Application of lactobacillus johnsonii in preparation of medicine for delaying senescence and / or improving cognitive impairment

Abstract

The invention relates to the technical field of medicines, in particular to application of lactobacillus johnsonii in preparation of medicines for delaying senescence and / or improving cognitive impairment, and the medicines have at least one of the following purposes: improving a cognitive function; prevention, alleviation, amelioration, adjuvant therapy or treatment of cognitive disorders; wherein the preservation number of the lactobacillus johnsonii is ATCC (American Type Culture Collection) 33200, and the preservation number of the lactobacillus johnsonii is CGMCC 33200. According to the invention, the brand new medicinal application of the lactobacillus johnsonii with the preservation number of ATCC 33200 in improvement of cognitive function or treatment of cognitive impairment is discovered and confirmed for the first time, so that the application range of the lactobacillus johnsonii is further expanded.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of Lactobacillus johnsonii in the preparation of anti-aging and / or cognitive impairment. Background Technology

[0002] Lactobacillus johnsonii ( Lactobacillus johnsonii Lactobacillus johnsonii is a recognized safe probiotic and has been included in China's "List of Microbial Strains that Can Be Used in Food". Currently, research and application of Lactobacillus johnsonii mainly focus on its effects on relieving anxiety and depression, improving gut health, antioxidation, anti-inflammation, and regulating immune function. Although Lactobacillus johnsonii has some applications in the food industry, its current scope of application is still relatively limited.

[0003] Therefore, further developing and expanding the application scope of Lactobacillus johnsonii is of great significance for its application in the development of pharmaceuticals, health products, or functional foods. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides the application of *Lactobacillus johnsonii* in the preparation of products for delaying aging and / or improving cognitive impairment. This invention is the first to discover and confirm a novel pharmaceutical use of *Lactobacillus johnsonii* with accession number ATCC 33200 in improving cognitive function or treating cognitive impairment, further expanding the application scope of *Lactobacillus johnsonii*.

[0005] In a first aspect, the present invention provides the use of *Lactobacillus johnsonii* in the preparation of pharmaceutical products. According to embodiments of the present invention, the pharmaceutical product has at least one of the following uses: improving cognitive function; preventing, alleviating, improving, adjunctive treatment of, or treating cognitive impairment; wherein the *Lactobacillus johnsonii* has the accession number ATCC 33200 (…). Lactobacillus johnsonii ).

[0006] According to embodiments of the present invention, the above-described uses may also have the following additional technical features: According to an embodiment of the present invention, the cognitive impairment is a cognitive impairment caused by aging.

[0007] According to an embodiment of the present invention, the cognitive impairment is a cognitive impairment induced by D-galactose.

[0008] According to an embodiment of the present invention, the improvement of cognitive function includes improving one or more of learning memory, spatial memory, working memory, and motor exploration ability.

[0009] In a second aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: live cells of *Lactobacillus johnsonii*; wherein the *Lactobacillus johnsonii* has the accession number ATCC 33200.

[0010] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0011] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a schematic diagram of the construction of the D-galactose-induced aging mouse model and the Lactobacillus johnsonii intervention experiment in Example 1 of the present invention; Figure 2 The figures shown are the open field test results for each group of mice in Example 1 of this invention. A represents the total distance traveled by each group of mice in the open field test; B represents the total distance traveled (mm) by each group of mice in the open field test; C represents the average speed (mm / s) of each group of mice in the open field test; D represents the fast movement time (s) of each group of mice in the open field test; E represents the slow movement time (s) of each group of mice in the open field test; * indicates... P <0.05, ** is P <0.01, *** is P <0.001; Figure 3 The figures shown are the results of the novel object recognition experiment for each group of mice in Example 1 of this invention. In Figure A, A is a schematic diagram of novel object recognition for each group of mice; in Figure B, B is a statistical result of the novel object recognition coefficients for each group of mice; and * indicates... P <0.05; Figure 4 The figures shown are the Y-maze-novel arm experiment results for each group of mice in Example 1 of this invention. A is a schematic diagram of the Y-maze for each group of mice; B is a statistical result of the time (s) for each group of mice to enter the novel arm; C is a statistical result of the number of times each group of mice entered the novel arm; * indicates... P <0.05, ** is P <0.01; Figure 5 The figures shown are the results of the Y-maze-autonomous alternation experiment for mice in each group in Example 1 of this invention, where * indicates... P <0.05. Detailed Implementation

[0012] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0013] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0014] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0015] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0016] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0017] use This invention discloses the use of *Lactobacillus johnsonii* in the preparation of pharmaceuticals. According to embodiments of the invention, the pharmaceutical product has at least one of the following uses: improving cognitive function; preventing, alleviating, improving, adjunctive treatment of, or treating cognitive impairment; wherein the *Lactobacillus johnsonii* (… Lactobacillus johnsonii The accession number for this work is ATCC 33200.

