CN115887505A - Effect of animal bifidobacterium J-12 on intervening oral ulcer - Google Patents
Effect of animal bifidobacterium J-12 on intervening oral ulcer Download PDFInfo
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- CN115887505A CN115887505A CN202211733557.7A CN202211733557A CN115887505A CN 115887505 A CN115887505 A CN 115887505A CN 202211733557 A CN202211733557 A CN 202211733557A CN 115887505 A CN115887505 A CN 115887505A
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- oral
- ulcer
- expression
- subject suffering
- bifidobacterium animalis
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Classifications
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- 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
Abstract
The invention relates to the field of microorganisms and discloses an effect of bifidobacterium animalis J-12 on intervening oral ulcer; the preservation number of the bifidobacterium animalis is CGMCC No.25005. The animal bifidobacterium J-12 can reduce the morbidity risk of the oral ulcer and has a positive effect on the intervention of the oral ulcer.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to application of bifidobacterium animalis (Bifidobacterium animalis) or a preparation thereof in preparing a product for preventing and treating oral ulcer.
Background
The oral ulcer is a common oral mucosa disease, has high morbidity and is widely covered by people. The disease is complicated, potential induction factors comprise genetic factors, immune factors, pathogen infection, oxidative stress injury, vitamin and mineral deficiency, digestive tract diseases and the like, the pathogenesis of the disease is not completely clear up to now, and no specific medicine exists at present. Studies have shown that oral ulcers are closely linked to the oral flora, e.g. deregulation of Veillonella (Veillonella), streptococcus (Streptococcus) in the human mouth is associated with the progression of oral ulcers.
Probiotics are living microorganisms that are beneficial to the health of the host. A great deal of published evidence suggests that preparations containing dead cells and their metabolites function in exerting the relevant biological responses to restore normal gut homeostasis, in many cases similar to the use of live cells. The dead cells inactivate the bacteria primarily by heat treatment, releasing bacterial components with key immunomodulatory and pathogen-antagonistic properties. Bacterial components such as lipoteichoic acid, peptidoglycan, exopolysaccharides, etc. are considered to be major factors of these properties in the heat-sterilizing formulation.
The combination of bacillus subtilis, bifidobacterium bifidum, lactobacillus acidophilus and enterococcus faecalis can reduce experimental oral mucositis of immunosuppressed rats; the combination of bifidobacterium longum, lactobacillus lactis and enterococcus faecalis can obviously enhance the immune response of patients, and reduce the severity of oral mucositis caused by radiotherapy and chemotherapy of nasopharyngeal carcinoma by changing intestinal flora; the mixed preparation of bifidobacterium longum, lactobacillus bulgaricus and streptococcus thermophilus can obviously reduce the pain level of candida-related stomatitis and tongue mucous membrane congestion after being applied for two weeks. The bifidobacterium animalis RH exopolysaccharide component separated from feces of centenarians of Guangxi Bama has direct and effective antioxidant activity.
Therefore, the probiotics and the preparation thereof are a new method for relieving oral ulcer at present, but the bifidobacterium animalis and the preparation thereof have less research on the intervention effect of the oral ulcer.
Disclosure of Invention
The invention aims to provide application of bifidobacterium animalis (bifidum animalis) or a preparation thereof in preparing products for preventing and treating oral ulcer.
In order to achieve the above objects, the present invention provides use of bifidobacterium animalis (bifidobacterium animalis) or a formulation thereof for preparing a product for preventing and treating oral ulcer;
the preservation number of the bifidobacterium animalis is CGMCC No.25005.
The inventor establishes a golden hamster oral ulcer model by a methyl viologen buccal membrane injection method, discusses whether bifidobacterium animalis J-12 (Bifidobacterium animalis J-12) is feasible to improve oral ulcer by regulating intestinal homeostasis in a gastric lavage mode, whether viable bacteria and inactivated bacteria of bifidobacterium animalis have anti-inflammatory and anti-oxidation effects at the same time and whether the effects are the same, and further discusses possible action sites of bifidobacterium animalis intervening oral ulcer, and finds that both viable bacteria of gastric lavage J-12 and bacteria after thermal inactivation can reduce proinflammatory factor IL-10 and oxidation indexes GSH and MDA contents in serum. The J-12 live bacteria and the heat inactivated bacteria can reduce the expression of MMP-9 by reducing the expression of NF-kB to reduce the inflammation level, thereby reducing the expression of anti-inflammatory factors LXA4 and PGE 2; reducing expression of PARP by reducing expression of Caspase-3, thereby reducing DNA damage of ulcer tissues; intervene in the course of canker sores by regulating the structure of the intestinal flora. Namely, the animal bifidobacterium J-12 can reduce the morbidity risk of the oral ulcer and has a positive effect on the intervention of the oral ulcer.
