CN118021850A - Lactobacillus paracasei and application thereof in preparation for relieving inflammatory diseases - Google Patents
Lactobacillus paracasei and application thereof in preparation for relieving inflammatory diseases Download PDFInfo
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- 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
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- 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
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- 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]
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- 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
- C12R2001/225—Lactobacillus
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus paracasei and application thereof in preparations for relieving inflammatory diseases. The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.24626. The strain has strong gastric juice tolerance and bile salt tolerance, the survival rate in gastric juice can reach 82.19%, and the growth efficiency in bile salt can reach 16.47%; the strain can obviously reduce the damage of colon tissues of the organism, obviously reduce the levels of inflammatory factors LPS, IL-6 and TNF-alpha in serum of the organism and improve the level of IL-10 so as to relieve the systemic inflammation of the organism.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus paracasei and application thereof in preparations for relieving inflammatory diseases.
Background
Inflammatory bowel disease (inflammatory bowel disease, IBD) is a chronic and non-specific intestinal inflammatory disease that accumulates primarily in the colorectal, ileum, mainly including ulcerative colitis (ulcerativecolitis, UC) and Crohn's Disease (CD), with clinical manifestations of abdominal pain, diarrhea and mucopurulent bloody stool. At present, the pathogenesis of the traditional Chinese medicine is not completely defined, and the traditional Chinese medicine tends to cause chronic inflammation of intestinal tracts due to abnormal immune response triggered by genetic, dietary or environmental factors acting on genetically susceptible hosts. IBD was first found in western countries, and in recent decades as industrialization and urbanization progresses, IBD has also emerged in emerging developing countries, now becoming a global disease. The number of IBD patients in our country has increased dramatically over the last 20 years, and the disease process is characterized by progressive and destructive nature. The data show that 5% -10% of patients with chronic enteritis are converted into intestinal cancers, while more than 25 years of long-term patients with enteritis are converted into intestinal cancers, and about 40% of patients with chronic enteritis are converted into intestinal cancers, so that great threat is brought to people's health and colonitis patients.
Currently, the treatment of IBD is mainly directed to inducing remission and preventing disease recurrence as a therapeutic direction, starting from aminosalicylates and antibiotics, and gradually developing into corticosteroids, immunomodulators to biological agents, which, although achieving therapeutic success, mostly come with serious side effects that increase the risk of disease recurrence and the medical burden of managing IBD.
With the increasing research of intestinal microorganisms in the pathogenesis of IBD, safe and side-effect-free probiotics are of great interest. Therefore, it is necessary to develop a microorganism and a microecological preparation which can treat inflammatory bowel disease.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide a probiotic lactobacillus paracasei (Lactobacillus paracasei) (hereinafter also referred to as lactobacillus paracasei TY-G05) that can treat inflammatory bowel disease, has good gastric juice and bile salt tolerance, can significantly alleviate enteritis symptoms, and can also alleviate systemic inflammation by significantly reducing the levels of inflammatory factors LPS, IL-6 and TNF- α in the serum and increasing IL-10 levels.
In order to achieve the above purpose, the present invention may adopt the following technical scheme:
In one aspect, the invention provides lactobacillus paracasei (Lactobacillus paracasei) which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24626.
In another aspect, the invention provides a composition that may include one or more of the following: (a) lactobacillus paracasei as described above; (b) a lysate of lactobacillus paracasei as described above; (c) a culture of lactobacillus paracasei as described above; (d) the fermentation broth of Lactobacillus paracasei.
In a further aspect the invention provides a formulation comprising lactobacillus paracasei as described above or a composition as described above, and a carrier, wherein the carrier is a pharmaceutically acceptable carrier or an edible carrier.
In a further aspect, the invention provides the use of a lactobacillus paracasei as described above or a composition as described above in the manufacture of a medicament for the treatment of inflammatory diseases.
The lactobacillus paracasei preservation information in the invention is as follows: preservation mechanism: china general microbiological culture Collection center (CGMCC); preservation address: the dynasty district North Star, department 1, hospital 3 in Beijing; preservation date: 2022, 4, 1; preservation number: cgmccno.24626; classification naming: lactobacillus paracasei (Lactobacillus paracasei).
The beneficial effects of the invention include:
(1) The lactobacillus paracasei TY-G05 provided by the invention has stronger gastric juice tolerance capability and bile salt tolerance capability, the survival rate in gastric juice can reach 82.19%, and the growth efficiency in bile salt can reach 16.47%.
