Lactobacillus plantarum TY-P22 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum TY-P22 and application thereof, and the preservation number of the lactobacillus plantarum TY-P22 is CGMCC No: 30875. The lactobacillus plantarum TY-P22 has good artificial gastric juice resistance and bovine bile salt resistance, has the functions of remarkably improving the body immunity and improving body intestinal injury, and particularly has a relatively strong improvement effect on body immunity reduction and body intestinal injury caused by cyclophosphamide.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus plantarum TY-P22 and an application thereof. Background Art

[0002] Cyclophosphamide is a widely used chemotherapy drug for cancer treatment and immunosuppression, although its effectiveness in treatment is widely recognized. However, under the action of liver microsomal enzymes in the body, cyclophosphamide can have toxic effects on rapidly proliferating healthy cells, killing cancer cells while also damaging immune cells and intestinal epithelial cells, which weakens the body's immune defense capabilities and destroys the integrity of intestinal epithelial cells, leading to intestinal dysbiosis and impaired intestinal barrier function. Patients show a significant decrease in immunity and are susceptible to infection by pathogens. At the same time, the intestinal barrier function is impaired, causing various gastrointestinal adverse reactions, such as nausea, vomiting, diarrhea, intestinal inflammation, etc. Therefore, how to alleviate the side effects of immunosuppression and intestinal damage caused by cyclophosphamide has become an important challenge in clinical treatment.

[0003] Probiotics are active microbial preparations that are beneficial to the host and conducive to the ecological balance of intestinal flora. When ingested in effective amounts, they stimulate the host immune system and enhance the host immune response, thereby helping the host resist various diseases. In recent years, studies have shown that specific probiotics can significantly alleviate immunosuppression and intestinal damage by regulating immune responses and maintaining intestinal microecological balance, providing a new auxiliary strategy for improving the prognosis of patients with immunosuppression caused by cyclophosphamide.

[0004] Therefore, it is necessary to discover a probiotic that can enhance the body's immune ability and improve intestinal damage. Summary of the invention

[0005] The present invention is a strain of bacteria separated from the feces of the elderly in the longevity area of ​​Jiangjin. After identification, it belongs to Lactobacillus plantarum, which can improve the body's immune ability and improve the body's intestinal damage, especially has a strong improvement effect on the reduced immunity and intestinal damage caused by cyclophosphamide.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] The present invention provides a Lactobacillus plantarum TY-P22, whose deposit number is CGMCC No: 30875.

[0008] Another aspect of the present invention provides a composition comprising the Lactobacillus plantarum TY-P22 of the present invention.

[0009] Preferably, the above composition may include a fermentation product or a culture.

[0010] In another aspect, the present invention provides a preparation comprising the composition of the present invention.

[0011] Preferably, the above-mentioned preparation can be an inactivated bacterium agent or a microecological bacterium agent.

[0012] Preferably, the above-mentioned preparation includes food, medicine or health care product.

[0013] In another aspect, the present invention provides a use of the Lactobacillus plantarum TY-P22 of the present invention or the composition of the present invention in the preparation of a preparation for improving the immune ability of an organism and / or improving intestinal damage of an organism.

[0014] Preferably, the above application includes: application of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for improving decreased immunity and / or intestinal damage caused by cyclophosphamide.

[0015] Preferably, the above application includes one or more combinations of the following applications:

[0016] (a) Use of Lactobacillus plantarum TY-P22 or a composition in the preparation of a preparation for increasing the body's weight loss caused by cyclophosphamide;

[0017] (b) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for improving an immune organ index of an organism;

[0018] (c) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for increasing the number of immune cells in a body;

[0019] (d) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for improving pathological damage to intestinal tissues of a body;

[0020] (e) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for promoting the expression of intestinal immune-related cytokines;

[0021] (f) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for promoting the expression of tight junction protein Claudin-1.

[0022] Preferably, in the above application,

[0023] Application (b) includes: application of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for alleviating atrophy of spleen and thymus immune organs;

[0024] In application (c), the body's immune cells include monocytes and basophils;

[0025] In application (e), the relevant cytokines include one or more of IL-2, IL-10, IL-1β or INF-γ.

