Bacillus licheniformis F0726 for improving neuroinflammation of Alzheimer disease and application of bacillus licheniformis F0726

Abstract

The invention provides bacillus licheniformis F0726 for improving neuroinflammation of Alzheimer's disease and application of the bacillus licheniformis F0726. Relates to the microbial field. Experiments prove that the bacillus licheniformis F0726 can improve spatial learning and memory impairment of AD model mice and reduce TNF-alpha and IL-1beta in serum of the AD mice in Alzheimer's disease model mice induced by injecting A beta oligomer into the brain; according to the invention, the content of IL-10 is increased, the alteration of AD mouse intestinal flora is improved, and a new way and potential probiotic resources are provided for developing medicines related to prevention and treatment of Alzheimer's disease.

Description

Technical Field

[0005] The invention belongs to the field of microorganisms, and specifically relates to a strain of Bacillus licheniformis F0726 for improving neuroinflammation in Alzheimer's disease and an application thereof. Background Art

[0006] Bacillus licheniformis is an aerobic Gram-positive bacterium widely found in various ecological environments such as water bodies and soil. It is a common endophytic bacterium in plants, especially in the roots and stems of plants. Bacillus licheniformis competes with pathogens for nutrients and living space, produces metabolites that inhibit the growth of pathogens, secretes lytic substances that dissolve the cell walls or cell membranes of pathogens, and even promotes plant growth and induces plant resistance, thereby playing its role in preventing and curing diseases. Bacillus licheniformis does not pollute the environment, is harmless to humans and animals, has strong environmental adaptability, can produce spores with strong stress resistance, and is extremely easy to isolate and culture. It is considered to be an ideal plant disease biocontrol bacterium.

[0007] Alzheimer's disease (AD) is the most common type of dementia. It is a degenerative disease of the central nervous system characterized by progressive cognitive dysfunction and behavioral impairment. In recent years, there have been more and more studies on the treatment of AD, but the treatment of AD is still difficult. AD patients generally have symptoms such as cognitive impairment, behavioral disorders, memory loss, language disorders, and personality changes. Studies have found that the pathological characteristics of patients with Alzheimer's disease include senile plaques formed by Aβ deposition, neurofibrillary tangles produced by hyperphosphorylation of tau protein, and neuronal loss mainly in the hippocampal cortex. At present, the main treatment drugs for Alzheimer's disease are acetylcholinesterase inhibitors (tacrine, donepezil, capracin, galantamine, etc.) and aspartate receptor antagonists (memantine), but these drugs will more or less produce some side effects, such as strong hepatotoxicity, nausea and vomiting, etc. Summary of the invention

[0008] The present invention aims to provide a strain of Bacillus licheniformis F0726 capable of improving neuroinflammation in Alzheimer's disease and its application. The Bacillus licheniformis F0726 can improve the spatial learning ability and memory impairment of AD mice, improve the level of inflammatory factors in the serum of AD mice, and regulate the homeostasis of the intestinal flora of AD mice.

[0009] The present invention provides a strain of Bacillus licheniformis F0726, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 18, 2024; the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou; the deposit number is GDMCC No.64432.

[0010] The above-mentioned method for culturing Bacillus licheniformis comprises the following steps:

[0011] (1) inoculating the above-mentioned Bacillus licheniformis into a 5 ml LB liquid test tube, and culturing at 37° C. and 200 r / min for 12 hours to prepare a seed solution;

[0012] (2) The seed liquid prepared in step (1) was inoculated into 100 mL of LB liquid fermentation medium at a volume ratio of 3%, and cultured at 37° C. and 200 r / min for 24 h; the LB medium had the following components per liter: 10 g of peptone, 5 g of yeast powder, and 10 g of sodium chloride, and water was added to make the volume to 1000 ml.

[0013] (3) Dilute the fermentation solution to a viable bacterial count of 1×10 8 CFU / mL~1×10 10 CFU / mL: Further, dilute with normal saline.

[0014] Furthermore, the Bacillus licheniformis F0726 is used to improve spatial learning and memory disorders in Alzheimer's disease mice.

[0015] Furthermore, the Bacillus licheniformis F0726 is used to increase the IL-10 content in the serum of Alzheimer's disease mice.

[0016] Furthermore, the Bacillus licheniformis F0726 is used to reduce the TNF-α content in the serum of Alzheimer's disease mice.

[0017] Furthermore, the Bacillus licheniformis F0726 is used to reduce the IL-1β content in the serum of Alzheimer's disease mice.

[0018] Furthermore, the Bacillus licheniformis F0726 is used to improve intestinal flora imbalance in Alzheimer's disease mice.