(2) The lactobacillus paracasei TY-G05 provided by the invention can obviously reduce colon tissue injury of an organism, obviously reduce the levels of inflammatory factors LPS, IL-6 and TNF-alpha in serum of the organism and improve the level of IL-10 so as to relieve systemic inflammation of the organism.
Drawings
FIG. 1 is a colony morphology diagram of an isolated strain;
FIG. 2 is a graph of gram stain results;
FIG. 3 is a morphology of each set of colon;
FIG. 4 is a graph of colon length comparisons for each group;
FIG. 5 is a graph of colon weight/colon length comparison for each group;
FIG. 6 is a comparison of colon pathology sections of each group;
FIG. 7 is a graph showing the comparison of LPS levels in each group;
FIG. 8 is a graph comparing IL-6 levels of each group;
FIG. 9 is a graph comparing TNF- α levels of each group;
FIG. 10 is a graph comparing IL-10 levels of each group;
In fig. 4, 5, 7, 8, 9 and 10, the symbol "×" indicates that there is a significant difference (p < 0.05) between the two groups.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.
The term "formulation" herein is not only a pharmaceutical formulation for pharmaceutical use but also includes edible food formulations or nutraceutical formulations.
An embodiment of the invention provides lactobacillus paracasei (Lactobacillus paracasei), namely lactobacillus paracasei TY-G05, which is preserved in the China general microbiological culture collection center with the preservation number of CGMCC No.24626.
The Lactobacillus paracasei TY-G05 was derived from the Homoxas of Qinghai province, and was detected as a gram-positive bacterium (G +) by a gram-staining result and was determined as a rod-like shape; and the 16S rDNA sequence is amplified by PCR, the 16S rDNA sequence of the lactobacillus paracasei TY-G05 is obtained by measuring that the 16S rDNA sequence contains a sequence shown as SEQ ID NO.1 and carrying out homology analysis. In addition, the gastric juice has stronger gastric juice tolerance capability and bile salt tolerance capability, the survival rate in gastric juice can reach 82.19 percent, and the growth efficiency in bile salt can reach 16.47 percent, namely the gastric juice has good survival rate in the digestive tract of the organism.
Another embodiment of the present invention provides a composition that may include one or more of the following in combination: (a) Lactobacillus paracasei TY-G05 as described above; (b) a lysate of Lactobacillus paracasei TY-G05 as described above; (c) a culture of Lactobacillus paracasei TY-G05 as described above; (d) fermentation broth of Lactobacillus paracasei TY-G05 as described above.
In the above-mentioned composition, as described above, lactobacillus paracasei TY-G05 has a good survival rate in the digestive tract of the body, and can be prepared into a composition which can be used for eating or medicine. In addition, when lactobacillus paracasei TY-G05 is prepared into a composition, the bacterium may be directly introduced into the composition as a live bacterium to act, or may be inactivated by a conventional method and then introduced into the composition as an inactivated bacterium to act; it is also possible to introduce a lysate of the bacterium into the composition for its effect; or introducing the product such as protein, peptide, secretion or metabolite obtained by culturing the strain into the composition to play a role; the fermentation liquid after fermentation of the bacteria can also be introduced into the composition to play a role. In a specific use process, the bacteria can be prepared into a composition to play a role by selecting different forms according to specific requirements.
In some embodiments, one or more of the probiotics, prebiotics, dietary fibers, and Chinese patent medicines are also included in the above composition.
It should be noted that the lactobacillus paracasei TY-G05 and its different forms may also be used in combination with one or more of probiotics, dietary fibers and pharmaceutically active compounds; for example, lactobacillus paracasei TY-G05 may be used in combination with bacillus subtilis, bifidobacterium or lactobacillus, etc., such that the composition has the efficacy of both lactobacillus paracasei TY-G05 and other probiotics; for another example, lactobacillus paracasei TY-G05 can be used in combination with a prebiotic that can provide an energy source for Lactobacillus paracasei TY-G05, and which has a high effect on Lactobacillus paracasei TY-G05; for another example, the lactobacillus paracasei TY-G05 can be used together with dietary fiber, and the dietary fiber can assist the lactobacillus paracasei TY-G05 to perform colonization, so that the effect of the lactobacillus paracasei TY-G05 is improved; for another example, lactobacillus paracasei TY-G05 can be used in combination with a Chinese patent drug to form a composition, and the functions of the Lactobacillus paracasei TY-G05 and the Chinese patent drug are exerted.
