CN113897302B - Bifidobacterium capable of relieving colitis and application thereof - Google Patents
Bifidobacterium capable of relieving colitis and application thereof Download PDFInfo
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- CN113897302B CN113897302B CN202110903416.4A CN202110903416A CN113897302B CN 113897302 B CN113897302 B CN 113897302B CN 202110903416 A CN202110903416 A CN 202110903416A CN 113897302 B CN113897302 B CN 113897302B
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- bifidobacterium pseudocatenulatum
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Abstract
The invention discloses a rapid screening method of bifidobacterium capable of relieving colitis and application thereof, belonging to the technical field of biology. The Bifidobacterium pseudocatenulatum is preserved in China center for type culture Collection, Wuhan, with a preservation date of 2021 year, 7 months and 5 days and a preservation number of CCTCC M2021820. The bifidobacterium pseudocatenulatum can remarkably improve the weight loss, the colon length and the disease activity index of a mouse during DSS induced colitis, enhance the integrity of an intestinal tract barrier, and inhibit tissue damage, Myeloperoxidase (MPO) activity, PGE2 level and proinflammatory factors. Compared with the model group mice, the bifidobacterium pseudocatenulatum KLDS N2 remarkably down-regulates the expression of proinflammatory cytokines mRNA of the DSS-stimulated mice and up-regulates the expression of mucin and tight junction protein mRNA.
Description
The technical field is as follows: the invention relates to bifidobacterium odonta capable of relieving colitis and application thereof, belonging to the technical field of biology.
Technical background:
ulcerative colitis is a chronic idiopathic inflammatory bowel disease. The early clinical manifestations of the disease are blood-type diarrhea, and the disease continues to cause symptoms of diarrhea, hematochezia, weight loss, vomiting and the like, and also causes complications such as toxic colon dilatation, intestinal perforation, polyp, enteritis and the like. The incidence of ulcerative colitis is on the rise in our country.
Ulcerative colitis is primarily characterized by inflammation of the colorectal mucosal and submucosal layers. Normally, the body is protected by the physical barrier composed of intestinal epithelial cells and surface mucus, the chemical barrier composed of immune-mediated antibacterial substances and the microbial barrier composed of intestinal probiotics, and patients with ulcerative colitis have intestinal epithelial injury, body immune disorder and microbial invasion, which worsen each other.
There are currently no effective measures for the prevention and treatment of ulcerative colitis, and the existing therapeutic drugs are: aminosalicylates, glucocorticoids, immunosuppressants and the like all have certain side effects, so that the research of a new alternative treatment method is very important.
Disclosure of Invention
The invention provides an application of bifidobacterium pseudocatenulatum in preparing a product for preventing and/or treating colitis, and the bifidobacterium pseudocatenulatum can be used for preparing the product for preventing and/or treating colitis.
The Bifidobacterium pseudocatenulatum KLDS N2 is derived from healthy infant feces, and the 16S rDNA sequence of the strain is as follows by sequencing analysis (SEQ ID NO: 1): AATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCGACTCCTCGCATGGGGTGTCGGGAAAGATTTCATCGGTATGGGATGGGGTCGCGTCCTATCA
GGTAGTCGGCGGGGTAACGGCCCACCGAGCCTACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACG
GGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGACGGCCTTCGGGTTGTAAACCGCTTTTGATCGG
GAGCAAGCCTTCGGGTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTAT
TGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAG
GGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGC
GAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGC
TAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTC
GATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACAGCCGTAGAGATATGGCCTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTC
GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTGTGTTGCCAGCACGTCATGGTGGGAACTCACGGGGGACCGCC
GGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGAC
ACGGCGACGTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATTGGAGTCTGCAACCCGACTCC, the sequence obtained by sequencing is compared with the nucleic acid sequence in NCBI database, the result shows that the strain is Bifidobacterium pseudocatenulatum, named Bifidobacterium pseudocatenulatum KLDS N2, which is preserved in China center for type culture Collection, Wuhan, with the preservation date of 2021 year, month and day and the preservation number of CCTCC M2021820. The Bifidobacterium pseudocatenulatum KLDS N2 (hereinafter referred to as N2) has a protruding colony on an MRS solid culture medium, and has a smooth, round and water-wet surface, white and semitransparent color and a diameter of 1-2 mm.
In one embodiment of the invention, the colitis is ulcerative colitis.
The invention also provides a product for preventing and/or treating colitis, which contains the bifidobacterium pseudocatenulatum.