Biological preservation
The strain Bifidobacterium animalis is preserved in the ordinary microorganism center of China Committee for culture Collection of microorganisms (address: no. 3 of West Lu No. 1 of Beijing Kogyo-the sunward area, microbiol research institute of Chinese academy of sciences, postal code: 100101) 6.6.6.2022 (the preservation unit is abbreviated as CGMCC), and the preservation number is CGMCC No.25005, which is abbreviated as J-12.
Drawings
FIG. 1 is a photograph of HE staining of a microtus oral mucosal tissue section; wherein (A) is Normal group, (B) is Model group, (C) is J-12L group, and (D) is J-12D group;
figure 2 shows GSH (E), MDA (F) and SOD (G) levels in serum;
FIG. 3 shows LXA4 (H) and PGE2 (I) levels in mucosal tissues of oral ulcers in rats;
FIG. 4 shows levels of proinflammatory cytokines IL-1 β (J) and IL-6 (K) in hamster serum;
FIG. 5 shows photographs of NF-. Kappa.B, MMP-9 immunohistochemistry of the oral ulcer mucosal tissue of hamster (scale: 100 μm);
FIG. 6 shows a photograph of Caspase3, PARP immunohistochemistry of the mucosal tissue of a hamster oral ulcer (scale: 100 μm);
FIG. 7 shows phylogenetic level of oral flora (L) and intestinal flora (M) community composition analysis;
FIG. 8 shows NMDS plots of oral flora (N) and intestinal flora (O) specifices levels;
FIG. 9 shows a circle of oral bacterial species visualization;
fig. 10 shows a visualized circle of gut species;
FIG. 11 shows a db-RDA analysis of the oral (P), intestinal (Q) flora levels of the hamster;
figure 12 shows a heat map of the correlation between oral (R) and intestinal (S) flora and immune and inflammatory factors in rats.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For numerical ranges, each range between its endpoints and individual point values, and each individual point value can be combined with each other to give one or more new numerical ranges, and such numerical ranges should be construed as specifically disclosed herein.
The invention provides application of animal bifidobacterium (Bifidobacterium animalis) or a preparation thereof in preparing a product for preventing and treating oral ulcer;
the preservation number of the bifidobacterium animalis is CGMCC No.25005.
Preferably, the bifidobacterium animalis is present in the formulation in the form of viable and/or dead bacteria.
Preferably, the dead bacteria are heat-inactivated bacteria (the heat-inactivation condition preferably comprises a temperature of 110-125 ℃ and a time of 10-25 min).
The bifidobacterium animalis can be cultured and fermented in a manner conventional in the art and will not be described herein.
Preferably, the administration mode of the bifidobacterium animalis or the preparation thereof is gastrointestinal administration, and can be at least one of intragastric gavage, direct oral administration and feed addition, and is more preferably oral administration. The dose of Bifidobacterium animalis or its preparation can be adjusted according to different types of subjects, and will not be described herein, for example, when the subject is murine, the application amount/frequency can be 10 8 CFU/only/day, 10 9 CFU/only/day, 10 10 CFU/any one value per day or a range of any two of the above values.
The subject may be a mammal, such as a human, a murine, etc.
Preferably, the oral ulcer is an oral ulcer caused by oxygen radical injury. The animal model for embodying the oral ulcer caused by the oxygen free radical injury is preferably a golden hamster oral ulcer model established by a methylviologen buccal injection method.
Preferably, the use comprises modulating gut flora architecture in a subject suffering from a dental ulcer; more preferably, the method comprises reducing the abundance of Candidatus _ Saccharomyces and Clostridium UCG-014 in the intestine of a subject suffering from a dental ulcer and increasing the abundance of Allobaculum.
