CN115569153A - Method for improving ulcerative colitis by using bacterial strains producing different short-chain fatty acids - Google Patents
Method for improving ulcerative colitis by using bacterial strains producing different short-chain fatty acids Download PDFInfo
- Publication number
- CN115569153A CN115569153A CN202211246638.4A CN202211246638A CN115569153A CN 115569153 A CN115569153 A CN 115569153A CN 202211246638 A CN202211246638 A CN 202211246638A CN 115569153 A CN115569153 A CN 115569153A
- Authority
- CN
- China
- Prior art keywords
- propionibacterium freudenreichii
- group
- clostridium butyricum
- bifidobacterium bifidum
- dss
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Rheumatology (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pathology (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Endocrinology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Pain & Pain Management (AREA)
- Diabetes (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The present invention develops a method for improving ulcerative colitis by using strains producing different short-chain fatty acids. The three strains are Bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 respectively. The effect of a tested strain on UC is evaluated by utilizing a DSS-induced mouse model, and three strains are found to remarkably reduce spleen enlargement, disease Activity Index (DAI), colon shortening, myeloperoxidase (MPO) activity, the concentrations of proinflammatory factors IL-8, IL-1 beta and TNF-alpha, TLR-2 and TLR-4 expression and over-expression of ROCK-1 and Axin 2; the expression of the colon tight junction protein (ZO-1, occludin and Claudin-1), the mRNA level of TLR-5, LGR5 and Rspo3, the alpha diversity Chao1 index and the Simpson index are obviously improved. Furthermore, the use of bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and clostridium butyricum C1-6 resulted in a significant increase in the level of SCFAs of the colon content (P < 0.05), with propionibacterium freudenreichii B1 having the most significant effect.
Description
Technical Field
The invention belongs to the technical field of microorganisms and immunoregulation, and mainly relates to a method for improving ulcerative colitis by using strains producing different short-chain fatty acids.
Background
Inflammatory Bowel Disease (IBD) is a chronic, recurrent inflammatory disease that includes Crohn's Disease (CD) and Ulcerative Colitis (UC). The etiology of IBD is complex, and immune imbalance, altered intestinal barrier permeability, microbial population status, genetic factors and environmental factors all contribute significantly to the development and progression of the disease. At present, the treatment of IBD is dominated by anti-inflammatory drugs and intestinal immunomodulation, and biotherapeutics with anti-TNF drugs are also in use. However, due to the complexity and diversity of IBD conditions, many patients do not respond to clinically approved drugs, and new therapeutic approaches need to be further explored.
With the intensive research on the intestinal flora, the intestinal flora is found to play an important role in the occurrence and development of UC. In one aspect, an imbalance in gut flora homeostasis induces changes in the gut epithelial mucosa and modulates inflammatory activity by modulating pro-inflammatory or anti-inflammatory cytokines. On the other hand, the composition and abundance of the intestinal microflora structure has changed significantly in UC, resulting in a decrease in beneficial bacteria and an increase in harmful bacteria. Probiotics generally promote health by regulating the homeostasis of intestinal microorganisms, and lactobacilli, bifidobacteria and various probiotic compounds are widely used in the study of UC relief. In addition, short Chain Fatty Acids (SCFA), which are metabolites and components of probiotics, also known as metagens, mainly acetate, propionate and butyrate, can be sensed by G protein-coupled receptors and thus participate in downstream immune responses in the gut. One previous study found that there was a reduction in propionic and butyric acid producing bacteria in patients with inflammatory bowel disease, suggesting that SCFA may play a role in reducing UC. The bifidobacterium bifidum H3-R2 with high acetic acid yield and potential probiotic functions, the propionibacterium freudenreichii B1 with high propionic acid yield and the clostridium butyricum C1-6 with high butyric acid yield are screened from early-stage laboratories in the experiment. The evaluation of the effects and mechanisms of the three strains with high yield of different SCFAs on the improvement of the ulcerative colitis has very important practical significance and application value.
Disclosure of Invention
The invention provides three strains which produce different short-chain fatty acids, and the initial UC prevention and UC alleviation effects of the three strains are judged through animal experiments.