In one embodiment of the invention, the viable count of Bifidobacterium pseudocatenulatum in the product is not less than 1X 10 9 CFU/mL. In one embodiment of the invention, the viable count of Bifidobacterium pseudocatenulatum in the product is not less than 5X 10 9 CFU/mL. In bookIn one embodiment of the invention, the product is a pharmaceutical product.
In one embodiment of the invention, the pharmaceutical product is a bacterial suspension.
In one embodiment of the present invention, the bacterial suspension is prepared by inoculating the bifidobacterium pseudocatenulatum into a culture medium for culture to obtain a seed solution; inoculating the seed solution into a culture medium for culturing to obtain a culture solution; centrifuging the culture solution, and collecting bacterial sludge; and washing the bacterial sludge with normal saline, and then re-suspending to obtain bacterial suspension.
In one embodiment of the present invention, the seed solution is inoculated into a culture medium at an inoculum size of 1-3% (v/v) for culture.
In one embodiment of the present invention, the seed culture medium is MRS solid culture medium and the fermentation culture medium is MRS liquid culture medium.
In one embodiment of the present invention, the MRS liquid medium is a cysteine hydrochloride-added MRS liquid medium.
In one embodiment of the present invention, the cysteine hydrochloride is added in an amount of 0.04 to 0.06% by mass.
In one embodiment of the invention, the seed solution is inoculated into the MRS liquid culture medium by 1-3% of inoculation amount to culture under the culture conditions that: anaerobic culture is carried out for 24 h-36 h at 35-38 ℃, centrifugation is carried out for 15min at 8000rmp, bacterial sludge is collected, and the bacterial sludge is washed for 3-4 times by using normal saline and then resuspended.
Advantageous effects
(1) The Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 provided by the invention is separated from intestinal flora of healthy infants, the strain has no toxic or side effect on human bodies, and in-vitro research shows that the strain has good tolerance capability and adhesion capability on digestive tracts and has the potential of being planted in intestinal tracts of human bodies. Can inhibit inflammation, protect intestinal barrier function, and improve intestinal content of mice. Therefore, the medicament prepared from the Bifidobacterium pseudocatenulatum N2 has certain advantages compared with the traditional medicament for treating colitis, and the strain can be used for preparing probiotic preparations and the like, thereby having wide market prospect.
(2) The Bifidobacterium pseudocatenulatum N2 provided by the invention can obviously reduce the release levels of NO, TNF-alpha, IL-1 beta and IL-6 in RAW264.7 cells stimulated by LPS.
(3) The Bifidobacterium pseudocatenulatum N2 provided by the invention can significantly reduce the TEER value of Caco-2 cells stimulated by LPS, and shows that the Bifidobacterium pseudocatenulatum N2 has good protection effect on the integrity of Caco-2 epithelial cells.
(4) By adopting the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 provided by the invention, the disease activity index of a mouse during DSS induced colitis can be obviously reduced, and the reduction of the body weight and the shortening of the colon can be reduced.
(5) The Bifidobacterium pseudocatenulatum N2 provided by the invention can significantly reduce the expression of MPO, PEG2 and COX-2 in colon tissues of mice during DSS induced colitis.
(6) The Bifidobacterium pseudocatenulatum N2 provided by the invention can obviously reduce the concentration of proinflammatory cytokines (TNF-alpha, IL-1 beta and IL-6) in colon tissues of mice during DSS induced colitis, and inhibit the gene expression quantity of TNF-alpha, IL-1 beta and IL-6.
(7) By adopting the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 provided by the invention, the damage of the colon tissue of a mouse during DSS induced colitis can be obviously reduced.
(8) The Bifidobacterium pseudocatenulatum N2 provided by the invention can significantly increase the mRNA expression levels of mouse colon MUC-2, tight junction protein ZO-1 and Occludin during DSS induced colitis.
(9) By adopting the Bifidobacterium pseudocatenulatum N2 provided by the invention, the relative abundance of Lactobacillus and Akkermansia in intestinal tracts of mice can be remarkably increased during DSS-induced colitis.
(10) The content of short-chain fatty acids (acetic acid and butyric acid) in the intestinal tract of a mouse can be remarkably increased during the DSS-induced colitis by adopting the Bifidobacterium pseudocatenulatum N2 provided by the invention.
Drawings
FIG. 1: gut tolerance and adhesiveness of different bifidobacterium strains.
FIG. 2: effect of different bifidobacterium strains on LPS-induced secretion of inflammation-associated cytokines by RAW264.7 cells.
FIG. 3: effect of different bifidobacteria on the TEER values of LPS-induced Caco-2 cells.
FIG. 4: body weight, DAI index and colon length changes during DSS modelling in different groups of mice.