In a further embodiment the invention provides a formulation comprising lactobacillus paracasei TY-G05 as described above or a composition as described above, and a carrier, wherein the carrier is a pharmaceutically acceptable carrier or an edible carrier.
The composition containing the lactobacillus paracasei TY-G05 and the different forms thereof can be added with a medicinal carrier or an edible carrier to prepare a pharmaceutical or an edible food or a health care product. Pharmaceutically acceptable or edible carriers are known in the art and may be selected according to the dosage form, for example, diluents (e.g., starch, dextrin, sucrose or glycation, etc.), absorbents (e.g., calcium sulfate, dibasic calcium phosphate or light magnesium oxide, etc.), binders (e.g., povidone, syrup or hypromellose, etc.), wetting agents (e.g., water, etc.), or disintegrants (e.g., dry starch, sodium carboxymethyl starch, or crospovidone, etc.) may be used primarily for preparing tablets; for example, the preparation of liquid agents mainly uses compatibilizers, suspending agents, emulsifying agents or coloring agents, etc.
In some embodiments, the above formulations may include pills, capsules, powders, gels, and granules in addition to tablets and liquids. The solid dosage forms such as tablets, pills, granules or capsules can be in the form of probiotic tablets, probiotic sugar pills, probiotic powder or probiotic capsules; the liquid agent can be in the form of probiotic beverage and other products; the gel can be in the form of probiotic jelly, probiotic milk cap or set yoghurt.
In a further embodiment, the invention provides the use of the Lactobacillus paracasei TY-G05 or the composition in preparing a medicament for treating inflammatory diseases. It should be noted that the lactobacillus paracasei TY-G05 can regulate the level of inflammatory factors in the organism, such as significantly reduce the level of LPS, IL-6 and TNF-alpha and significantly increase the level of IL-10, thereby achieving the purpose of relieving inflammation, i.e. the lactobacillus paracasei TY-G05 can be prepared into a medicament for treating inflammatory diseases.
In some embodiments, the inflammatory disease is inflammatory bowel disease, including primarily ulcerative colitis and Crohn's disease. It should be noted that the lactobacillus paracasei TY-G05 can obviously increase the colon length of the DSS-induced colitis model and obviously slow down the colon mucosa injury of the DSS-induced colitis model, and the lactobacillus paracasei TY-G05 can be prepared into a medicament for treating inflammatory bowel disease.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
In the following examples, MRS liquid medium is available from Beijing land bridge technologies Inc.
Example 1 TY-G05 isolation and purification and identification
(1) Experimental materials
Collecting self-made qula of Qinghai province herdsman: taking self-made triton in the herdsman by using a sterile small spoon, putting the triton into a 15mL sterile cap-screwing centrifuge tube, screwing the cap, putting the cap into a refrigerator, and immediately carrying out purification and separation of lactobacillus after transporting the cap back to a laboratory.
(2) Isolation and purification of TY-G05
Under aseptic conditions, 5g of a triton sample is added into sterilized 45mL of skim milk, the sterilized 45mL of skim milk is placed in a 37 ℃ incubator for enrichment culture, after the curdled milk is demulsified, under aseptic conditions, 1mL of the demulsified sample is sucked into 9mL of sterile normal saline, the sample is uniformly mixed by vortex to obtain 10 -1 mL of sample diluent, 10 times of gradient dilution is sequentially carried out until the sample diluent is 10 -7, 100 mu L of diluent at 10 -5、10-6 and 10 -7 dilution is selected, the diluent is uniformly coated on an MRS flat plate, and the sample is subjected to inversion culture at 37 ℃ for 48 hours; after the culture is finished, observing colony morphology on the MRS plate, picking medium-sized, raised, slightly white or slightly yellow, moist, clean-edged and circular colonies, and purifying the strain by a plate streaking method, and repeating the streaking operation until the purified strain is obtained.