[0026] The preservation information of Lactobacillus plantarum TY-P22 in the present invention is as follows: preservation institution: General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC); address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; preservation date: June 7, 2024; preservation number: CGMCC No. 30875; classification name: Lactobacillus plantarum.

[0027] The beneficial effects of the present invention include at least:

[0028] (1) The Lactobacillus plantarum TY-P22 provided by the present invention has good resistance to artificial gastric juice and ox bile salt, indicating that it can survive well in gastric juice.

[0029] (2) The Lactobacillus plantarum TY-P22 provided by the present invention has an excellent protective effect on weight loss in mice caused by intraperitoneal injection of cyclophosphamide.

[0030] (3) The Lactobacillus plantarum TY-P22 provided by the present invention can improve the immune organ index of immunosuppressed mice, inhibit spleen and thymus atrophy caused by cyclophosphamide, and may alleviate the immune dysfunction caused by radiochemotherapeutic drugs.

[0031] (4) The Lactobacillus plantarum TY-P22 provided by the present invention can increase the number of immune cells in immunosuppressed mice and improve the body's systemic immune function.

[0032] (5) The Lactobacillus plantarum TY-P22 provided by the present invention can improve pathological damage to mouse colon tissue.

[0033] (6) The Lactobacillus plantarum TY-P22 provided by the present invention can enhance the intestinal immune function by promoting the mRNA expression of inflammatory factors.

[0034] (7) The Lactobacillus plantarum TY-P22 provided by the present invention repairs and enhances the intestinal mucosal barrier function by enhancing the expression of tight junction proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the phylogenetic relationship diagram of Lactobacillus plantarum TY-P22;

[0036] Figure 2 The effect of Lactobacillus plantarum TY-P22 on the body weight of mice;

[0037] Figure 3 The effect of Lactobacillus plantarum TY-P22 on the spleen index of mice;

[0038] Figure 4 The effect of Lactobacillus plantarum TY-P22 on the thymus index of mice;

[0039] Figure 5 The effect of Lactobacillus plantarum TY-P22 on the number of peripheral blood leukocytes in mice;

[0040] Figure 6 The effect of Lactobacillus plantarum TY-P22 on the number of neutrophils in peripheral blood of mice;

[0041] Figure 7 The effect of Lactobacillus plantarum TY-P22 on the number of peripheral blood lymphocytes in mice;

[0042] Figure 8 The effect of Lactobacillus plantarum TY-P22 on the number of peripheral blood mononuclear cells in mice;

[0043] Fig. 9 The effect of Lactobacillus plantarum TY-P22 on the number of eosinophils in the peripheral blood of mice;

[0044] Fig.10 The effect of Lactobacillus plantarum TY-P22 on the number of peripheral blood basophils in mice;

[0045] Fig.11 The effect of Lactobacillus plantarum TY-P22 on the colon tissue morphology of mice;

[0046] Fig.12 The effect of Lactobacillus plantarum TY-P22 on the expression level of IL-2 in the intestinal tissue of mice;

[0047] Fig.13 The effect of Lactobacillus plantarum TY-P22 on the expression level of IL-10 in the intestinal tissue of mice;

[0048] Fig.14 The effect of Lactobacillus plantarum TY-P22 on the expression level of IL-1β in the intestinal tissue of mice;

[0049] Fig.15 The effect of Lactobacillus plantarum TY-P22 on the expression level of INF-γ in the intestinal tissue of mice;

[0050] Fig.16 To investigate the effect of Lactobacillus plantarum TY-P22 on the expression level of tight junction protein Claudin-1 in the intestinal tissue of immunocompromised mice;

[0051] Figures 2 to 16In the table, *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05, all of which indicate significant differences compared with the cyclophosphamide model group; data are presented as Mean ± SEM. DETAILED DESCRIPTION

[0052] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0053] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. 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, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0054] The Latin name of "Lactobacillus plantarum" in the present invention is the old Latin name of Lactobacillus plantarum, and its new Latin name is "Lactiplantibacillus plantarum".