The technical problem to be solved by the invention is realized by the following technical scheme: a method for ameliorating ulcerative colitis using strains that produce different short chain fatty acids, comprising the steps of: (1) culture of the strain: bifidobacterium H3-R2 was cultured in De Man, rogosa and Sharpe (MRS) medium supplemented with 0.05% L-cysteine hydrochloride (mMRS) at 37 ℃ for 16 hours, propionibacterium freudenreichii B1 was cultured in glucose medium at 30 ℃ for 24 hours, and Clostridium butyricum C1-6 was cultured in Reinforced Clostridium Medium (RCM) at 37 ℃ for 12 hours. Two subcultures were performed before the experiment. All strains were washed twice with PBS and adjusted to 10 by PBS resuspension 9 CFU/mL, for subsequent in vivo experiments. (2) The effect of the test strains on UC was evaluated using a DSS-induced mouse model: male Specific Pathogen Free (SPF) C57BL/6J mice, 8 weeks old, were used and reared at 23. + -. 3 ℃ with 55. + -. 10% humidity and 12h alternating light. The 48 mice were randomized into 6 groups after 1 week of acclimation: normal control group (NC), DSS group (DSS), positive control group (PC), bifidobacterium H3-R2 group (BB), propionibacterium freudenreichii B1 group (PF), and Clostridium butyricum C1-6 group (CB). Spleen index, disease Activity Index (DAI), colon tissue status, colon tissue Myeloperoxidase (MPO) Activity, cytokine levels in Colon, claudin and inflammation-related Gene expression, pathway protein Table for different groups of miceAnd measuring short-chain fatty acid (SCFA) in the mouse intestinal flora and the feces, and performing statistical analysis.
The bifidobacterium bifidum H3-R2, the propionibacterium freudenreichii B1 and the clostridium butyricum C1-6 are automatically separated by a laboratory and are reserved in an important laboratory of the dairy science education department of northeast university of agriculture.
The mouse spleen index is as follows: the bifidobacterium bifidum H3-R2, the propionibacterium freudenreichii B1 and the clostridium butyricum C1-6 can obviously relieve the enlargement of the spleen of a mouse caused by DSS stimulation.
The Disease Activity Index (DAI) is: DSS treatment increased mouse DAI significantly above normal groups. The use of Bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 can significantly reduce this index.
The colon tissue state is as follows: the application of the bifidobacterium bifidum H3-R2, the propionibacterium freudenreichii B1 and the clostridium butyricum C1-6 can effectively relieve the colitis symptoms caused by DSS, recover the colon morphology, reduce inflammatory cell infiltration, improve crypt displacement and enrich goblet cells. However, the effects of the propionibacterium freudenreichii B1 and the clostridium butyricum C1-6 on recovering the form and the crypt depth are better than those of the bifidobacterium bifidum H3-R2 to a certain extent.
The activity of the colon tissue Myeloperoxidase (MPO) is as follows: the use of Bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 significantly reduced the increase in MPO viability in the colon of mice caused by DSS.
The levels of cytokines in the colon are: the use of Bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 can reduce the content of proinflammatory factors IL-8, IL-1 beta and TNF-alpha and obviously increase the content of anti-inflammatory factors IL-10. The three strains are shown to relieve the DSS-induced colitis in mice by modulating inflammatory factors.
The expression of the tight junction protein and the inflammation related gene is as follows: the use of Bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 can increase colonic Claudin expression (ZO-1, occludin and Claudin-1) and improve intestinal barrier function. The combined results of the expression of the three proteins show that Propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 are more effective in protecting the intestinal barrier than Bifidobacterium bifidum H3-R2. The three strains can obviously reduce the expression of TLR-2 and TLR-4, obviously improve the expression of TLR-5 and increase the mRNA level of LGR5 and Rspo 3. The B1 group of P.freudenreichii showed the most significant effect, suggesting that it may have greater potential for ameliorating colitis.
The pathway protein is expressed as: the use of bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 all down-regulated the over-expression of ROCK-1 and Axin2, but only the Propionibacterium freudenreichii B1 group and the Bifidobacterium bifidum H3-R2 group were significantly different (P < 0.05) compared with the DSS group, indicating that these two strains regulate inflammation through ROCK-1 pathway and stimulate the regeneration of damaged intestinal epithelial cells through Wnt/beta-catenin pathway.
