Lactobacillus reuteri with anti-hypoxia function and application

Abstract

The invention belongs to the technical field of biological medicines, and discloses a strain of Lactobacillus reuteri with an anti-hypoxia function, the name of the Lactobacillus reuteri is 40509a, the classification name of the Lactobacillus reuteri is Limosilbacillus reuteri, the preservation number of the Lactobacillus reuteri is CGMCC No.31996, the preservation date of the Lactobacillus reuteri is September 18, 2024, the preservation unit is China General Microbiological Culture Collection Center, the preservation number of the Lactobacillus reuteri is CGMCC No.31996, the preservation number of the Lactobacillus reuteri is CGMCC No.31996, and the preservation number of the Lactobacillus reuteri is CGMCC No.31996. The medicine is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The lactobacillus reuteri 40509a can prolong the survival time of a CD-1 hypoxic mouse, inhibit the content of HIF1-alpha and PHD in serum of the hypoxic mouse so as to achieve the anti-hypoxic function, and can improve the activity of PC12 hypoxic cells and improve the expression of NSE and NGF of hypoxic cells so as to achieve the anti-hypoxic function.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and relates to a functional food preparation technology, in particular to a strain of Lactobacillus reuteri with anti-hypoxia function and its application. Background Art

[0002] Members of the genus Lactobacillus have long been considered one of the most abundant microorganisms in the human gastrointestinal tract and are important beneficial bacteria in the human intestine. Lactobacillus reuteri 40509a is a new probiotic strain isolated and screened by our research group from yak yogurt with independent intellectual property rights. There are currently no reports on the study of the effects of this genus on anti-hypoxic mice and cell improvement.

[0003] Yak is a precious animal belonging to the Bovine subfamily. It is a domestic animal that can graze at high altitudes of 3,000-6,000 meters. The climate conditions in the Qinghai-Tibet Plateau are harsh, with strong radiation, high altitude and lack of oxygen. Most domestic animals cannot graze on plateau pastures. Yak is the main animal species in plateau grassland animal husbandry, using 1.323 billion mu of high-altitude grassland that other cattle species cannot use, and is an indispensable part of the Qinghai-Tibet Plateau ecosystem.

[0004] Oxygen is an indispensable substance for the human body. Cells maintain oxygen balance to ensure normal physiological functions. Under normal oxygen conditions, human cells consume oxygen and nutrients to synthesize energy ATP; oxygen is also involved in various key biochemical reactions of cells. In the absence of oxygen, human cells activate a series of signaling pathways, including hypoxia-inducible factors, energy metabolism pathways, and cell stress pathways, thereby inducing cellular stress responses to hypoxia.

[0005] The core molecule of the cell's response to hypoxia is the hypoxia-inducible factor (HIF). The HIF signaling pathway has the functions of sensing metabolic changes caused by cell hypoxia, regulating cell proliferation, and inducing inflammatory responses. Hypoxia activates related signaling pathways, which are mainly controlled by the stability of HIF. Under normoxic conditions, the proline residues of the HIF-α subunit are hydroxylated by oxygen-dependent prolyl-4-hydroxylase (PHD). The HIF signaling pathway is closely related to the occurrence and development of many diseases, including cardiovascular and cerebrovascular diseases, metabolic diseases, tissue inflammation, and infectious diseases. The discovery of the HIF pathway explains how cells sense changes in oxygen and mediate downstream signaling pathways, providing a new target for the prevention and treatment of human diseases.

[0006] Therefore, the development of probiotics that can improve the body's ability to resist hypoxia is of great significance to current human health. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a strain of Lactobacillus reuteri with anti-hypoxia function and its application.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] A strain of Limosilactobacillus reuteri with anti-hypoxia function, named 40509a, classified as Limosilactobacillus reuteri, deposited on September 18, 2024, deposited at General Microbiology Center of China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The use of Lactobacillus reuteri as described above in the preparation of anti-hypoxia functional products.

[0011] Furthermore, the functional products include functional foods and / or medicines.

[0012] The use of Lactobacillus reuteri as described above in the preparation of medicines for treating radiation enteritis.

[0013] Furthermore, the live bacterial liquid of Lactobacillus reuteri 40509a can prolong the survival time of CD-1 hypoxic mice.