[0019] The invention provides a method for preparing Bacillus licheniformis F0726 into a dosage form of medicine, health care product or food, and a method for preparing a dosage form of Bacillus licheniformis F0726 having a viable bacterial count of 1×10 8 CFU / mL~1×10 10 CFU / mL.

[0020] The technical solution of the present invention has the following advantages:

[0021] The present invention provides a strain of Bacillus licheniformis F0726 capable of reducing neuroinflammation in Alzheimer's disease, and relates to the field of microorganisms. The present invention proves through experiments that Bacillus licheniformis F0726 can improve the spatial learning and memory disorders of AD model mice induced by brain injection of Aβ oligomers, reduce TNF-α and IL-1β in the serum of AD mice; increase IL-10 content, and improve the intestinal flora imbalance of AD mice, providing a new way for the development of drugs related to the prevention and treatment of Alzheimer's disease, as well as a potential probiotic resource. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is the movement path diagram of the water maze experiment of AD mice treated with Bacillus licheniformis F0726;

[0024] Figure 2 The statistical results of the effect of Bacillus licheniformis F0726 intervention on the escape time of the water maze test in AD mice;

[0025] Figure 3 The statistical results of the effect of Bacillus licheniformis F0726 intervention on the number of crossing platforms in the water maze test of AD mice;

[0026] Figure 4 The statistical results of Bacillus licheniformis F0726 intervention treatment on serum IL-10 content in AD mice;

[0027] Figure 5 The statistical results of Bacillus licheniformis F0726 intervention treatment on TNF-α content in serum of AD mice;

[0028] Figure 6 The statistical results of Bacillus licheniformis F0726 intervention treatment on serum IL-1β content in AD mice;

[0029] Figure 7 The results of Bacillus licheniformis F0726 intervention treatment on the species richness of intestinal flora in AD mice. DETAILED DESCRIPTION

[0030] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0031] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the art can be used. The raw materials or instruments used are all conventional products that can be obtained commercially, including but not limited to the raw materials or instruments used in the examples of this application.

[0032] Studies have shown that the increase in Aβ protein is the main cause of AD. Therefore, the current brain injection of Aβ42 oligomers can induce AD ​​symptoms in mice. This injection method can simulate changes at the molecular and behavioral levels, such as weakened learning and memory abilities. This modeling method has a short modeling time, but there are large individual differences, and the injection method is more skill-intensive. The increase in the content of Aβ protein in the mouse brain can induce a series of other symptoms, such as oxidative stress, inflammatory response, etc., which ultimately lead to cell death.

[0033] The following will explain in detail a strain of Bacillus licheniformis F0726 that can improve neuroinflammation in Alzheimer's disease and its application in combination with specific experiments.

[0034] Example 1

[0035] 1. Experimental Materials and Instruments

[0036] 1.1 Experimental animals

[0037] C57BL / 6 male mice, 8 weeks old, weighing 20-25 g, were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. There were 6 mice per cage, and all were housed in an environment with a temperature of 22-25°C, a 12-h circadian rhythm, and free access to food and water.

[0038] 1.2 Drugs and main reagents

[0039] Bacillus licheniformis F0726 has been deposited in Guangdong Microbiological Culture Collection Center with the deposit number: GDMCC No. 64432; Aβ42 protein used for modeling was purchased from Shanghai Jier Biochemical Co., Ltd. IL-10, TNF-α, and IL-1β detection kits were purchased from Solarbio.

[0040] 2.1 Isolation and preparation of Bacillus licheniformis

[0041] Wine lees samples were collected from a winery in Tianjin, quickly placed in an ice box and sent to the laboratory for strain isolation. The wine lees samples were treated in a 60°C water bath for 10 minutes, and then the extract was diluted in a gradient and evenly applied to the LB solid culture medium plate, cultured at 37°C for 24 hours, and a single colony was picked and inoculated on the LB culture medium plate for continued culture. After repeated streaking and purification on the LB culture medium plate, multiple pure colonies were obtained. The pure cultured strains were inoculated into liquid LB culture medium for culture, and then 30% glycerol was added and stored in a -80°C refrigerator. One of the strains was named F0726.

[0042] Strain identification (BGI): Genomic DNA was extracted and PCR amplification was performed using a primer pair consisting of 16sF and 16sR (16sF: 5'-AGAGTTTGATCCTGGCTCAG-3'; 16sR: 5'-AGAAAGGAGGTGATCCAGC-3', etc.). The PCR reaction system and reaction procedure are detailed in Table 1.