[0018] Therefore, this invention innovatively reveals a new pharmaceutical use of Lactobacillus johnsonii with accession number ATCC 33200 in improving cognitive impairment, giving new value to this known and safe probiotic, and providing a new drug development strategy that is derived from the human symbiotic flora, has a natural effect and high safety for improving cognitive function and solving cognitive impairment.

[0019] It should be noted that in this invention, the Lactobacillus johnsonii with accession number ATCC 33200 is a standard reference strain deposited and provided by the American Type Culture Collection (ATCC). Its safety has been widely recognized and it has been included in the "List of Microbial Strains that Can Be Used in Food" issued by the National Health Commission of China. This provides a very high safety basis for its development into a drug or health food for long-term use.

[0020] According to an embodiment of the present invention, the cognitive impairment is aimed at age-related cognitive decline. Therefore, a medicine containing *Lactobacillus johnsonii* can effectively intervene in cognitive degenerative changes caused by overall aging of the body.

[0021] In this article, the term "age-related cognitive impairment" refers to a syndrome of cognitive decline caused by degenerative changes in the central nervous system as an organism naturally ages. Its clinical manifestations mainly include a decline in the ability to learn new information, difficulties in memory consolidation and retrieval (learning and memory), a decline in spatial orientation and navigation (spatial memory), a weakening of short-term information processing and retention (working memory), and a decrease in autonomous exploration behavior and motor activity.

[0022] According to an embodiment of the present invention, the cognitive impairment is specifically targeted at cognitive impairment induced by D-galactose. Thus, a drug containing the *Lactobacillus johnsonii* can specifically reverse the accelerated aging process induced by D-galactose and its associated cognitive impairment.

[0023] In this paper, the term "D-galactose-induced cognitive impairment" refers to the cognitive dysfunction exhibited by an animal model (such as rodents) established through long-term, excessive administration of D-galactose to simulate accelerated aging. The mechanism of this model is that excessive D-galactose metabolism in vivo produces excessive reactive oxygen species (ROS), triggering oxidative stress, chronic inflammation, and mitochondrial dysfunction, thereby simulating neuronal damage and brain tissue pathological changes similar to natural aging in a short period of time. It should be noted that the cognitive impairment characteristics exhibited by this model include, but are not limited to: a decreased recognition index in the new object recognition test (suggesting impaired learning and memory), a decreased rate of spontaneous alternation and a reduced time for exploring new and different arms in the Y-maze test (suggesting impaired spatial and working memory), and a decrease in total movement distance and average speed in the open field test (suggesting impaired motor exploration ability).

[0024] According to embodiments of the present invention, the improvement in cognitive function includes improving one or more of learning memory, spatial memory, working memory, and motor exploration ability. Therefore, a drug containing the *Lactobacillus johnsonii* can improve various indicators related to cognitive function.

[0025] Pharmaceutical Composition This invention proposes a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises live cells of *Lactobacillus johnsonii*, wherein the *Lactobacillus johnsonii* has the accession number ATCC 33200. Thus, by adding live cells of *Lactobacillus johnsonii*, the pharmaceutical composition of this invention can be used to improve cognitive function; prevent, alleviate, improve, or assist in the treatment of cognitive impairment, etc. Furthermore, compared with traditional chemical drugs, it has significant advantages such as fewer potential side effects, high biocompatibility, and good patient acceptance, and has broad application prospects.

[0026] In this article, the term "live cells" refers to intact Lactobacillus johnsonii cells with metabolic activity and reproductive capacity. These cells exist in, but are not limited to, the following forms: (1) fermentation broth form, which refers to a fermentation culture medium containing a large number of live cells, including the cells and the fermentation medium; (2) cell suspension form, which refers to a suspension formed by resuspending fermented cells in physiological saline, buffer solution, or a protective agent (such as skim milk or sugars) after centrifugation, washing, and other steps; and (3) lyophilized powder form, which refers to a dry powder preparation made by dehydrating a cell suspension using freeze-drying technology, wherein the cells are in a dormant state but can recover their metabolic and reproductive capacity after rehydration.

