Bifidobacterium bifidum and macrophage co-culture, preparation method and application

Abstract

The present invention discloses a co - culture of Bifidobacterium bifidum and macrophages, a preparation method and an application, belonging to the field of biotechnology. The co - culture of Bifidobacterium bifidum and macrophages of the present invention is the product after co - culturing Bifidobacterium bifidum BD - 1 and macrophages. A preparation method of the co - culture of Bifidobacterium bifidum and macrophages disclosed by the present invention comprises the following steps: co - culture the bacterial suspension of Bifidobacterium bifidum BD - 1 and the macrophage suspension, then centrifuge and take the supernatant to obtain the product. The present invention discloses the application of the above co - culture in the preparation of products for promoting the growth and development of nerve cells. The co - culture of the present invention has an obvious promoting effect on the development indexes such as mRNA expression and protein translation of nerve cells, and can promote the morphological development of nerve cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a co-culture of Bifidobacterium bifidum and macrophages, a preparation method and an application thereof. Background Art

[0002] Intestinal microbiota is a very large family. Probiotics are an important part of intestinal microbiota and play important roles in anti-inflammation, anti-obesity, immune regulation, promoting neural development, etc. The gut microbiota can participate in brain neural development through the microbiota-gut-brain axis, and the gut microbiota directly affects the immune system. Probiotic-mediated immune activation may be a pathway for transmitting the effects of microorganisms to the central nervous system. After oral administration, probiotics enter the intestine, adhere to the lamina propria, lymph nodes, etc., and are recognized by macrophages and dendritic cells in the relevant lymphoid tissues in the intestine, stimulating the secretion and expression of cytokines such as IL-6, IL-10, IL-1β, etc., and then leading to functional changes in organs outside the gastrointestinal tract including the brain.

[0003] The neurodevelopmental level of infants is related to the quality of life of children throughout their lives, which not only affects children's future academic performance and mental health in adulthood, but also is related to a series of potential long-term adverse outcomes. Early life is both a critical period for human neurodevelopment and a critical period for gut microbiota establishment. The gut microbiota colonized in infancy may promote the development and functional expression of the nervous system and is crucial for their health.

[0004] There is still a gap in the field of probiotics promoting early-life neurodevelopment, and there is relatively little overall research on the prevention of long-term adverse neurodevelopmental outcomes by supplementing probiotics with neurodevelopment-regulating effects in early life. The applicant's previous study, "Lactobacillus Rhamnosus GG and Bifidobacterium Bifidum TMC3115 Can Affect the Development of Hippocampal Neurons Cultured in Vitro in a Strain-dependent Manner" (Ruyue Cheng et al., Probiotics Antimicrob Proteins. 2020 Jun; 12(2):589-599. doi: 10.1007 / s12602-019-09571-4.), explored whether Lactobacillus rhamnosus GG (LGG) and Bifidobacterium bifidum TMC3115 (TMC3115) would affect the development of hippocampal neurons in vitro morphologically or physiologically. The results showed that LGG and TMC3115 could affect neuronal viability and hippocampal synapse and functional development in a strain-dependent manner, but had no effect on the development of nerve cells. Patent CN114886929A discloses that Bifidobacterium bifidum B11 can significantly increase the cell viability of hippocampal neurons and enhance the relative expression levels of the genes of brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) and increase the secretion of BDNF whether it is active or inactivated. Similarly, this patent also does not mention the effect of Bifidobacterium bifidum B11 on the morphological development of nerve cells.

[0005] Therefore, providing a substance that can not only increase the viability of nerve yuan, enhance the relative mRNA expression levels of neuron-related genes, but also promote the morphological development of nerve cells has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] One of the objectives of the present invention is to provide a co-culture of Bifidobacterium bifidum and macrophages, which can significantly promote the development indicators such as mRNA expression and protein translation of nerve cells, and can promote the morphological development of nerve cells.

[0007] Another objective of the present invention is to provide a preparation method of the co-culture of Bifidobacterium bifidum and macrophages.

[0008] A third objective of the present invention is to provide the application of the co-culture of Bifidobacterium bifidum and macrophages.

[0009] To achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0010] A co - culture of Bifidobacterium bifidum and macrophages disclosed by the present invention, wherein the co - culture is the product after co - culturing Bifidobacterium bifidum BD - 1 and macrophages.

[0011] The Bifidobacterium bifidum BD - 1 of the present invention is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The address of the depositary institution is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is: March 11, 2022; the deposit number is CGMCC NO.24517, and the taxonomic name is: Bifidobacterium bifidum; the Latin name is Bifidobacterium bifidum.