[0014] Furthermore, the live bacterial liquid of Lactobacillus reuteri 40509a can inhibit the levels of HIF1-α and PHD in the serum of CD-1 hypoxic mice.

[0015] Furthermore, the inactivated bacterial solution of Lactobacillus reuteri 40509a can prolong the survival time of CD-1 hypoxic mice, improve the content of HIF1-α and PHD, and it is found that it can inhibit the expression of the above hypoxia factors.

[0016] Furthermore, the inactivated bacterial solution of Lactobacillus reuteri 40509a can significantly improve the viability of cobalt chloride-induced hypoxic PC12 cells, namely rat adrenal medullary pheochromocytoma cells.

[0017] Furthermore, the inactivated bacterial solution of Lactobacillus reuteri 40509a can significantly improve the activity of nerve growth factor NGF (Neurotrophin) in hypoxic PC12 cells induced by cobalt chloride, and it was found that it can improve the expression of NGF and NSE in hypoxic PC12 cells.

[0018] Furthermore, the inactivated bacterial solution of Lactobacillus reuteri 40509a can significantly improve the activity of neurofilament protein NSE (Neurofilament protein) in cobalt chloride-induced hypoxic PC12 cells.

[0019] The advantages and positive effects achieved by the present invention are:

[0020] 1. The present invention has discovered an application of lactic acid bacteria in anti-hypoxia. Experiments were conducted using Lactobacillus reuteri 40509a, which showed that it can prolong the survival time of CD-1 hypoxic mice and inhibit the levels of HIF1-α and PHD in the serum of hypoxic mice to achieve the function of anti-hypoxia.

[0021] 2. The present invention has discovered an application of lactic acid bacteria in anti-hypoxia. Experiments were conducted using Lactobacillus reuteri 40509a, and the results showed that it can improve the vitality of PC12 hypoxic cells and improve the expression of NSE and NGF in hypoxic cells to achieve the function of anti-hypoxia.

[0022] 3. The strain used in the present invention is a food-grade probiotic, which is highly safe for conducting relevant drug research.

[0023] 4. The present invention provides new theoretical guidance for the treatment of hypoxia-related diseases.

[0024] 5. The experiment of the present invention shows that Lactobacillus reuteri can prolong the survival time of CD-1 hypoxic mice, inhibit the HIF1-α and PHD content in the serum of hypoxic mice, improve the vitality of PC12 hypoxic cells, improve the expression of NSE and NGF in hypoxic cells, and achieve the function of anti-hypoxia. Lactobacillus reuteri 40509a of the present invention can be used in the fields of food, medicine, etc., and can be used to develop related functional foods and medicines.

[0025] 6. The Lactobacillus reuteri 40509a of the present invention can prolong the survival time of CD-1 hypoxic mice, inhibit the HIF1-α and PHD content in the serum of hypoxic mice to achieve the function of anti-hypoxia, and can improve the vitality of PC12 hypoxic cells and improve the expression of NSE and NGF in hypoxic cells to achieve the function of anti-hypoxia. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Gram microscopic image, sequence and homology comparison of Lactobacillus reuteri 40509a used in the present invention;

[0027] Figure 2 This is a comparison chart of the survival time of animals in a hypoxia experiment at normal pressure after being treated with Lactobacillus reuteri 40509a in the present invention;

[0028] Figure 3 This is a graph showing the expression levels of HIF1-α and PHD detected by ELISA after treatment with Lactobacillus reuteri 40509a in the present invention;

[0029] Figure 4 This is a diagram for exploring the optimal dosage and time of cobalt chloride-induced hypoxic injury in PC12 cells in the present invention;

[0030] Figure 5 This is a CCK8 data diagram for exploring the anti-hypoxic dose and time of improving the viability of hypoxic PC12 cells after treatment with Lactobacillus reuteri 40509a in the present invention;

[0031] Figure 6 This is a diagram showing the expression of NSE and NGF detected by ELISA after treatment with Lactobacillus reuteri 40509a in the present invention.

