Application of a type of animal lactobacillus in the preparation of anti-aging drugs

Abstract

This invention discloses the application of *Lactobacillus animalis* ATCC35046 in the preparation of anti-aging drugs. The *Lactobacillus animalis* ATCC35046 involved in this invention is colonized in dental plaque of primates and in the intestines of dogs and mice. It can significantly reduce the expression of P16 or P21 in brain, bone, muscle, and skin tissues, significantly downregulate the level of aging-related secretory phenotypes in serum, and improve spatial memory, motor function, and muscle function. This provides a theoretical reference and guidance for the development of multi-target, multifunctional anti-aging probiotic preparations.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of a type of animal lactobacillus. Background Technology

[0002] Aging is a complex biological process accompanied by the progressive decline in the function of multiple tissues and organs, and is associated with an increased risk of various chronic diseases. Its core characteristics include the accumulation of cellular aging markers, intensified inflammatory responses, and tissue functional degeneration, severely impacting individual health and quality of life. At the level of brain aging, the senescence of nerve cells leads to a decline in cognitive function, while the abnormally high expression of cell cycle inhibitors such as P16 and P21 is a key molecular marker of cellular aging. Bone aging is characterized by decreased bone density, destruction of bone microstructure, and decreased biomechanical properties, easily leading to osteoporosis and fracture risk. Activation of P16 and P21 in bone tissue inhibits osteoblast function, exacerbating bone loss. Muscle aging is characterized by reduced muscle mass and decreased muscle strength, closely related to muscle cell senescence and an inflammatory environment. As the body's largest barrier organ, skin aging manifests as decreased elasticity and wrinkle formation; the accumulation of cellular aging markers is one of the core mechanisms driving skin aging. Inflammation also plays a crucial role in the aging process. Elevated levels of pro-inflammatory factors such as IL-1α and TNF-α in serum create an "inflammatory aging" state, further accelerating the aging process of various tissues and organs. These pro-inflammatory factors are also classified as age-related secretory phenotypes. In recent years, in addition to genetic and environmental factors, gut microbiome dysbiosis has been proven to be a key driver of accelerated aging. Gut microbiome imbalance can trigger a series of detrimental changes, including chronic low-grade inflammation ("inflammatory aging"), increased oxidative stress, decreased immune function, and metabolic disorders—all core hallmarks of aging. Current anti-aging strategies mainly include antioxidants, hormone replacement therapy, and stem cell therapy, but these methods have limitations in effectiveness, high cost, and significant side effects. Summary of the Invention

[0003] This invention provides an application of animal lactobacillus in the preparation of anti-aging drugs, in order to solve the technical problems mentioned in the background art, such as limited effectiveness, high cost, and large side effects of existing anti-aging methods.

[0004] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0005] The application of a type of animal lactobacillus in the preparation of anti-aging drugs, wherein the animal lactobacillus is ( ) Lactobacillus animalis ATCC35046.

[0006] Existing technologies have disclosed the applications of *Lactobacillus animalis* in stimulating macrophage differentiation, promoting dendritic cell maturation, and enhancing immunity, and have been shown to alleviate symptoms of hormone-induced osteonecrosis in animal models. However, its research and application in delaying aging and improving age-related symptoms have not been reported. The inventors accidentally discovered that this specific species of *Lactobacillus animalis* can utilize its regulation of the microbiome-gut-organ axis to combat multiple aging markers. In vivo experiments in aged C57BL / 6 mice confirmed that its bacterial suspension, after gavage intervention, can significantly reduce the expression levels of P16 or P21 in the brain, bone, muscle, and skin tissues, with the most significant improvement in skin tissue aging; it can effectively increase femoral bone mineral density, improve tibial biomechanical properties, and enhance bone load-bearing capacity; simultaneously, it can reduce serum IL-1α and TNF-α secretion levels, alleviating inflammatory aging states; in behavioral experiments, it can significantly improve spatial memory, exercise endurance, and muscle grip strength in mice, comprehensively improving age-related behavioral decline.

[0007] As a further preferred embodiment of the above technical solution, the anti-aging drug includes drugs that improve aging, drugs that improve aging-related behavioral performance, and drugs that reduce serum aging-related secretory phenotype levels.

[0008] As a further preferred embodiment of the above technical solution, the aging mentioned in the drug for improving aging includes bone aging, brain aging, muscle tissue aging, and skin aging.