[0027] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0028] Example 1: Construction of a D-galactose-induced aging mouse model and Lactobacillus johnsonii intervention experiment Five-week-old male C57BL / 6 mice were acclimatized for one week before the experiment began. They were randomly divided into four groups (n=11): a blank control group (Control group), a model group (D-gal group), a Lactobacillus johnsonii group, and a control group (D-gal group). L. johnsonii group) and heat-inactivated Lactobacillus johnsonii group (Inactivated- L. johnsoniiMice in each group were first given antibiotic solution (0.2 g / L ampicillin, 0.1 g / L vancomycin, 0.2 g / L metronidazole, 0.2 g / L neomycin) for 2 weeks to clear the intestinal flora. After 2 weeks, the treatment for each group was as follows: Blank control group: After continuous gavage of 200 μL / day of PBS for one week, 200 mg / kg of NaCl (solvent is deionized water) was injected subcutaneously every day. During this period, the gavage treatment continued until 13 weeks. Model group: After continuous gavage of 200 μL / day of PBS for one week, subcutaneous injection of 200 mg / kg D-gal (Sigma, catalog number G0750-50G) was started daily. During this period, the gavage treatment continued until 13 weeks. Lactobacillus johnsonii group: continuous gavage 200 μL / day, concentration 1×10 9 One week after administering CFU / mL Lactobacillus johnsonii bacterial culture (accession number ATCC 33200, Ningbo Mingzhou Biotechnology Co., Ltd., catalog number B86021), patients were given subcutaneous injections of 200 mg / kg D-gal daily, while gavage continued for up to 13 weeks. Heat-inactivated Lactobacillus johnsonii group: continuous gavage 200 μL / day, concentration 1×10 9 One week after administering CFU / mL heat-inactivated Lactobacillus johnsonii culture (heat inactivation treatment: 95℃ for 30 min), patients were given subcutaneous injections of 200 mg / kg D-gal daily, with gavage treatment continuing for up to 13 weeks.

[0029] During the experiment, mice in each group had free access to food and water, and their weight, food and water intake, and growth were monitored weekly. After 12 weeks, feces were collected, appearance scores were assessed, grip strength was measured using a dynamometer, and behavioral tests (including open field, new object recognition, and Y-maze) were performed. At the end of the experiment, each mouse was euthanized, and its liver and brain tissues were collected. One part of the tissues was used to prepare pathological sections, and the tissue samples were fixed in 4% paraformaldehyde solution. The other part was flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0030] Construction of a D-galactose-induced aging mouse model and experimental results of Lactobacillus johnsonii intervention Figure 1 .

[0031] 1. Open field experiment Each group of mice was placed in the center of a 50×50×50 cm open space box and allowed to explore freely for 5 minutes. Then, the timing was started, and the movement trajectory, total distance traveled (mm), average speed (mm / s), fast movement time (s), and slow movement time (s) of the mice were recorded within 10 minutes. After the test, the mice were removed and placed in a new cage. They were not put back into the original cage to avoid affecting the behavior of the mice being tested. The equipment was then cleaned with 75% ethanol. Each mouse was tested 5 times.

[0032] The results of the open field experiment for each group of mice are shown below. Figure 2 .

[0033] The results showed that, compared with the control group mice, the total open field distance, average speed, and fast movement time of the D-gal-induced mice were significantly reduced, while the slow movement time was significantly increased. However, intervention with Lactobacillus johnsonii bacterial solution could significantly reverse this trend (Lactobacillus johnsonii group), while heat-inactivated Lactobacillus johnsonii bacterial solution did not have this effect (heat-inactivated Lactobacillus johnsonii group), indicating that only live Lactobacillus johnsonii can play this role.

[0034] 2. New Object Recognition Experiment Each group of mice was placed in the center of a 50×50×50 cm open space box and allowed to explore freely for 5 minutes. The mice were then removed and placed in new cages, not returned to their original cages to avoid affecting their behavior. The equipment was then cleaned with 75% ethanol. After all mice had finished exploring, two identical objects (ensuring they were odorless and could not be moved) were placed on one side of the test box, 10 cm from the side walls. The mice were placed in the center of the test box, equidistant from the two objects, and allowed to explore freely for 10 minutes. Exploration was defined as the mice touching the objects with their mouths or noses or coming within approximately 2-3 cm of the objects. The equipment was cleaned with 75% ethanol after each mouse's exploration. One hour later, one of the previously used objects (the old object) and another new object were placed on one side of the test box, 10 cm from the side walls. The mice were placed in the center of the test box, equidistant from the two objects, and allowed to explore freely for 10 minutes. After this, the mice were removed and placed in new cages, not returned to their original cages to avoid affecting their behavior. The equipment was then cleaned with 75% ethanol.

[0035] The formula for calculating the coefficient of the new object is shown in Equation 1.