[0032] A strain of Limosilactobacillus reuteri with anti-hypoxia function, named 40509a, classified as Limosilactobacillus reuteri, deposited on September 18, 2024, deposited at General Microbiology Center of China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0034] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0035] A strain of Limosilactobacillus reuteri with anti-hypoxia function, named 40509a, classified as Limosilactobacillus reuteri, deposited on September 18, 2024, deposited at General Microbiology Center of China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0036] The use of Lactobacillus reuteri as described above in the preparation of anti-hypoxia functional products.

[0037] Preferably, the functional product includes functional food and / or medicine.

[0038] The use of Lactobacillus reuteri as described above in the preparation of medicines for treating radiation enteritis.

[0039] Preferably, the live bacterial liquid of Lactobacillus reuteri 40509a can prolong the survival time of CD-1 hypoxic mice.

[0040] Preferably, the live bacterial liquid of Lactobacillus reuteri 40509a can inhibit the levels of HIF1-α and PHD in the serum of CD-1 hypoxic mice.

[0041] Preferably, the inactivated bacterial solution of Lactobacillus reuteri 40509a can prolong the survival time of CD-1 hypoxic mice, improve the content of HIF1-α and PHD, and it is found that it can inhibit the expression of the above hypoxia factors.

[0042] Preferably, the inactivated bacterial solution of Lactobacillus reuteri 40509a can significantly improve the viability of cobalt chloride-induced hypoxic PC12 cells, i.e., rat adrenal medullary pheochromocytoma cells.

[0043] Preferably, the inactivated bacterial solution of Lactobacillus reuteri 40509a can significantly improve the activity of nerve growth factor NGF (Neurotrophin) in hypoxic PC12 cells induced by cobalt chloride, and it is found that it can improve the expression of NGF and NSE in hypoxic PC12 cells.

[0044] Preferably, the inactivated bacterial solution of Lactobacillus reuteri 40509a can significantly improve the activity of neurofilament protein NSE (Neurofilament protein) in cobalt chloride-induced hypoxic PC12 cells.

[0045] Specifically, the relevant preparation and detection are as follows:

[0046] Example 1 Isolation and purification of lactic acid bacteria

[0047] Weigh 25g of yogurt samples collected from the Qinghai-Tibet Plateau in China, mix with 225mL of sterile saline, and dilute continuously to appropriate multiples. Take 100μL of the dilutions and spread them on MRS agar plates, invert and culture them in anaerobic bags at 37℃ for 24h, and observe the growth of colonies. Take a single colony, examine it microscopically using Gram staining, observe the cell morphology, and select Gram-positive bacteria as the target strain. Purify the selected strains repeatedly on MRS agar plates until there are no other bacteria, and record the status of different strains.

[0048] 16S rDNA sequence analysis and identification: The bacterial genomic DNA extraction kit (Beijing Solarbio Co., Ltd.) was used to extract the strain genome and used it as a template for PCR amplification. The PCR reaction used 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as upstream and downstream primers to amplify the 16S rDNA sequence for strain identification. The amplified product was sent to Tianjin GENEWIZ Biotechnology Co., Ltd., China for sequencing, and the sequencing sequence result was (i.e., the gene sequence of Lactobacillus mucosa 40509a of Reuteri). Sequence homology was compared and analyzed by Basic Local Alignment Search Tool (BLAST, www.ncbi.nlm.nih.gov / blast / ).The known strain with the highest similarity to them was selected for similarity analysis, and it was found that the strain was highly similar to Lactobacillus reuteri.

[0049] pass Figure 1 It can be seen that strain 40509a appears purple after Gram microscopy, is a Gram-positive bacterium, and is rod-shaped. BLAST homology comparison revealed that it is Lactobacillus reuteri.

