Extracellular vesicle of bifidobacterium animalis BL99 and application of extracellular vesicle in treatment and / or prevention of nerve injury

Abstract

The invention relates to an extracellular vesicle of bifidobacterium animalis BL99 and application of the extracellular vesicle in treatment and / or prevention of nerve injury, and particularly provides application of the extracellular vesicle of the bifidobacterium animalis subsp. Lactis BL-99 in preparation of medicine, common food, health food, food additives and / or dietary supplements, and the medicine is used for treating and / or preventing nerve injury. The common food, the health food, the food additive and / or the dietary supplement are used for relieving nerve injury, and the preservation number of the animal bifidobacterium subsp. Lactis BL-99 is CGMCC (China General Microbiological Culture Collection Center) No.15650. The animal bifidobacterium subsp. Lactis BL-99 extracellular vesicle has the effect of relieving nerve injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an extracellular vesicle of animal bifidobacterium BL99 and application thereof in treating and / or preventing nerve injury. BACKGROUND

[0002] Inflammatory nerve injury refers to inflammation of nerve tissue, which is caused by many reasons, including infection, traumatic brain injury, toxic metabolites of mental illness, or autoimmunity. Such injury can further cause a series of nervous system diseases, including neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease). In the central nervous system (CNS), including the brain and spinal cord, microglia are resident innate immune cells that respond to signals in the central nervous system. The central nervous system is a typical immune-privileged site, because peripheral immune cells are usually blocked by the blood-brain barrier (BBB). When the BBB is weakened, microglia produce reactive oxygen species (ROS) and release signals to call peripheral immune cells to produce inflammatory responses. Therefore, exploring the effects of new active ingredients on the brain nerves is of great significance for developing new treatment programs for nerve inflammation. SUMMARY

[0003] To solve the above problems, the present application provides an extracellular vesicle of animal bifidobacterium BL-99 and its extraction and preparation method, and finds that it has the effect of relieving nerve injury.

[0004] Therefore, in the first aspect of the present application, the present application provides the use of an extracellular vesicle of animal bifidobacterium BL-99 in the preparation of a drug, a general food, a health food, a food additive and / or a dietary supplement, the drug is used for treating and / or preventing nerve injury, and the general food, the health food, the food additive and / or the dietary supplement are used for relieving nerve injury, the preservation number of the animal bifidobacterium BL-99 is CGMCC No. 15650.

[0005] In the second aspect of the present application, the present application provides the use of a composition in the preparation of a drug, a general food, a health food, a food additive and / or a dietary supplement, the composition comprises an extracellular vesicle of animal bifidobacterium BL-99, the drug is used for treating and / or preventing nerve injury, and the general food, the health food, the food additive and / or the dietary supplement are used for relieving nerve injury, the preservation number of the animal bifidobacterium BL-99 is CGMCC No. 15650.

[0006] In a third aspect of the present application, the present application provides use of a metabolite of Bifidobacterium animalis lactis BL-99 in preparation of a medicine, a general food, a health food, a food additive and / or a dietary supplement, wherein the metabolite of Bifidobacterium animalis lactis BL-99 comprises an extracellular vesicle of Bifidobacterium animalis lactis BL-99, the medicine is used for treating and / or preventing nerve injury, the general food, the health food, the food additive and / or the dietary supplement are used for relieving nerve injury, and the deposit number of the Bifidobacterium animalis lactis BL-99 is CGMCC No.15650.

[0007] In a fourth aspect of the present application, the present application provides use of a lysate or a lysate of an extracellular vesicle of Bifidobacterium animalis lactis BL-99 in preparation of a medicine, a general food, a health food, a food additive and / or a dietary supplement, wherein the lysate or the lysate of the extracellular vesicle of Bifidobacterium animalis lactis BL-99 is obtained by lysing or lysing the extracellular vesicle of Bifidobacterium animalis lactis BL-99, the medicine is used for treating and / or preventing nerve injury, the general food, the health food, the food additive and / or the dietary supplement are used for relieving nerve injury, and the deposit number of the Bifidobacterium animalis lactis BL-99 is CGMCC No.15650.

