., grant Nos. 31770066, 31470218), the Open fund for Discipline Construction, Institute of Physical Science and Information Technology of Anhui University (Y.W.), and the Outstanding Talents Program of Anhui University, China (Y.W.).
Name | Company | Catalog Number | Comments |
0.01 M PBS (powder, pH7.2–7.4) | Solarbio | P1010-2L | |
Absolute ethyl alcohol | Hushi | 64-17-5 | |
Acid alcohol fsat differentiation solution | Beyotime | No.C0163S | |
Agar | Sangon Biotech | 9002-18-0 | |
Ampicillin | Solarbio | 69-53-4 | Store at 2–8 °C |
Anaerobic incubator | Long Yue | LA1-3T | |
Dextran sulfate sodium salt colitis grade | MP Biomedicals | 160110 | |
Electrothermal incubator | SANFA | HP-050A | |
General purpose tissue fixator | biosharp | BL539A | |
Glycerinum | Hushi | 56-81-5 | |
Hematoxylin and eosin staining kit | Beyotime | No.C0105 | |
Kisser's Mounting Medium | Beyotime | No.C0181 | |
Metronidazole | Solarbio | 443-48-1 | Store at 2–8 °C |
Neomyein sulfate | Solarbio | 1405-10-3 | Store at 2–8 °C |
Oscillating incubator | Shanghai Zhichu | ZQLY-180S | |
Sodium chloride | Sangon Biotech | 7647-14-5 | |
Stool occult blood test paper | Baso | BA2020B | |
Tryptone | OXOID | 2285856 | |
Vancomycin | Solarbio | 1404-93-9 | Store at 2–8 °C |
Xylene | Hushi | 1330-20-7 | |
Yeast extract | Sangon Biotech | 8013-01-2 |
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