[0055] In the present invention, "enhancing the body's immune ability" refers to restoring the body to a healthy immune state, and does not mean additionally improving the immune ability on the basis of a healthy state.

[0056] The embodiment of the present invention provides a Lactobacillus plantarum TY-P22, whose preservation number is CGMCC No: 30875.

[0057] It should be noted that the plant lactobacillus TY-P22 in the present invention is isolated from the feces of a 76-year-old male in the longevity area of ​​Jiangjin, Chongqing, and has no side effects; the plant lactobacillus TY-P22 is sequenced and its 16sRNA sequence is shown in SEQ ID NO: 1. It has good resistance to artificial gastric juice and ox bile salts, and can be used as a beneficial bacteria to colonize in the stomach and intestines.

[0058] The embodiment of the present invention also provides a composition comprising the Lactobacillus plantarum TY-P22 of the present invention.

[0059] It should be noted that, in order to improve the application effect of Lactobacillus plantarum TY-P22, Lactobacillus plantarum TY-P22 can be used in the form of a composition.

[0060] In some specific examples, the composition can be a fermentation product or a culture.

[0061] It should be noted that the above-mentioned composition can be in the form of a fermentation product or culture containing Lactobacillus plantarum TY-P22, wherein the fermentation product refers to various substances produced by the fermentation process using Lactobacillus plantarum TY-P22 under suitable culture conditions; the culture refers to a strain culture, which refers to a microbial community obtained by cultivation and reproduction under artificially specified conditions.

[0062] An embodiment of the present invention also provides a preparation comprising the composition of the present invention.

[0063] It should be noted that the plant lactobacillus TY-P22 in the present invention can be prepared into a stable preparation for use, and the preparation form is a form known in the art, such as powder, tablet, liquid dosage form, granule or capsule dosage form, etc. There are differences in the auxiliary materials added to different dosage forms, which can be added according to what is known in the art.

[0064] In some specific examples, the above-mentioned preparation can be an inactivated bacterial agent or a microecological bacterial agent.

[0065] It should be noted that the above-mentioned preparations can be preparations prepared from inactivated bacteria agents, or can be microecological preparations prepared from live bacteria. The specific dosage form can be selected according to specific needs, and the preparation method can be selected by those known in the art.

[0066] In some specific examples, the preparation includes food, medicine or health care product.

[0067] It should be noted that, in terms of the application scope of the preparation, the above-mentioned preparation can be prepared into functional foods without therapeutic effects, such as probiotic beverages, dairy beverages (such as yogurt), and can also be prepared into probiotic biscuits, probiotic powder and other foods; of course, it can also be prepared into medicines with therapeutic effects with pharmaceutical excipients; similarly, it can be prepared into health products with certain health functions, which can be prepared specifically according to the effects to be achieved.

[0068] The embodiment of the present invention also provides a use of the Lactobacillus plantarum TY-P22 of the present invention or the composition of the present invention in the preparation of a preparation for improving the immune ability of an organism and / or improving intestinal damage of an organism.

[0069] It should be noted that the plant lactobacillus TY-P22 of the present invention or the composition of the present invention can be used in the preparation of a preparation, and the preparation can be a food, a medicine or a health product; it should be understood that the plant lactobacillus TY-P22 of the present invention, as a probiotic, has the properties of a functional food, a therapeutic drug and a health product. When it plays the functional role of a food, the above-mentioned preparation is a food, when it plays a therapeutic role, the above-mentioned preparation is a medicine, and when it plays a health care role, the above-mentioned preparation is a health product.

[0070] It should also be noted that, as mentioned above, the above-mentioned preparations can be powders, tablets, liquid dosage forms, granules or capsule dosage forms, etc., and the excipients added to different dosage forms are different and can be added according to what is known in the art.

[0071] In some specific examples, the above application includes: application of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for improving the reduced immunity and / or intestinal damage caused by cyclophosphamide.

[0072] It should be noted that, as described above, the plant lactobacillus TY-P22 or composition of the present invention can improve the body's immune ability and / or improve the body's intestinal damage, and is mainly suitable for reduced immunity and / or intestinal damage caused by cyclophosphamide; when used in reduced immunity and / or intestinal damage caused by cyclophosphamide, it can significantly improve immune ability and improve intestinal damage.