FIG. 5: MPO, PGE2 and COX-2mRNA expression levels in colon tissue of different groups of mice.
FIG. 6: the proinflammatory factor content and the mRNA expression quantity in colon tissues of mice of different groups.
FIG. 7: colon tissues HE, AB and immunofluorescence staining of different groups of mice.
FIG. 8: the expression level of MUC-2 and claudin in colon of different groups of mice.
FIG. 9: intestinal microorganisms of different groups of mice.
FIG. 10: dominant flora analysis and KEGG pathway analysis on intestinal microorganism composition of mice of different groups
FIG. 11: the intestinal tract short-chain fatty acid content of mice of different groups.
FIG. 12: experimental protocol and treatment pattern for each group of mice.
In fig. 1 to 11, the difference in letters indicates a significant difference.
Detailed Description
The invention is further elucidated with reference to a specific embodiment and a drawing.
Caco-2 cells and RAW264.7 cells referred to in the examples below were purchased from the Chinese academy of sciences; the mice referred to in the examples below were 7 week old male SPF (Specific pathogen free) grade C57BL/6J mice, purchased from vita River laboratory animal technologies; the ELISA kits involved in the following examples were purchased from Nanjing construction Co., Ltd; sodium dextran sulfate (DSS) referred to in the examples below was purchased from mbpdio corporation, usa; bifidobacterium strain BB-12 in the examples is a commercially available Bifidobacterium strain, and F2, G2 and the like are derived from infant feces.
The media involved in the following examples are as follows:
MRS liquid medium: 10g/L of tryptone, 10g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 2g/L of anhydrous sodium acetate, 0.5g/L of magnesium sulfate heptahydrate, 0.25g/L of manganese sulfate monohydrate, 2g/L of diammonium hydrogen citrate, 2.6g/L, Tween 801 mL/L of dipotassium hydrogen phosphate trihydrate and 0.5g/L of cysteine hydrochloride.
MRS solid medium: 10g/L of tryptone, 10g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 2g/L of anhydrous sodium acetate, 0.5g/L of magnesium sulfate heptahydrate, 0.25g/L of manganese sulfate monohydrate, 2g/L of diammonium hydrogen citrate, 2.6g/L, Tween 801 mL/L of dipotassium hydrogen phosphate trihydrate, 0.5g/L of cysteine hydrochloride and 20g/L of agar.
The detection methods referred to in the following examples are as follows:
detection method of digestive tract tolerance:
adjusting the strain to 1.0X 10 9 And (5) placing the CFU/mL into gastric juice, incubating for 2h, transferring into intestinal juice, incubating for 2h, and calculating the survival rate of the strain. The survival rate of the experimental strains was calculated as follows:
note: wherein N1 is the surviving strain after treatment, and N0 is the total strain without treatment.
Adhesion capability to Caco-2 cells:
caco-2 cells (3.0X 10) 5 ) The cells were inoculated into culture plates (12 wells) and cultured for 7 days. 1mL of Bifidobacterium suspension (1.0X 10) was added to each well 8 CFU/mL) for 2 h. After 4 washes with PBS, cells were trypsinized for 10min and digestion was stopped with 0.25mL fetal bovine serum. Cells were collected and plated on MRSL plates and adhesion to bifidobacteria was calculated as follows:
Adhesion(％)＝(the number of adhered bacteria/1.0×10 8 )×100％
effect of each bifidobacterium on LPS-stimulated RAW264.7 cell NO levels:
at 1.0 × 10 4 RAW264.7 cells were cultured at a concentration of cells/mL and plated on 96-well plates. After 24h, cells were pretreated with Bifidobacterium (MOI 1:100) for 1h, and then stimulated with LPS (1. mu.g/mL) for 24 h. The culture solution was collected, and the NO accumulation was measured with a kit (Nanjing Kangji Co., Ltd., Nanjing, China).
Effect of each bifidobacterium on LPS-stimulated RAW264.7 cellular immune factor levels:
RAW264.7 cells were cultured as described above. After 1h addition of the strain, the cells were stimulated with 1. mu.g/mL LPS for 24 h. The supernatants were then collected and the concentrations of TNF-. alpha.IL-1. beta. and IL-6 were determined using an ELISA kit.
Effect of each bifidobacterium on TEER value of LPS-stimulated Caco-2:
caco-2 cells (3X 10) 5 one/mL) were seeded on Transwell inlays and cultured for 15d until the cells were fully differentiated. After 3 PBS washes, the strain was pretreated for 1h and induced with LPS (1. mu.g/mL) for 24 h. TEER values were measured using Millipore MERS00002 Millicell-ERS.