[0009] As a further preferred embodiment of the above technical solution, when the drug for improving aging is used to improve brain aging, it reduces the expression level of the aging indicator P21 in brain tissue; when the drug for improving aging is used to improve bone aging, it increases bone density, improves bone load-bearing function, and reduces the expression level of the aging indicator P16 in bone tissue; when the drug for improving aging is used to improve muscle tissue aging, it reduces the expression level of the aging indicator P16 in muscle tissue; and when the drug for improving aging is used to improve skin aging, it reduces the expression level of the aging indicator P21 in skin tissue.

[0010] As a further preferred embodiment of the above technical solution, the age-related behavioral manifestations mentioned in the improvement of age-related behavioral manifestations include at least one of spatial memory ability, exercise endurance, and muscle grip strength.

[0011] As a further preferred embodiment of the above technical solution, the serum aging-related secretion phenotype levels mentioned in the drug for reducing serum aging-related secretion phenotype levels include IL-1α and TNF-α.

[0012] As a further preferred embodiment of the above technical solution, the anti-aging drug is in the form of at least one of pharmaceuticals, food, or health products.

[0013] The present invention has the following beneficial effects: This invention provides a *Lactobacillus animalis* ATCC35046 and its application in the preparation of anti-aging drugs. *Lactobacillus animalis* ATCC35046 is colonized in dental plaque of primates and in the intestines of dogs and mice. In vivo experiments in aged C57BL / 6 mice have verified that it can significantly reduce the expression of P16 or P21 in brain, bone, muscle, and skin tissues, significantly downregulate the level of age-related secretory phenotypes in serum, and improve spatial memory, motor function, and muscle function. This provides a theoretical reference and guidance for developing multi-target, multifunctional anti-aging probiotic preparations using *Lactobacillus animalis* ATCC35046. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 The effect of the bacterial suspension of Example 1 on the behavioral performance of aged mice (statistical graph), where A is the escape latency of the Barnes maze test, B is the suspension time of the suspension test, and C is the dwell time of the rotundus test; Figure 2 The effect of the bacterial suspension of Example 1 on P21 expression in the brain tissue of aged mice (left: immunofluorescence staining image, right: statistical graph); Figure 3 The effect of the bacterial suspension of Example 1 on P16 expression in bone tissue of aged mice (left: immunohistochemical staining image; right: statistical graph). Figure 4 The effect of the bacterial suspension of Example 1 on P16 expression in muscle tissue of aged mice (left: immunohistochemical staining image; right: statistical graph). Figure 5 The effect of the bacterial suspension of Example 1 on P21 expression in the skin tissue of aged mice (left: immunohistochemical staining image; right: statistical graph). Figure 6 The effect of the bacterial suspension of Example 1 on femoral bone mineral density in aged mice (left: computed tomography scan, right: statistical graph). Figure 7 The effect of the bacterial suspension of Example 1 on serum IL-1α and TNF-α levels in aged mice (statistical graph). Detailed Implementation

[0016] The following detailed description is based on embodiments of the present invention, but the present invention can be implemented in many different ways as defined and covered by the claims.

[0017] Example 1: Preparation of Lactobacillus animalis ATCC35046 suspension: After activating the bacteria, they were inoculated into MRS medium and cultured anaerobicly at 37°C until the bacteria reached the logarithmic growth phase (OD value approximately 1, corresponding to a concentration of approximately 2 × 10⁻⁶). 8 After centrifuging at 4000g for 15 min to collect the bacterial pellet (CFU / mL), the bacterial cells were washed three times with sterile PBS buffer, and finally resuspended in PBS to adjust the bacterial concentration to 1.5 × 10⁻⁶ CFU / mL. 9 CFU / mL, to obtain a bacterial suspension (to be used immediately).

[0018] Example 2: This embodiment uses the *Lactobacillus animalis* ATCC35046 suspension from Example 1 to conduct an intervention experiment on aging mice with *Lactobacillus animalis* ATCC35046: Sixteen 15-month-old SPF-grade male C57BL / 6 mice were randomly divided into a control group and an experimental group, with eight mice in each group.