(3) Morphological structure observations
Inoculating the strain purified in the step (2) into 5mL of MRS liquid culture medium, and culturing at 37 ℃ for 18h; centrifuging 1mL of bacterial liquid at the rotating speed of 12000r/min for 1min, washing twice with sterile physiological saline, adding the same volume of sterile physiological saline to resuspend the bacterial body, finally uniformly coating a small amount of bacterial liquid on a glass slide by using an inoculating loop, fixing, performing gram staining, microscopic examination and photographing; cells stained with gram-positive bacteria (G +) appear bluish-purple and cells stained with gram-negative bacteria (G -) appear red; observing and recording bacterial colony morphology and gram staining results;
The bacterial colony morphology is shown in figure 1, single bacterial colony is formed in a solid culture medium after the bacterial colony is purified, the bacterial colony morphology is consistent, and the bacterial colony is hemispherical, slightly white, smooth and moist in surface and flat in edge;
The result of gram staining is shown in FIG. 2, and after gram staining, the morphology of purple cells was observed under a microscope (the original image was purple in FIG. 2, and the shape was in the form of a rod, and it was determined as gram-positive bacteria (G +).
(4) PCR amplification of 16S rDNA sequence
The 16SrDNA gene amplification was performed using a 25. Mu.L reaction system and the following PCR amplification procedure, 25. Mu.L reaction system: template 1. Mu.L, upstream primer (sequence shown as SEQ ID NO. 2) (10. Mu.M) 1. Mu.L, downstream primer (sequence shown as SEQ ID NO. 3) (10. Mu.M) 1. Mu.L, 2 XTaqPCRMasterMix 12.5. Mu.L, with sterile ultrapure water to 25. Mu.L; PCR amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min for 35 cycles; end extension at 72 ℃ for 10min; after the sequence is amplified, sequencing a PCR amplified product which is qualified in detection by entrusting a biological engineering (Shanghai) stock company, and measuring the sequence as shown in SEQ ID No. 1; searches and homology analysis were performed in GenBank using BLAST (http:// www.ncbi.nlm.nih.gov/BLAST), and the homology analysis results showed that TY-G05 was Lactobacillus paracasei.
Gastrointestinal survival verification of example 2 TY-G05
(1) Experimental materials
Lactobacillus paracasei TY-G05: the strain is separated from Qinghai Laback mountain herdsman, self-made by Qula, and preserved in China general microbiological culture Collection center with a preservation number of CGMCC No.24626.
(2) Survival of TY-G05 in pH3.0 artificial gastric juice
The probiotics entering the human body have corresponding functional activities, have good digestive tract tolerance, pass through the stomach (the pH of gastric juice is about 3.0, the retention time is 1-3 h) before entering the human intestinal tract to perform the functions, and the strong acid stomach environment is not beneficial to the survival of the probiotics, so the probiotics have the primary condition of being capable of tolerating the gastric juice in the human body.
Inoculating TY-G05 into MRS liquid culture medium according to an inoculum size of 2%, culturing at 37 ℃ for 18h, taking 10mL, centrifuging at 4000r/min for 10min, collecting bacterial precipitate, washing with sterile physiological saline for 2 times, and re-suspending in an equal volume of sterile physiological saline to obtain bacterial suspension; mixing the prepared bacterial suspension with artificial gastric juice (0.2% NaCl and 0.35% pepsin 1:10000, adjusting pH=3.0 with 1mol/L HCl, filtering and sterilizing for later use) according to the proportion of 1:9, uniformly mixing, placing in a constant temperature oscillator, culturing for 3 hours at 37 ℃ at the rotating speed of 100r/min, and respectively measuring the viable count of 0 hours and 3 hours by adopting a pouring plate method; the survival rate of the strain was calculated to withstand pH3.0 artificial gastric juice according to formula (1).
The experimental and calculation results show that the survival rate of TY-G05 cultured in artificial gastric juice with pH=3.0 for 3 hours is 82.19%, and the gastric juice survival rate is higher.
(3) Growth efficiency of TY-G05 in 0.3% bile salts
After the probiotics which survive gastric juice treatment enter the intestinal tract, the probiotics can be inhibited and poisoned by bile salts in the small intestine, so that the tolerance of the strain to the bile salts is also one of important indexes for screening the probiotics, and the mass concentration of the bile salts of a human body fluctuates within the range of 0.03% -0.3%.