Preferably, the use comprises modulating the oral flora structure of a subject suffering from a dental ulcer; more preferably, the method comprises decreasing the abundance of Streptococcus _ respircuri and Fusobacterium-nucleus-svbsp. _ polynorbium in the oral cavity of a subject suffering from an oral ulcer, and increasing the abundance of Bacteroides _ dorei.
Preferably, the use comprises reducing the severity of oxidative damage in a subject suffering from a dental ulcer, more preferably comprises reducing the levels of MDA and GSH in a subject suffering from a dental ulcer.
Preferably, the use comprises down-regulating the level of inflammation in a subject suffering from a dental ulcer; more preferably, the expression of NF-kB is reduced, and the expression of anti-inflammatory factors LXA4 and PGE2 is reduced.
Preferably, the use comprises reducing the expression of a proinflammatory factor, preferably IL-1 β, in a subject having a canker sore.
Preferably, the use comprises reducing DNA damage of ulcerated tissue in a subject suffering from a dental ulcer, more preferably comprises reducing expression of Caspase-3 and expression of PARP.
The present invention will be described in detail below by way of examples.
In the following examples, chloral hydrate was purchased from Beijing Bailingwei technologies, inc., methylphospholin was purchased from Sigma (USA), nuclear factor kappa B (nuclear factor kappa-B, NF-kappa B), matrix metalloproteinase-9 (matrix metalloproteinases-9, MMP-9), cysteine protease-3 (Caspase-3) antibody was purchased from Proteintetech (Wuhan Sanying), adenosine diphosphate ribopolymerase (PARP) antibody was purchased from Beijing Boohinson Biotech, inc., and other common reagents were purchased from conventional commercial sources.
Bifidobacterium animalis J-12 (J-12 for short) with preservation number of CGMCC N0.25005.
Data are presented as Mean ± standard deviation (Mean ± SD). One-way ANOVA (One-way ANOVA) was performed on each group using statistical software SPSS23.0, and P <0.05 was considered statistically different.
Preparation example 1 animal experiment grouping, modeling and sampling
Male LVG Syrian Golden yellow rats (LVG Golden Syrian Hamster) of six weeks of age, body weight (110. + -.20) g/rat, purchased from Beijing Wittiulihua laboratory animals technology Co., ltd, were raised under 12h light alternation, temperature was maintained at 20. + -. 2 ℃ and humidity was maintained at 45-50%.
The hamster is domesticated in a feeding room for one week and then randomly divided into 4 groups, namely a Normal control group (Normal), a Model group (Model), a J-12 viable bacterium group (J-12L) and a J-12 viable bacterium inactivated group (J-12D), wherein 7 Normal groups are provided, and 10 groups are provided for the rest groups respectively. The normal saline solution is 1mL per day for Norma1 group and Model group, and the concentration is 10 for 1mL per day for J-12L group 9 CFU/mL J-12 viable bacteria solution, and 1mL for intragastric administration in J-12D groupJ-12 at a concentration to inactivate bacteria. Performing stomatocace modeling 15 days after gastric lavage, i.e. first injecting 10% chloral hydrate (the injection amount is 0.3-0.5% of the body weight) into the abdominal cavity of an anesthetized rat, injecting 0.25mL PBS buffer (pH = 7.4) into the buccal cavity of a Normal group of the rat, and injecting 0.25mL methylviologen (10 mmol/L dissolved in PBS) into the buccal cavity of the rest 3 groups of the rat; the day of injection until the rats were sacrificed, each group remained on daily gavage intervention. Anesthetizing the hamster on the 4 th day after the model building, taking blood, scraping the inside of the oral cavity (ulcer part) of the hamster by using an oral swab, and shearing a swab head and putting the swab head into a centrifugal tube filled with nucleic acid protective solution; cutting the focus tissue or normal mucosa of oral ulcer, and freezing one part of the focus tissue or normal mucosa in 4% paraformaldehyde and the other part of the focus tissue or normal mucosa. Separating serum from whole blood, subpackaging and freezing at-80 ℃, dissecting, taking colon segment tissue contents, subpackaging in two 2mL centrifuge tubes (collecting about 0.5g per tube), and freezing for later use.