[0073] In some specific examples, the above application includes one or more combinations of the following applications:

[0074] (a) Use of Lactobacillus plantarum TY-P22 or a composition in the preparation of a preparation for slowing down weight loss caused by cyclophosphamide;

[0075] (b) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for improving an immune organ index of an organism;

[0076] (c) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for increasing the number of immune cells in a body;

[0077] (d) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for improving pathological damage to intestinal tissues of a body;

[0078] (e) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for promoting the expression of intestinal immune-related cytokines;

[0079] (f) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for promoting the expression of tight junction protein Claudin-1.

[0080] It should be noted that the plant lactobacillus TY-P22 or the composition in the present invention can achieve the purpose of improving the reduced immunity and / or intestinal damage caused by cyclophosphamide by slowing down the body's weight loss caused by cyclophosphamide, improving the body's immune organ index, increasing the body's immune cell number, improving the body's intestinal tissue pathological damage, promoting the body's intestinal immune-related cytokine expression and promoting the body's tight junction protein Claudin-1 expression.

[0081] In some specific examples, in the above applications,

[0082] Application (b) includes: application of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for alleviating spleen and thymus immune organ atrophy; specifically, the above-mentioned improvement of the body's immune organ index mainly includes the preparation for alleviating spleen and thymus immune organ atrophy;

[0083] In application (c), the body's immune cells include monocytes and basophils; specifically, Lactobacillus plantarum TY-P22 can especially increase the number of monocytes and basophils in peripheral blood;

[0084] In application (e), the relevant cytokines include one or more of IL-2, IL-10, IL-1β or INF-γ.

[0085] It should also be noted that in the Lactobacillus plantarum TY-P22 or the composition of the present invention, the number of viable bacteria of Lactobacillus plantarum TY-P22 is preferably ≥ 1×10 8 CFU / g or 1×10 8 CFU / mL.

[0086] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0087] 1. Screening and identification of Lactobacillus plantarum TY-P22

[0088] (I) Screening

[0089] The sample was derived from the feces of a 76-year-old male in the Changshou area of ​​Jiangjin, Chongqing. After pretreatment, it was stored in a -80°C refrigerator in 20% glycerol. After being taken out and thawed, the sample was mixed and 1 mL of the sample was added to 9 mL. It was gradient diluted with 0.9% saline, and the appropriate gradient dilution was selected and spread on MRS solid culture medium. It was cultured at 37°C for 48 hours, and the colonies were picked and streaked on MRS solid culture medium for purification. Single colonies were picked and transferred to MRS liquid culture medium for bacterial enrichment and preserved in 30% glycerol to obtain strain TY-P22.

[0090] (II) Identification

[0091] The 16S rDNA of TY-P22 was amplified and sequenced. The 16S rDNA sequence contained the sequence shown in SEQ ID NO.1. The sequence was compared with the nucleic acid sequence in NCBI. The result showed that the strain was Lactobacillus plantarum, and was named Lactobacillus plantarum TY-P22. The phylogenetic relationship between Lactobacillus plantarum TY-P22 and other strains is shown in Figure 1 shown.

[0092] 2. Artificial gastric juice tolerance test of Lactobacillus plantarum TY-P22

[0093] Lactobacillus plantarum TY-P22 was inoculated in MRS liquid culture medium at a rate of 3%, cultured at 37°C for 18h, and activated for three generations; 2mL of the culture was collected by centrifugation at 8000r / min for 10min, washed twice with sterile PBS solution, and resuspended in an equal volume of sterile saline to obtain a bacterial suspension; the prepared bacterial suspension was mixed with artificial gastric juice (mass fraction 0.2% NaCl and mass fraction 0.35% pepsin, volume ratio of 1:10000, pH=3.0 adjusted with 1mol / L HCl, filtered and sterilized for later use) at a ratio of 1:9 (v / v), mixed well, and cultured in a constant temperature oscillator at 37°C and 100r / min for 3h, and the number of viable bacteria at 0h and 3h was determined by the pour plate method; the survival rate of the strain tolerant to pH 3.0 artificial gastric juice was calculated according to formula (1):

[0094]

[0095] The results of the artificial gastric juice resistance experiment showed that the survival rate of Lactobacillus plantarum TY-P22 in artificial gastric juice at pH 3.0 was 19.75±0.91%, indicating that Lactobacillus plantarum TY-P22 has the characteristics of resistance to artificial gastric juice.