[0019] The experimental group was treated with gavage of Lactobacillus ATCC35046 suspension, at a dose of 3 × 10⁻⁶ per animal per gavage. 8 CFU (i.e., 200µL bacterial suspension); the control group was given an equal volume of sterile PBS by gavage twice a week for 8 consecutive weeks. During the intervention, mice had free access to food and water, and the environment was maintained at a temperature of 22-25℃, humidity of 40%-60%, and normal circadian rhythm. Example 3: This embodiment conducted experiments on mice in each group from Example 2 to verify the effect of Lactobacillus animalis ATCC35046 on improving spatial memory ability in aging mice (results correspond to...). Figure 1 A): Barnes' maze experiment: (1) The intervention was conducted in week 6 of Example 2. The Barnes maze device was a white circular platform 140cm high and 100cm in diameter, made of odorless ABS plastic. Twenty equidistant circular holes, each 5cm in diameter, were evenly spaced along the platform's edge. One hole (called the target hole) connected to a dark box (i.e., the target box), while the remaining 19 holes were empty and not connected to any container. The target box was a drawer-type structure for easy access by animals and was not visible from the platform surface. Four quadrants were set outside the platform, with different shapes and colors of graphics in each quadrant serving as spatial visual cues. The platform was rotatable to eliminate odor interference.

[0020] (2) One day before the experiment, acclimatization training was conducted. The mice were gently placed into the target box from the target hole and allowed to acclimatize for 4 minutes. Then, the mice were placed in a transparent plastic cylinder (20 cm in diameter and 27 cm in height) in the center of the platform and their movement was restricted for 30 seconds before the cylinder was removed. The experimental time was recorded immediately. If the mouse actively entered the target box (i.e., all four limbs were inside), it was considered a successful escape and it was allowed to stay in the box for 30 seconds. If it could not find the target box within 5 minutes, the experimenter would guide it into the box and allow it to stay for 30 seconds. During the experimental intervals, the platform surface was wiped with alcohol and wet wipes to eliminate odor interference.

[0021] (3) For the formal experimental phase, adaptive training was conducted for 5 consecutive days, once a day. Before each experiment, the platform was randomly rotated to one or more holes, but the target box was kept in a fixed position to eliminate the mice's olfactory-dependent localization. During the training process, the escape latency (the time required from release to entering the target box) and the number of errors (the number of times the mouse explored non-target holes) were recorded in the same way. If the mouse successfully escaped in each experiment, it was allowed to stay in the box for 30 seconds.

[0022] (4) In the memory retention test after training, the mice were released from the center of the platform again, and their ability to accurately find the target hole and enter the target box without prompting was recorded. The experimental data included escape latency and the number of errors, which were used to assess the mice's spatial learning and memory abilities. The mice's behavior was recorded and analyzed using VisuTrack animal behavior analysis software (Shanghai Xinruan Information Technology Co., Ltd.).

[0023] result: Figure 1 The results of the Barnes maze test showed that, compared with the control group, the experimental group mice had a significantly shorter latency to find the escape hole and a significantly fewer number of errors (P<0.01), indicating that Lactobacillus animalis ATCC35046 suspension can improve the spatial memory ability of mice.

[0024] Example 4: This embodiment conducts experiments on mice in each group from Example 2 to verify the effect of Lactobacillus animalis ATCC35046 on improving exercise endurance and muscle grip strength in aging mice: Wire-hanging experiment: conducted at week 7 of the intervention (results correspond to...) Figure 1 B).

[0025] Experimental methods: (1) One hour before the start of the experiment, the mice to be tested were placed in the test room to allow them to adapt to the environment; (2) Fix a metal rod 50cm long and 9mm in diameter at a height of 40cm above the ground, and place a cage filled with soft bedding directly below it. (3) Place the mouse’s forelimbs on the metal rod so that it can grasp the metal rod with its forelimbs; (4) Record the time the mice were suspended on the metal rod. Each mouse was tested 3 times a day for 5 days.

[0026] Rotating bar test: conducted in week 8 of the intervention. (Results correspond to...) Figure 1 C) Experimental methods: (1) One hour before the start of the experiment, the mice to be tested were placed in the test room to allow them to adapt to the environment.

[0027] (2) Turn on the rotator and make it rotate at a constant speed of 10 rpm. Place the mouse on the rotator and make sure it can move normally on the rotator. Then let the rotator rotate at a constant acceleration of 2 rpm / s.

[0028] (3) The time taken for the mouse to fall off the spinner was recorded from the start of the spinner's acceleration. Each mouse was tested 3 times a day for 5 days.