Method for detecting Disease Activity Index (DAI):
the DAI score includes three aspects of body weight change, hematochezia status and stool characteristics (specific scoring criteria are shown in table 1). During modeling, the weight of the mouse is measured every day, the occult blood condition and the stool hardness of the mouse are detected, and the score is calculated according to the table 1, wherein DAI is the sum of the weight change score, the stool blood score and the stool character score. The fecal occult blood condition is measured by an occult blood kit, and the specific operation is carried out according to the reagent instruction.
TABLE 1 disease Activity index Scoring criteria
The detection method of the colon length comprises the following steps:
after the mice were sacrificed, the entire colon (end of cecum to anus) was removed and the length was measured.
And (3) determination of colon tissue biochemical indexes:
fresh colon specimens were homogenized with MPO buffer and changes in MPO levels were determined. Colon homogenates were taken and tested for TNF-. alpha.IL-1. beta., IL-6 and IL-10 levels using an ELISA kit.
HE staining and AB staining:
after sacrifice, colon tissue was soaked in 10% formalin for HE and AB staining.
And (3) performing immunofluorescence detection:
10 μm colon was superfliced and incubated with antibodies ZO-1 and MUC-2 overnight at 4 ℃. Subsequently, the samples were counterstained with FITC-labeled antibody and CY 3-labeled antibody, respectively, for 60 min. Images were obtained at the same site of each colon specimen using a microscope BX53(400 x).
Real-time quantitative polymerase chain reaction (RT-qPCR) analysis:
to detect the mRNA expression of colonic claudin and inflammatory cytokines, RNA was extracted using the kit and the level of acquisition was detected using Nanodrop. cDNA was synthesized using PrimeScriptTM RT kit and subsequently amplified with Go Taq qPCR Master Mix. The expression of the target gene was detected by the Quant-Studio 3Real-Time PCR system (Applied Biosystems, Foster City) by the SYBR Green Premix Ex TaqII method. And calculating the folding change of the target gene by adopting a 2 delta Ct method.
Colon content microbiological analysis:
the colon contents were collected, DNA extracted using the FastDNA SPIN kit, and amplified. Sequencing was complete and analyzed.
Analysis of short-chain fatty acids of colon contents:
a sample (80mg) was treated with a feces treatment apparatus (HALO-F100), and 500. mu.L of the treated sample was taken and mixed with a crotonic monophosphate solution. After centrifugation at 4 deg.C (5000g,20min), the supernatant was filtered through a 0.22 μm filter for detection.
Example 1: screening, identification, culture, observation and preservation of Bifidobacterium pseudocatenulatum N2
1. Screening
Taking 1g of a healthy infant feces sample from a Harbin region, coating the sample in an MRS solid culture medium (containing rapid screening) after gradient dilution, placing the sample in an anaerobic environment at 37 ℃ for culturing for 72 hours, and observing and recording the colony morphology; selecting a colony with a wet surface, a bulge and white and yellow color, streaking on an MRS solid culture medium, carrying out purification culture under the anaerobic condition at 37 ℃, and repeating the operation for 3 times to obtain a purified single colony; and selecting a single colony, streaking the single colony on an MRS solid culture medium, and carrying out anaerobic culture at 37 ℃ for 36 h.
2. Identification
Extracting the genome of the screened strain, amplifying and sequencing the 16S rDNA of the strain (the nucleotide sequence of the 16S rDNA obtained by amplification is shown as SEQ ID NO. 1), and comparing the obtained sequence with the nucleic acid sequence in NCBI-Blast to show that the strain is bifidobacterium pseudocatenulatum which is named as bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2;
the primers used for 16S rDNA amplification are as follows:
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；
1492R：5’-TACGGCTACCTTGTTACGACTT-3’；
the 16S rDNA amplification procedure was as follows:
5min at 95 ℃; 35 cycles (95 ℃ 30 s; 55 ℃ 30 s; 72 ℃ 2 min); 10min at 72 ℃. 4. Preserving and picking a single colony of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2, inoculating into an MRS liquid culture medium, and culturing for 24h at 37 ℃ under an anaerobic condition to obtain a bacterial liquid; adding sterilized 40% (v/v) glycerol into the obtained bacterial liquid, mixing, and storing in glycerol tube at-80 deg.C. Meanwhile, the bifidobacterium pseudocatenulatum is preserved in China center for type culture Collection, Wuhan, with the preservation date of 2021 year, 7 months and 5 days and the preservation number of CCTCC M2021820.