TY-G05 was inoculated in an MRS liquid medium at an inoculum size of 2%, cultured at 37℃for 18 hours, and the culture solution was inoculated in an MRS-THIO medium (MRS liquid medium with 0.2% sodium thioglycolate added to 0.2%) containing 0.0% and 0.3% pig bile salts at an inoculum size of 2%, respectively, mixed uniformly, placed in a constant temperature shaker, and cultured at 37℃for 24 hours at a rotational speed of 100 r/min. Taking an unvaccinated MRS-THIO culture medium as a blank control, and respectively measuring OD 600 values of a culture solution containing 0.0% and 0.3% of pig bile salt; the growth efficiency of the strain in bile salts was calculated according to formula (2).
The experimental and calculation results show that the growth efficiency of TY-G05 in bile salt with the concentration of 0.3% is 16.47%, and the bile salt tolerance is good.
Example 3 TY-G05 verification of the alleviation of enteritis
(1) Experimental materials
Lactobacillus paracasei TY-G05: the strain is separated from Qinghai Laback mountain herdsman, self-made by Qula, and preserved in China general microbiological culture Collection center with a preservation number of CGMCC No.24626.
(2) Grouping and processing of laboratory animals
7 Week old C57BL/6 male mice (20G-25G) were kept in standardized laboratories at room temperature 25+ -2deg.C, relative humidity 50% + -5%, 12h light/12 h darkness, and after one week of adaptive feeding, the experiment was started, after the end of the adaptation period, the mice were randomly divided into a normal group, an enteritis group and a TY-G05 treatment group, 10 each.
The experimental period is 5 weeks, during the experimental period, all groups of mice are free to ingest, and the modeling medicine is Dextran Sodium Sulfate (DSS); normal groups of mice were free to drink water (without DSS) daily until the end of the experiment; the remaining groups of mice were free to drink water (without DSS) for the first, second and four weeks, 2% DSS for the third week and 4% DSS for the fifth week. TY-G05 treatment group was perfused with 10 9 CFU/kg. BW TY-G05 bacteria solution during the whole experimental period, and the lavage reagent was 10mL/kg. BW physiological saline. Experiment 35d, mice began to fasted, after 16h of fasted, eyes were taken and the mice were sacrificed and colon tissues of the mice were collected.
(3) Colon length and colon weight determination
After the mice were sacrificed, the whole section of colon intestine from the distal cecum to the anus was cut, and the length was measured and weighed for recording.
(4) Pathological section observation of colon tissue
The fixed colon tissue is dehydrated, transparent, waxed, embedded and sliced, then HE stained, and finally the pathological changes of the tissue are observed under a microscope (50×).
(5) ELISA method for determining serum cytokine level of mouse
The blood of the mice was allowed to stand for 2 hours, and the serum was obtained after centrifugation at 3000r/min for 15 minutes, and the concentrations of endotoxin (LPS), interleukin-6 (interleukin-6, IL-6), anti-tumor necrosis factor (tumornecrosis factor. Alpha., TNF-. Alpha.) and interleukin-10 (interleukin-10, IL-10) in the serum of the mice were measured. Experiments were performed using an enzyme-linked immunosorbent assay kit from Shanghai enzyme-linked biotechnology limited and with reference to the instructions of the corresponding kit. OD values of the standard solutions were measured at 450nm and the serum LPS, IL-6, TNF-. Alpha.and IL-10 levels were calculated from the standard curves.
(6) Experimental results and analysis
① Relief of enteritis by TY-G05
In enteritis, the reduction of colon length and edema are typical features and are also important indicators reflecting the degree of inflammation, and as can be seen from fig. 3 and fig. 4, the colon length of mice in enteritis groups is significantly reduced compared with that of normal groups, which indicates that the DSS induced colitis model is successful. The colon length of the enteritis mice treated by TY-G05 is obviously increased, which proves that the TY-G05 treatment has a preventive effect on colon shortening of the mice. In enteritis experiments, the colon edema of mice is usually used as an evaluation index, the greater the ratio, the more serious the edema and inflammation degree, because the edema can increase the weight of the colon, and enteritis mice are often accompanied with the characteristic of colon shortening, the ratio is large, and compared with the normal group ratio, the ratio of the enteritis group is obviously increased, which indicates that the colon edema of the mice in the enteritis group is more serious than the normal group; and after being treated by TY-G05, the colonic edema condition of the enteritis mice is improved, and a significant difference is formed between the colonic edema condition and the enteritis group, which shows that the TY-G05 treatment has a good improvement effect on colonic edema. The above results indicate that TY-G05 can alleviate DSS-induced enteritis.