Example 1
This example illustrates HE stained section experiments of mucosal tissue of oral ulcers.
The fixed mucosa tissues of the oral ulcer are embedded and sliced by paraffin, then the slices are sequentially dewaxed and dehydrated in a gradient manner, then hematoxylin and eosin staining are carried out, finally the slices are dehydrated and sealed, and observation and photographing are carried out under a microscope, and the result is shown in figure 1.
In FIG. 1, red arrows point to inflammatory cell infiltration, black arrows point to mucosal epithelial cell necrosis, nuclear fragmentation, yellow arrows point to loose connective tissue alignment, blue arrows point to hemorrhage, purple arrows point to regenerated muscle fibers, green arrows point to connective tissue hyperplasia, chocolate arrows point to capillary extravasation expansion, orange arrows point to local epidermal layer loss (scale for Normal and Model groups: 200 μm; scale for J-12L and J-12D groups: L00 μm).
As shown in FIG. 1, the Normal group has intact epidermal layer structure, abundant collagen fibers in the dermal layer, abundant muscle fibers in the hypodermal layer, and no obvious inflammation. Compared with the Normal group, the epidermal layer and the dermal layer of the Model group are subjected to cell necrosis, nuclear fragmentation or lysis; severe edema, loose arrangement of connective tissue, scattered inflammatory cell infiltration, and more bleeding were observed in the subcutaneous tissue, indicating successful modeling of the oral ulcer. More new muscle fibers in subcutaneous tissues, a small amount of hyperplastic connective tissues around the subcutaneous tissues and scattered inflammatory cell infiltration can be seen in the J-12L group visual field, and moderate edema and loose arrangement of the connective tissues can be seen at the same time; local epidermis layer is missing. The results in the field of J-12D were similar to those of J-12L, but no local epidermal layer was missing and a small amount of capillary extravasated blood was visible. Wound healing is a complex process involving hemostasis, inflammation, proliferation and tissue remodeling. The connective tissue has strong regeneration capacity, and the wound healing is completed through the proliferation of the connective tissue, so that the proliferation phenomena of regenerated muscle fibers and the connective tissue are observed in the J-12L group and the J-12D group, which indicates that the animal bifidobacterium J-12 can promote the healing of the ulcer surface of the oral mucosa.
Example 2
This example illustrates the determination of serum SOD activity, MDA and GSH concentrations.
Superoxide dismutase (SOD) is the first line of defense against oxygen radicals. Aerobic organisms receive a certain level of reactive oxygen species, i.e., superoxide and hydrogen peroxide, formed by physiological oxidative stress, which can cause lipid peroxidation and cell damage. Wherein the hydrogen peroxide can be reduced by reduced Glutathione (GSH) with antioxidant effect. In addition, lipid peroxidation is enhanced in the oxidative stress process, so that lipid peroxidation final products such as malonic acid (MDA) and the like are generated, and the MDA can be attached to self biomolecules, so that a new self epitope is generated, and potential adverse biological reactions can be induced. Thus, the increase in SOD and GSH suggests that the body stimulates the increase in antioxidant substances due to the generation of oxidative stress; MDA reflects the degree of damage due to oxidative stress.
The kit (Biyunnan biology) is adopted to detect SOD activity, GSH and MDA levels in serum, and the specific operation steps are carried out according to the corresponding kit instructions.
As shown in FIG. 2, there were significant differences (p < 0.05) between groups except for SOD level; the levels of GSH in serum of mice in the Model group were significantly increased (P < 0.05) compared to the Normal group, and the levels of GSH in both the J-12L group and the J-12D group were significantly lower than those in the Model group; the MDA levels in the Model group were significantly higher than in the Normal group, and the MDA levels in both the J-12L and J-12D groups were significantly lower (P < 0.05) than in the Model group. Four groups of treatments had no significant differences in SOD levels. The results show that both the J-12 dead bacteria and the J-12 live bacteria can reduce the damage degree of the oxidative stress of the golden hamster so as to reduce the level of antioxidant substances in vivo, namely the J-12 has a positive effect on reducing the oxidative damage of organisms.