Example 2: preparation of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 bacterial suspension (1) bacterial solution of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 was dipped from glycerol tube and streaked on MRS solid medium, anaerobic culture was performedCulturing at 37 deg.C for 48h to obtain single colony; and selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 48h at 37 ℃ in an anaerobic environment for activation culture, and repeating the operation for 3 times to obtain activated bacterial liquid. (2) Inoculating the activated bacterial liquid obtained in the step (1) into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), culturing at 37 ℃ for 24h to obtain a fermentation liquid, centrifugally collecting the bacteria from the fermentation liquid, re-suspending the bacteria by using normal saline, and adjusting the viable count to be 5 multiplied by 10 9 CFU/mL, and preparing a bacterial suspension.
Example 3: bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 resistance to the digestive tract: adjusting the strain to 1.0X 10 9 CFU/mL, incubated in gastric juice for 2h, then transferred to intestinal juice and incubated for 2 h. The survival rate of the experimental strains was calculated as follows:
note: wherein N1 is the surviving strain after treatment, and N0 is the total strain without treatment.
Bifidobacterium pseudocatenulatum N2 shows a high survival rate in the digestive tract, and has no significant difference from BB-12. This result shows that Bifidobacterium pseudocatenulatum (Bifidobacterium pseudostellatum) N2 survived in the digestive tract.
Example 4: adhesion capacity of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 to Caco-2 cells: caco-2 cells (3.0X 10) 5 ) The cells were inoculated into culture plates (12 wells) and cultured for 7 days. 1mL of Bifidobacterium suspension (1.0X 10) was added to each well 8 CFU/mL) for 2 h. After 4 washes with PBS, cells were trypsinized for 10min and digestion was stopped with 0.25mL fetal bovine serum. Cells were collected and plated on MRSL plates, calculated as follows:
Adhesion(％)＝(the number of adhered bacteria/1.0×10 8 )×100％
the adhesion rate of Bifidobacterium pseudocatenulatum N2 was 53.34%, which was significantly higher than BB-12 (28.08%). This result indicates that Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 has potential as a probiotic.
Example 5: effect of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 on LPS-stimulated RAW264.7 cell NO accumulation and proinflammatory factor levels:
at 1.0 × 10 4 RAW264.7 cells were cultured at a concentration of cells/mL and plated on 96-well plates. After 24h, cells were pretreated with Bifidobacterium (MOI 1:100) for 1h, and then stimulated with LPS (1. mu.g/mL) for 24 h. The culture medium was collected and the amount of accumulated NO and the concentrations of TNF-. alpha., IL-1. beta. and IL-6 were determined by ELISA kits.
Fig. 2 shows that the culture solution of RAW264.7 cells stimulated by LPS has a significant increase in NO and proinflammatory factors, while Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 can significantly reduce the levels of NO and proinflammatory factors of RAW264.7 cells stimulated by LPS. This result indicates that Bifidobacterium pseudocatenulatum (Bifidobacterium pseudostellatum) N2 has the potential to inhibit inflammation.
Example 6: effect of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudoticum) N2 Bifidobacterium on TEER value of LPS-stimulated Caco-2:
caco-2 cells (3X 10) 5 one/mL) were seeded on Transwell inlays and cultured for 15d until the cells were fully differentiated. After 3 PBS washes, the strain was pretreated for 1h and induced with LPS (1. mu.g/mL) for 24 h. TEER values were measured using Millipore MERS00002 Millicell-ERS.
FIG. 3 shows that LPS stimulated Caco-2 cells significantly reduced the TEER value of the cells, while Bifidobacterium pseudocatenulatum N2 significantly reduced the TEER value of the Caco-2 cells stimulated by LPS, i.e., Bifidobacterium pseudocatenulatum N2 protected the integrity of the intestinal epithelial cells.
Example 7: bifidobacterium pseudocatenulatum N2 for relieving symptoms of DSS-induced colitis mice
The method comprises the following steps:
40 healthy male C57BL/6J mice at 7 weeks of age were randomly divided into 4 groups, which were designated as: a blank Control group (Control), a construction kit (DSS), a Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 dry preparation group (N2) and a Bifidobacterium BB-12 dry preparation group (BB-12); the experimental protocol and treatment of each group of mice with 10 mice per group was as follows (as shown in figure 12):
the method for treating the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 intervention group (N2) and the Bifidobacterium BB-12 intervention group (BB-12) comprises the following steps: on the 1 st to 14 th days of the experiment, the bifidobacterium pseudocatenulatum N2 intervention group (N2) and the bifidobacterium BB-12 intervention group (BB-12) were respectively subjected to intragastric administration by 5X 10 times per day 9 CFU/mL of 200uL and 5X 10 suspension of N2 bacteria 9 200 mu L of BB-12 bacterial suspension of CFU/mL, and freely drinking distilled water; the experiment was continued on days 14-21 by gavage with Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 dry stock group (N2) and Bifidobacterium BB-12 dry stock group (BB-12) on days 5X 10 9 200 mu L of bacterial suspension of N2 and BB-12 in CFU/mL bacterial quantity, and freely drinking 3% DSS solution;
the processing method of the building module (DSS) comprises the following steps: on the 1 st to 14 th days of the experiment, 200 mu L of skim milk is infused into the stomach of the DSS group every day, and distilled water is freely drunk; on the 14 th to 21 th days of the experiment, 200 mu L of skim milk is infused into the DSS group every day, and 3 percent DSS solution is freely drunk; the blank Control group (Control) processing method comprises the following steps: in the experimental process, 200 μ L of skim milk was infused into the control group daily, and distilled water was freely drunk.