② Effects of TY-G05 on colon tissue of enteritis mice
Colonic mucosa injury is a typical feature of a DSS induced colonic mucosa model, and as can be seen from fig. 6, the colonic mucosa and crypt of a normal group are complete, the lamina propria has no inflammatory cell infiltration phenomenon, and goblet cells are continuously distributed; however, the intestinal inflammation colon mucosa is seriously damaged, the goblet cells are reduced, the phenomenon of the resident layer cells infiltration is serious, and the intestinal wall is thickened, which proves that the DSS induced colonitis model is successful. The colon mucosa injury and inflammatory cell infiltration of the enteritis mice treated by TY-G05 are both relieved, which proves that the TY-G05 treatment can improve the colon injury caused by DSS and has the promotion effect on preventing colonitis.
③ Effects of TY-G05 on serum cytokine levels in enteritis mice
Systemic inflammatory response is closely related to the gastrointestinal tract, when the gastrointestinal tract is inflamed, intestinal mucosa is damaged, intestinal permeability is increased, excessive LPS passes through intestinal walls to enter the systemic circulation, and the organism is induced to produce inflammatory cytokines, so that the systemic inflammatory response is aggravated. IL-6 is mainly derived from activated T cells, mononuclear macrophages, th2 cells, vascular endothelial cells and the like, and can promote cell synthesis of acute proteins and accumulation of T cells at an inflammation site, thereby causing intestinal inflammation. When intestinal inflammation occurs, IL-6 level of inflammatory organism is higher than that of normal organism. TNF-alpha is mainly produced by monocytes and macrophages, is the earliest and most important inflammatory mediator in the inflammatory reaction process, promotes the occurrence of inflammation in enteritis, has the effects of enhancing vascular endothelial cell permeability and aggregating inflammatory cells at the inflammation site, causes inflammatory cell infiltration and tissue edema, and aggravates the inflammation. In the aspect of inflammatory response, IL-10 inhibits activation, migration and adhesion of inflammatory cells by down-regulating T lymphocyte activity, and simultaneously IL-10 can inhibit synthesis and release of inflammatory factors, thereby playing an anti-inflammatory role.
FIGS. 7, 8, 9, and 10 show LPS, IL-6, TNF- α, and IL-10 levels, respectively, in the serum of mice of each group. Compared with the normal group, the serum LPS, IL-6 and TNF-alpha levels of the enteritis group mice are obviously increased, the IL-10 level is obviously reduced (P is less than 0.05), the serum LPS, IL-6 and TNF-alpha levels of the mice are obviously reduced after TY-G05 treatment, and the IL-10 level is obviously increased (P is less than 0.05). The above results demonstrate that enteritis mice exhibit increased systemic inflammation, while TY-G05 can reduce the increase in inflammation caused by enteritis.
As can be seen from the animal experiment results, the DSS induces the mice to have enteritis, the lactobacillus paracasei TY-G05 relieves the enteritis, and the systemic inflammation caused by the enteritis is reduced.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (9)
1. Lactobacillus paracasei (Lactobacillusparacasei) is characterized in that the lactobacillus paracasei is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24626.
2. Lactobacillus paracasei according to claim 1, characterized in that its 16SrDNA sequence comprises the sequence shown in SEQ ID No. 1.
3. A composition comprising one or more of the following in combination: (a) lactobacillus paracasei according to claim 1 or 2; (b) A lysate of lactobacillus paracasei according to claim 1 or 2; (c) A culture of lactobacillus paracasei according to claim 1 or 2; (d) Lactobacillus paracasei fermentation broth according to claim 1 or 2.
4. The composition of claim 3, further comprising one or more of a combination of probiotics, prebiotics, dietary fiber, and chinese patent medicine.
5. Formulation comprising lactobacillus paracasei according to claim 1 or 2 or a composition according to claim 3 or 4, and a carrier, wherein the carrier is a pharmaceutically acceptable carrier or an edible carrier.
6. The formulation of claim 5, wherein the formulation is a tablet, pill, capsule, powder, gel, granule, or liquid.
7. Use of lactobacillus paracasei according to claim 1 or 2 or a composition according to claim 3 or 4 for the manufacture of a medicament for the treatment of inflammatory diseases.
8. The use according to claim 7, wherein the inflammatory disease is inflammatory bowel disease.
9. The use according to claim 8, wherein the inflammatory bowel disease is ulcerative colitis or crohn's disease.
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