The intestinal flora of the mouse is as follows: the use of bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 raises the Chao1 index and Simpson index, increasing the relative abundance of Actinomycetes phylum. At the genus level, the abundance of Prevotellaceae UCG-001, turcibacter, clostridium-UCG-014 and Murebacteriaceae can be remarkably increased, and the abundance of Escherichia coli, shigella, staphylococci and enterobacteriaceae can be reduced.
The short-chain fatty acid (SCFA) in the excrement is as follows: the use of bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and clostridium butyricum C1-6 resulted in a significant increase in the level of SCFAs of the colon content (P < 0.05), with the most significant effect of propionibacterium freudenreichii B1 group.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts;
FIG. 1 is a graph of the effect of strains on DSS-induced mice; (a) weight change; (B) a DAI score; (C) colon length; (D) spleen index; (E) an image of colon tissue stained with H & E;
FIG. 2 is a graph showing the effect of strains on MPO activity and cytokine production in the colon;
FIG. 3 is the effect of the strain on the restoration of the epithelial barrier;
FIG. 4 is a graph of the effect of strains on Toll-like receptor, LGR5 and Rspo3 mRNA expression;
FIG. 5 shows the effect of the strain on ROCK-1 and Wnt/beta-catenin signaling pathways;
FIG. 6 is the effect of strains on gut microbiota;
FIG. 7 is a graph of the effect of the strain on SCFAs levels in colon contents;
Detailed Description
The following detailed description of specific embodiments of the invention refers to the accompanying drawings.
A method for ameliorating ulcerative colitis using strains that produce different short chain fatty acids, comprising the steps of:
(1) Culturing the strain: bifidobacterium H3-R2 was cultured in De Man, rogosa and Sharpe (MRS) medium supplemented with 0.05% L-cysteine hydrochloride (mMRS) at 37 ℃ for 16 hours, propionibacterium freudenreichii B1 was cultured in glucose medium at 30 ℃ for 24 hours, and Clostridium butyricum C1-6 was cultured in Reinforced Clostridium Medium (RCM) at 37 ℃ for 12 hours. Two subcultures were performed before the experiment. All strains were washed twice with PBS and adjusted to 109CFU/mL by PBS resuspension for subsequent in vivo experiments. (2) The effect of the test strains on UC was evaluated using a DSS-induced mouse model: male pathogen-free (SPF) C57BL/6J mice 8 weeks old were used and were raised at 23. + -. 3 ℃ and 55. + -. 10% humidity for 12h with alternating light. The 48 mice were randomized into 6 groups after 1 week of acclimation: normal control group (NC), DSS group (DSS), positive control group (PC), bifidobacterium H3-R2 group (BB), propionibacterium freudenreichii B1 group (PF), and Clostridium butyricum C1-6 group (CB). The NC group was fed standard normal mouse diet throughout the experiment, while the other five groups provided free of DSS-supplemented drinking water from day 1 to day 7. DSS was dissolved in drinking water at a concentration of 2.5%. From day 8 to day 14, the DSS group was gavaged with 200. Mu.L of sterile physiological saline daily, the PC group was gavaged with 75mg/kg/d daily, mesalamine (5-AS) dissolved in sterile PBS, and the BB, PF and CB groups were gavaged with 200. Mu.L of 109CFU/ml Bifidobacterium H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6, respectively, daily. At the end of the experiment, mice were sacrificed. Spleen index, disease Activity Index (DAI), colon tissue status, colon tissue Myeloperoxidase (MPO) activity, cytokine levels in colon, claudin and inflammation-related gene expression, pathway protein expression, mouse intestinal flora, short Chain Fatty Acid (SCFA) in stool were determined and statistically analyzed in different groups of mice.
The bifidobacterium bifidum H3-R2, the propionibacterium freudenreichii B1 and the clostridium butyricum C1-6 are provided by the collection center of industrial microorganism strains in key laboratories of the department of dairy science education of the northeast university of agriculture.
Example 1
After the mice were sacrificed, spleens of the mice were collected by dissection, and the spleens were weighed (mg) after sucking the residual blood with filter paper, and divided by the body weight (g) of the mice, respectively, to calculate a spleen index. The use of bifidobacterium bifidum H3-R2 (BB group), propionibacterium freudenreichii B1 (PF group) and Clostridium butyricum C1-6 (CB group) can significantly alleviate the mouse splenomegaly caused by DSS stimulation. The results are shown in FIG. 1.