[0096] 3. Experiment on tolerance of ox bile salts by Lactobacillus plantarum TY-P22

[0097] Lactobacillus plantarum TY-P22 was inoculated in MRS liquid culture medium at a rate of 3%, cultured at 37°C for 18 hours, and activated for three generations; 2 mL of the culture was collected by centrifugation at 8000 r / min for 10 minutes, washed twice with sterile PBS solution, and resuspended in an equal volume of sterile physiological saline containing 0.0% and 0.3% ox bile salt, mixed and placed in a constant temperature oscillator at 37°C and centrifuged at 100 r / min for 2 hours, and the number of viable bacteria in the 0.0% and 0.3% ox bile salt culture solution was determined by the pour plate method; the survival rate of the strain in bile salt was calculated according to formula (2):

[0098]

[0099] The results of the artificial gastric juice resistance experiment showed that the survival rate of Lactobacillus plantarum TY-P22 in 0.3% ox bile salt was 3.49±0.46%, indicating that Lactobacillus plantarum TY-P22 has the property of ox bile salt resistance.

[0100] 4. Animal Experiments

[0101] (I) Preparation of Lactobacillus plantarum TY-P22 bacterial suspension

[0102] Lactobacillus plantarum TY-P22 was inoculated into MRS solid medium and cultured at 37°C for 48 hours, and its colonies were observed and its bacterial bodies were observed under a microscope. Then, it was inoculated into MRS liquid medium and cultured at 37°C for 18 hours, and then transferred into fresh MRS liquid medium and cultured under the same conditions for 18 hours. The fermentation liquid was centrifuged at 6000g for 15 minutes, and the bacterial bodies were washed with 0.9% saline and centrifuged again at 6000g for 10 minutes to obtain bacterial bodies, which were resuspended with 30% sucrose solution and frozen at -80°C for use. Before gavage, the bacterial bodies were resuspended with sterile saline and the number of viable bacteria was adjusted to 5×10 8 CFU / cell, and made into bacterial suspension.

[0103] (II) Animals and breeding

[0104] The experimental animals were female SPF-grade BALB / c mice (6 weeks old, weighing 19 g-20 g), purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.; the mice were housed in the Experimental Animal Center of Jiangnan University (temperature 21°C-25°C, relative humidity 50%-60%, 12 h day and night cycle) and had free access to water.

[0105] (III) Experimental process

[0106] After 7 days of adaptive feeding, the experimental animals were randomly divided into a blank control group (blank group), a cyclophosphamide model group (model group), a Lactobacillus plantarum TY-P22 group (TY-P22 group, 1×10 9 CFU / d), with 6 mice in each group; among them, the blank group and the model group were gavaged with sterile saline, and the Lactobacillus plantarum TY-P22 group was gavaged with Lactobacillus plantarum TY-P22, with an gavage dose of 200 μL / mouse / day, for 28 consecutive days; on the 22nd and 23rd days, the mice in the blank group were intraperitoneally injected with 200 μL of saline, and the mice in the model group and the Lactobacillus plantarum TY-P22 group were intraperitoneally injected with the same volume of cycloheximide (80 mg / kg / d, based on body weight); after the experiment, the mice were weighed, blood was collected from the eyeballs, and the mice were killed by dislocation, the thymus and spleen were weighed, and the spleen index and thymus index were calculated; the colon tissue was taken for subsequent HE staining and determination of immune regulation-related inflammatory factors.

