Intestinal Rosebawnella strain as well as application and composition of intestinal Rosebawnella strain

Abstract

The invention belongs to the technical field of biological medicines, and relates to an intestinal Rosebariella sp. Strain as well as an application and a composition of the intestinal Rosebariella sp. Strain. The bacterial strain is Roseburia intestinalis, is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on July 22, 2024, and has the preservation number of CGMCC NO.46067. The invention also discloses a preparation method of the Roseburia intestinalis strain. The intestinal Rosebariella sp. Strain provided by the invention has an effect of reducing Abeta in host brain hippocampus and prefrontal cortex, alleviates learning memory and cognitive dysfunction of mice with Alzheimer's disease, and plays a role in alleviating Alzheimer's disease.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to an intestinal Roseburia rosea strain and a use and a composition thereof. Background Art

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease, with clinical manifestations of progressive memory and cognitive impairment, behavioral changes, etc. The core pathological characteristics of AD are the abnormal aggregation of extracellular β-amyloid protein (Aβ), and the formation of neurofibrillary tangles by hyperphosphorylated tau protein in cells, which ultimately lead to synaptic dysfunction and neuronal death. Current studies believe that AD is not only caused by local lesions in the brain, but also that intestinal flora disorders are an important factor leading to AD. Intestinal microorganisms participate in the occurrence and development of AD through the "gut-brain axis", including Aβ deposition, increased tau protein phosphorylation, neuroinflammation, metabolic dysfunction and oxidative stress. Studies have confirmed that individuals with intestinal microecological disorders caused by intestinal diseases have a higher incidence of AD. Changes in the composition and diversity of intestinal flora increase the permeability of the intestinal barrier and the production of inflammatory factors, which in turn activate microglia and cause neuronal death. In addition, reactive microglia activate astrocytes, exacerbating neuroinflammation and damage to the blood-brain barrier, affecting cortical myelination, inhibiting hippocampal neurogenesis, and ultimately causing cognitive function and memory impairment.

[0003] The pathological connection between intestinal flora imbalance and AD and its potential mechanism have become a hot topic in recent years. In-depth research on the causal relationship between intestinal flora and AD and its host interaction mechanism, and the development of new probiotics that are effective in alleviating AD have practical application value. Summary of the invention

[0004] In order to effectively treat AD, a neurodegenerative disease, the present invention provides an intestinal Roseborgia strain, which is an intestinal Roseborgia strain. Roseburia intestinalis , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 22, 2024, and the deposit number is CGMCC NO.46067.

[0005] The invention also provides the use of the strain, which is used for preparing a composition for treating Alzheimer's disease.

[0006] The present invention further provides a composition, which contains the intestinal Roseburia strain.

[0007] Compared with the prior art, the beneficial effects of the present invention are: the intestinal Roseburia strain provided by the present invention can be used to prepare a composition for treating Alzheimer's disease, has a reducing effect on Aβ in the host's brain hippocampus and prefrontal cortex, alleviates the learning, memory and cognitive dysfunction of AD mice, and plays a role in alleviating AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is a colony morphology diagram of the strain HA3781 in the example of the present invention; Figure 2 3 is a transmission electron microscopic morphology image of the strain HA3781 in the embodiment of the present invention; Figure 3 This is a flow chart of the live bacteria gavage animal experiment of the strain HA3781 in the embodiment of the present invention; Figure 4 Schematic diagram of the results of the Y-maze spatial recognition experiment of experimental mice, including: (a) the number of explorations of the new arm; (b) the distance explored in the new arm; **P < 0.01, ***P < 0.001; Figure 5 This is a schematic diagram of the results of the novel object recognition experiment of experimental mice - recognition index, ** P < 0.01, **** P < 0.0001; Figure 6 The expression of Aβ and Aβ1-42 proteins in the hippocampus of experimental mice, including: (a) Aβ and Aβ1-42 protein expression strip diagram in the hippocampus; (b) grayscale analysis result of Aβ protein expression in the hippocampus; (c) grayscale analysis result of Aβ1-42 protein expression in the hippocampus; * P < 0.05, ** P < 0.01; Figure 7 The results of HE staining of the colon of experimental mice. DETAILED DESCRIPTION