Example 2
From 8 to 14 days, we measured the DAI score, which is assessed by combining weight loss, fecal consistency and fecal blood score. DSS treatment increased mouse DAI significantly above normal groups. The use of Bifidobacterium bifidum H3-R2 (BB group), propionibacterium freudenreichii B1 (PF group) and Clostridium butyricum C1-6 (CB group) significantly reduced this index. The results are shown in FIG. 1.
Example 3
The fixed samples were dehydrated in a dehydrator by ethanol washing, then paraffin embedded and cut into 4 μm sections, deparaffinized, stained with hematoxylin-eosin (H & E), and finally histopathological changes of colon between different groups were observed under a high power microscope. The application of the bifidobacterium bifidum H3-R2, the propionibacterium freudenreichii B1 and the clostridium butyricum C1-6 can effectively relieve the symptoms of the colitis caused by DSS, recover the colon form, reduce the infiltration of inflammatory cells, improve the displacement of crypts and enrich goblet cells. However, the effect of Propionibacterium freudenreichii B1 (PF group) and Clostridium butyricum C1-6 (CB group) on recovering morphology and crypt depth was superior to that of Bifidobacterium bifidum H3-R2 (BB group) to some extent. The results are shown in FIG. 1.
Example 4
Colon tissue was homogenized by adding reagents in the kit at a weight to volume ratio of 1. The MPO activity in the homogenate was measured according to the instructions of Nanjing-built MPO (colorimetric) kit. The use of bifidobacterium bifidum H3-R2 (BB group), propionibacterium freudenreichii B1 (PF group) and Clostridium butyricum C1-6 (CB group) can significantly reduce the MPO activity of the colon of UC mice. The results are shown in FIG. 2.
Example 5
Colonic Tissue was weighed and RNA extracted using the FastPure Cell/Tissue Total RNA Isolation Kit V2 according to the manufacturer's instructions. GoScript was used according to the manufacturer's instructions TM Reverse Transcription was performed using Reverse Transcription Mix Kit. Use of
The qPCRMaster Mix Kit performs PCR on the PikoReal 96 real-time PCR system. The relative expression levels of the mRNAs of ZO-1, occludin, claudin-1, TLR2, TLR4, TLR5, rspo3 and LGR5 were calculated according to the 2- Δ Δ Ct method and compared with the expression level of the GAPDH housekeeping gene. ZO-1 was observed to be mainly distributed on the membranes of the regenerated colonic epithelium in BB, PF and CB groups, the results of IHC staining and the measurement of the relative mRNA amount of occludin and claudin-1 were consistent with those of ZO-1, DSS was decreased, all the strain-treated groups were increased, and the effect was not significantly different from that of the PC group. Propionibacterium freudenreichii B1 (PF group) and Clostridium butyricum C1-6 (CB group) were more effective in protecting the intestinal barrier than Bifidobacterium bifidum H3-R2 (BB group). The results are shown in FIG. 3.
Example 6
Immunohistochemistry (IHC) measures the expression of ZO-1, occludin and claudin-1 in colon tissues. According to the experimental method described previously, the embedded paraffin was cut into 4 μm sections by microtome, then deparaffinized, hydrated, placed in a mixture of antigen retrieval solutions, and incubated with 3% hydrogen peroxide solution to block endogenous peroxidase. After incubation in 3-vol BSA blocking solution, sections were incubated with primary antibodies to ZO-1, occludin and Claudin-1 in that order, and then the corresponding secondary antibodies were incubated with the samples overnight. The reaction was terminated with 2% PBST after color development with DAB. And finally, observing a DAB-stained positive area under a microscope after hematoxylin staining and dehydration treatment. Compared with the DSS group, the expression of all strains TLR-2 and TLR-4 is obviously reduced, the expression of TLR-5 is obviously improved, and the difference between TLR-1mRNA expression groups has no statistical significance. The mRNA levels of LGR5 and Rspo3 were increased in all strain-treated groups, while propionibacterium freudenreichii B1 (PF group) showed the most significant effect, suggesting that it may have greater potential to rescue the damaged intestine. The results are shown in FIG. 4.