[0029] result: Figure 1 The results of the wire-hanging and rotisserie experiments showed that, compared with the control group, the experimental group mice spent a longer time suspended on the metal rod (P<0.01), and their muscle strength was effectively improved; the experimental group mice spent a significantly longer time on the rotisserie than the control group (P<0.001), and their exercise endurance was significantly improved.

[0030] Example 5: This embodiment tests and analyzes the expression and analysis of aging markers P16 and P21 in various tissues of mice in each group in Example 2 (results correspond to...). Figure 2-5 ): Experimental methods: Brain, femur, quadriceps femoris muscle and back skin tissue of each mouse in Example 2 were taken, fixed with 4% paraformaldehyde, dehydrated with graded alcohol, embedded in paraffin, and prepared into 4μm thick sections. Immunohistochemistry and immunofluorescence were used to detect the expression of P16 and P21 proteins in each tissue.

[0031] The specific steps are as follows: Immunohistochemistry: 1) Dewaxing and rehydrating paraffin sections: Place sections in a 65ºC oven for 2 hours, dewax with xylene three times for 20 minutes each time, then place sections in a gradient of 100%, 95%, 90%, 80%, and 70% ethanol solutions for 3 minutes each, followed by PBS soaking for 3 minutes three times. 2) Antigen retrieval: Boil sodium citrate antigen retrieval working solution in a microwave oven, place sections in the solution, and microwave on low for 10 minutes. After retrieval, allow sections to cool naturally at room temperature. 3) Membrane permeation: Soak sections in PBS three times for 3 minutes each time. Place sections in PBST solution containing 0.3% Triton X-100 for membrane permeation at room temperature for 10 minutes. 4) Blocking endogenous peroxidase: Soak sections in PBS three times for 3 minutes each time, draw circles around the tissue with an immunohistochemical pen, and place sections in a humidified chamber. Add an appropriate amount of 3% hydrogen peroxide solution to each tissue to completely cover it. Block at room temperature for 20 minutes. 5) Blocking non-specific protein binding sites: Immerse sections in PBS for 3 minutes three times, remove excess liquid, and add an appropriate amount of 10% goat serum to each tissue to completely cover it. Block at room temperature for 20 minutes. 6) Primary antibody incubation: Immerse sections in PBS for 3 minutes three times, remove excess liquid, and add an appropriate amount of the corresponding primary antibody to each tissue to completely cover it. Incubate overnight at 4ºC. 7) Secondary antibody incubation: Immerse sections in PBS for 3 minutes three times, remove excess liquid, and add an appropriate amount of goat anti-rabbit or goat anti-mouse secondary antibody to each tissue according to the species of the primary antibody. Incubate at room temperature for 1 hour. 8) Staining: Immerse sections in PBS for 3 minutes three times, remove excess liquid. Add an appropriate amount of DAB staining solution to the tissue and observe the staining degree under a microscope. Immerse the sections in PBS to terminate the staining process. 9) Counterstaining: Immerse the sections in hematoxylin staining solution for 3 minutes, gently rinse off excess staining solution with running water, immerse the sections in differentiation solution for 3 seconds, rinse with running water, and then immerse in reverse blue solution for 7 seconds. After rinsing the sections with running water, place them in PBS. 10) Gradient dehydration: Immerse the sections sequentially in 70%, 80%, 90%, and 100% alcohol for dehydration, then dry them in a 37ºC oven. 11) Clear the sections with xylene, mount them with neutral resin, photograph them under an optical microscope, and count positive cells.

[0032] Immunofluorescence: 1) Dewaxing, rehydration, antigen retrieval, and membrane perforation are the same as those described in the immunohistochemical staining section. 2) Blocking: After membrane perforation, soak the sections in PBS for 3 minutes three times. Remove the sections and place them in a humidified chamber. Draw circles around the tissue with an immunohistochemical pen, and add an appropriate amount of 10% donkey serum to each tissue to completely cover it. Block at room temperature for 20 minutes. 3) Primary antibody incubation: Soak the sections in PBS for 3 minutes three times. Remove excess liquid from the sections. Add an appropriate amount of the corresponding primary antibody to each tissue to completely cover it. Incubate overnight at 4ºC. 4) Secondary antibody incubation: Soak the sections in PBS for 3 minutes three times. Remove excess liquid from the sections. Add an appropriate amount of the corresponding fluorescent secondary antibody to each tissue according to the species of the primary antibody. Incubate at room temperature for 1 hour. Ensure that the reagents and sections are protected from light during the dilution and addition of the secondary antibody and the subsequent incubation process. 5) Mounting: Soak the sections in PBS for 3 minutes three times. Remove excess liquid from the sections. Add an appropriate amount of mounting medium containing DAPI to the tissue and cover with a coverslip. After the edges of the slide have dried, seal them with clear nail polish. This step requires complete protection from light. 6) Use an Apotome fluorescence microscope to take photographs and count positive cells.