[0107] (IV) Effect of Lactobacillus plantarum TY-P22 on alleviating cyclophosphamide-induced immunosuppression in mice

[0108] (1) Changes in mouse body weight

[0109] The mice were weighed at the end of the experiment on day 28. Figure 2 As shown, compared with the blank group, intraperitoneal injection of cyclophosphamide caused a significant decrease in mouse body weight, and oral gavage of Lactobacillus plantarum TY-P22 group was able to significantly inhibit the decrease in mouse body weight, indicating that Lactobacillus plantarum TY-P22 has a protective effect on the weight loss of mice caused by intraperitoneal injection of cyclophosphamide.

[0110] (2) Mouse immune organ index

[0111] After the mice were killed, the thymus and spleen were carefully removed, rinsed with PBS buffer, and dried with filter paper; then the thymus and spleen were weighed and recorded, and the ratio of thymus or spleen mass (mg) to body mass (g) was calculated.

[0112] The results are as follows Figure 3 and Figure 4 As shown in the figure, compared with the blank control group, the spleen index and thymus index of the model group mice were significantly reduced (P<0.001); compared with the model group, the spleen index (P<0.05) and thymus index (P<0.01) of the Lactobacillus plantarum TY-P22 group mice increased to varying degrees, especially the thymus index was almost the same as the blank group. The above results show that Lactobacillus plantarum TY-P22 can improve the immune organ index of immunocompromised mice, inhibit the spleen and thymus atrophy caused by cyclophosphamide, and alleviate the immunodeficiency caused by radiochemotherapeutic drugs.

[0113] (3) Changes in the number of immune cells in the peripheral blood of mice

[0114] After blood sampling from the eyeball, the blood was collected into a blood collection tube containing EDTA anticoagulant, and then the white blood cells in the mouse whole blood were counted using a fully automatic blood cell analyzer.

[0115] The results are as follows Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 and Fig.10As shown in the figure, compared with the blank control group, the number of leukocytes, lymphocytes, neutrophils, monocytes, eosinophils and basophils in the peripheral blood of the cyclophosphamide-treated group was significantly reduced (P<0.05); intragastric administration of Lactobacillus plantarum TY-P22 could counteract the decrease of monocytes and basophils caused by cyclophosphamide. The above results show that Lactobacillus plantarum TY-P22 can increase the number of immune cells in immunocompromised mice to a certain extent and improve the systemic immune function of the body.

[0116] (V) Effect of Lactobacillus plantarum TY-P22 on alleviating cyclophosphamide-induced intestinal damage in mice

[0117] (1) HE staining to detect pathological damage of colon tissue

[0118] Fresh intestinal colon tissues of the above-mentioned groups of mice were taken, and the colon tissue samples were fixed overnight with 4% paraformaldehyde solution, dehydrated with ethanol solutions of different concentrations (100%, 95%, 85%, 70%), embedded in paraffin after being transparentized with xylene, and cut into 5 μm thick slices. The slices were baked, dewaxed to water, stained with hematoxylin, differentiated with 0.5% hydrochloric acid alcohol, blued with 0.5% ammonia water, dehydrated with 95% ethanol, stained with eosin, and dehydrated with ethanol. A drop of sealing glue was dripped in the center of the paraffin slice to seal the slice, and the slice was observed after it was dried; after hematoxylin-eosin staining, it was observed with an inverted microscope.

[0119] The results are as follows Fig.11 As shown, the colon tissue structure of the blank group mice was intact and normal, with neatly arranged crypts and no inflammatory cell infiltration; the colon tissue structure of the mice in the cyclophosphamide-treated group was obviously damaged, with a large number of missing crypt structures, and the intestinal villi were short and sparse or even fell off; the integrity of the colon tissue structure of the mice in the Lactobacillus plantarum TY-P22 group was improved, indicating that Lactobacillus plantarum TY-P22 can alleviate the intestinal damage caused by cyclophosphamide by maintaining the integrity of the intestinal epithelial structure.