During the molding period, the body weight of each group of mice and disease activity index DAI index change body weight and colon length and state of each group of mice are detected (the detection result is shown in figure 4).
After the molding is finished, the blank Control group (Control), the molding group (DSS), the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 dry pretreatment group (N2) and the Bifidobacterium BB-12 dry pretreatment group (BB-12) were killed respectively. The results show that the weight of the DSS induced 4d mice is reduced, the weight of the DSS group before slaughter is obviously reduced, and the weight is reduced by 13.27% compared with the control group. The Bifidobacterium pseudocatenulatum N2 pretreatment can significantly improve the weight change of the mice stimulated by the DSS, and the effect is better than that of the control strain BB-12. Compared with a control group, the DAI index of the DSS group is obviously increased, and the DAI index of mice induced by the DSS can be obviously reduced by Bifidobacterium pseudocatenulatum (N2). The mice treated with DSS had significantly shortened colon length, bleeding colon, red color and sparse content. Therefore, the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 has better improvement effect on mice with DSS-induced colitis.
Example 8: effect of Bifidobacterium pseudocatenulatum N2 on inflammatory-related cytokines in colitis mice
The detailed description is the same as the procedure in example 7;
after completion of the molding, the mice obtained in example 7 were sacrificed, and colon tissues were taken and subjected to MPO, PGE2 activity and COX-2mRNA expression level using the kit (see FIG. 5 for the results of the assay).
Compared with the control group, the MPO activity of mice in the DSS group is obviously increased. Bifidobacterium pseudocatenulatum N2 can significantly inhibit the activity of MPO in colon tissues of mice induced by DSS, and the effect is superior to BB-12.
Compared with the control group, the level of PGE2 in mice in the DSS group is obviously increased. Bifidobacterium pseudocatenulatum N2 can significantly reduce the level of DSS-induced mouse colon tissue PGE2, and the effect is better than BB-12. Similarly, Bifidobacterium pseudocatenulatum N2 was able to significantly reduce the amount of mRNA expression of COX-2 in the colon tissue of DSS-induced mice (see FIG. 5 for the test results).
Therefore, the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 provided by the invention reduces the activity of the cell factor related to the inflammation of the colon tissue of the DSS-stimulated mouse.
Example 9: the effect of Bifidobacterium pseudocatenulatum N2 on colonic pro-inflammatory factors in colitis mice.
The detailed description is the same as the procedure in example 7;
mice were sacrificed after the completion of the molding obtained in example 7, and distal colon tissues were taken to detect the contents of proinflammatory factors TNF-. alpha., IL-1. beta., and IL-6 by ELISA kit, and the mRNA expression levels of the proinflammatory factors TNF-. alpha., IL-1. beta., and IL-6 were detected by RT-PCR (see FIG. 6 for the detection results).
Colonic tissue was taken and RNA was extracted using a kit and levels were obtained using Nanodrop. cDNA was synthesized using PrimeScriptTM RT reagent kit and subsequently amplified with Go Taq qPCR Master Mix. The expression of the target gene is detected by a Quant-Studio 3Real-Time PCR system through a SYBR Green Premix Ex TaqII method. And calculating the folding change of the target gene by adopting a 2 delta Ct method.
Compared with a control group, the proinflammatory factors of colon tissues of mice stimulated by DSS are remarkably increased, the proinflammatory cytokine level induced by DSS is remarkably reduced by the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 and BB-12, and the proinflammatory cytokine level induced by DSS is better than that induced by the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2. This finding is consistent with the changes in the levels of these cytokine mRNA in colon tissue (see figure 6 for test results).
Therefore, Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 can significantly reduce the proinflammatory cytokines induced by the DSS in the colon tissue of the mouse.