[0050] Example 2 Animal grouping and normobaric hypoxia animal experiment

[0051] CD-1 male mice, 6 weeks old, weighing 15-18 g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. (License: SCXK (Xiang) 2017-0006). The animals were randomly divided into a blank group (gavage with normal saline, 0.1 ml / 10 g, after 7 days of adaptive feeding), a model group (no treatment), a positive drug group (30 mg / kg salidroside, after 7 days of adaptive feeding), a low-dose Lactobacillus reuteri 40509a (5*10 7 CFU / mL, 0.1ml / 10g, gavage treatment after 7 days of adaptive feeding), medium (5*10 8 CFU / mL, 0.1ml / 10g, gavage treatment after 7 days of adaptive feeding), high dose (5*10 9 CFU / mL, 0.1ml / 10g, gavage treatment after 7 days of adaptive feeding). Raised in an SPF animal laboratory at 18℃-22℃. Gavage for 35 days. One hour after the last gavage, the experiment was carried out using the normobaric hypoxia model. Each group of mice was placed in a frosted wide-mouth bottle filled with soda lime (the volume ratio of soda lime to bottle volume was 1:50), sealed with vaseline, and covered tightly to prevent air leakage. The time when the mice died due to hypoxia was observed using the mouse respiratory arrest as an indicator.

[0052] pass Figure 2 As shown, the survival time of mice in the high-dose 40509a group was about 40 minutes, which was significantly more resistant to hypoxia than about 23 minutes in the normal group, and could prolong the survival time of hypoxic mice.

[0053] Example 3 ELISA to detect the levels of hypoxia-related factors in mouse serum

[0054] 1. Blood was collected from the eyeballs of CD-1 experimental mice 35 days after intragastric administration in Example 2 above. The serum samples were kept at room temperature for 30 min, and the levels of HIF1-α and PHD in the serum were determined according to the instructions of Mouse HIF-1α ELISA Kit and Mouse PHD ELISA Kit.

[0055] 2. Determine OD using UV spectrophotometer450 .

[0056] Test results such as Figure 3 As shown, from Figure 3 It can be seen that there is no obvious trend in the ELISA detection of HIF1-α and PHD in the high-dose group and the positive drug (salidroside) group, HIF1-α is maintained between 0.32-0.34ng / ml, and PHD is maintained at 0.17ng / ml, which has a very significant anti-hypoxia trend compared with the normal group (0.36ng / ml, 0.185ng / ml); this once again confirms that Lactobacillus reuteri 40509a has anti-hypoxia ability.

[0057] Example 4 CCK8 detection of hypoxic cell viability

[0058] 1. Rat adrenal medullary pheochromoma cells (PC12) were placed in RPMI 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / L streptomycin. The cells were cultured in a 37°C, 5% CO2 incubator. The cells adhered to the wall and the cell growth was observed under a microscope. When the cells grew to 90%, they were passaged according to different experimental requirements.

[0059] 2. CoCl2-induced hypoxic injury in PC12 cells

[0060] PC12 cells in the growth phase were taken to prepare a cell suspension, 5000 cells / well were inoculated in a 96-well plate and cultured overnight. A control group and different CoCl2 concentration hypoxia groups (100, 200, 400, 800 μmol / L) were set up and cultured for 12, 24, and 48 hours to determine the optimal modeling conditions.

[0061] from Figure 4 It can be seen that the inhibitory effect of cobalt chloride on PC12 cells was most obvious at 400 μmol / L and 24 h, which was the optimal modeling condition.

[0062] 3. CCK-8 method to detect cell activity

[0063] Purpose:

[0064] The purpose of this experiment was to evaluate the effect of inactivated bacterial solution on cell survival in a hypoxia-induced PC12 cell model using the CCK-8 method, in order to explore the protective or toxic effects of the substance under hypoxic conditions.

[0065] step:

[0066] (1) Preparation of inactivated bacteria solution:

[0067] According to the experimental design, prepare a working solution of appropriate concentration of the inactivated bacteria to be tested. Ensure the solubility and stability of the substance. For specific strain concentrations, see (2).

[0068] (2) Cell processing:

[0069] a. PC12 cells were seeded at 5000 cells / well in a 96-well plate and cultured overnight until the cells were completely attached;

[0070] b. When the cells grow to 50%, group them as follows:

[0071]

[0072] c. Use DMEM sugar-free medium to prepare the above substances according to the corresponding concentrations, 100 μL / well to culture cells, and continue to culture in a 37°C, 5% CO2 incubator for 24 or 48 hours to fully expose the cells to the functional substances.

[0073] d. After the incubation is completed, remove the cell culture plate from the incubator;

[0074] e. Discard the old culture medium, add fresh culture medium containing 10% CCK-8, and place in a 37°C, 5% CO2 incubator for another 1-4 hours.