[0012] In some embodiments of the present invention, the co - culture is the supernatant after co - culturing Bifidobacterium bifidum BD - 1 and macrophages.

[0013] In some embodiments of the present invention, the Bifidobacterium bifidum BD - 1 is viable bacteria or inactivated bacteria.

[0014] In some embodiments of the present invention, the macrophages are macrophages RAW264.7.

[0015] A method for preparing a co - culture of Bifidobacterium bifidum and macrophages disclosed by the present invention comprises the following steps: co - culture the bacterial suspension of Bifidobacterium bifidum BD - 1 and the macrophage suspension, then centrifuge and take the supernatant to obtain the co - culture.

[0016] The culture conditions in the present invention are conventional culture.

[0017] In one embodiment of the present invention, the culture is carried out at 37 ± 0.5 °C and 5% CO2.

[0018] Preferably, the culture time is 1 - 96 hours, more preferably 8 - 48 hours, further preferably 12 - 36 hours, and still further preferably 24 hours.

[0019] In some embodiments of the present invention, the amount of bacteria in the bacterial suspension of Bifidobacterium bifidum BD - 1 is 5×10 5 ~5×10 9 CFU / mL, preferably 5×10 7 CFU / mL.

[0020] In some embodiments of the present invention, the macrophages in the macrophage suspension are 5×10 3 ~5×10 7 cells / mL, preferably 5×10 5 cells / mL.

[0021] In some embodiments of the present invention, the volume ratio of the macrophage suspension to the bacterial suspension of Bifidobacterium bifidum BD-1 is 5 to 20:1, preferably 10:1.

[0022] The application of the above-mentioned co-culture of Bifidobacterium bifidum and macrophages disclosed by the present invention is the application in the preparation of products for promoting the growth and development of nerve cells.

[0023] The application of the above-mentioned co-culture of a Bifidobacterium bifidum and macrophages disclosed by the present invention is the application in the preparation of products for promoting early-life nerve development;

[0024] Preferably, it is the application in the preparation of drugs or foods for promoting early-life nerve development.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The co-culture of Bifidobacterium bifidum BD-1 and macrophages of the present invention can significantly improve the cell viability of primary hippocampal neurons, increase the relative mRNA expression levels of NGF, CREB, Drebrin, CFOS, PSD95, and SYP genes, and promote the expression of proteins related to the presynaptic membrane and postsynaptic membrane. And the applicant surprisingly found that the co-culture of Bifidobacterium bifidum BD-1 and macrophages of the present invention also has an unexpected effect of promoting the morphological development of nerve cells.

[0027] The co-culture of the present invention can be used to prepare related probiotic products for promoting early-life nerve and brain development. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Attached Figure 1 is a graph showing the effect of the co-culture of the present invention on the viability of primary hippocampal neurons; where *P < 0.05, ***P < 0.001;

[0029] Attached Figure 2 is a graph showing the results of the investigation of the immune activation of macrophages RAW264.7 by the co-culture of the present invention; if there is a statistical significance between groups, it is represented by different letters;

[0030] Attached Figure 3 is a graph showing the effect of the co-culture of the present invention on the mRNA expression of primary hippocampal neurons; if there is a statistical significance between groups, it is represented by different letters;

[0031] Appendix Figure 4 This is the result diagram of the effect of the co-culture of the present invention on the development of the pre- and post-synaptic membranes of primary hippocampal neurons; if there is a statistical significance between groups, it is represented by different letters.

[0032] Appendix Figure 5 This is the result diagram of the effect of the co-culture of the present invention on the protein expression of primary hippocampal neuron development.

[0033] Appendix Figure 6 This is the result diagram of the effect of the co-culture of the present invention on the growth of primary hippocampal neurons.

[0034] Among them, in Appendix Figure 2 -Appendix Figure 6 - Figure 6 , different letters indicate that there is a statistical significance in the differences between groups. Detailed implementation manners

[0035] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention. Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments and equipment used in this application can be obtained through market purchase or can be prepared by existing methods.

[0036] Example 1

[0037] This example discloses a preparation method of the co-culture of Bifidobacterium bifidum and macrophages of the present invention, which is specifically as follows:

[0038] 1. Preparation of bacterial liquid: Take the BD-1 bacterial suspension frozen at -80°C and spread it on the TOS agar medium. After subculturing until stable, scrape the strain and dissolve it in sterile physiological saline to make a bacterial suspension with a bacterial amount of 5×10 7 CFU / mL.