[0120] (2) Expression levels of immune inflammatory factors in colon tissue

[0121] The total RNA of colon tissue was extracted by Trizol method. 100 mg of mouse colon tissue was added with 1 mL of Trizol reagent and ground in a tissue homogenizer. Then, the mixture was centrifuged at 4°C and 3000 rpm / min for 10 min and the supernatant was aspirated. RNA was converted into cDNA using StarScript reverse transcription kit. The gene expression level was detected by real-time quantitative PCR. PCR system: 7.6 μL ddH2O, 10 μL Mix (2×SYBR Green qPCR Mix), 0.4 μL ROX (50×ROX ReferenceDye), 0.5 μL upstream primer, 0.5 μL downstream primer (primer sequence see Table 1 below), 1 μL cDNA template. The housekeeping gene β-actin was used as the internal reference. The relative expression of mRNA was calculated by relative quantification method. The expression of all inflammatory factor genes to be detected was expressed by Ct value and expressed by 2 -ΔΔCt To calculate the relative mRNA expression levels of inflammatory factors in the model group and Lactobacillus plantarum TY-P22 group.

[0122] Table 1 Primer sequence information

[0123]

[0124] The expression of IL-2, IL-10, IL-1β and INF-γ mRNA was as follows Fig.12 , Fig.13 , Fig.14 and Fig.15 As shown in the figure, compared with the blank group, the mRNA expression levels of the above four inflammatory factors in the cyclophosphamide model group were significantly decreased; compared with the cyclophosphamide group, the mRNA expression levels of IL-2, IL-10, IL-1β and INF-γ in the colon tissue of the Lactobacillus plantarum TY-P22 group were significantly upregulated, indicating that Lactobacillus plantarum TY-P22 can enhance the intestinal immune function by promoting the mRNA expression of inflammatory factors.

[0125] (3) Expression level of tight junction protein Claudin-1 in colon tissue

[0126] According to the method described in (2) above, the expression level of tight junction protein Claudin-1 was detected by fluorescence quantitative analysis. The primer sequences of Claudin-1 were: Forward 5'-GGGGACAACATCGTGACCG-3', Reverse 5'-AGGAGTCGAAGACTTTGCACT-3'.

[0127] The results are as follows Fig.16As shown, compared with the blank group, cyclophosphamide treatment significantly reduced the mRNA expression of the colon tissue immune factor Claudin-1 (P<0.05), and Lactobacillus plantarum TY-P22 treatment significantly reversed the cyclophosphamide-induced decrease in Claudin-1 expression, indicating that Lactobacillus plantarum TY-P22 can repair and enhance the intestinal mucosal barrier function by enhancing the expression of tight junction proteins.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A Lactobacillus plantarum TY-P22, whose deposit number is CGMCC No: 30875.

2. A composition comprising the Lactobacillus plantarum TY-P22 according to claim 1.

3. The composition according to claim 2, characterized in that The composition includes a fermentation or culture.

4. A preparation comprising the composition of claim 2 or 3.

5. The preparation according to claim 4, characterized in that The preparation is an inactivated bacteria agent or a microecological bacteria agent.

6. The preparation according to claim 4 or 5, characterized in that Preparations include food, medicine or health products.

7. Use of the Lactobacillus plantarum TY-P22 according to claim 1 or the composition according to claim 2 or 3 in the preparation of a preparation for improving the body's immune ability and / or improving the body's intestinal damage.

8. The use according to claim 7, characterized in that: The application includes: application of Lactobacillus plantarum TY-P22 or the combination in preparing a preparation for improving the reduced immunity and / or intestinal damage caused by cyclophosphamide.

9. The use according to claim 7 or 8, characterized in that: Applications include one or a combination of the following: (a) Use of Lactobacillus plantarum TY-P22 or a composition in the preparation of a preparation for increasing the body's weight loss caused by cyclophosphamide; (b) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for improving an immune organ index of an organism; (c) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for increasing the number of immune cells in a body; (d) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for improving pathological damage to intestinal tissues of a body; (e) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for promoting the expression of intestinal immune-related cytokines; (f) Use of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for promoting the expression of tight junction protein Claudin-1.

10. The use according to claim 9, characterized in that: in, Application (b) includes: application of Lactobacillus plantarum TY-P22 or the composition in the preparation of a preparation for alleviating atrophy of spleen and thymus immune organs; In application (c), the body's immune cells include monocytes and basophils; In application (e), the relevant cytokines include one or more of IL-2, IL-10, IL-1β or INF-γ.

Images