Example 3
This example illustrates the measurement of LXA4 and PGE2 levels in mucosal tissues of oral ulcers.
LXA4 is a double acting mediator that activates specific cellular pathways through FPR2/ALX, resulting in anti-inflammatory and pro-catabolic effects. PGE2 not only reduces inflammation, but also has significant therapeutic potential for tissue regeneration by macrophages.
Taking oral mucosa tissue of hamster, preparing tissue homogenate, and measuring the concentrations of Lipoxin A4 (Lipoxin A4, LXA 4) and prostaglandin E2 (prostaglandin E2, PGE 2) in the tissue homogenate by enzyme-linked immunosorbent assay (ELISA) respectively; and determining the total protein concentration of the tissue homogenate to correct the content of LXA4 and PGE2, and the specific operation steps are carried out according to the corresponding kit instructions.
As can be seen from fig. 3 (H), model group LXA4 levels were increased to some extent without significant difference compared to Normal group. LXA4 levels were significantly reduced in both the J-12L and J-12D groups compared to the Model group (p < 0.05). Both the dead and live J-12 bacteria are shown to reduce the level of inflammation in vivo, resulting in a reduced degree of LXA4 activation.
As can be seen from FIG. 3 (I), PGE2 levels were significantly elevated in the Model and J-12D groups (p < 0.05), and there was no significant change in PGE2 levels in the J-12L group, but slightly higher than in the Normal group. The Model group had the highest level of PGE2, indicating that it was most severely damaged by inflammation and that the body expressed high levels of PGE2 to combat inflammation. The level of inflammation in golden yellow hamster is reduced due to the intervention of J-12, so that the expression of PGE2 level in J-12 treatment group is lower than that in Model group.
Example 4
This example illustrates the determination of the proinflammatory cytokine levels in serum.
Interleukin-I beta (Interleukin-1 beta, IL-1 beta) is a major stimulator of regional and systemic inflammation and is a major pro-inflammatory mediator; interleukin 6 (Interleukin-6, IL-6) is a typical cytokine for maintaining homeostasis. When homeostasis is disrupted by infection or tissue damage, IL-6 is immediately produced and helps the host defend against this acute stress by activating the acute phase and immune response.
IL-6 and IL-1. Beta. In serum were assayed by ELISA (Cosubject organism) and the specific procedures were performed according to the corresponding instructions.
As can be seen in FIG. 4, at the IL-1. Beta. Level (J), the Model group was significantly higher than the Normal group, the J-12L group, and the J-12D group, indicating that the J-12 treatment had some reduction in the level of inflammation; at IL-6 levels (K), the Model cohort was significantly higher than the Normal cohort (P < 0.05), and the IL-6 levels were significantly higher in the J-12L cohort and the J-12D cohort than the Normal cohort (P < 0.05) and higher in the Model cohort IL-6 levels.
It has been experimentally demonstrated that IL-6 and Candidatus Saccharomyces are negatively associated in a diabetic nephropathy rat model. The Model group and the Normal group in this experiment were not significantly different at the species level, and the relative abundance of this species was significantly increased (p < 0.05) in the Model group relative to the J-12 treated group, thus not excluding the possibility of candidatus saccharomonas inhibiting IL-6 production in this experiment.
Example 5
This example illustrates immunohistochemical sectioning experiments of ulcerated oral mucosal tissue.
Sequentially dewaxing and gradient dehydrating paraffin sections, performing antigen retrieval by using a citric acid antigen retrieval buffer solution (pH = 6.0), putting the sections into a 3% hydrogen peroxide solution, incubating for 25min in a dark place to block endogenous peroxidase, performing operation steps of sealing, primary antibody incubation, secondary antibody incubation and the like, finally performing DAB color development and cell nucleus counterstaining, dehydrating and sealing the sections, and observing and photographing under a microscope.