[0039] 2. Preparation of cell suspension: After resuscitation, the macrophage RAW264.7 is subcultured until stable to make a cell suspension with a cell density of 5×10 5 cells / mL.

[0040] 3. Co-culture: Mix the macrophage suspension and the bacterial suspension in a volume ratio of 10:1 and culture them. The culture conditions are: co-culture in a 37°C, 5% CO2 cell incubator for 24 hours, then collect the cell supernatant, centrifuge, and take the centrifuged supernatant to obtain the co-culture.

[0041] Example 2

[0042] This example discloses a method for preparing a co-culture of Bifidobacterium bifidum and macrophages of the present invention. Compared with Example 1, the Bifidobacterium bifidum BD-1 in this example is an inactivated bacterium, specifically as follows:

[0043] 1. Preparation of inactivated bacterial suspension: Take the BD-1 bacterial suspension stored at -80°C and spread it on TOS agar medium. After subculturing until stable, scrape the strain and dissolve it in sterile physiological saline to prepare a bacterial suspension with a bacterial count of 5×10 7 cells / mL. Heat it in a water bath at 65°C for 2 h to prepare an inactivated bacterial suspension.

[0044] 2. Preparation of cell suspension: After resuscitation and subculturing until stable, the macrophages RAW264.7 are made into a cell suspension with a density of 5×10 5 cells / mL.

[0045] 3. Co-culture: Mix the macrophage suspension and the inactivated bacterial suspension in a volume ratio of 10:1 and culture them. The culture conditions are: co-culture in a cell incubator at 37°C and 5% CO2 for 24 h, then collect the cell supernatant, centrifuge, and take the centrifuged supernatant to obtain the product.

[0046] Test Example 1

[0047] This test example discloses an investigation test on the promotion of neuronal development by the co-culture of Bifidobacterium bifidum and macrophages of the present invention.

[0048] 1. Preparation and inactivation of bacterial suspension

[0049] Take the BD-1 bacterial suspension stored at -80°C and spread it on TOS agar medium. After subculturing until stable, scrape the strain and dissolve it in sterile physiological saline to prepare a bacterial suspension with a bacterial count of 5×10 7 cells / mL. Half of it is heated in a water bath at 65°C for 2 h to prepare an inactivated bacterial suspension.

[0050] 2. Co-culture of bacterial suspension and macrophages RAW264.7

[0051] After resuscitation and subculturing until stable, the macrophages RAW264.7 are inoculated into a 24-well plate at a density of 5×10 5 cells / mL and 1 mL / well. Add the above-mentioned BD-1 live bacteria and inactivated bacterial suspension into the 24-well plate, 100 μL per well, and set three parallel wells. In addition, set two groups of positive controls and one group of blank control, namely 2 μg / mL PGN (peptidoglycan), 2 μg / mL LPS (lipopolysaccharide), and blank medium, 100 μL per well.

[0052] The cell supernatants were collected after co - culturing 24 - well plates in a 37°C, 5% CO₂ cell incubator for 24 h. After centrifugation, the subsequent co - culture intervention substances were obtained, which were respectively designated as: live BD - 1 - CM, inactivated BD - 1 - CM, PGN - CM, LPS - CM, CON - CM. Total RNA was extracted and reverse - transcribed.

[0053] 3. Preparation of primary hippocampal neurons

[0054] Within 24 h after birth, the hippocampal tissues of SD rats were dissected after decapitation. After trypsin digestion, the cell density was adjusted to 1×10 6 cells / mL with the planting solution, inoculated into 6 - well plates at a density of 2 mL / well and into 96 - well plates at a density of 100 μL / well, and cultured in a 37°C, 5% CO₂ cell incubator. After 24 h, it was replaced with serum - containing culture medium 1 containing 1% N2, 2% B 27 and double - antibody solution. After 48 h, it was replaced with serum - containing culture medium 2 containing 1% N2, 2% B 27 , 1.5‰ cytosine arabinoside and double - antibody solution. After 48 h, it was replaced with serum - free medium containing 1% N2. The whole process lasted for seven days until the neurons differentiated into obvious but not fully mature synaptic structures.

[0055] 4. Determination of the co - culture survival rate of co - culture supernatant and primary hippocampal neurons

[0056] When the primary hippocampal neurons were cultured to the seventh day, 10 μL of the above - mentioned co - culture intervention substances were added to the 96 - well plates, that is, the intervention amount of the co - culture intervention substances was 10%. Six parallel wells were set up in each group, and a control group without intervention and a blank group without cells were also set up. After incubating in a 37°C, 5% CO₂ cell incubator for 22 h, 10 μL of CCK8 reagent was added to each well, and the cell survival rate was measured after continuous incubation in the dark for 2 hours.