[0043] Table 1

[0044]

[0045]

[0046] Then the amplified product was sequenced, and the 16S rDNA sequence was compared and analyzed for homology in the NCBI library using the BLAST program, and the homology with the Bacillus licheniformis KK7-12 gene for 16S ribosomal RNA, partial sequence reached 99%.

[0047] Based on the above identification results, strain F0726 was identified as Bacillus licheniformis.

[0048] (1) Inoculating Bacillus licheniformis F0726 into a 5 mL LB liquid test tube, culturing at 37° C. and 200 rpm for 12 hours to prepare a seed solution;

[0049] (2) inoculating 3% of the seed solution prepared in step (1) into 100 ml of LB liquid fermentation medium at a volume ratio, and culturing at 37° C. and 200 r / min for 24 h;

[0050] (3) Dilute the fermentation broth with saline until the viable count is 1×10 8 CFU / mL~1×

[0051] 10 10 CFU / mL.

[0052] 2.2 Establishment of AD mouse model induced by brain injection of Aβ42 and experimental grouping

[0053] The mice were randomly divided into a normal group, a model group, and a Bacillus licheniformis group, with 8 mice in each group.

[0054] The AD model group mice and the drug-treated group mice were injected with Aβ42 oligomer protein into the brain to establish the AD mouse model; the normal group mice only received the same volume of normal saline injection into the brain.

[0055] The mice in the drug-treated group were intragastrically administered with Bacillus licheniformis one week before brain injection, with a dose of 1×10 9 CFU / mL, the dose was 0.2ml / d / mouse for 21 consecutive days. The model group mice and the normal group mice were gavaged with the same volume of normal saline every day.

[0056] 2.3 Water maze experiment

[0057] The Morris water maze system mainly includes a water maze device, a water maze image automatic acquisition system and a software analysis system. The pool is divided into four areas, namely the first quadrant (northeast), the second quadrant (southeast), the third quadrant (southwest) and the fourth quadrant (northwest). Before the experiment, a certain amount of water was injected into the pool, the heating device was turned on, and the water temperature was controlled at 25±1℃. On the first day, the platform was placed in the middle of the fourth quadrant 1.5cm above the water surface, and the mice were placed in the second quadrant facing the pool wall, so that the mice could find the platform in the pool. If the platform was not found, the mice were artificially guided to stay on the platform for 10s. After two to six days, the platform was placed 1.5cm underwater and the operation of the first day was repeated. The time required for the mouse to find the hidden platform and stay on it for 5s was recorded as the incubation period. Each mouse was trained four times a day, with an interval of 15-20min between each training to ensure that the mouse had enough rest time.

[0058] 24 hours after the last hidden platform navigation experiment, keep the control, water depth, light, temperature, humidity and other external conditions unchanged, and only remove the water maze platform. Place the mouse facing the wall of the second quadrant of the water maze pool and gently put it into the water. Record the mouse's swimming trajectory in the water within 90 seconds, the percentage of the distance in the fourth quadrant, and the number of times it crosses the platform position.

[0059] 2.4 Mouse serum sampling

[0060] Blood was collected from mice after euthanasia, placed in a 4°C refrigerator for 30 min, and centrifuged at 3000-3500 rpm for 15 min. The supernatant was serum and stored in a -80°C refrigerator.

[0061] 2.5 Determination of inflammatory factors in serum

[0062] The experiment was carried out according to the steps of the kit, the protein concentration of the sample was measured, and the IL-10 content, IL-1β activity and TNF-α content of the sample were further calculated based on the protein concentration.

[0063] 2.6 Stool sample processing and DNA extraction, PCR amplification, quantification and sequencing

[0064] After feces were collected, they were immediately stored in a -80°C refrigerator. The fecal genome was extracted using the FastDNA Spin kit (MP Biomedicals, catalog no. 6560-200) and analyzed and counted using the MicroCloud platform.

[0065] 2.7 Statistical analysis

[0066] The experimental data were processed with Graphpad Prism 8.0 software. All data were expressed as mean ± standard deviation. The comparison between two groups of data was performed with t test. Multiple groups of data were analyzed with analysis of variance (ANOVA), and the Q test was used for significant differences. P < 0.05 was considered to be significantly different.