The immunohistochemical Score is obtained by respectively scoring the staining degree (0-3 points) and the positive rate (0-4 points) of the immunohistochemical Score by an IRS (Immunoreactive Score) scoring method and multiplying the results to obtain a comprehensive Score (0-12 points); the scoring criteria are as follows, and the degree of staining is scored as the staining profile exhibited by the protein of interest: no coloration is 0 minutes, light yellow is 1 minute, tan is 2 minutes, and tan is 3 minutes; positive rates were scored as the positive rate of cells in the section: 0-5% for 0 min, 6-25% for 1 min, 26-50% for 2 min, 51-75% for 3 min, and >75% for 4 min. A composite score of 0 was negative (1); 1-3 points were weakly positive (+); 3.1-5 as positive (++); 5.1-7 were classified as strongly positive (+++).
(1) Expression of NF-kB and MMP-9 in ulcerated mucosal tissue in a model of canker sore
The MMP-9 gene promoter has NF-kB binding sites. NF-. Kappa.B is an important transcription factor, and activation of its signaling pathway initiates inflammatory responses. It has been shown that MMP-9 up-regulation mediated by NF- κ B occurs in response to MMP-9-induced increase in intestinal permeability via activation of NF- κ B in the Caco-2 monolayer and increased TJ permeability in HT-29 intestinal epithelium induced by aluminum. 15-deoxy-D12, 14-prostaglandin J2 activates MCF-7 cells by regulating NF-kB/AP-1, thereby inhibiting MMP-9 expression induced by 12-O-tetradecanoyl-13-acetate. Thus, there is a positive relationship between MMP-9 expression mediated by NF-. Kappa.B upregulation and disease.
FIG. 5 shows photographs of NF-. Kappa.B, MMP-9 immunohistochemistry of the mucosal tissue of a hamster oral ulcer (scale: 100 μm); table 1 shows the immunohistochemical scoring table. As can be seen from Table 1, there were significant differences (p < 0.05) between the Normal group and the Model group, J-12L group, and J-12D group, indicating that the methyl viologen buccal membrane injection method successfully modeled and initiated the inflammatory response in the rats. Both NF-. Kappa.B and MMP-9 levels were reduced in the J-12 treated group compared to the Model group, and it was found that intervention of J-12 had a positive effect on reduction of the level of inflammation.
TABLE 1 immunohistochemical scoring Table
(2) Level of DNA repair
Apoptosis is a stage of oral wound healing, and secondary necrosis of apoptotic epithelial cells leads to extracellular leakage of epithelial-derived danger signals, including HMGB-1.HMGB-1 is considered to be an important facilitator of sepsis, collagen diseases, atherosclerosis, cancer, arthritis, acute lung injury, epilepsy, myocardial infarction, local and systemic inflammation, and the like. Caspase-3 is considered to be an important effector protease, which is cleaved and activated during apoptosis. Caspase-3 in turn cleaves a variety of cellular substrates, most notably PARI. PARP has the function of repairing single-stranded DNA damage, and cleaved PARP is an important marker of apoptosis.
FIG. 6 shows typical photographs of CaSpaSE3 and PARP immunohistochemistry of oral ulcer mucosal tissue in rats (scale: 100 μm); table 2 shows the immunohistochemical scoring table.
As can be seen from the quantitative scores in Table 2, the Model group and the J-12L and J-12D groups are significantly different from the Normal group (p < 0.05), i.e., the success of oral ulcer modeling by methyl viologen buccal injection is achieved. The Model group Caspase-3 and PARP protein expression levels were the highest in the four groups, and there was a significant difference (p < 0.05) from the J-12 treated group, indicating that the Model group suffered the most severe DNA damage, while the intervention available for J-12 could mitigate the DNA damage caused by modeling.
TABLE 2 immunohistochemical scoring Table
Example 5
This example illustrates oral flora and gut composition measurements.
Respectively extracting total DNA in a mouth swab sample and a colon content sample of a hamster, performing 16SrDNA high-throughput sequencing (Shanghai Meiji biological medicine science and technology Co., ltd.) by adopting an IlluminaPE300 sequencing platform, and performing bioinformatics and data statistical analysis such as OUT clustering, species annotation, diversity analysis and the like on sequencing data.
It can be seen from fig. 7 that the dominant flora at phylum level was not different between the oral (L) and intestinal (M) flora in the four groups of samples, and the relative abundance of each flora was different. From phylogenetic analysis of the oral flora, it can be seen that they include Firmicutes, bacteroidetes, proteobacteria, fusobacterium, etc. The predominant flora of the intestine includes Bacteroides (Bacteroides), firmicutes (Firmicutes), actinomycetes (Actinobacillus), with Bacteroides and Firmicutes dominating the intestinal flora.