[0008] In a fifth aspect of the present application, the present application provides the extracellular vesicle of Bifidobacterium animalis lactis BL-99, the metabolite of Bifidobacterium animalis lactis BL-99, the lysate or the lysate of the extracellular vesicle of Bifidobacterium animalis lactis BL-99, or a composition for treating and / or preventing nerve injury or relieving nerve injury, wherein the composition or the metabolite of Bifidobacterium animalis lactis BL-99 comprises the extracellular vesicle of Bifidobacterium animalis lactis BL-99, the lysate or the lysate of the extracellular vesicle of Bifidobacterium animalis lactis BL-99 is obtained by lysing or lysing the extracellular vesicle of Bifidobacterium animalis lactis BL-99, and the deposit number of the Bifidobacterium animalis lactis BL-99 is CGMCC No.15650.

[0009] In a sixth aspect, the present invention provides a method for treating and / or preventing or alleviating nerve damage, comprising administering to a subject in need an effective amount of extracellular vesicles of *Bifidobacterium lactis* subsp. lac ...

[0010] In a seventh aspect, the present invention provides a method for inhibiting / alleviating inflammation of nerve cells, comprising administering to nerve cells an effective amount of extracellular vesicles of *Bifidobacterium lactis* subsp. *animal* BL-99, metabolites of *Bifidobacterium lactis* subsp. *animal* BL-99, a solution or lysate of extracellular vesicles of *Bifidobacterium lactis* subsp. *animal* BL-99, or a composition thereof, wherein the composition or the metabolites of *Bifidobacterium lactis* subsp. *animal* BL-99 contain extracellular vesicles of *Bifidobacterium lactis* subsp. *animal* BL-99, and the solution or lysate of the extracellular vesicles of *Bifidobacterium lactis* subsp. *animal* BL-99 is obtained by dissolving or lysing the extracellular vesicles of *Bifidobacterium lactis* subsp. *animal* BL-99 has the accession number CGMCC No. 15650.

[0011] The *Bifidobacterium lactis* subspecies BL-99 was deposited on April 26, 2018, at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with the classification name *Bifidobacterium lactis* and accession number CGMCC No. 15650. For further details, please refer to CN110964657B.

[0012] In some embodiments, the dosage of the extracellular vesicles of Bifidobacterium animalis subsp. lactis BL-99 is 50-400 ng / μL, such as 50 ng / μL, 100 ng / μL, 200 ng / μL or 400 ng / μL, preferably 200 ng / μL.

[0013] In some implementations, the nerve injury is an inflammatory nerve injury.

[0014] In some implementations, the inflammatory nerve injury is LPS-induced inflammatory nerve injury.

[0015] In some embodiments, the Bifidobacterium animalis lactis BL-99 extracellular vesicles are prepared by the following method:

[0016] 1) subjecting a bacterial suspension of Bifidobacterium animalis lactis BL-99 to low-speed centrifugation and high-speed centrifugation in sequence, and taking the supernatant;

[0017] 2) subjecting the supernatant to ultrafiltration centrifugation, and taking the filtrate;

[0018] 3) subjecting the filtrate to ultrahigh-speed centrifugation, and taking the supernatant;

[0019] 4) subjecting the supernatant to ultrafiltration, and the filtrate contains the Bifidobacterium animalis lactis BL-99 extracellular vesicles.

[0020] In some embodiments, the pore size of the ultrafiltration centrifugation is 100 KDa.

[0021] In some embodiments, the ultrafiltration centrifugation is performed at a speed of 4000-6000 g (such as 4000 g, 4200 g, 4400 g, 4600 g, 4800 g, 5000 g, 5200 g, 5400 g, 5600 g, 5800 g, or 6000 g, preferably 5000 g).

[0022] In some embodiments, the ultrafiltration centrifugation is performed for 5-15 min (such as 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min), preferably 10 min.

[0023] In some embodiments, the ultrafiltration centrifugation is performed using an ultrafiltration centrifuge tube (such as an Amicon Ultra-15 ultrafiltration centrifuge tube).

[0024] In some embodiments, the low-speed centrifugation is performed in two times, the first low-speed centrifugation is performed at 3500-4000 g (such as 3500 g, 3600 g, 3700 g, 3800 g, 3900 g, or 4000 g, preferably 3600 g) for 20-40 min (such as 20 min, 25 min, 30 min, 35 min, or 40 min, preferably 30 min), and the second low-speed centrifugation is performed at 5000-5500 g (such as 5000 g, 5100 g, 5200 g, 5300 g, 5400 g, or 5500 g, preferably 5000 g) for 20-40 min (such as 20 min, 25 min, 30 min, 35 min, or 40 min, preferably 30 min).