[0067] 3. Experimental results

[0068] 3.1 Water maze test results

[0069] Comparison of the average escape latency of each experimental group over 6 days. Figure 2 As shown. Figure 3 The experimental results showed that after 6 days of training, the difference between the model group and the wild group became more and more obvious, indicating that the brain injection of Aβ42 oligomers was successful in modeling. The latency time of mice in the model group was significantly higher than that in the drug-treated group, and the latency of each group treated with drug therapy shortened with the increase in the number of training days. And compared with the control group, the drug-treated group showed significant differences on the 5th and 6th days. This shows that the administration of Bacillus licheniformis provided by the present invention has an improving effect on the memory behavior ability of mice. After removing the platform, compared with the model group, the number of times the mice in the drug-treated group crossed the platform in the Morris water maze increased significantly. The results of the spatial exploration experiment showed that Bacillus licheniformis F0726 has a trend of improving the spatial position memory ability of AD model mice. See the results. Figure 1 .

[0070] 3.2 Effect of Bacillus licheniformis F0726 on serum IL-10 levels in mice

[0071] Figure 4The statistical results of the IL-10 content in mouse serum show that compared with the wild-type group, the IL-10 content in the model group mice was lower, indicating that brain injection of Aβ oligomers can induce AD ​​symptoms, while Bacillus licheniformis F0726 increased the IL-10 content in mouse serum. This indicates that Bacillus licheniformis F0726 can reverse the IL-10 content in the serum of AD model mice and alleviate the neuroinflammation caused by Aβ oligomers.

[0072] 3.3 Effect of Bacillus licheniformis F0726 on TNF-α content in mouse serum

[0073] Figure 5 The statistical results of the SOD enzyme activity in the serum of mice show that compared with the wild-type group, the TNF-α content of the model group mice was significantly increased, while the intervention of Bacillus licheniformis F0726 significantly increased the reduction of TNF-α content in the model mice. This indicates that Bacillus licheniformis F0726 can reduce the TNF-α content in the serum of AD model mice and alleviate the neuroinflammation caused by Aβ oligomers.

[0074] 3.4 Effect of Bacillus licheniformis F0726 on serum IL-1β levels in mice

[0075] Figure 6 The statistical results of the IL-1β content in the serum of mice show that compared with the wild-type group, the IL-1β content in the model group mice increased significantly, while the intervention with Bacillus licheniformis F0726 significantly attenuated the increase in the IL-1β content in the model group mice. This indicates that Bacillus licheniformis F0726 can reduce the IL-1β content in the serum of AD model mice and alleviate the neuroinflammation caused by Aβ oligomers.

[0076] 3.5 Effects of Bacillus licheniformis F0726 on the intestinal flora of mice

[0077] Figure 7 The statistical results of the species richness of intestinal flora in mouse feces show that compared with the wild group, the Bacteroidetes increased and the Firmicutes decreased in the feces of mice in the model group, while the intervention with Bacillus licheniformis F0726 reversed this trend to a certain extent, making the intestinal flora tend to be closer to that of normal mice.

[0078] In summary, Bacillus licheniformis F0726 can improve the spatial learning and memory disorders and autonomous exploration ability of AD model mice induced by brain injection of Aβ oligomers, increase the IL-10 in mouse serum, reduce TNF-α and IL-1β, and regulate the intestinal flora of AD mice. This proves that Bacillus licheniformis F0726 has a good preventive and therapeutic effect on Alzheimer's disease, can be used to prepare drugs, health products or foods related to the prevention or treatment of Alzheimer's disease, and has potential probiotic value.

[0079] Obviously, the above examples are only examples for clear explanation, and are not intended to limit the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. A strain of Bacillus licheniformis F0726 for improving neuroinflammation in Alzheimer's disease, characterized in that: The Bacillus licheniformis F0726 has been deposited in Guangdong Microbiological Culture Collection Center with the deposit number: GDMCC No.64432.

2. The use of Bacillus licheniformis F0726 according to claim 1, characterized in that: The application is the application of the Bacillus licheniformis F0726 in the preparation of a drug for preventing and treating Alzheimer's disease, wherein the number of viable bacteria of the Bacillus licheniformis F0726 in the drug is 1×10 8 CFU / mL~1×10 10 CFU / mL.

3. The use according to claim 2, characterized in that: The Bacillus licheniformis F0726 is used for preparing a medicine for improving spatial learning and memory disorders of Alzheimer's disease.

4. The use according to claim 2, characterized in that: The Bacillus licheniformis F0726 is used for preparing a drug for improving the level of inflammatory factors in serum of Alzheimer's disease.

5. The use according to claim 2, characterized in that: The Bacillus licheniformis F0726 is used for preparing a drug for regulating the homeostasis of intestinal flora in Alzheimer's disease.

6. The use of Bacillus licheniformis F0726 according to claim 1, characterized in that: The application includes: application of the Bacillus licheniformis F0726 in the preparation of medicines, foods and health products for improving learning and memory ability or regulating intestinal flora.

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