[0057] 5. RNA extraction and RT - PCR

[0058] When the primary hippocampal neurons were cultured to the seventh day, 200 μL of the above - mentioned co - culture intervention substances were added to the 6 - well plates, with three replicates in each group. After incubating in a 37°C, 5% CO₂ cell incubator for 24 h, total RNA of macrophages and primary hippocampal neurons was extracted according to the instructions of the cell total RNA isolation kit, and then according to iScript TMInstructions for cDNA Synthesis Kit: Prepare a reverse transcription reaction system with a total volume of 20 μL. Perform RT-PCR assays on cytokines IL-6, IL-10, IL-1β, TNF-α and neurodevelopmental indicators NGF, CREB, Drebrin, CFOS, PSD95, SYP. The primer sequences are shown in Table 1. The PCR reaction system is 5 μL of supermix, 1 μL of each upstream and downstream primer, 3 μL of double-distilled water, and 1 μL of template, with a total system of 10 μL. The reaction program is pre-denaturation at 98°C for 30 s; denaturation at 98°C for 15 s, annealing at 60°C for 20 s, for 39 cycles.

[0059] Table 1. RT-qPCR primer sequences

[0060]

[0061]

[0062] 6. Immunofluorescence staining of developmental proteins

[0063] When the primary hippocampal neurons are cultured to the seventh day, add 200 μL of the above co-culture intervention in a 6-well plate, with three replicates in each group. After incubating for 24 h in a cell incubator at 37°C and 5% CO2, fix the primary hippocampal neurons on cell slides with 4% paraformaldehyde. Perform immunofluorescence staining on the presynaptic and postsynaptic membrane development indicators PSD95, SYP and the neuronal maturity indicators MAP2, DCX respectively. After permeabilization, blocking, incubating with primary antibody, incubating with secondary antibody, TSA staining, washing, blocking, and nuclear staining according to the TSA double-staining procedure, seal the slides with a fluorescence mounting medium and collect images under a microscope.

[0064] 7. Results

[0065] 7.1 Cell viability

[0066] The results are as shown Figure 1 After culturing the co-culture supernatant of live and inactivated BD-1 bacteria with primary hippocampal neurons and comparing with the blank control and positive control groups, the cell viability of neurons increased. It indicates that the co-culture of Bifidobacterium bifidum BD-1 of the present invention with macrophages can promote the survival rate of neurons.

[0067] 7.2 Immunostimulation of macrophage RAW264.7

[0068] As shown Figure 2As shown, after co - culturing live and heat - inactivated Bifidobacterium bifidum BD - 1 with macrophage RAW264.7, compared with the blank control group, the expression of macrophages was activated, and the mRNA expression levels of cytokines IL - 6, IL - 10, IL - 1β, and TNF - α increased significantly. The activation effect was different from that of the positive control group, showing strain specificity. It indicates that the co - culture of Bifidobacterium bifidum BD - 1 of the present invention with macrophages can promote the immune activation of macrophage RAW264.7.

[0069] 7.3 Effects of co - culture intervention substances on mRNA expression of primary hippocampal neurons

[0070] As attached Figure 3 As shown, after the supernatant of co - culture of live Bifidobacterium bifidum BD - 1 with macrophage RAW264.7 (BD - 1 live - bacteria - CM) was used to intervene in primary hippocampal neurons, compared with the blank control group and two positive control groups, the nerve cells entered an activated state (increased CFOS mRNA expression), transcriptional regulation was enhanced (increased Drebrin and CREB mRNA expression), and the expression of nerve growth factor NGF mRNA increased.

[0071] After the supernatant of co - culture of heat - inactivated Bifidobacterium bifidum BD - 1 with macrophage RAW264.7 (BD - 1 heat - inactivated - bacteria - CM) was used to intervene in primary hippocampal neurons, compared with the blank control group and the PGN - CM positive control group, the expressions of NGF mRNA, CREB mRNA, Drebrin mRNA, and CFOS mRNA all increased; compared with the LPS - CM positive control, the expressions of NGF mRNA, Drebrin mRNA, and CFOS mRNA all increased.

[0072] The results show that the co - cultures of live / heat - inactivated Bifidobacterium bifidum BD - 1 of the present invention with macrophages can all promote the mRNA expression of primary hippocampal neurons.