In order to determine whether the four treatments cause significant difference on the oral cavity and intestinal flora structures of the gerbils, non-metric multidimensional scaling (NMDS) analysis is carried out on gerbil oral cavity and intestinal flora community data based on the Bray-Curtis-faith algorithm at the specificity level, and the result is shown in fig. 8, wherein the samples of different treatment groups of the gerbil oral flora have large overlap, which indicates that similar Species exist in the four groups of samples at the specificity level and no significant difference exists. Intestinal flora samples were at Species level: the intestinal flora structure of the Model group and the Normal group is significantly different.
To determine the source of the above differences, a visual circle of species was constructed and species with greater relative abundance differences between groups were statistically analyzed. Tables 3 and 4 list the selection of species with relative abundance ranking top ten and significant differences between groups among oral, intestinal species. FIG. 9 shows a circle of oral bacterial species visualization; fig. 10 shows a visualized circle of gut bacteria species.
TABLE 3 oral flora dominant species relative abundance ratio table
As can be seen from Table 3, streptococcus _ respirauli, fusobacterium _ nucleosum _ subsp _ polymorphum are two species of bacteria that significantly increased in the oral cavity of the Model group rats, and Bacteroides _ dorei is a species of bacteria that significantly decreased in the oral cavity of the Model group rats.
Fusobateriumnuclearum (f. Nuclearum) participates in the formation of typical dental plaque, causing periodontal disease; is considered to be a key promoter of colon cancer (CRC). Streptococcus _ respiraculi currently lacks disease-related studies and is considered to belong to a new Streptococcus genus. This may prove to be a health threat to the imbalance of the oral flora caused by oral ulcers. The relative abundance of oral flora of the J-12 treated hamster is close to the normal level, which indicates that the J-12 has the capability of regulating the imbalance of the oral flora so as to reduce the occurrence probability of oral and systemic diseases.
Bacteroides _ dorei is an intestinal microorganism isolated from human stool samples, inhibiting the growth of Clostridium difficile and reducing lipopolysaccharide production and atherosclerosis, and mice inoculated with this bacterium in animal experiments were protected from influenza infection and negatively associated with SARS-COV-2. Dorei was detected in the oral cavity in this experiment, and is presumed to be a cause of the fluidity of microorganisms in the environment.
TABLE 4 intestinal flora dominant bacteria relative abundance ratio table
As can be seen in Table 4, the species significantly regulated by gavage were norak _ f _ Erysipelotrichaceae, allobaculum, candidatus _ Saccharomyces, clostridium _ UCG-014. Where the effect of treatment in group J-12 on the relative abundance of norak _ f _ Erysipelotrichaceae was reversed, group J-12L was upregulated, and group J-12D was downregulated as well as the Model group.
The regulatory effects of J-12 treatment on the three bacteria were consistent, and the J-12 treated group increased the relative abundance of Allobaculum and decreased the relative abundance of Candidatus-Saccharomyces and Clostridium-UCG-014, all in close proximity to the control group, compared to the model group.
Allobaculum has been identified as a short chain fatty acid producing strain with a negative correlation to different proinflammatory markers. Candidatus _ Saccharionoas is a opportunistic pathogen that increases significantly in the gut of gout patients, which is significantly elevated in a rat model of diabetic nephropathy, and studies have shown that Candidatus _ Saccharionoas the dominant bacterium is involved in inflammatory mucosal disease.
Clostridium _ UCG-014 is a pro-inflammatory bacterium that may play a significant positive role in type 2 diabetes and has a strong positive correlation with fasting blood glucose levels.
As can be seen from tables 3 and 4, the J-12D group and the J-12L group have a regulatory ability for the changes in the abundance of the oral and intestinal flora caused by oral ulcer and the regulatory ability of the J-12D group is stronger than that of the J-12L group.