[0036] Formula 1 In Equation 1, TN and TF represent the time taken by the mouse to explore new objects and old objects, respectively.

[0037] The results of the novel object recognition experiment for each group of mice are shown below. Figure 3.

[0038] The results showed that compared with the control group, the recognition coefficient of mice induced by D-gal was significantly reduced, indicating that D-gal induced a decrease in learning and memory abilities and cognitive impairment in aging mice. However, intervention with Lactobacillus johnsonii bacterial solution could significantly reverse this trend (Lactobacillus johnsonii group), and the recognition coefficient of mice in this group tended to be at the level of the control group. The recognition coefficient of mice treated with heat-inactivated Lactobacillus johnsonii bacterial solution was similar to that of the D-gal group (heat-inactivated Lactobacillus johnsonii group). This indicates that only live Lactobacillus johnsonii can effectively improve cognitive impairment and enhance learning and memory in mice.

[0039] 3. Y Maze (1) New heteroarm experiment The three arms of the Y-maze were randomly designated as the novel arm, the start arm, and the other arms. The novel arm was blocked by a partition in the first phase (training period) and opened in the second phase (testing period). The start arm was the arm in which the mouse entered the Y-maze. Throughout the experiment, the start arm and the other arms remained open, allowing the mouse to enter and exit freely.

[0040] The novel arm was closed, and mice were allowed to explore freely in the other two arms for 10 minutes. After 1 hour, the barrier was opened, and each mouse was allowed to explore freely for 5 minutes. The number of times and the time each mouse entered each arm were recorded. After each mouse finished, it was placed in a new cage, and the equipment was cleaned with 75% alcohol to eliminate odor.

[0041] The results of the Y-maze-novel heteroarm experiment for each group of mice are shown below. Figure 4 .

[0042] The results showed that compared with the Control group mice, the D-gal group mice had significantly reduced time spent in the neo-arm and the number of times they entered the neo-arm, while the Lactobacillus johnsonii group mice had increased time spent in the neo-arm and the number of times they entered the neo-arm. However, the heat-inactivated Lactobacillus johnsonii group mice and the D-gal group mice had similar time spent in the neo-arm and the number of times they entered the neo-arm, with no significant difference. This indicates that only live Lactobacillus johnsonii can improve the spatial memory ability and cognitive impairment in mice.

[0043] (2) Autonomous alternation experiment The spontaneous alternation rate of mice reflects their learning and memory abilities. When cognitive impairment occurs, the spontaneous alternation rate will decrease. Therefore, each mouse was placed in the same arm (starting arm) of the Y maze and continuously alternating spontaneously for 8 minutes. The spontaneous alternation rate was measured. After each mouse finished, it was placed in a new cage. The equipment was cleaned with 75% alcohol to eliminate odor.

[0044] The formula for calculating the autonomous alternation rate is shown in Equation 2.

[0045] Formula 2 In Equation 2, the total number of alternations is the number of times the mouse enters all three arms of the Y maze (arm A (abbreviated as A), arm B (abbreviated as B), and arm C (abbreviated as C)) in sequence, such as ABC, ACB, BAC, BCA, CAB, CBA; the maximum number of alternations is the total number of arm entries minus 2.

[0046] The results of the Y-maze-autonomous alternation experiment for each group of mice are shown in the figure. Figure 5 .

[0047] The results showed that compared with the Control group, the spontaneous alternation rate of mice in the D-gal group was significantly reduced, while the spontaneous alternation rate of mice in the Lactobacillus johnsonii group was significantly increased. The spontaneous alternation rate of mice in the heat-inactivated Lactobacillus johnsonii group was similar to that of mice in the D-gal group, with no significant difference. This indicates that only live Lactobacillus johnsonii can improve the spatial memory and motor ability of mice.

[0048] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0049] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of Lactobacillus johnsonii in the preparation of pharmaceuticals, characterized in that, The drug has at least one of the following uses: Improve cognitive function; To prevent, alleviate, improve, or assist in the treatment of cognitive impairment; Among them, the Lactobacillus johnsonii ( Lactobacillus johnsonii The accession number for this work is ATCC 33200.

2. The use according to claim 1, characterized in that, The cognitive impairment refers to cognitive impairment caused by aging.

3. The use according to claim 1, characterized in that, The cognitive impairment refers to cognitive impairment induced by D-galactose.

4. The use according to claim 1, characterized in that, The improvement in cognitive function includes one or more of the following: learning memory, spatial memory, working memory, and motor exploration ability.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: Live cells of Lactobacillus johnsonii; The Lactobacillus johnsonii has the accession number ATCC 33200.

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