Example 10: bifidobacterium pseudocatenulatum N2 is used for protecting intestinal barrier of colitis mice.
The detailed description is the same as the procedure in example 7;
after completion of the molding obtained in example 7, the mouse was sacrificed, and the colon of the mouse was subjected to HE staining, AB staining and immunofluorescence staining.
The immunofluorescence staining method is as follows: 10 μm colon supersections were taken and incubated overnight at 4 ℃ with antibodies ZO-1 and MUC 2. Subsequently, the samples were counterstained with FITC-labeled antibody and Cy 3-labeled antibody, respectively, for 60 minutes. Images were obtained at the same site of each colon specimen using a microscope BX53(400 x) (see fig. 7 for test results).
As shown in fig. 7, DSS mice had damaged crypts in colon tissue, decreased goblet cell numbers, less mucus in the mucus layer, and impaired inflammatory cell infiltration, while the control mice had intact crypts and abundant goblet cells and mucin in the mucus layer.
As shown in FIG. 7, the expression of ZO-1 and MUC2 was down-regulated in colon tissue of mice in the DSS group compared to the control group.
And the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 restores the changes of ZO-1 and MUC2 under the stimulation of DSS and has better effect than BB-12.
Therefore, Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 is able to ameliorate DSS-induced colitis by enhancing colonic barrier integrity.
Example 11: the effect of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 on the expression levels of mucin and claudin mRNA in colitis mouse.
The detailed description is the same as the procedure in example 7;
after completion of the molding, the mice obtained in example 7 were sacrificed, and the colon tissue was used for the measurement of mRNA expression in the same manner as in example 9 (see FIG. 8 for the measurement results).
Compared with the control group, the expression of mRNA of the nian protein and the tight junction protein in the colon tissue of the mice in the DSS group is obviously reduced. The expression level of ZO-1mRNA was significantly increased after dry prognosis by Bifidobacterium pseudocatenulatum N2, and the expression level of Bifidobacterium pseudocatenulatum N2 was superior to BB-12 in recovering these adverse changes (see FIG. 8 for the test results).
Therefore, Bifidobacterium pseudocatenulatum N2 enhances the intestinal barrier of DSS-induced mice by up-regulating the mRNA expression of MUC2, ZO-1, Occludin and Claudin-1.
Example 12: the effect of Bifidobacterium pseudocatenulatum N2 on intestinal microorganisms in colitis mice.
The detailed description is the same as the procedure in example 7;
after completion of the molding, the mice obtained in example 7 were sacrificed, and the intestinal contents were subjected to the 16S rDNA assay (see FIG. 9, FIG. 10).
Compared with the control group, the intestinal microorganisms of the mice in the DSS group are remarkably increased in phylum level, the relative abundance of firmicutes is remarkably increased, and the relative abundance of Verrucomicrobia and Bacteroides is obviously reduced. At the genus level, the DSS group had a significant increase in Bacteroides (Bacteroides) and pseudomonas aeruginosa (Blautia) and a significant decrease in lactobacilli and akkermansia. These changes were recovered by Bifidobacterium pseudocatenulatum N2 (see FIG. 9).
As shown in FIG. 10, Lefse analysis based on LDA shows that Bacteroides, Blautia, and Ruminococcus belong to Firmicutes, Desulovibrio belongs to Desulovibrio, which is the dominant flora of the DSS group, and Lactobacillus belongs to Firmicutes, and Akkermansia belongs to Verrucomicrobia is more abundant in Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2.
The effect of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 on the DSS-stimulated mouse intestinal flora pathway KEGG pathway was evaluated using the picrast assay.
As shown in fig. 10, glycosyltransferase, glyoxylate and dicarboxylate metabolism and pantothenate and COA biosynthetic pathways associated with SCFAs levels were significantly elevated and bacterial secretion systems, sulfur metabolism and valine, leucine and isoleucine biosynthesis were significantly reduced compared to the DSS group.
Therefore, Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 was able to repair the differences in the mouse intestinal microbiota and metabolic pathways induced by DSS.
Example 13: effect of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) N2 on intestinal short-chain fatty acid levels in colitis mice.
The detailed description is the same as the procedure in example 7;
after completion of the molding, the mice obtained in example 7 were sacrificed, and the intestinal contents were collected and subjected to measurement of short-chain fatty acids (see FIG. 11).
Compared with the control group, the content of intestinal short-chain fatty acids (acetic acid, propionic acid and butyric acid) in mice in the DSS group is remarkably reduced, the content of acetic acid and butyric acid in the DSS-stimulated mice can be remarkably increased by Bifidobacterium pseudocatenulatum (N2) and BB-12, and the effect of Bifidobacterium pseudocatenulatum (N2) is caused by the BB-12 (the detection result is shown in figure 11).
Therefore, Bifidobacterium pseudocatenulatum N2 can increase the content of functional metabolites in the intestinal tract of mice with ulcerative colitis.
Example 14: effect of oral administration of a formulation containing Bifidobacterium pseudocatenulatum N2 on colitis treatment
Compared with the control group, the DAI index of the DSS group is obviously increased, and the DAI index of mice induced by the DSS can be obviously reduced by the bifidobacterium pseudocatenulatum N2 by replacing the intragastric administration in the example 7 with oral administration. The mice treated with DSS had significantly shortened colon length, bleeding colon, red color and sparse content. Therefore, the bifidobacterium pseudocatenulatum N2 has a better improvement effect on mice with DSS-induced colitis.
Similarly, the gavage of example 7 was replaced by oral administration of a suspension containing Bifidobacterium pseudocatenulatum N2, and the mice were sacrificed after the end of molding and the results were similar to those of the gavage of examples 8-13, as determined in examples 8-13. Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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<120> bifidobacterium capable of relieving colitis and application thereof
<141> 2021-06-14
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cccaccgagc ctacgacggg tagccggcct gagagggcga ccggccacat tgggactgag 180
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gagcaagcct tcgggtgagt gtacctttcg aataagcacc ggctaactac gtgccagcag 360
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gcggttcgtc gcgtccggtg tgaaagtcca tcgcttaacg gtggatctgc gccgggtacg 480
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tatcgggaag aacaccaatg gcgaaggcag gtctctgggc cgttactgac gctgaggagc 600
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ctggggagta cggccgcaag gctaaaactc aaagaaattg acgggggccc gcacaagcgg 780
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Claims (7)
1. Bifidobacterium pseudocatenulatum KLDS N2 is preserved in China center for type culture Collection, Wuhan, with a preservation date of 2021 year, 7 months and 5 days and a preservation number of CCTCC M2021820.
2. Use of Bifidobacterium pseudocatenulatum in the preparation of a medicament for prophylaxis and/or ∑ treatment according to claim 1Or the application in the medicine for treating ulcerative colitis, which is characterized in that the viable count of the bifidobacterium pseudocatenulatum in the medicine is not less than 1 multiplied by 10 9 CFU/mL。
3. Use of Bifidobacterium pseudocatenulatum in the preparation of a medicament for the prevention and/or treatment of ulcerative colitis according to claim 2, wherein the viable count of Bifidobacterium pseudocatenulatum is not less than 5X 10 9 CFU/mL。
4. Use of Bifidobacterium pseudocatenulatum in the manufacture of a medicament for the prevention and/or treatment of ulcerative colitis according to any of claims 2 to 3, wherein said medicament is a bacterial suspension.
5. Use of Bifidobacterium pseudocatenulatum for the preparation of a medicament for the prevention and/or treatment of ulcerative colitis according to claim 4, wherein said bacterial suspension is prepared by a process comprising: inoculating the bifidobacterium pseudocatenulatum of claim 1 into a culture medium for culture to obtain a seed solution; inoculating the seed solution into a culture medium for culture to obtain a culture solution; centrifuging the culture solution, and collecting bacterial sludge; and washing the bacterial sludge with normal saline, and then re-suspending to obtain bacterial suspension.
6. A pharmaceutical product for preventing and/or treating ulcerative colitis, comprising Bifidobacterium pseudocatenulatum according to claim 1, wherein the viable count of Bifidobacterium pseudocatenulatum is not less than 5X 10 9 CFU/mL。
7. A method for producing a pharmaceutical product for preventing and/or treating ulcerative colitis, said pharmaceutical product comprising the Bifidobacterium pseudocatenulatum of claim 1, wherein the viable count of said Bifidobacterium pseudocatenulatum is not less than 5X 10 9 CFU/mL。
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| CN114933994B (en) * | 2022-06-02 | 2023-12-12 | 南昌大学 | Composition with ulcerative colitis relieving function and application thereof |
| CN115651856B (en) * | 2022-06-14 | 2023-04-18 | 东北农业大学 | Combined bifidobacterium capable of relieving mouse intestinal injury caused by lipopolysaccharide |
| CN116178509B (en) * | 2022-08-09 | 2024-05-28 | 东北农业大学 | Preparation method and application of bifidobacterium surface protein |
| CN117363543B (en) * | 2023-10-30 | 2024-04-23 | 中国农业大学 | Exopolysaccharide pseudocatenin bifidobacterium pseudocatenin with enteritis relieving function and application thereof |
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