Example 7
Western blotting was used to detect RHO kinase (ROCK 1) and the Wnt/beta-catenin pathway negative regulator Axin2 protein. Reference Experimental method Li H et al 22 Colon tissue was pulverized in liquid nitrogen, mixed with complete RIPA buffer, centrifuged at 13000r/min for 10 minutes at 4 ℃ in an ice bath, and the supernatant was taken for measurement of protein concentration. Proteins were separated by gel electrophoresis and transferred to PVDF membrane. Membranes were blocked with 5% skim milk for 2 hours at room temperature, then a dilution of anti-ROCK-1 antibody and anti-Axin 2 antibody was added. Membranes were incubated overnight at 4 ℃, washed, immersed in diluted goat anti-rabbit IgG antibody, incubated in ECL luminescence solution for 2 minutes, and then scanned by a full-automatic chemiluminescence image analysis system. Different strains can down-regulate the overexpression of ROCK-1 and Axin2, but only PF group and BB group are significantly different from DSS group (P < 0.05), which indicates that Propionibacterium freudenreichii B1 (PF group) and Bifidobacterium bifidum H3-R2 (BB group) regulate inflammation through ROCK-1 pathway and stimulate the regeneration of damaged intestinal epithelial cells through Wnt/beta-catenin pathway. The results are shown in FIG. 5.
Example 8
Total bacterial genomic DNA in the colon contents was extracted using the Fast DNA SPIN extraction kit. The V3-V4 region of the bacterial 16S rRNA gene was PCR amplified using forward and reverse primer 338F 806R. PCR amplicons were purified with AgencourtAmure Beads and quantified using the PicoGreen dsDNAassay Kit. After a single quantification step, equal amounts of amplicons were pooled and paired-end 2 × 300bp sequencing was performed using the illumina MiSeq platform and MiSeq regent kit v3 of shanghai personal biotechnology limited (shanghai, china). A microbial ecological quantitative analysis (QIIME, v 1.8.0) pipeline was used to process the sequencing data. In the PC, BB, PF and CB groups, the Chao1 index and the Simpson index are increased in different degrees, wherein the enrichment of the flora in the PC group is increased most, and the effect difference of the Propionibacterium freudenreichii B1 (PF group) and the Clostridium butyricum C1-6 (CB group) is not obvious. At the genus level, prevotellaceae UCG-001, turcibacter, clostridium-UCG-014 and Murebacteriaceae were elevated and E.coli-Shigella, staphylococci and Enterobacter were reduced in abundance. The results are shown in FIG. 6.
Example 9
Add 200 μ L of deionized water to the colon contents (0.4 g) and mix well. Then 200. Mu.L of 50% sulfuric acid, 25. Mu.L of 500mg/L internal standard (cyclohexanone) solution and 0.5mL of ether were added, homogenized for 1min, and centrifuged at 12000rpm at 4 ℃ for 20min. The supernatant was extracted and detected by gas chromatography-mass spectrometer. The chromatographic system is an Agilent DB-WAX capillary column (30 m multiplied by 0.25mm multiplied by 0.25 mu m), high-purity helium (the purity is not lower than 99.999%) is taken as carrier gas, the flow rate is 1.0mL/min, the temperature of a sample inlet port is 220 ℃, the sample inlet amount is 1 mu L, the delay time of a solvent is 2.2min, and no cracking occurs. The mass spectrometry system is an electron impact ion source (EI), the ion source temperature is 230 ℃, and the interface temperature is 220 ℃. The levels of colon content SCFAs were significantly elevated in BB, PF and CB groups compared to DSS groups (P < 0.05), with propionibacterium freudenreichii B1 (PF group) effects being most significant. The results are shown in FIG. 7.
Claims (8)
1. A method for ameliorating ulcerative colitis using strains that produce different short chain fatty acids, comprising the steps of: (1) culturing of the strain: bifidobacterium H3-R2 was cultured in De Man, rogosa and Sharpe (mMRS) medium supplemented with 0.05% L-cysteine hydrochloride at 37 ℃ for 16 hours, propionibacterium freudenreichii B1 was cultured in glucose medium at 30 ℃ for 24 hours, and Clostridium butyricum C1-6 was cultured in Reinforced Clostridium Medium (RCM) at 37 ℃ for 12 hours. Two subcultures were performed before the experiment. All strains were washed twice with PBS and adjusted to 10 by PBS resuspension 9 CFU/mL, for subsequent in vivo experimentsAnd (6) testing. (2) Evaluating the effect of the test strain on UC using a DSS-induced mouse model: male pathogen-free (SPF) C57BL/6J mice 8 weeks old were used and were raised at 23. + -. 3 ℃ and 55. + -. 10% humidity for 12h with alternating light. The 48 mice were randomized into 6 groups after 1 week of acclimation: normal control group (NC), DSS group (DSS), positive control group (PC), bifidobacterium H3-R2 group (BB), propionibacterium freudenreichii B1 group (PF), and Clostridium butyricum C1-6 group (CB). Spleen index, disease Activity Index (DAI), colon tissue status, colon tissue Myeloperoxidase (MPO) activity, cytokine levels in colon, claudin and inflammation-related gene expression, pathway protein expression, mouse intestinal flora, short Chain Fatty Acid (SCFA) in stool were determined and statistically analyzed in different groups of mice.