[0009] The present invention is described more completely and clearly below in conjunction with the accompanying drawings and specific embodiments. The described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0010] Improved GAM liquid medium (g / L): 0.5 g D-mannose, 0.5 g D-fructose, 0.5 g galactose, 0.5 g trehalose, 0.5 g inulin, 0.5 g palatinose, 0.5 g rhamnose, 0.5 g arginine, 0.5 g tryptophan, 0.5 g cysteine, heat and stir to fully dissolve. Add 60 g mGAM broth, 2.46 g sodium acetate, 2 g sodium bicarbonate, 1.5% agar powder on the solid plate (1% agar powder on the solid slope), heat and stir to dissolve with a temperature-controlled magnetic stirrer, remove the rotor, add 5% (v / v) clarified rumen fluid, 0.001 g resazurin, 0.01 g hematoxylin, distilled water to 1L, pH 7.2±0.1, sterilize at 115℃ for 25 minutes before use.

[0011] Example 1 Acquisition of strain HA3781 1. Isolation and identification of strains The strain was anaerobically isolated by the inventor of the present invention on August 8, 2023 from fresh feces provided by the feces donor after signing an informed consent form, and was marked as strain HA3781.

[0012] Colony morphology: In modified GAM broth medium (each liter of modified GAM broth medium contains: 0.5 g D-mannose, 0.5 g D-fructose, 0.5 g galactose, 0.5 g trehalose, 0.5 g inulin, 0.5 g palatinose, 0.5 g rhamnose, 0.5 g arginine, 0.5 g tryptophan, 0.5 g cysteine), heat and stir to fully dissolve. Add 60 g mGAM broth, 2.46 g sodium acetate, 2 g sodium bicarbonate, 1.5% agar powder to the solid plate (1% agar powder to the solid slope), heat and stir to dissolve with a temperature-controlled magnetic stirrer, remove the rotor, and add 5% (v / v) clarified rumen fluid, 0.001 g resazurin, 0.01 g hematoxylin, and distilled water to 1L, pH 7.2±0.1, sterilized at 115℃ for 25 minutes before use. ) grows fast, cultured at 37℃ under anaerobic conditions for 1-2 days, colony diameter is 1-2 mm, white, smooth, such as Figure 1 As shown. Under transmission electron microscopy, the cell morphology was observed to be slightly curved rod-shaped with flagella, such as Figure 2 shown.

[0013] Molecular identification method: Full sequence analysis of 16S rRNA, the sequence of which is shown in the sequence table SEQ ID NO.1. The Latin classification is named Roseburia intestinalis, the corresponding Chinese name is Enterobacteriaceae, and the strain HA3781 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 22, 2024, with the deposit number CGMCC NO.46067, and the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The 16S rRNA sequence homology of the strain HA3781 and Enterobacteriaceae is 99.86%.

[0014] 2. Cultivation and preservation methods of strain HA3781 The strain HA3781 of the present invention is activated on a modified GAM solid medium, anaerobically cultured at 37°C for 1-2 days, and then a single colony is picked and inoculated into a modified GAM liquid medium for seed liquid culture at 37°C for 1-2 days. Subsequently, the seed liquid is inoculated into a modified GAM liquid medium with the same ingredients at an inoculation amount of 1-5%, and anaerobically cultured at 37°C for 1-2 days.

[0015] The long-term preservation method of the strain HA3781 of the present invention is as follows: the bacterial solution is centrifuged at 4°C and 4000 rpm for 15 minutes to collect the precipitated cells, and after washing with a PBS buffer solution, the cells are resuspended in a solution containing 15%-25% skim milk powder at a ratio of 5:1, freeze-dried and sealed for preservation, or frozen in liquid nitrogen in a preservation solution containing 30% glycerol and stored at -80°C.

[0016] Example 2 Use and effect of strain HA3781 of the present invention 1. Animal Experiment Design The experiment used 5xFAD mice, male, 4 months old. The frequency of gavage was once a day, and the treatment group was gavaged 10 times a day. 9 The control group was given an equal dose of PBS buffer solution every day. The total experimental period was 8 weeks. Figure 3 shown.