[0075] (3) Absorbance measurement:

[0076] After the incubation period, measure the absorbance of each well at 450 nm using a microplate reader. Make sure to follow the instructions of the CCK-8 kit for the measurement.

[0077] (4) Data analysis:

[0078] a. Calculate the cell viability of each treatment group based on the absorbance value.

[0079] b. Draw a concentration-effect curve to evaluate the effect of functional substances on cell survival rate.

[0080] Test results such as Figure 5 As shown, from Figure 5 It can be seen that the 7.0±0.2, 8.0±0.2, and 9.0±0.2Log CFU / ml groups can improve the hypoxic damage of PC12 cells induced by cobalt chloride and have significant differences at 24h and 48h; while 10.0±0.2Log CFU / ml and 11.0±0.2Log CFU / ml may cause cell toxicity due to excessively high bacterial concentrations, severely inhibiting cell proliferation and growth.

[0081] Example 5 ELISA detection of neuron damage factors in PC12 cells

[0082] 1. Add 800 μL of PC12 cell suspension of the above concentration to a 6-well plate. Pre-culture in an incubator for 24 hours (37°C, 5% CO2). Perform hypoxia modeling intervention and therapeutic intervention as described in Example 4. Incubate the culture plate in an incubator at 37°C for 24 hours. Collect the cell suspension, centrifuge at 10000g for 10 minutes at 4°C, collect the cell pellet and lyse by ultrasonication, centrifuge again according to the above method after lysis, and collect the supernatant for subsequent detection.

[0083] 2. Determine OD using UV spectrophotometer 450 .

[0084] Test results such as Figure 6 As shown, from Figure 6 As can be seen, ELISA detected NGF and NSE, and the results showed that 7.0±0.2, 8.0±0.2, and 9.0±0.2Log CFU / ml could improve the expression of NGF and NSE in PC12 hypoxic damaged cells. The improvement effect of the medium-dose group 8.0±0.2Log CFU / ml was the most obvious, with NGF expression at around 13ng / ml and NSE expression at around 17ng / ml, which was significantly different from the model group (10ng / ml, 13ng / ml). This once again confirmed that Lactobacillus reuteri 40509a has excellent anti-hypoxia ability.

[0085] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A strain of Lactobacillus reuteri having an anti-hypoxia function, characterized in that: Its name is 40509a, the classification name is Limosilactobacillus reuteri, the preservation number is CGMCC No.31996, the preservation date is September 18, 2024, and the preservation unit is General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. Use of Lactobacillus reuteri as claimed in claim 1 in preparing anti-hypoxia functional products.

3. The use according to claim 2, characterized in that: The functional products include functional foods and / or medicines.

4. Use of Lactobacillus reuteri as claimed in claim 1 in the preparation of a drug for treating radiation enteritis.

5. The use according to any one of claims 1 to 3, characterized in that: The live bacterial liquid of Lactobacillus reuteri 40509a can prolong the survival time of CD-1 hypoxic mice.

6. The use according to any one of claims 1 to 3, characterized in that: The live bacterial liquid of Lactobacillus reuteri 40509a can inhibit the content of HIF1-α and PHD in the serum of CD-1 hypoxic mice.

7. The use according to any one of claims 1 to 3, characterized in that: The inactivated bacterial liquid of Lactobacillus reuteri 40509a can prolong the survival time of CD-1 hypoxic mice and improve the contents of HIF1-α and PHD.

8. The use according to any one of claims 1 to 3, characterized in that: The inactivated bacterial solution of Lactobacillus reuteri 40509a can significantly improve the viability of hypoxic PC12 cells induced by cobalt chloride, namely, rat adrenal medullary pheochromocytoma cells.

9. The use according to any one of claims 1 to 3, characterized in that: The inactivated bacterial solution of Lactobacillus reuteri 40509a can significantly improve the activity of nerve growth factor NGF in hypoxic PC12 cells induced by cobalt chloride.

10. The use according to any one of claims 1 to 3, characterized in that: The inactivated bacterial solution of Lactobacillus reuteri 40509a can significantly improve the activity of neurofilament protein NSE in hypoxic PC12 cells induced by cobalt chloride.

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