2. The method of claim 1, wherein the bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and clostridium butyricum C1-6 are isolated by the laboratory and deposited in the important laboratory of the dairy science education department of northeast university of agriculture.
3. The method of claim 1, wherein the spleen index, disease Activity Index (DAI) and colon tissue status of the mouse are as follows: the application of the bifidobacterium bifidum H3-R2, the propionibacterium freudenreichii B1 and the clostridium butyricum C1-6 can obviously relieve the enlargement of the spleen of a mouse caused by DSS stimulation and obviously reduce the Disease Activity Index (DAI). The three strains can effectively relieve colitis symptoms caused by DSS, recover colon morphology, reduce inflammatory cell infiltration, improve crypt displacement and enrich goblet cells. However, the effects of the propionibacterium freudenreichii B1 and the clostridium butyricum C1-6 on recovering the form and the crypt depth are better than those of the bifidobacterium bifidum H3-R2 to a certain extent.
4. The method of claim 1, wherein the colon tissue Myeloperoxidase (MPO) activity and cytokine levels are: the use of Bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 significantly reduced the increase in MPO viability in the colon of mice caused by DSS. Can reduce the content of proinflammatory factors IL-8, IL-1 beta and TNF-alpha and obviously increase the content of anti-inflammatory factors IL-10. It is shown that the DSS-induced colitis of mice can be relieved by adjusting inflammatory factors.
5. The method of claim 1, wherein the tight junction protein and the inflammation-related gene are expressed as: the use of Bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 can increase colonic Claudin expression (ZO-1, occludin and Claudin-1) and improve intestinal barrier function. The combined results of the expression of the three proteins indicate that Propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 are more effective in protecting the intestinal barrier than Bifidobacterium bifidum H3-R2. The three strains can be used to obviously reduce the expression of TLR-2 and TLR-4, obviously increase the expression of TLR-5 and increase the mRNA level of LGR5 and Rspo 3. The B1 group of P.freudenreichii showed the most significant effect, suggesting that it may have greater potential for ameliorating colitis.
6. The method of claim 1, wherein the pathway protein is expressed as: the use of bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 all down-regulated the over-expression of ROCK-1 and Axin2, but only the Propionibacterium freudenreichii B1 group and the Bifidobacterium bifidum H3-R2 group were significantly different (P < 0.05) compared with the DSS group, indicating that these two strains regulate inflammation through ROCK-1 pathway and stimulate the regeneration of damaged intestinal epithelial cells through Wnt/beta-catenin pathway.
7. The method of claim 1, wherein the intestinal flora of the mouse is selected from the group consisting of: the use of bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and Clostridium butyricum C1-6 raises the Chao1 index and Simpson index, increasing the relative abundance of Actinomycetes phylum. At the genus level, the abundance of Prevotellaceae UCG-001, turcibacter, clostridium-UCG-014 and Murebacteriaceae can be remarkably increased, and the abundance of Escherichia coli, shigella, staphylococci and enterobacteriaceae can be reduced.