[0017] End point sampling: 1) Plasma samples (anticoagulated blood was centrifuged and the supernatant was collected); 2) Brain tissue was separated and quickly frozen in liquid nitrogen, and then transferred to -80℃ for cryopreservation; 3) The small intestine, cecum, colorectum and cecal contents were separated and quickly frozen in liquid nitrogen; 4) 2-3 mice were randomly selected from each group for cardiac perfusion, and the small intestine and colorectum were fixed with polyformaldehyde and then frozen and sectioned, and immunofluorescence staining was performed after sectioning.

[0018] 2. Detection indicators (1) Behavioral indicators: Y-maze-spatial recognition test to detect the cognitive function of mice, and novel object recognition test to detect the learning and memory ability of mice; (2) The levels of Aβ and Aβ1-42 in the hippocampus of the brain; (3) Integrity of colon mucosal tissue.

[0019] 3. Experimental results (1) Oral administration of live bacteria of strain HA3781 alleviates cognitive dysfunction in AD mice The Y-maze spatial recognition experiment can detect the behavior and cognitive process of animals in a spatial environment. Figure 4 As shown in the figure, the number of explorations and exploration distances of the new arm of AD mice were significantly lower than those of WT mice. Oral administration of live bacteria of strain HA3781 had no significant effect on the new arm exploration behavior of WT mice. In AD mice, the number of explorations and exploration distances of the new arm were further reduced after oral administration of live bacteria of strain HA3781 compared with the control group. This indicates that oral administration of live bacteria of strain HA3781 improves cognitive dysfunction in AD mice.

[0020] (2) Oral administration of live bacteria of strain HA3781 improves the learning and memory ability of AD mice The novel object recognition experiment can test the learning and memory ability of animals. Figure 5 As shown: The number of novel object recognitions of AD mice was significantly lower than that of WT mice. Oral administration of live bacteria of strain HA3781 had no significant effect on the novel object exploration behavior of WT mice. In AD mice, the number of explorations of mice to novel objects further increased after oral administration of live bacteria of strain HA3781 compared with the control group. This indicates that oral administration of live bacteria of strain HA3781 can improve the learning and memory ability of AD mice.

[0021] (3) Oral administration of live bacteria of strain HA3781 reduces Aβ content in the hippocampus of AD mice Abnormal Aβ aggregation is one of the core pathological features of AD. Aβ aggregation begins to appear in the brain of 5xFAD mice after 2 months of age. Aβ1-42 is the more neurotoxic subtype of Aβ. The content of Aβ and Aβ1-42 in the hippocampus of mice was detected. Figure 6 As shown in the figure, the levels of Aβ and Aβ1-42 in WT mice were low and their expression was not detected. In contrast, the expression of Aβ and Aβ1-42 in the hippocampus of AD mice was significantly higher than that of WT mice. After oral administration of live bacteria of strain HA3781, the expression of Aβ and Aβ1-42 in the hippocampus of AD mice was significantly reduced.

[0022] (4) Oral administration of live bacteria of strain HA3781 alleviates colon mucosal damage in AD mice The results of HE staining of mouse colon are as follows Figure 7 As shown in the figure, the colonic epithelial mucosa of WT mice was intact, with abundant goblet cells, regular and neatly arranged crypts, and no inflammatory cell infiltration. The colonic epithelial mucosa of AD mice was intact, but the crypts were loosely arranged and lymphocyte infiltration was present. After oral administration of live bacteria of strain HA3781, intestinal inflammation and colonic mucosal damage in AD mice were improved.

Claims

1. An intestinal Roseburia strain, characterized in that: Roseborgia intestinalis Roseburia intestinalis, It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 22, 2024, and the deposit number is CGMCC NO.46067.

2. The use of the intestinal Roseburia strain according to claim 1, characterized in that: The strain is used for preparing a composition for treating Alzheimer's disease.

3. A composition, characterized in that: The composition comprises the enteric Roseburia strain of claim 1.

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