In order to further study the relevance of the oral and intestinal bacterial communities of the four treated rats to other physiological indexes, distance-based redundancy analysis (db-RDA) was performed by using the Bray-Curtis algorithm. As shown in FIG. 11, the factors that have a great influence on the structure of the oral flora community of the hamster are GSH, MDA, NF-kB and MMP-9, and the influence sequence is GSH > MDA > NF-kB > MMP-9; factors which have great influence on the intestinal flora community structure are GSH, MDA and PGE2, and the influence sequence is GSH > MDA > PGE2. Selecting strains belonging to the top 50 of the genus level, and evaluating the correlation between the strains and inflammatory factors and immune factors by using a sperman correlation coefficient. From fig. 12 it can be seen that MDA has a strong correlation with both oral and intestinal flora.
Correlation analysis showed the strongest correlation between MDA and flora, consistent with db-RDA results.
By adopting the technical scheme of the invention, the intervention of the oral ulcer is feasible by live bifidobacteria J-12 bacteria or inactivated J-12 bacteria of the gavage animal, the intervention mechanism is that the live bifidobacteria J-12 bacteria and the inactivated J-12 bacteria intervene the oral ulcer by adjusting intestinal flora, and the effect exhibited by the inactivated J-12 bacteria is optimal. The J-12 viable bacteria and the J-12 inactivated bacteria can reduce the level of oxidative stress damage without significant difference, and the results show that the J-12 viable bacteria and the J-12 inactivated bacteria have indirect anti-inflammatory and antioxidant effects.
In the invention, J-12 intervenes in the healing of oral ulcer caused by methyl viologen through three paths. First is the oxidative stress pathway: by reducing expression of NF-kB, expression of MMP-9 is down-regulated, thereby reducing the level of inflammation. Secondly, the expression of Caspase-3 is reduced, the apoptosis is reduced, the expression of DNA repair protein PARP is further reduced, and the cell damage caused by methyl viologen is reduced. And finally, the relative abundance of pathogenic bacteria in the intestinal tract is reduced, and the relative abundance of beneficial bacteria is up-regulated to maintain the steady state of intestinal flora, strengthen the gastrointestinal tract barrier and regulate the immune system.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. Use of Bifidobacterium animalis (Bifidobacterium animalis) or a preparation thereof for the manufacture of a product for the prevention and treatment of oral ulcer;
the preservation number of the bifidobacterium animalis is CGMCC No.25005.
2. Use according to claim 1, wherein the Bifidobacterium animalis is present in the preparation in the form of viable and/or dead bacteria;
preferably, the dead bacteria are heat-inactivated bacteria.
3. Use according to claim 1 or 2, wherein the bifidobacterium animalis or a formulation thereof is administered parenterally, preferably orally.
4. The use of any one of claims 1-3, wherein the oral ulcer is an oral ulcer caused by oxygen radical damage.
5. The use of any one of claims 1-4, wherein the use comprises modulating gut flora architecture in a subject with a dental ulcer,
preferably comprising decreasing the abundance of Candidatus Saccharioninas and Clostridium UCG-014, increasing the abundance of Allobaculum, in the intestine of a subject suffering from a dental ulcer.
6. The use of any one of claims 1-5, wherein the use comprises modulating the oral flora structure of a subject suffering from a dental ulcer,
preferably, the method comprises reducing the abundance of Streptococcus _ respircium and Fusobacterium-nucleus-svbsp. _ polynorbium in the oral cavity of a subject suffering from an oral ulcer and increasing the abundance of Bacteroides _ dorei.
7. The use of any one of claims 1-6, wherein the use comprises reducing the severity of oxidative damage in a subject suffering from a dental ulcer, preferably comprising reducing the levels of MDA and GSH in a subject suffering from a dental ulcer.
8. The use of any one of claims 1-7, wherein the use comprises down-regulating the level of inflammation in a subject with a canker sore; preferably, the expression of NF-kB is reduced, and the expression of anti-inflammatory factors LXA4 and PGE2 is reduced.
9. The use of any one of claims 1 to 8, wherein the use comprises reducing the expression of a pro-inflammatory factor in a subject suffering from a canker sore, preferably the pro-inflammatory factor is IL-1 β.
10. Use according to any of claims 1-9, wherein the use comprises reducing DNA damage of ulcer tissue in a subject suffering from a dental ulcer, preferably comprising reducing the expression of Caspase-3 and the expression of PARP.
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