[0033] Result: As Figure 3 As shown, the results of immunohistochemical and immunofluorescence staining experiments showed that, compared with the control group, the rates of P16 and P21 positive cells in the brain, femur, quadriceps femoris muscle and skin tissue of mice in the experimental group were significantly reduced. The rate of positive cells in the skin tissue decreased the most, indicating that the animal lactobacillus ATCC35046 suspension can effectively resist aging, and its effect on improving skin aging is the most obvious.

[0034] Example 6: This embodiment tested the bone mineral density and bone biomechanics of mice in each group in Example 2 (results correspond to...). Figure 6 ): Experimental methods: Femurs of mice in each group in Example 2 were taken, and bone density at the distal 1 / 3 of the femur was detected using a Micro-CT tomographic imaging system; a three-point bending test was conducted using a biomechanical testing machine to detect the maximum bending load of the tibia.

[0035] Results: Compared with the control group, the experimental group of mice showed significantly increased bone mineral density (P<0.05) and significantly increased maximum tibial load (P<0.05), indicating that Lactobacillus animalis ATCC35046 suspension can effectively increase bone mass, improve bone biomechanical properties, and enhance bone load-bearing capacity.

[0036] Example 7: This example detected the serum inflammatory factors IL-1α and TNF-α in mice from each group in Example 2 (results correspond to...). Figure 7 ): Experimental method: Blood was collected from the orbital cavity of mice in each group before sacrifice in Example 2. The blood was centrifuged at 3000 r / min for 15 min. The concentrations of IL-1α and TNF-α in the serum were detected using an ELISA kit. The operation was strictly performed in accordance with the kit instructions.

[0037] Results: Compared with the control group, the concentrations of IL-1α and TNF-α in the serum of mice in the experimental group were significantly reduced, indicating that the animal lactobacillus ATCC35046 suspension has a good anti-inflammatory effect and can alleviate inflammatory senescence.

[0038] The above are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and modifications obtained without departing from the inventive concept should also be considered within the scope of protection of the present invention.

[0039] 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 application of a type of animal lactobacillus in the preparation of anti-aging drugs, characterized in that, The animal lactobacillus is animal lactobacillus ATCC35046.

2. The application of *Lactobacillus animalis* according to claim 1 in the preparation of anti-aging drugs, characterized in that, The anti-aging drugs include drugs that improve aging, drugs that improve age-related behavioral performance, and drugs that reduce serum levels of age-related secretory phenotypes.

3. The application of *Lactobacillus animalis* according to claim 2, characterized in that, The aging mentioned in the drugs for improving aging includes bone aging, brain aging, muscle tissue aging, and skin aging.

4. The application of *Lactobacillus animalis* according to claim 3, characterized in that, When the aging-improving drug is used to improve brain aging, it reduces the expression level of the aging indicator P21 in brain tissue; when the aging-improving drug is used to improve bone aging, it increases bone density, improves bone load-bearing function, and reduces the expression level of the aging indicator P16 in bone tissue; when the aging-improving drug is used to improve muscle tissue aging, it reduces the expression level of the aging indicator P16 in muscle tissue; when the aging-improving drug is used to improve skin aging, it reduces the expression level of the aging indicator P21 in skin tissue.

5. The application of *Lactobacillus animalis* according to claim 3, characterized in that, The age-related behavioral manifestations mentioned in the improvement of age-related behavioral sciences include at least one of spatial memory ability, exercise endurance, and muscle grip strength.

6. The application of *Lactobacillus animalis* according to claim 3, characterized in that, The serum aging-related secretory phenotype levels mentioned in the drugs that reduce serum aging-related secretory phenotype levels include IL-1α and TNF-α.

7. The application of *Lactobacillus animalis* according to claims 1-6, characterized in that, The anti-aging drugs are in the form of pharmaceuticals, food, or health products.

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