[0025] In some embodiments, the high-speed centrifugation is performed at 10000-12000g (such as 10000g, 10200g, 10400g, 10600g, 10800g, 11000g, 11200g, 11400g, 11600g, 11800g or 12000g, preferably 11000g) for 50-70min (such as 50min, 55min, 60min, 65min or 70min, preferably 60min).

[0026] In some embodiments, the ultra-high-speed centrifugation is performed twice, the first ultra-high-speed centrifugation is performed at 110000-130000g (such as 112000g, 114000g, 116000g, 118000g, 120000g, 122000g, 124000g, 126000g, 128000g or 130000g, preferably 120000g) for 80-100min (such as 80min, 85min, 90min, 95min or 100min, preferably 90min), and the second ultra-high-speed centrifugation is performed at 110000-130000g (such as 112000g, 114000g, 116000g, 118000g, 120000g, 122000g, 124000g, 126000g, 128000g or 130000g, preferably 120000g) for 80-100min (such as 80min, 85min, 90min, 95min or 100min, preferably 90min).

[0027] In some embodiments, the ultrafiltration is achieved by using an ultrafiltration membrane.

[0028] In some embodiments, the pore size of the ultrafiltration is 0.22um.

[0029] In some embodiments, the low-speed centrifugation is performed at a temperature of 4℃.

[0030] In some embodiments, the high-speed centrifugation is performed at a temperature of 4℃.

[0031] In some embodiments, the ultra-high-speed centrifugation is performed at a temperature of 4℃.

[0032] In some embodiments, in step 4), after the supernatant is subjected to ultrafiltration, further comprising: diluting the filtrate with PBS buffer, and the PBS dilution liquid contains the animal Bifidobacterium lactis BL-99 extracellular vesicles.

[0033] In some embodiments, the bacterial suspension of Bifidobacterium animalis lactis BL-99 is obtained by pre-fermentation of the Bifidobacterium animalis lactis BL-99. In some embodiments, the fermentation is performed in MRS liquid medium at 37°C.

[0034] In some embodiments, the extracellular vesicles of Bifidobacterium animalis lactis BL-99 have a particle size of 50-100 nm (e.g., 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, or 100 nm, e.g., 85-90 nm).

[0035] The term "treatment" generally refers to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. As used herein, "treatment" covers any treatment of a patient, including: (a) preventing the disease or symptom that is susceptible to the treatment but has not yet developed; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.

[0036] The term "subject" refers to a vertebrate. In certain embodiments, the vertebrate refers to a mammal. Mammals include, but are not limited to, farm animals (such as cows), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human.

[0037] The term "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a substance / molecule of the present application can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule to elicit a desired response in the individual. A therapeutically effective amount also encompasses an amount of the substance / molecule that has therapeutic benefits outweighing any toxic or deleterious consequences. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, but not necessarily, since a prophylactic dose is used in subjects prior to or at the early stages of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0038] Beneficial effects

[0039] 1. The present application provides an extracellular vesicle of Bifidobacterium animalis lactis BL-99 and its extraction and preparation method, and it is found to have the effect of relieving nerve injury (such as inflammatory nerve injury).

[0040] 2、The animal Bifidobacterium lactis BL-99 extracellular vesicle provided by the application can inhibit nerve inflammation and relieve nerve damage (such as inflammatory nerve damage) by regulating the levels of growth factors, anti-inflammatory factors and pro-inflammatory factors.

[0041] 3、The animal Bifidobacterium lactis BL-99 extracellular vesicle provided by the application can inhibit nerve inflammation and relieve nerve damage (such as inflammatory nerve damage) by reducing the production of active oxygen.

[0042] 4、The animal experiment proves that the animal Bifidobacterium lactis BL-99 extracellular vesicle provided by the application can effectively relieve brain nerve damage (such as inflammatory nerve damage). BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 It is a BL-99 bacterial source vesicle extraction and separation method schematic diagram in the embodiment one of the application.

[0044] Figure 2 It is a BL-99-MVs protein concentration comparison chart extracted by different methods in the embodiment one of the application.

[0045] Figure 3 It is a BL-99-MVs particle size chart extracted by different methods in the embodiment one of the application.

[0046] Figure 4 It is a BL-99-MVs morphological structure chart under a transmission electron microscope extracted by different methods in the embodiment one of the application.

[0047] Figure 5 It is a BL-99-MVs relative quantity comparison chart extracted by different methods in the embodiment one of the application.

[0048] Figure 6 It is a different concentration animal Bifidobacterium BL-99 extracellular vesicle influence result chart on LPS-induced inflammatory HT22 cell survival rate in the embodiment three of the application; "*" is p<0.05; "**" is p<0.01; "***" is p<0.001.

[0049] Figure 7 It is a 200ng / ul concentration animal Bifidobacterium BL-99 extracellular vesicle influence detection result chart on growth factors in HT22 cells in the embodiment four of the application; "*" is p<0.05; "**" is p<0.01; "***" is p<0.001.

[0050] Figure 8The figure shows the detection results of the influence of 200 ng / μl concentration of Bifidobacterium animalis subsp. lactis BL-99 extracellular vesicles on inflammatory factors in HT22 cells in Example Four of the present application; "*" represents p<0.05; "**" represents p<0.01; "***" represents p<0.001.

[0051] Figure 9 The figure shows the detection results of the influence of 200 ng / μl concentration of Bifidobacterium animalis subsp. lactis BL-99 extracellular vesicles on reactive oxygen species (ROS) in HT22 cells in Example Five of the present application; "*" represents p<0.05; "**" represents p<0.01; "***" represents p<0.001.

[0052] Figure 10 The figure shows the intervention of the Ctrl group, the LPS-encephalitis group, and the MV group in Example Six of the present application.

[0053] Figure 11 The figure shows the typical water maze trajectory of mice in each group in Example Six of the present application.

[0054] Figure 12 The figure shows the typical new object recognition trajectory of mice in each group in Example Six of the present application. DETAILED DESCRIPTION

[0055] The embodiments of the present application will be described in detail below with reference to the examples and drawings, but those skilled in the art will understand that the following examples and drawings are only used to illustrate the present application, and are not a limitation on the scope of the present application. According to the following detailed description of the drawings and preferred embodiments, various purposes and advantages of the present application will become apparent to those skilled in the art. Unless otherwise specified, each raw material and reagent can be commercially available. Among them, Bifidobacterium animalis subsp. lactis BL-99 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on April 26, 2018 (address: No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences), and the classification and naming is Bifidobacterium lactis; the preservation number is CGMCC No. 15650. For example, specific information can be found in CN110964657B.

[0056] Example One:

[0057] In this example, an extracellular vesicle of Bifidobacterium animalis subsp. lactis BL-99 was prepared, and the protein concentrations of two preparation methods were compared. As shown in Figure 1 , compared with Method Two, Method One is the same as Method Two in all other steps and operations except that it does not use an ultrafiltration centrifuge tube (pore size 100 KDa, Amicon Ultra-15) for ultrafiltration centrifugation (5000g, 10min).

[0058] Using animal Bifidobacterium lactis BL-99 to ferment (37℃ culture in MRS liquid medium), 1L of bacterial suspension was obtained.

[0059] Method one: the bacterial suspension of animal Bifidobacterium was sequentially subjected to low-speed centrifugation (first time, 3600g, 30min; second time, 5000g, 30min) and high-speed centrifugation (11000g, 60min) at 4℃, and the supernatant was extracted after centrifugation. The supernatant was subjected to twice ultra-high-speed centrifugation (120000g, 90min) at 4℃, and the supernatant was extracted. The supernatant was subjected to ultrafiltration with an ultrafiltration membrane (0.22um), and the filtrate was diluted with PBS buffer, and the PBS dilution was the extracellular vesicles of BL-99.

[0060] Method two: the bacterial suspension of animal Bifidobacterium was sequentially subjected to low-speed centrifugation (first time, 3600g, 30min; second time, 5000g, 30min) and high-speed centrifugation (11000g, 60min) at 4℃, and the supernatant was extracted after centrifugation. The supernatant was subjected to ultrafiltration centrifugation (5000g, 10min) with an ultrafiltration centrifuge tube (pore size 100KDa, Amicon Ultra-15), and relatively pure supernatant was obtained. The supernatant after ultrafiltration centrifugation was subjected to twice ultra-high-speed centrifugation (120000g, 90min) at 4℃, and the supernatant was extracted. The supernatant was subjected to ultrafiltration with an ultrafiltration membrane (0.22um), and the filtrate was diluted with PBS buffer, and the PBS dilution was the extracellular vesicles of BL-99.

[0061] The detection method of protein concentration refers to GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Food". The detection results are shown in Table 1. Figure 2

[0062] From the detection results, the protein concentration of BL-99-MVs extracted by method one was 1.29ug / ul; the protein concentration of BL-99-MVs extracted by method two was 1.36ug / ul, which was significantly higher than that of method one.

[0063] Example Two:

[0064] The BL-99-MVs extracted by method one and method two described in Example one were subjected to particle size analysis, respectively. Particle size analysis was realized by using a particle size analyzer. The particle size distribution detection method was: 700μL of 20μg / mL animal Bifidobacterium BL-99 extracellular vesicles was taken in a particle size dish for particle size detection, and was gradually diluted until the particle size did not change as the final result. The analysis results are shown in Table 2. Figure 3 ​​

[0065] Figure 2 shows the BL-99-MVs extracted by method one and method two under the transmission electron microscope. Figure 3 Figure 2 shows that the BL-99-MVs extracted by method one have a particle size of 141.9 nm, while the BL-99-MVs extracted by method two have a particle size of 87.11 nm.

[0066] Figure 3 shows the morphological structure of the BL-99-MVs extracted by method one and method two described in Example One under the transmission electron microscope. Figure 4 The transmission electron microscope morphological detection method is as follows: 20 μL of animal Bifidobacterium BL-99 extracellular vesicles are taken on a 300-mesh copper grid, naturally dried, 20 μL of phosphotungstic acid negative staining is added, naturally dried, and then detected on the machine.

[0067] Figure 4 shows the BL-99-MVs extracted by method one and method two under the transmission electron microscope. Figure 4 Figure 4 shows that the BL-99-MVs extracted by method one and method two have a round structure as a whole, but the particle sizes are obviously different.

[0068] Comprehensive transmission electron microscope images and particle size analysis show that the BL-99-MVs extracted by method two have a smaller particle size and better dispersibility compared with the BL-99-MVs extracted by method one.

[0069] The BL-99-MVs extracted by method one and method two described in Example One were measured for the number and size of BL-99-MVs using flow imaging method (reference literature Inamdar S, Nitiyanandan R, Rege K. Emerging applications of exosomes in cancer therapeutics and diagnostics [J]. Bioeng Transl Med, 2017, 2(1): 70.). Figure 5 and Table 1.

[0070] Figure 5 Table 1 shows that the number of extracellular vesicles extracted by method two is about 1.6 times the number of extracellular vesicles extracted by method one (“**” is p<0.01), and it can be seen that the number of extracellular vesicles extracted by method two is significantly higher than that extracted by method one.

[0071] Table 1: Comparison of the number of extracellular vesicles extracted by different methods

[0072]

[0073] Example Three:

[0074] This example tests the effect of animal Bifidobacterium BL-99 extracellular vesicles extracted by method two in Example One on the relative survival rate of nerve cells HT22 cells.

[0075] The mouse hippocampal neuron cells (HT22, purchased from iCell (Bioscience Inc., Shanghai, China)) and normal epithelial cell lines (purchased from iCell (Bioscience Inc., Shanghai, China)) were recovered and subcultured for more than three generations, then inoculated in the well plate, adherent culture for 12 h, and then starved for 12 h. At 24 h of culture, different concentrations of BL-99 extracellular vesicles were added to the BL-99 vesicle + LPS-induced inflammation group. After 36 h of culture, the LPS-induced inflammation group and the BL-99 vesicle + LPS-induced inflammation group were subjected to LPS induction treatment (for details, refer to CN102807557B), to induce inflammation of mouse hippocampal neuron cells HT22. Continue to culture to 60 h. Sampling was started after LPS induction treatment to determine the corresponding indicators.

[0076] Blank group (N): normal mouse hippocampal neuron cells HT22

[0077] LPS-induced inflammation group: 36 h of LPS-induced neuroinflammation in mouse hippocampal neuron cells HT22

[0078] Low-dose BL-99 vesicle + LPS-induced inflammation group: 24 h of BL-99 extracellular vesicles (50 ng / μL) was added, and 36 h of LPS-induced neuroinflammation in mouse hippocampal neuron cells HT22.

[0079] Medium-dose BL-99 vesicle + LPS-induced inflammation group: 24 h of BL-99 extracellular vesicles (100 ng / μL) was added, and 36 h of LPS-induced neuroinflammation in mouse hippocampal neuron cells HT22.

[0080] High-dose BL-99 vesicle + LPS-induced inflammation group: 24 h of BL-99 extracellular vesicles (200 ng / μL) was added, and 36 h of LPS-induced neuroinflammation in mouse hippocampal neuron cells HT22;

[0081] Higher-dose BL-99 vesicle + LPS-induced inflammation group: 24 h of BL-99 extracellular vesicles (400 ng / μL) was added, and 36 h of LPS-induced neuroinflammation in mouse hippocampal neuron cells HT22.

[0082]

[0083] The total number of cells and the number of dead cells were detected according to GB 4789.35-2016 "National Food Safety Standard Food Microbiological Examination Lactic Acid Bacteria Examination". The experimental results are shown in Table 1. Figure 6

[0084] ​From the results, it can be seen that the relative survival rate of HT22 cells induced by LPS to cause inflammatory damage is significantly reduced, the addition of low-dose, medium-dose, high-dose and higher-dose BL-99 extracellular vesicle groups can significantly improve the relative survival rate of HT22 cells induced by inflammation, and the survival rate is the highest when the dose is 200 ng / μL.

[0085] Example Four:

[0086] This example tests the effect of animal bifidobacterium BL-99 extracellular vesicles extracted by method two in example one on inflammatory factors of HT-22 cells.

[0087] There are many causes of nerve damage, and nerve inflammation is considered to be the main cause of nerve damage. After the mouse hippocampal neuron cells (HT22, purchased from iCell (Bioscience Inc., Shanghai, China)) and the normal epithelial cell line (purchased from iCell (Bioscience Inc., Shanghai, China)) were recovered and passaged for more than three generations, they were inoculated in the well plate respectively, and adherent culture was carried out for 12 h, and then starved culture was carried out for 12 h. At 24 h of culture, BL-99 vesicles + LPS-induced inflammation group was added with BL-99 extracellular vesicles. After culture for 36 h, LPS-induced inflammation group and BL-99 vesicles + LPS-induced inflammation group were subjected to LPS induction treatment (for specific method, refer to CN102807557B), to cause inflammation of mouse hippocampal neuron cells HT22. Sampling was carried out at 60 h of culture. ELISA kit was used to detect the expression levels of Ki67, IL-10, TNF-α and IL-17 respectively.

[0088] Blank group (N): normal mouse hippocampal neuron cells HT22

[0089] LPS-induced inflammation group: 36 h of LPS-induced nerve inflammation of mouse hippocampal neuron cells HT22

[0090] BL-99 vesicles + LPS-induced inflammation group: 24 h of addition of BL-99 extracellular vesicles (200 ng / μL), 36 h of LPS-induced nerve inflammation of mouse hippocampal neuron cells HT22.

[0091] Figure 7 It is shown that, compared with the blank group, the expression of cell growth factor Ki67 of the LPS-induced group is significantly reduced; compared with the LPS-induced nerve inflammation group, BL-99-MVs can significantly improve the level of cell growth factor Ki67 after LPS-induced inflammation.

[0092] Figure 8As shown, compared with the blank group, the expression of the anti-inflammatory factor IL-10 in the LPS induction group was significantly reduced, and the expression of the pro-inflammatory factors TNF-a and IL-17 was significantly increased; compared with the LPS induction neuroinflammation group, the BL-99-MVs can significantly increase the level of the anti-inflammatory factor IL-10 in the cells after LPS induction inflammation, and can significantly reduce the level of the pro-inflammatory factors TNF-a and IL-17.

[0093] The experimental results show that the BL-99-MVs can inhibit neuroinflammation by regulating the levels of growth factors, anti-inflammatory factors and pro-inflammatory factors.

[0094] Example Five:

[0095] This example tests the effect of the animal bifidobacterium BL-99 extracellular vesicles extracted by the method two in Example One on the antioxidant activity of HT-22 cells.

[0096] Reactive oxygen species (ROS) are important signaling molecules for regulating metabolism and inflammatory response, and are normally maintained at a low level in the body, and accumulation thereof in the body can cause neurodegeneration and neuroinflammation.

[0097] The mouse hippocampal neuron cells (HT22, purchased from iCell (Bioscience Inc., Shanghai, China)) and normal epithelial cell lines (purchased from iCell (Bioscience Inc., Shanghai, China)) were recovered and subcultured for more than three generations, and then inoculated in the well plates, adherent cultured for 12 h and starved for 12 h. At 24 h of culture, the BL-99 vesicles + LPS induction inflammation group was added with the BL-99 extracellular vesicles. At 36 h of culture, the LPS induction inflammation group and the BL-99 vesicles + LPS induction inflammation group were subjected to LPS induction treatment (for details, refer to CN102807557B), to induce inflammation of the mouse hippocampal neuron cells HT22. At 60 h of culture, the cells were collected, and the reactive oxygen species detection kit was used for sample detection.

[0098] Blank group (N): normal mouse hippocampal neuron cells HT22

[0099] LPS induction inflammation group: 36 h of LPS induction neuroinflammation of the mouse hippocampal neuron cells HT22

[0100] BL-99 vesicles + LPS induction inflammation group: 24 h of addition of the BL-99 extracellular vesicles (200 ng / μL), 36 h of LPS induction neuroinflammation of the mouse hippocampal neuron cells HT22.

[0101] Figure 9It is shown that compared with the blank group, the ROS production of the LPS-induced inflammation group is significantly increased; compared with the LPS-induced neuroinflammation group, BL-99-MVs can significantly reduce the intracellular ROS (reactive oxygen species) production. The results show that BL-99-MVs can inhibit nerve cell damage by reducing the production of reactive oxygen species.

[0102] Example Six:

[0103] This example tests the alleviating effect of animal bifidobacterium BL-99 extracellular vesicles extracted by method two in example one on brain neuroinflammatory damage of mice (Balb / c mice).

[0104] The mice are divided into Ctrl group, LPS-brain inflammation group, and MV group, and the corresponding intervention conditions are as shown in Figure 10

[0105] The specific administration of each group is as follows:

[0106] The blank control group (Ctrl group): physiological saline is used for gavage (3 mg / kg);

[0107] The model group (LPS-brain inflammation group): physiological saline is used for gavage (3 mg / kg), and LPS (1 mg / kg) is injected for 28-30 days;

[0108] The BL99-MV group (BL-99 vesicles + LPS-brain inflammation group): BL99-MV (200 ul, 3 mg / kg) is used for gavage daily, and LPS (1 mg / kg) is injected for 28-30 days.

[0109] The experimental results are as shown in Figure 11 and Figure 12

[0110] Figure 11 From the activity trajectory of the mice, it can be seen that compared with the normal control group, the number of platform crossings of the mice in the model group is significantly reduced, and the movement trajectory in the platform quadrant is also reduced; the mice in the BL99-MV group show a significant increase in the number of platform crossings, and the activity in the target quadrant is also increased. The results show that after BL99-MV intervention, the movement trajectory of the mice is better improved, and the number of platform crossings and the activity in the platform quadrant are increased.

[0111] Figure 12 From the activity trajectory of the mice, it can be seen that compared with the normal control group, the mice in the model group are reluctant to explore new things, and there is basically no movement trajectory in the platform quadrant; although the mice in the BL99-MV group do not show high performance in exploring new things, the activity in the target quadrant is significantly increased. The results show that after BL99-MV intervention, the activity of the mice in the target quadrant is significantly increased.

[0112] ​​In summary, after BL99-MV intervention, the brain nerve inflammatory injury of mice can be effectively alleviated.

[0113] It is to be understood that the application described herein is not limited to particular methodologies, protocols or reagents, as such can vary. The discussion and examples provided herein are presented to describe particular embodiments presented and are not intended to limit the scope of the application, which is limited only by the claims.

Claims

1. Use of extracellular vesicles of Bifidobacterium animalis lactis BL-99 in the manufacture of a medicament for treating and / or preventing nerve injury, and / or a common food, a health food, a food additive and / or a dietary supplement for alleviating nerve injury, wherein the Bifidobacterium animalis lactis BL-99 has a preservation number of CGMCC No. 15650.

2. Use of a composition comprising extracellular vesicles of Bifidobacterium animalis lactis BL-99 in the manufacture of a medicament for treating and / or preventing nerve injury, and / or a common food, a health food, a food additive and / or a dietary supplement for alleviating nerve injury, wherein the Bifidobacterium animalis lactis BL-99 has a preservation number of CGMCC No. 15650.

3. Use of metabolites of Bifidobacterium animalis lactis BL-99 in the manufacture of a medicament for treating and / or preventing nerve injury, and / or a common food, a health food, a food additive and / or a dietary supplement for alleviating nerve injury, wherein the metabolites of Bifidobacterium animalis lactis BL-99 comprise extracellular vesicles of Bifidobacterium animalis lactis BL-99, and wherein the Bifidobacterium animalis lactis BL-99 has a preservation number of CGMCC No. 15650.

4. Use of a lysate or a lysate of extracellular vesicles of Bifidobacterium animalis lactis BL-99 in the manufacture of a medicament for treating and / or preventing nerve injury, and / or a common food, a health food, a food additive and / or a dietary supplement for alleviating nerve injury, wherein the lysate or the lysate of extracellular vesicles of Bifidobacterium animalis lactis BL-99 is obtained by lysing or lysing extracellular vesicles of Bifidobacterium animalis lactis BL-99, and wherein the Bifidobacterium animalis lactis BL-99 has a preservation number of CGMCC No. 15650.

5. Use according to any one of claims 1 to 4, wherein, The nerve injury is inflammatory nerve injury.

6. Use according to any one of claims 1 to 5, wherein, The extracellular vesicles of Bifidobacterium animalis lactis BL-99 are prepared by the following method: 1) sequentially subjecting a bacterial suspension of Bifidobacterium animalis lactis BL-99 to low-speed centrifugation and high-speed centrifugation, and taking the supernatant; 2) subjecting the supernatant to ultrafiltration centrifugation, and taking the filtrate; 3) subjecting the filtrate to ultrahigh-speed centrifugation, and taking the supernatant; 4) subjecting the supernatant to ultrafiltration, and the filtrate comprises the extracellular vesicles of Bifidobacterium animalis lactis BL-99.

7. Use according to claim 6, wherein, The method further has one or more technical features selected from (i)-(iv): (i) the pore size of the ultrafiltration centrifugation is 100 KDa; (ii) the ultrafiltration centrifugation is performed at a rotation speed of 4000-6000 g (preferably 5000 g); (iii) the time of the ultrafiltration centrifugation is 5-15 min, preferably 10 min; (iv) the ultrafiltration centrifugation treatment is achieved by using an ultrafiltration centrifuge tube (such as Amicon Ultra-15 ultrafiltration centrifuge tube).

8. Use according to any one of claims 6-7, wherein, The method further has one or more technical features selected from the following (i)-(v): (i) the low speed centrifugation treatment is performed twice, the first low speed centrifugation is performed at 3500-4000g (preferably 3600g) for 20-40min (preferably 30min), and the second low speed centrifugation is performed at 5000-5500g (preferably 5000g) for 20-40min (preferably 30min); (ii) the high speed centrifugation treatment is performed at 10000-12000g (preferably 11000g) for 50-70min (preferably 60min); (iii) the ultra-high speed centrifugation treatment is performed twice, the first ultra-high speed centrifugation is performed at 110000-130000g (preferably 120000g) for 80-100min (preferably 90min), and the second ultra-high speed centrifugation treatment is performed at 110000-130000g (preferably 120000g) for 80-100min (preferably 90min); (iv) the ultrafiltration treatment is achieved by using an ultrafiltration membrane; (v) the pore size of the ultrafiltration treatment is 0.22um.

9. Use according to any one of claims 6-8, wherein, The method further has one or more technical features selected from the following (i)-(iv): (i) the low speed centrifugation treatment is performed at a temperature of 4℃; (ii) the high speed centrifugation treatment is performed at a temperature of 4℃; (iii) the ultra-high speed centrifugation treatment is performed at a temperature of 4℃; (iv) in step 4), after the supernatant is subjected to ultrafiltration treatment, further comprising: diluting the filtrate with PBS buffer, and the PBS dilution liquid contains the animal Bifidobacterium lactis BL-99 extracellular vesicles.

10. Use according to any one of claims 1 to 9, wherein, The use further has one or more technical features selected from the following (i)-(ii): (i) the particle size of the animal Bifidobacterium lactis BL-99 extracellular vesicles is 50-100nm; (ii) the bacterial suspension of the animal Bifidobacterium lactis BL-99 is obtained by pre-fermentation of the animal Bifidobacterium lactis BL-99.

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