[0073] 7.4 Effects of co - culture intervention substances on the development of pre - and post - synaptic membranes of primary hippocampal neurons

[0074] As attached Figure 4 As shown, after the supernatant of co - culture of live and heat - inactivated Bifidobacterium bifidum BD - 1 with macrophage RAW264.7 was used to intervene in primary hippocampal neurons, compared with the blank control group, the expressions of genes related to pre - synaptic and post - synaptic membranes of nerve cells, PSD95 and SYP mRNA, increased significantly, and the protein expression increased most significantly in the BD - 1 live - bacteria - CM group.

[0075] 7.5 Effects of co - culture intervention substances on the protein expression of primary hippocampal neuron development

[0076] As attached Figure 5As shown, after the co-culture supernatant of live and heat-inactivated Bifidobacterium bifidum BD-1 and macrophages RAW264.7 was used to intervene in primary hippocampal neurons, compared with the blank control group and two positive control groups, live BD-1-CM enhanced the expression of the neuronal maturation protein MAP2 and weakened the expression of the immature neuronal protein DCX. Live BD-1-CM significantly promoted the development and maturation of neurons. Compared with the positive control groups, heat-inactivated BD-1-CM also promoted the expression of the neuronal maturation protein MAP2.

[0077] 7.6 Effects of co-culture intervention substances on the growth of primary hippocampal neurons

[0078] As shown in the appendix Figure 6 After the co-culture supernatant of live Bifidobacterium bifidum BD-1 and macrophages RAW264.7 (live BD-1-CM) was used to intervene in primary hippocampal neurons, compared with the blank control group and two positive control groups, the axon length, soma area, number of primary roots, total length of primary processes, average synaptic length, and number of branching points of primary hippocampal neurons were all better.

[0079] After the co-culture supernatant of heat-inactivated Bifidobacterium bifidum BD-1 and macrophages RAW264.7 (heat-inactivated BD-1-CM) was used to intervene in primary hippocampal neurons, compared with the two positive control groups, the axon length, soma area, number of primary roots, total length of primary processes, and number of branching points of primary hippocampal neurons were all better.

[0080] The results show that the co-culture of Bifidobacterium bifidum BD-1 of the present invention and macrophages can promote the morphological development of the hippocampus.

[0081] The above embodiments are only preferred embodiments of the present invention and are not used to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. Use of a co - culture of Bifidobacterium bifidum and macrophages, characterized in that, Use in the preparation of products for promoting the growth and development of nerve cells; the co-culture is the product after co-culturing Bifidobacterium bifidum ( Bifidobacterium bifidum) the product after co-culturing BD-1 and macrophages; The Bifidobacterium bifidum ( Bifidobacterium bifidum) The bacterial count in the BD-1 bacterial suspension is 5×10 7 CFU / mL; The macrophages in the macrophage suspension are 5×10 5 cells / mL; The volume ratio of the macrophage suspension to the bacterial suspension of Bifidobacterium bifidum ( Bifidobacterium bifidum) BD-1) is 10:

1.

2. Use of a co-culture of Bifidobacterium bifidum and macrophages, characterized in that, Use in the preparation of drugs or foods for promoting early neurodevelopment; the co-culture is the product of co-culturing Bifidobacterium bifidum ( Bifidobacterium bifidum) the product after co-culturing BD-1 and macrophages; The Bifidobacterium bifidum ( Bifidobacterium bifidum) The bacterial count in the BD-1 bacterial suspension is 5×10 7 CFU / mL; The macrophages in the macrophage suspension are 5×10 5 cells / mL; The volume ratio of the macrophage suspension to the bacterial suspension of Bifidobacterium bifidum ( Bifidobacterium bifidum) BD-1) is 10:

1.

3. Use of a co-culture of Bifidobacterium bifidum and macrophages according to claim 1 or 2, characterized in that, The co-culture is the supernatant of the co-culture of Bifidobacterium bifidum ( Bifidobacterium bifidum) BD-1 and macrophages).

4. Use of a co-culture of Bifidobacterium bifidum and macrophages according to claim 1 or 2, characterized in that, The Bifidobacterium bifidum ( Bifidobacterium bifidum) BD-1 is a live bacterium or an inactivated bacterium.

5. Use of a co - culture of Bifidobacterium bifidum and macrophages according to claim 1 or 2, characterized in that, The macrophage is macrophage RAW264.

7.

6. Use of a co - culture of Bifidobacterium bifidum and macrophages according to claim 1 or 2, characterized in that, The preparation method of the co-culture of Bifidobacterium bifidum and macrophages comprises the following steps: Bifidobacterium bifidum ( Bifidobacterium bifidum ) After co - culturing the bacterial suspension of BD - 1 with the macrophage suspension, centrifuge and take the supernatant to obtain it.

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