8. The method of using bacterial strains producing different short chain fatty acids for ameliorating ulcerative colitis according to claim 1, wherein said fecal Short Chain Fatty Acids (SCFAs) are: the use of bifidobacterium bifidum H3-R2, propionibacterium freudenreichii B1 and clostridium butyricum C1-6 resulted in a significant increase in the level of SCFAs of the colon content (P < 0.05), with the most significant effect of propionibacterium freudenreichii B1 group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211246638.4A CN115569153A (en) | 2022-10-12 | 2022-10-12 | Method for improving ulcerative colitis by using bacterial strains producing different short-chain fatty acids |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211246638.4A CN115569153A (en) | 2022-10-12 | 2022-10-12 | Method for improving ulcerative colitis by using bacterial strains producing different short-chain fatty acids |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115569153A true CN115569153A (en) | 2023-01-06 |
Family
ID=84584487
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211246638.4A Pending CN115569153A (en) | 2022-10-12 | 2022-10-12 | Method for improving ulcerative colitis by using bacterial strains producing different short-chain fatty acids |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115569153A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115011516A (en) * | 2022-06-13 | 2022-09-06 | 东北农业大学 | Synbiotic yoghourt with constipation relieving effect and preparation method thereof |
-
2022
- 2022-10-12 CN CN202211246638.4A patent/CN115569153A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115011516A (en) * | 2022-06-13 | 2022-09-06 | 东北农业大学 | Synbiotic yoghourt with constipation relieving effect and preparation method thereof |
| CN115011516B (en) * | 2022-06-13 | 2023-11-14 | 东北农业大学 | Synbiotic yogurt with constipation relieving effect and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2754367C2 (en) | Faecalibacterium prausnitzii and desulfovibrio piger for use in treatment or prevention of diabetes and intestinal diseases | |
| CN114164134B (en) | Bifidobacterium longum subspecies longum with functions of preventing and relieving colitis symptoms and application thereof | |
| CN113897302B (en) | Bifidobacterium capable of relieving colitis and application thereof | |
| WO2019199895A1 (en) | Microbial consortia | |
| Xie et al. | Effects of phycocyanin in modulating the intestinal microbiota of mice | |
| CN110023484B (en) | Bifidobacterium pseudocatenulatum as well as culture method and application thereof | |
| CN115569153A (en) | Method for improving ulcerative colitis by using bacterial strains producing different short-chain fatty acids | |
| Hong et al. | Lactobacillus salivarius and Lactobacillus agilis feeding regulates intestinal stem cells activity by modulating crypt niche in hens | |
| Gresse et al. | Pathogen challenge and dietary shift alter microbiota composition and activity in a mucin-associated in vitro model of the piglet colon (MPigut-IVM) simulating weaning transition | |
| WO2018112740A1 (en) | Lactobacillus gasseri, culture method therefor and application thereof | |
| Chen et al. | The gut microbiome promotes arsenic metabolism and alleviates the metabolic disorder for their mammal host under arsenic exposure | |
| JP2023538016A (en) | Faecalibacterium prausnitzii strain and uses thereof | |
| CN112535693A (en) | Mixed lactobacillus for preventing and treating ulcerative colitis and application thereof | |
| WO2008064521A1 (en) | Lactobacillus fermentum cms-h002 and use thereof | |
| CN115011518A (en) | Lactobacillus mixture with effect of relieving colorectal cancer related to colitis and application thereof | |
| CN111733137A (en) | Method for screening medicine capable of preventing and/or treating alcoholic liver injury | |
| CN114990004B (en) | Secretory immunoglobulin A-packaged lactobacillus reuteri and application thereof in preventing and treating gestational diabetes | |
| CN114438236B (en) | Application of intestinal desulfurization vibrio in screening high risk group of cholecystolithiasis and prevention and treatment medicines | |
| Xu et al. | Milk-derived Lactobacillus with high production of short-chain fatty acids relieves antibiotic-induced diarrhea in mice | |
| CN117965401B (en) | Lactobacillus rhamnosus AFY01 and application of product thereof in inflammatory colon cancer | |
| CN116004486B (en) | Bacteroides fragilis BFS17 for relieving irritable bowel syndrome and intestinal tract hypersensitivity and application thereof | |
| Wang et al. | Garlic exosomes alleviate dextran sodium sulfate-induced colitis, in which peu-MIR2916-p3 acts as a key component boosting the growth of anti-colitis Bacteroides thetaiotaomicron | |
| RU2797466C2 (en) | Faecalibacterium prausnitzii and desulfovibrio piger for use in the treatment or prevention of diabetes and intestinal diseases | |
| Higgins | Investigating the Predatory Capability of Bdellovibrio bacteriovorus towards Colorectal Cancer Oncomicrobes in vitro | |
| Yu et al. | Comparison of immune responses and intestinal flora in epicutaneously sensitized BALB/c or C57BL/6 mouse models of food allergy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |