Application of indolebutyric acid combined with indolelactic acid in the preparation of health food for assisting memory improvement

Abstract

The present application relates to the technical field of health food, and particularly relates to application of indole butyric acid combined with indole lactic acid in preparation of health food for assisting in improving memory. It is found in the present application that IBA and ILA are combined to inhibit downstream inflammatory cascade reaction by regulating interaction between AhR and STAT1 signal pathways, to reduce in-vivo and in-vitro inflammatory levels, to reduce over-activation of microglial cells and astrocytes, to relieve body damage and inflammatory reaction of each tissue caused by obesity, and to further protect blood-brain barrier and intestinal barrier, to reduce central nervous system damage caused by obesity, to improve cognitive function and memory. IBA and ILA can be used for preparation of health food for assisting in improving memory.

Description

Technical Field

[0001] This invention relates to the field of health food technology, and in particular to the application of indolebutyric acid combined with indolelactic acid in the preparation of health foods that help improve memory. Background Technology

[0002] Studies have shown that a long-term high-fat, high-calorie diet not only induces obesity, diabetes, and other disorders of glucose and lipid metabolism, but is also a significant risk factor for cognitive impairment. This is because chronic inflammation caused by glucose and lipid metabolism disorders can further overactivate microglia, the brain's unique immune cells, and trigger downstream inflammatory signaling pathways, leading to the massive release of pro-inflammatory cytokines and inducing inflammatory damage to the central nervous system. Simultaneously, it can overactivate the complement system, causing synapses to be mislabeled and cleared, disrupting the integrity of neural networks, and ultimately leading to cognitive decline. Therefore, inhibiting inflammatory pathways in the body can reduce the inflammatory response caused by the accumulation of inflammatory factors, protect against neurological damage caused by metabolic disorders, thereby protecting cognitive function and improving memory.

[0003] Indolebutyric acid (IBA) and indolelactic acid (ILA) are a class of naturally occurring indole compounds, widely distributed in nature and produced by gut microbiota in humans and animals, possessing natural advantages such as wide availability and good biocompatibility. Existing studies have confirmed that these compounds have certain anti-inflammatory, antioxidant, and metabolic-regulating biological activities. However, to date, no research has publicly disclosed or suggested that IBA synergistically with ILA can be used to improve neuroinflammation and cognitive decline caused by glucose and lipid metabolism disorders. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides the application of indolebutyric acid (IBA) combined with indolelactic acid (ILA) in the preparation of health foods that aid in memory improvement. This invention discovers that the combined use of IBA and ILA can synergistically inhibit the interaction between the aryl hydrocarbon receptor (AhR) and transcription activator 1 (STAT1) signaling pathways and their downstream inflammatory pathways, thereby alleviating obesity-related cognitive impairment, neuroinflammation, and related tissue damage. This combination can be used to prepare health foods that aid in memory improvement.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of indolebutyric acid combined with indolelactic acid in the preparation of health food products that help improve memory.

[0006] This invention provides a health food product that helps improve memory, with active ingredients including indolebutyric acid and indolelactic acid.

[0007] Preferably, the mass ratio of indolebutyric acid to indolelactic acid in the health food is 1:5 to 5:1.

[0008] Preferably, the dosage form of the health food includes capsules, tablets, granules, or oral liquid.

[0009] This invention provides the use of indolebutyric acid in combination with indolelactic acid in the preparation of a medicament for the prevention or improvement of complications caused by metabolic syndrome, said complications including one or more of the following: 1) Cognitive impairment; 2) Neuroinflammation; 3) Damage to related tissues.

[0010] Preferably, the metabolic syndrome is a metabolic syndrome caused by a high-fat, high-calorie diet.

[0011] Preferably, the relevant tissue damage includes one or more of the following: intestinal mucosal barrier damage, hepatic steatosis, and inflammatory damage to brain tissue.

[0012] Preferably, the brain tissue inflammatory damage includes upregulating the expression of brain tissue inflammation-related genes; the brain tissue inflammation-related genes include Cxcl9, Cxcl10, Cxcl11 and Nos2 One or more of them.

[0013] Preferably, prevention or improvement of cognitive impairment caused by metabolic syndrome includes one or more of the following: reducing anxiety, restoring normal motor function, and improving learning and memory function.

[0014] Preferably, the neuroinflammation includes inflammatory damage caused by lipopolysaccharide pathway activation due to metabolic syndrome.

[0015] Beneficial effects: This invention discovers that the combined use of IBA and ILA can inhibit downstream inflammatory cascades by regulating the interaction between the AhR and STAT1 signaling pathways, reducing inflammation levels both in vivo and in vitro, alleviating excessive activation of microglia and astrocytes, and mitigating obesity-induced damage and inflammatory responses in various tissues. Furthermore, by protecting the blood-brain barrier and intestinal barrier, it reduces obesity-induced central nervous system damage, improves cognitive function, and enhances memory. IBA and ILA can be used to prepare health foods that aid in memory improvement. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The effects of combined IBA and ILA on LPS-induced inflammation in BV2 cells were investigated; specifically, a. the effects of different concentrations of IBA, ILA, and LPS on BV2 cell proliferation; b. inflammatory factors. Tnf-α , Il-1β andIl-6 c. mRNA expression levels; d. Protein levels of inflammatory factors TNF-α, Il-1β, and Il-6; relative to the control group. p <0.05, p <0.01; relative to the LPS treatment group, # p <0.05, ## p <0.01.

[0018] Figure 2 The study investigated the effects of combined IBA and ILA intervention on cognitive function in obese mice; specifically, a. the effect of combined IBA and ILA intervention on nesting behavior in obese mice; and b. the effect of combined IBA and ILA intervention on open field behavior in obese mice.

[0019] Figure 3 To investigate the effect of combined IBA and ILA intervention on the improved behavioral performance of obese mice in the elevated cross maze experiment.

[0020] Figure 4 The effects of combined IBA and ILA on the expression of inflammatory factors and blood-brain barrier-related genes mRNA in the hippocampus of obese mice.

[0021] Figure 5 The effects of combined IBA and ILA on the number of Nissl cells and the microstructure of the hippocampus in chronically obese mice were shown. The results included: a. Nissl staining of the hippocampus in each group of mice; b. Transmission electron microscopy (TEM) results of the hippocampus in each group of mice; c. Quantitative analysis of Nissl staining of the hippocampus in each group of mice; and de. Quantitative analysis of transmission electron microscopy (TEM) results of the hippocampus in each group of mice.

[0022] Figures 6-7 The effects of combined IBA and ILA on hippocampal microglia and astrocytes in chronically obese mice. Figure 6 In the middle, a. hippocampal tissues of mice in each group Iba1 a. mRNA expression level; b. Quantitative analysis results of Iba-1 fluorescence intensity based on immunofluorescence staining; c. Representative images of Iba1 protein immunofluorescence staining in the hippocampus of mice in each group; d. Morphological skeleton analysis results of Iba-1 positive cells. Figure 7 In the middle, a. hippocampal tissues of mice in each group Gfap a. mRNA expression level; b. Quantitative analysis results of Gfap fluorescence intensity based on immunofluorescence staining; c. Representative images of Gfap protein immunofluorescence staining in the hippocampus of mice in each group; d. Morphological skeleton analysis results of Gfap-positive cells.

[0023] Figures 8-9The combination of IBA and ILA reduces obesity-induced neuroinflammation by activating the AhR / STAT1 interaction signaling axis. Figure 8 In the middle, ab. KEGG pathway enrichment analysis of common targets: Toll-like receptor signaling pathway and neurodegenerative disease-related pathways; Figure 9 In the middle, a. hippocampal tissues of mice in each group AhR , Stat1 and its downstream inflammation-related genes ( Cxcl9 , Cxcl10 , Cxcl11 , Nos2 a. mRNA expression level of α, p-Stat1 and total Stat1 protein in hippocampus; b. expression and quantitative analysis of AhR, p-Stat1 and total Stat1 protein in hippocampus; c. comparison of CXCL9, CXCL10, CXCL11 and NOS2 protein content in brain tissue of mice in each group; e. molecular mechanism of IBA and ILA in reducing neuroinflammation and protecting cognitive function.

[0024] Figure 10 The effects of combined IBA and ILA on liver tissue pathology and inflammatory response in obese mice were investigated. Specifically: a. Representative pathological sections of liver tissue stained with hematoxylin and eosin (HE) from each group of mice; b–g. Inflammation-related genes in liver tissue from each group of mice. Tnf-α , Il-1β , Il-6 , Tlr4 , Myd88 and Cd14 mRNA expression levels.

[0025] Figure 11 The effects of combined IBA and ILA on inflammatory status and colonic barrier function in obese mice; where, a–f. colonic tissues of mice in each group. Tnf-α , Il-1β , Il-6 , Tlr4 , Myd88 and Cd14 mRNA expression levels; g–i. colon tissue of mice in each group Zo-1 , Claudin-4 and Occludin mRNA expression levels.

[0026] Figure 12 The effects of combined IBA and ILA on serum endotoxin and systemic inflammatory factor levels in chronically obese mice were investigated. Specifically, a) comparisons of serum lipopolysaccharide (LPS) levels in each group; b–d) comparisons of serum inflammatory factors TNF-α, IL-1β, and IL-6 in each group.

[0027] Figures 2-12 In the middle, relative to the NC group, p <0.05, p <0.01; relative to the HFD group, ## p <0.01. Detailed Implementation

[0028] This invention provides the application of indolebutyric acid combined with indolelactic acid in the preparation of health food products that help improve memory.

[0029] This invention discovers that the combined use of IBA and ILA can reduce the levels of inflammatory factors in tissues including the liver, brain, and colon by regulating the interaction between the AhR and STAT1 signaling pathways and their downstream pathways, thereby alleviating neuroinflammatory damage, preventing the reduction in the number of hippocampal synapses and / or the destruction of synaptic structure caused by obesity, helping to maintain synaptic function and promote the effective transmission of nerve signals between cells, protecting the blood-brain barrier, and improving brain cognitive function and memory. Thus, it is possible to prepare health foods that can help improve memory.

[0030] This invention provides a health food product that helps improve memory, with active ingredients including indolebutyric acid and indolelactic acid.

[0031] In one embodiment, the mass ratio of indolebutyric acid (IBA) to indolelacic acid (ILA) in the health food is 1:5 to 5:1. In another embodiment, the mass ratio of IBA to ILA in the health food is 1:2 to 2:1. In yet another embodiment, the mass ratio of IBA to ILA in the health food is 1:1. In one embodiment, the effective dose per unit of the health food is 120-600 mg (total of IBA and ILA), preferably 300-360 mg. This dose range is based on the effective dose in mouse experiments converted according to body surface area (60 mg / kg BW corresponds to an equivalent human dose of approximately 300 mg / 60kg adult), and estimated in vivo exposure corresponding to the effective concentration in cell experiments. The above ratios and dose ranges are within the range that can be expected through conventional optimization based on the experimental results of the 1:1 ratio in the examples. Those skilled in the art will understand that within the range of 1:5 to 5:1, IBA and ILA can still achieve the effect of assisting in improving memory.

[0032] As one implementation method, the dosage form of the health food includes capsules, tablets, granules, or oral liquids.

[0033] As one implementation method, the preparation methods of health food in different dosage forms are as follows: (1) Capsules: IBA and ILA are mixed at a mass ratio of 1:1 to obtain an active ingredient composition. Take 150 mg of this composition and mix it evenly with 45 mg of microcrystalline cellulose, 50 mg of pregelatinized starch, and 5 mg of magnesium stearate (to keep the total filling amount at about 250 mg). After sieving and granulation, the mixture is filled into hard capsule shells to make capsules containing a total active ingredient of 150 mg per capsule. It is recommended to take 2 capsules daily to achieve the preferred daily intake of 300 mg.

[0034] (2) Oral solution: Mix IBA and ILA at a mass ratio of 1:1. Take 300 mg of this composition, dissolve it in an appropriate amount of purified water, add 5 g of honey and 0.1 g of potassium sorbate, add water to make up to 10 mL, stir to dissolve, filter and sterilize, and then fill into a container to prepare an oral solution containing a total of 300 mg of active ingredients per 10 mL. It is recommended to take one vial daily to achieve the preferred daily intake of 300 mg.

[0035] (3) Granules: Mix IBA and ILA at a mass ratio of 1:1. Take 360 ​​mg of this composition and mix it evenly with 622 mg of maltodextrin, 10 mg of vitamin C, 5 mg of citric acid, and 3 mg of sucralose (ensuring a total weight of approximately 1000 mg per sachet for easy repackaging). Add an appropriate amount of ethanol to form a soft mass, sieve and granulate, dry, and then package into sachets to produce granules containing a total active ingredient content of 360 mg per sachet. It is recommended to take one sachet daily to achieve the preferred daily intake of 360 mg.

[0036] Mix IBA and ILA at a 1:1 mass ratio. Take 300 mg of this mixture and mix it thoroughly with 600 mg of maltodextrin, 30 mg of vitamin C, 15 mg of citric acid, and 5 mg of sucralose (ensuring a suitable taste). Add an appropriate amount of ethanol to form a soft mass, sieve and granulate, dry, and then package into sachets to produce granules containing a total of 300 mg of active ingredients per sachet. It is recommended to take one sachet daily to achieve the preferred daily intake of 300 mg.

[0037] It should be noted that the proportion, dosage and type of excipients of the active ingredients in the above embodiments can be adjusted according to the actual product requirements. The mass ratio of IBA to ILA can vary from 1:10 to 10:1, and its potential effective dose range is 120-600 mg, preferably 300-360 mg.

[0038] The health food of this invention is suitable for people with long-term high-fat diet, obesity, and metabolic syndrome, and is used to prevent or help improve memory.

[0039] This invention provides the use of indolebutyric acid in combination with indolelactic acid in the preparation of a medicament for the prevention or improvement of complications caused by metabolic syndrome, said complications including one or more of the following: 1) Cognitive impairment; 2) Neuroinflammation; 3) Damage to related tissues.

[0040] In one implementation, the metabolic syndrome is metabolic syndrome caused by a high-fat, high-calorie diet. The complications (neuroinflammation and cognitive impairment) caused by the metabolic syndrome described in this invention mainly refer to cognitive decline, anxiety-like behavior, learning and memory impairment, and related neuroinflammatory states caused by long-term high-fat diets, obesity, and metabolic syndrome.

[0041] As one implementation, the relevant tissue damage includes one or more of the following: intestinal mucosal barrier damage, hepatic steatosis, and inflammatory damage to brain tissue.

[0042] As one implementation, the brain tissue inflammatory damage includes upregulating the expression of brain tissue inflammation-related genes; the brain tissue inflammation-related genes include Cxcl9, Cxcl10, Cxcl11 and Nos2 One or more of them.

[0043] As one implementation method, preventing or improving cognitive impairment caused by metabolic syndrome includes one or more of the following: reducing anxiety, restoring normal motor function, and improving learning and memory function. As another implementation method, the drug prevents or improves cognitive impairment through the liver-brain axis and gut-brain axis pathways. Specifically, it improves colonic barrier function and metabolism by reducing hepatic steatosis and inflammatory damage.

[0044] In one embodiment, the neuroinflammation includes inflammatory damage resulting from lipopolysaccharide pathway activation caused by metabolic syndrome. In another embodiment, the neuroinflammation includes lipopolysaccharide-induced inflammatory damage to microglia caused by metabolic syndrome.

[0045] In one embodiment, the drug has one or more of the following effects: 1) It reduces lipopolysaccharide (LPS)-induced inflammation in BV2 cells and decreases the mRNA and protein expression levels of TNF-α, IL-1β, and IL-6; 2) Improve cognitive impairment caused by a high-fat diet, including improving nesting behavior, spontaneous activity, and anxiety-like behavior; 3) Reduces blood-brain barrier damage, upregulates the expression of tight junction proteins ZO-1, Claudin-4, and Occludin, and maintains the structure and function of the blood-brain barrier; 4) Maintain neuronal and synaptic structure: Protect the morphological structure of hippocampal neurons (increase the number of Nissl positive neurons), maintain synaptic integrity and function (improve the length of the active area and the width of the gap), and maintain the function of nerve signal transmission; 5) Inhibit excessive activation of glial cells: Regulate the mRNA and protein expression of microglia marker Iba-1 and astrocyte marker Gfap, inhibit the excessive activation of microglia and astrocytes, and promote their conversion to homeostatic phenotype; 6) Downregulates the expression of brain tissue inflammation-related genes Cxcl9, Cxcl10, Cxcl11, and Nos2; its mechanism of action involves upregulating the expression of AhR protein in brain tissue, inhibiting STAT1 phosphorylation, and then downregulating the expression of downstream chemokines (CXCL9, CXCL10, CXCL11) and inflammatory mediators (NOS2); 7) Improve liver pathological condition: reduce hepatic steatosis and inflammatory cell infiltration, and decrease the expression of hepatic inflammatory factors; 8) Reduces colonic tissue inflammation and barrier damage, upregulates colonic tight junction protein expression, and maintains intestinal barrier integrity; 9) Reduce serum lipopolysaccharide and pro-inflammatory factors TNF-α, IL-1β, and IL-6 levels.

[0046] Cellular experiments have confirmed that, at an equimolar total concentration (10 μM), the combined application of IBA and ILA (5 μM each) significantly inhibited LPS-induced TNF-α, IL-1β, and IL-6 protein levels in BV2 microglia compared to the single application of any one compound at the same total dose (10 μM). This indicates that the combination is not a simple additive effect, but rather exhibits a genuine synergistic effect in anti-inflammatory activity, constituting one of the core innovative aspects of this invention's composition compared to existing single-component technologies.

[0047] This invention discovers that the combined use of IBA and ILA can reduce the levels of inflammatory factors in tissues including the liver, brain, and colon by regulating the interaction between the AhR and STAT1 signaling pathways and their downstream pathways, thereby alleviating neuroinflammatory damage, preventing the reduction in the number of hippocampal synapses and / or the destruction of synaptic structure caused by obesity, helping to maintain synaptic function and promote the effective transmission of nerve signals between cells, protecting the blood-brain barrier, and improving cognitive function.

[0048] To further illustrate the present invention, the application of indolebutyric acid combined with indolelactic acid provided by the present invention in the preparation of health food that helps improve memory is described in detail below with reference to embodiments and accompanying drawings, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0049] Example (I) Cell Experiments 1. Experimental grouping, model establishment, and intervention Mouse BV2 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in a 37°C, 5% CO2 incubator. After being passaged three times in our laboratory, they were used for formal experiments.

[0050] (1) Cell counting and plating When the cell density reaches 80%, collect the cells, resuspend them in 2 mL of culture medium, and mix well to obtain a cell suspension. Add 10 μL of cells to a cell counting chamber and count the cells under a microscope. After counting, dilute the cells to 5 × 10⁻⁶ cells based on the cell count. 3 Cells / mL. The diluted cell suspension was seeded into 12-well plates, 800 μL per well, and incubated at 37°C for 6 h to allow cell adhesion. Experimental groups: NC group, model group, and groups treated with different concentrations of IBA, ILA, and IBA+ILA.

[0051] (2) Dosing treatment First, the appropriate concentrations of IBA and ILA, and the optimal dosage of LPS for cell proliferation were determined. In this experiment, the concentrations of IBA and ILA used alone, from low to high, were: 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM, without LPS treatment; the concentrations of LPS used, from low to high, were: 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL, without IBA or ILA treatment. After incubation for 24 h, the effects of different doses of IBA, ILA, and LPS on cell proliferation rate were detected using a CCK-8 assay kit. Based on the cell proliferation rate results, in subsequent experiments, the working concentration for IBA and ILA intervention alone was 10 μM. When IBA and ILA were used in combination, they were mixed at a 1:1 ratio to ensure that the final concentration of each drug in the mixed solution was 5 μM (i.e., the total concentration was 10 μM). The effect difference between single-drug and combination therapy at the same total dose was compared. LPS was used at a concentration of 500 ng / mL. After treatment for 24 h, cells and supernatant were collected. Cells were used for RNA extraction and q-PCR experiments to detect inflammatory factors. Tnf-α , Il-β , Il-6 The mRNA expression levels were determined, and further proteomics analysis was performed using shotgun / non-targeted mass spectrometry. The levels of inflammatory factors (TNF-α, IL-β, IL-6) in the cell culture supernatant were detected using ELISA.

[0052] (ii) Animal experiments 1. Grouping of experimental animals, establishment of models, and intervention This animal experimental research plan was approved by the Laboratory Animal Ethics and Use Committee (IACUC) of Shanghai Jiao Tong University (Research Plan No. A2024453-002).

[0053] Six-week-old male SPF-grade C57BL / 6j mice were purchased from the Shanghai branch of Jiangsu Jicui Pharmaceutical Experimental Animal Technology Co., Ltd. (License No.: SCXK(Su)2023-0009). After one week of acclimatization feeding, the experimental animals were randomly divided into 7 groups (n=10 mice / group), and the intervention methods for each group were as follows: Normal control group (NC): Administered basal feed daily and gavaged with the same amount of distilled water.

[0054] High-fat diet model group (HFD): fed a high-fat diet and the same amount of distilled water daily.

[0055] IBA intervention group (IBA): Mice were given a high-fat diet daily and were also given IBA aqueous solution by gavage at a dose of 60 mg / kg BW (equivalent to 360 mg per day for an adult weighing 60 kg).

[0056] ILA intervention group (ILA): Mice were given a high-fat diet daily and were also given ILA aqueous solution by gavage at a dose of 60 mg / kg BW.

[0057] IBA+ILA Synergistic Intervention Group (IBLA): Mice were given a high-fat diet daily and simultaneously administered a mixed aqueous solution of IBA+ILA by gavage. The IBA and ILA solutions were mixed in equal volumes at a 1:1 ratio. The sum of the gavage doses of IBA and ILA in mice was 60 mg / kg. In this mixed working solution, the final gavage dose of both IBA and ILA was 30 mg / kg.

[0058] IBA+ILA+CH-223191 intervention group (CH-223191): Mice were given a high-fat diet daily and were simultaneously administered a mixed aqueous solution of IBA+ILA+CH-223191 by gavage. The doses of IBA and ILA were 60 mg / kg·BW, and the dose of the AhR antagonist CH-223191 by gavage was 10 mg / kg·BW.

[0059] All groups were administered via gavage. An additional IBA+ILA intraperitoneal injection + high-fat diet group (IBLA_IP) was also included. The entire intervention lasted 42 days, with room temperature maintained at 25±1℃ and relative humidity at 45%~65%, and alternating light and dark cycles (6:00~18:00). Mice in all groups had free access to food and water during the experiment, and food intake, water intake, and weekly weight changes were recorded.

[0060] 2. Collection of laboratory animal samples Twelve hours before the end of the experiment, mice were fasted but allowed free access to water. The following day, mice were anesthetized with 2% isoflurane, and their cervical vertebrae were dislocated. Blood was collected via cardiac puncture, allowed to stand at room temperature for 2 hours, and then centrifuged (4°C, 3000 rpm, 15 min) to obtain serum. The heads were removed, and the hippocampus from each group was fixed in glutaraldehyde. The hippocampus from the other side, as well as the left and right brains, were placed into 2 mL centrifuge tubes and immediately frozen in liquid nitrogen. The abdominal cavity was opened, and liver tissue from the same location was fixed in paraformaldehyde. The liver and colon were washed with physiological saline, excess water was absorbed with clean filter paper, and the tissues were cut into small pieces and aliquoted into 2 mL centrifuge tubes, which were immediately frozen in liquid nitrogen. All frozen tissues were transferred to a -80°C freezer for storage. Serum was aliquoted into 1.5 mL centrifuge tubes and stored at -80°C.

[0061] 3. Behavioral experiments (1) Nest building experiment Nesting behavior is an important, species-typical instinctive behavior in rodents, closely related to their health, welfare, and neurobehavioral function. This experiment, by assessing the complexity and completeness of nest construction in mice, is often used to comprehensively evaluate the mice's motivation, fine motor skills, ability to perform instinctive behaviors, and overall health / disease status. In some neurological disease models (such as neurodegenerative diseases and brain injury), a decline in nesting ability can serve as an auxiliary, non-specific behavioral indicator of model severity or intervention effectiveness, but it is not a primary method for assessing specific cognitive functions (such as learning and memory).

[0062] Three days before the end of the experiment, the nesting ability of mice was tested. Before the experiment, mice were individually housed 24 hours in advance to acclimatize to the environment. After acclimatization, at 8 PM (the start of darkness), a 3g square compressed cotton pad was placed in each cage. 24 hours later (8 AM the following morning), the nesting results were observed without disturbing the mice, and a 5-point scale was used for scoring. The scoring criteria were as follows: 1 point: The cotton pad was barely touched (>90% intact). 2 points: The cotton pad was partially torn (50%-90% intact). 3 points: The cotton pad was significantly torn (50%-100% pulverized), but the cotton fibers were scattered and no obvious nest structure was formed. 4 points: The cotton pad was severely torn (>90% pulverized), and the cotton fibers were concentrated into a simple, flat nest that the mouse could sit in. 5 points: A complete nest was built, with the nest walls exceeding the height of the mouse when curled up, forming a good shelter.

[0063] (2) Open field experiment The open field test is primarily used to assess spontaneous activity, exploratory behavior, and anxiety-like levels in laboratory animals. The open field test was conducted in a four-channel mouse open field chamber (500×500×400 mm). A video recording system was fixed directly above the center of each open field channel to record the mice's movement distance, dwell time, and movement trajectory in different areas of the open field (e.g., periphery and central areas). In this study, the open field test was conducted 5 days before the end of the experiment. Before the experiment, mice were transferred to the open field behavior laboratory and allowed to acclimatize to the dark environment for 1–2 hours. At the start of the experiment, mice were placed in the center of the open field and allowed to explore freely for 5 minutes, during which their activity was recorded. After each mouse's test, the open field area was wiped and disinfected with 75% alcohol to eliminate interference such as odors left by previous test mice. Topscan software was used to record the mice's movement trajectory within the open field over 5 minutes, as well as the dwell time and movement distance in the central and periphery areas.

[0064] (3) Elevated cross maze experiment The elevated cross maze test is another gold standard test for assessing anxiety-like behavior in rodents. It assesses anxiety levels by utilizing the conflict between rodents' natural fear of suspended open spaces (apoptosis) and their instinctive desire to explore new environments. This study was conducted 3 days before the end of the experiment. The specifications of the elevated cross maze equipment were: arm width 5 cm, open and closed arm lengths both 75 cm, and the height of the elevated cross maze from the ground 50 cm. ① Set the corresponding parameters in the software and record the animal's number, date, status, and other information; ② Remove the experimental animal from its cage, with the animal facing away from the experimenter as much as possible, and gently place the animal in the central area of ​​the instrument, facing the open arm. Then, the experimenter quickly and quietly leaves; ③ Open the VisuTrack animal behavior analysis software to track the animal's trajectory within the elevated cross maze instrument, automatically calculate indicators, and set the experiment duration to 5 minutes. The elevated cross maze instrument was wiped clean with alcohol before and after the experiment to ensure it was clean and odorless.

[0065] 4. Morphological analysis of hippocampal tissue (1) Observe the number of Nissl bodies in the mouse hippocampus using Nissl staining. Hippocampal tissues from mice in each group were placed in 4% paraformaldehyde and fixed at 4 ℃ for 48 h. The hippocampal tissues were then prepared into paraffin sections. The specific experimental procedures are as follows: ① Dewaxing paraffin sections to water: Sections were sequentially immersed in environmentally friendly dewaxing and clearing solution I for 15 min, environmentally friendly dewaxing and clearing solution II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then washed with tap water; ② Nissl staining: Sections were immersed in the staining solution for 15 min, then washed with water; ③ Differentiation: After differentiation in the differentiation solution for 2 s, sections were washed with water; ④ Dehydration, clearing, and mounting: Rapid dehydration with anhydrous ethanol, clearing with clean xylene for 5 min, and mounting with neutral resin; ⑤ Microscopic examination, image acquisition and analysis, and analysis of tissue structure changes using the ImageJ online system.

[0066] (2) Observe the morphology of the hippocampus using transmission electron microscopy. Hippocampal tissues from mice in each group were fixed in 2.5% glutaraldehyde at 4°C for 48 h. After dehydration with ethanol of varying concentrations, the tissue blocks were hardened by immersion in acetone. Following this treatment, the samples were embedded in clear resin. Subsequently, samples were cut using an ultramicrotome to prepare 60–80 nm sections, which were then stained with uranium acetate-saturated ethanol solution. Finally, the structure of the hippocampus was observed and photographed using transmission electron microscopy, and structural changes were analyzed using the ImageJ online system.

[0067] 5. Detection of inflammatory factors and neuroinflammatory marker proteins in hippocampal tissue (1) The mRNA expression levels of inflammation-related genes and tight junction proteins in mouse hippocampus were detected by qPCR. ① Total RNA extraction: The TaKaRa MiniBEST Universal RNA Extraction Kit (9767, Takara Corporation, Japan) was used, following the kit instructions.

[0068] ② Total RNA reverse transcription: PrimeScript™ RT reagent Kit (Perfect Real Time) (RR037A, Takara Corporation, Japan) was used. The reverse transcription system and conditions were in accordance with the kit instructions.

[0069] Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific sequences are shown in Table 1.

[0070] Table 1 Primer sequences

[0071] Note: In Table 1, the primer numbers in the upstream primer series are SEQ ID NO.1-SEQ ID NO.18 from top to bottom, and the primer numbers in the downstream primer series are SEQ ID NO.19-SEQ ID NO.36 from top to bottom. β-Actin The upstream primer sequence number is SEQ ID NO.1. The downstream primer sequence number is SEQ ID NO.19.

[0072] ③ Amplification: qPCR was performed using the TB Green® Premix Ex Taq™ (Tli RNaseH Plus) kit (RR420A, Takara Corporation, Japan). A three-step method was used for quantitative real-time amplification of the qPCR system. Internal control was used... β-actin ( Actb Correcting the target gene mRNA expression level, the final result is expressed as 2. -ΔΔCt The calculation yielded the result.

[0073] (2) Immunofluorescence was used to detect the expression of Iba-1 and Gfap proteins in mouse hippocampus. Protein expression detection: Mouse hippocampal tissue was fixed in paraformaldehyde for 48 h, then paraffin blocks were prepared. Immunofluorescence detection of Iba-1 and Gfap was then performed. The specific experimental steps are as follows: Dewaxing paraffin sections to water: Sections were sequentially immersed in dewaxing solution I for 10 min, dewaxing solution II for 10 min, dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then washed with distilled water. Antigen retrieval: Slides were placed in PBS and washed three times on a decolorizing shaker, 5 min each time. Serum blocking: After slightly drying the sections, a circle was drawn around the tissue using a histochemical pen, and 3% BSA was added for blocking for 30 min. Primary antibody addition: Mouse anti-Iba-1 (1:250) and rabbit anti-Gfap (1:250) primary antibodies were mixed in equal proportions, and the prepared primary antibody was added. The sections were then incubated overnight at 4 ℃ in a humidified chamber. Secondary antibody addition: Slides were washed three times with a shaker in PBS for 5 min each time. Alexa Fluor 488-labeled goat anti-mouse IgG and CY3-labeled goat anti-rabbit IgG were added, and incubated at room temperature in the dark for 50 min. DAPI counterstaining of cell nuclei: Slides were washed three times with a shaker in PBS for 5 min each time. DAPI staining solution was added, and incubated at room temperature in the dark for 10 min. Autofluorescence quenching: Slides were washed three times with a shaker in PBS for 5 min each time. Autofluorescence quencher solution B was added for 5 min, followed by rinsing with running water for 10 min. Mounting: Slides were mounted with antifluorescence quenching mounting medium. Image acquisition: DAPI excitation wavelength 330 nm, emission wavelength 420 nm; Alexa Fluor 488 excitation wavelength 480 nm, emission wavelength 520 nm; CY3 excitation wavelength 550 nm, emission wavelength 570 nm.

[0074] 6. Cellular Astral DIA proteomics sequencing (1) Protein extraction and quantification: The samples were taken out of the -80 ℃ ultra-low temperature freezer and thawed slowly on ice. After thawing, 4 volumes of protein lysis buffer (containing 8 mol / L urea and 1% protease inhibitor) were added to each sample and mixed thoroughly. The mixture was placed on ice for ultrasonic lysis for 3 min. After ultrasonication, the samples were centrifuged at 12000 g for 5 min at 4 ℃. After centrifugation, the supernatant was collected and transferred to a new centrifuge tube to obtain the protein extract. The protein extract was quantified using the BCA method and the protein samples were detected by SDS-PAGE electrophoresis.

[0075] Protease desalting: Take equal amounts of protein from each sample and adjust the volume to be consistent with lysis buffer; add an equal volume of ultrapure water and mix well, then add trichloroacetic acid (TCA) to a final concentration of 20% (w / v), vortex mix, and precipitate at 4 ℃ for 2 h. Wash the protein precipitate three times with acetone, add dithiothreitol (DTT) to the washed protein to a final concentration of 5 mM, and reduce at 37 ℃ for 1 h; then add iodoacetamide (IAA) to a final concentration of 11 mM and incubate at room temperature in the dark for 45 min to complete alkylation. Add trypsin to the alkylated protein solution and digest overnight. The peptide samples after enzymatic hydrolysis were desalted using a C18 desalting pipette tip: the desalting column was activated with 100% acetonitrile and equilibrated with 0.1% formic acid solution; the peptide sample was added, and the desalting column was washed with 0.1% formic acid solution, the waste liquid was discarded, and the washing was repeated twice; finally, the eluent (containing 70% acetonitrile and 0.1% formic acid) was used for elution and the eluent was collected. The eluent was concentrated to dryness using a vacuum centrifuge and used for later use.

[0076] Liquid chromatography-mass spectrometry (LC-MS) detection: Each protein peptide sample was separated using a Vanquish Neo UHPLC system (nano-flow mode) (Thermo Fisher Scientific). The chromatographic column used was a Thermo Scientific™ EASY-Spray™ column (C18 150 μm × 15 cm × 2 μm, ES906), with column temperature controlled at 55 ℃ and autosampler temperature controlled at 5 ℃. Mobile phases: Mobile phase A was a 0.1% formic acid aqueous solution (volume fraction), and mobile phase B was an 80% acetonitrile aqueous solution containing 0.1% (volume fraction) formic acid. After chromatographic separation, the peptide samples were introduced into an Astral high-resolution mass spectrometer for DIA (data-independent acquisition) mode. The mass spectrometry detection parameters were set as follows: the precursor ion scan range was 380-980 m / z; the primary mass spectrometry resolution was 240,000 (with 200 m / z as a reference); the normalized AGC target was 500%; and the maximum ion implantation time (IT) was 5 ms. The secondary mass spectrometry (MS2) used DIA data acquisition mode, with the following specific parameter settings: 300 scan windows were set; the isolation window was 2 m / z; the high-energy collision dissociation energy (HCD Collision Energy) was 25 eV; the normalized AGC target was 500%; and the maximum ion implantation time (IT) was 3 ms.

[0077] Mass spectrometry data search: After mass spectrometry detection, a database search and analysis were performed. The database used in this study was uniprotkb_Mus_musculus_id10090_17224s_reviewed_2024_08_28.fasta. DIA-NN 1.9.2 was used for analysis. The main parameters of this software were set as follows: trypsin was selected as the enzyme type, with a maximum deletion cleavage site of 1; fixed modifications were aminomethylation (modification site: cysteine ​​C); dynamic modifications were oxidation (modification site: methionine M) and acetylation (modification site: protein N-terminus). The data screening criterion was a false detection rate (FDR) ≤ 1%. After obtaining qualified data using the above methods, bioinformatics methods were further used to visualize and analyze the obtained data to complete subsequent data interpretation.

[0078] 7. The mRNA expression levels of AhR and its downstream pathway genes in mouse hippocampus were detected by qPCR. The primer sequences are shown in Table 1.

[0079] 8. Detection of AhR protein expression level and Stat1 phosphorylation level in mouse brain tissue Total protein extraction from brain tissue: Add phosphatase inhibitor to RIPA lysis buffer at a volume ratio of 1:100 and mix well. Weigh 20 mg of brain tissue, add 400 µL of the above lysis buffer, freeze-mill, and incubate at 4 °C for 20 min for lysis. Centrifuge at 14000 g for 5 min at 4 °C, collect the supernatant, and determine the protein concentration.

[0080] BCA protein concentration determination: Prepare BCA working solution at a ratio of solution A:solution B = 50:1 (v / v) and vortex to mix. Take a 96-well plate and add protein standards (0, 1, 2, 4, 8, 12, 16, 20 µL) to each well. Add standard dilution to each well to a final concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. Dilute the protein sample to be tested 20 times at a volume ratio of 1:19, and add 20 µL to each well. Add 200 µL of BCA working solution to each well, mix gently, and incubate at 37 ℃ for 20 min. Measure the absorbance at 560 nm using a microplate reader, plot a standard curve, and calculate the actual concentration of the sample.

[0081] Protein SDS-PAGE electrophoresis: Take an appropriate amount of protein sample, add 5× loading buffer, vortex to mix, and boil at 95 °C for 10 min to denature. Centrifuge at 10000 g for 10 min at 4 °C, and collect the supernatant and cool to room temperature. Load 30 µg of protein into each well using a 10% SDS-PAGE gel. Electrophoresis conditions: 20 V for 20 min, 80 V for 20 min, 120 V for 30 min, until bromophenol blue migrates to about 2 cm from the bottom of the gel. Cut gels containing the molecular weight ranges of AhR, Stat1, and p-Stat1 according to the protein markers.

[0082] Transfer: After activating the PVDF membrane with anhydrous ethanol for 5 min, immerse it in transfer buffer. Assemble the transfer clamp in the following order: sponge-filter paper-gel-PVDF membrane-filter paper-sponge, removing any air bubbles. Transfer at a constant voltage of 100 V for 50 min (ice bath).

[0083] Immunological reaction: After transfer, the membrane was quickly washed once with TBST. Blocking was performed for 1 h at room temperature with 5% skim milk (prepared with TBST, w / v), followed by three TBST washes for 5 min each. Anti-AhR antibody, anti-total Stat1 antibody, and anti-p-Stat1 (Tyr701) antibody were added, and incubated overnight at 4°C (primary antibody diluted according to the antibody manufacturer's instructions, as shown in Table 2). After three TBST washes, the corresponding HRP-labeled secondary antibody was added, and incubation was performed for 1 h at room temperature, followed by three TBST washes.

[0084] ECL chemiluminescence: The membrane is laid flat, ECL luminescent solution is added evenly, and development is performed immediately.

[0085] Table 2 Antibody List

[0086] 9. Detection of AhR / Stat1 downstream protein expression levels in mouse brain tissue using ELISA. Accurately weigh 70 mg of brain tissue and add 700 μL of 1×PBS buffer to homogenize thoroughly. Incubate the homogenate at -20 °C overnight, followed by two freeze-thaw cycles to fully lyse the cell membranes. After centrifugation at 5000 g for 5 min at 4 °C, collect the supernatant for the detection of CXCL9, CXCL10, CXCL11, and NOS2 levels. The sample volume for each indicator is 100 μL. Finally, measure the absorbance at 450 nm and calculate the final concentration. All procedures were performed strictly according to the kit instructions.

[0087] 10. Pathological evaluation of mouse liver and detection of inflammatory factor mRNA expression levels (1) Pathological analysis Structural changes in mouse liver tissue were observed using hematoxylin and eosin (HE) staining. Liver tissue was fixed in 4% paraformaldehyde for 48 h, followed by routine paraffin embedding, sectioning, and HE staining. The specific steps are as follows: ① Tissue dehydration, clearing, and embedding: After fixation, the tissue was rinsed with running water, and representative sections were cut. The tissue was dehydrated in a gradient of ethanol (75%, 85%, 95%, 100% I, 100% II, 1–2 h each), cleared with xylene (I, II, 30–60 min each), and then embedded in paraffin and paraffin. Serial sections were prepared to a thickness of 4 μm, and after mounting, the sections were baked at 60 ℃ overnight.

[0088] ② Dewaxing and hydration: Dewaxing is performed by placing the sections in xylene I and II for 10–15 min each, followed by hydration with a gradient of ethanol (100% I, 100% II, 95%, 85%, and 75% ethanol, 5 min each), and rinsing with distilled water.

[0089] ③ Hematoxylin staining, differentiation, and blue inversion: Add hematoxylin staining solution and stain for 5–8 min, then rinse with running water. Differentiate with 1% hydrochloric acid ethanol (1 mL concentrated hydrochloric acid + 99 mL 70% ethanol) for a few seconds, then stop rinsing with running water. Blue inversion with 0.2% ammonia solution for 30 s–2 min, then rinse with running water.

[0090] (At this point, the cell nucleus appears blue and the cytoplasm is colorless, which can be confirmed by microscopic examination.) ④ Eosin staining: Add eosin staining solution and stain for 1–3 minutes, then gently rinse away excess staining solution with running water.

[0091] Dehydration, clearing, and mounting: Dehydrate with a gradient of ethanol (75%, 85%, 95% for 30 s each, 100% I and II for 1–2 min each), and clear with xylene (I and II for 5 min each). Allow to dry slightly, drop in neutral resin, and slowly cover with a coverslip from one side to mount (avoiding air bubbles).

[0092] ⑤ Microscopic analysis: After the mounting medium solidifies, observe and acquire images under an optical microscope to assess changes in liver tissue structure.

[0093] (2) Detection of mRNA expression levels of inflammatory factors. Primer sequences are shown in Table 1.

[0094] 11. Detection of expression levels of tight junction protein and inflammatory factor mRNA in mouse colon. Primer sequences are shown in Table 1.

[0095] 12. ELISA detection of serum lipopolysaccharide content and inflammatory factors in mice. In ELISA testing, serum requires no pretreatment and can be directly loaded at 100 μL. The experimental procedure should be strictly followed according to the kit instructions.

[0096] 13. Statistical Analysis Methods All data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software. One-way ANOVA was used for comparisons among multiple groups, followed by Fisher's Least Significant Difference (LSD) post-hoc test for multiple comparisons. p <0.05 indicates a statistically significant difference.

[0097] 14. Results Analysis The combined use of IBA and ILA reduced LPS-induced inflammation in BV2 cells (cell experiments). Figure 1 Figure a shows the effect of different concentrations of IBA, ILA, and LPS on cell proliferation rate. As can be seen from the figure, when the IBA concentration exceeds 10 μM and the ILA concentration exceeds 20 μM, the proliferation rate of BV2 cells can be significantly reduced. Therefore, in subsequent experiments, the IBA and ILA concentrations were selected as 10 μM, and the LPS concentration was 500 ng / mL.

[0098] like Figure 1 As shown in Figures b and c, compared with the control group (Con group), LPS stimulation alone (LPS group) significantly upregulated inflammatory factors in BV2 cells. Tnf-α , Il-1β and Il-6 mRNA expression levels and protein concentrations of TNF-α, IL-1β, and IL-6 ( p <0.01). IBA or ILA intervention alone can significantly reduce LPS-induced [therapeutic effects]. Tnf-α and Il-1β mRNA expression level ( p <0.01), but neither treatment alone significantly downregulated Il-6 mRNA expression levels or the protein concentrations of the three inflammatory factors. p>0.01). In contrast, the combined intervention of IBA and ILA significantly reduced the protein concentrations of TNF-α, IL-1β, and IL-6. These results indicate that IBA or ILA alone can only partially inhibit LPS-induced inflammatory responses in BV2 cells, while the combined intervention shows a superior anti-inflammatory effect. Further analysis showed that, under the premise of maintaining a consistent total dosage (10 μM), the combined treatment group (5 μM each) had a significantly stronger inhibitory effect on the protein levels of TNF-α, IL-1β, and IL-6 than the single treatment groups (10 μM IBA or 10 μM ILA). This result excludes the possibility of enhanced effect due to increased total dosage, strongly demonstrating that IBA and ILA have a significant synergistic effect in anti-inflammation, which is the scientific basis for the composition of this invention to achieve better efficacy with lower dosage.

[0099] The combination of IBA and ILA improves cognitive function in obese mice (animal experiments). (1) Nest building experiment like Figure 2 As shown in Figure a, compared with the normal control group (NC group), the untorn nestlet weight of mice in the high-fat diet group (HFD group) and the CH-223191 group was significantly increased. p <0.01, and the Deacon nest score decreased significantly. p <0.01 indicates that high-fat diet-induced cognitive impairment can significantly weaken nesting behavior in mice. Oral administration of IBA or ILA alone can significantly reverse the HFD-induced decline in nesting ability, as evidenced by a significant reduction in the weight of unbuilt nesting material. p <0.01), among which ILA intervention alone also significantly improved nesting scores ( p <0.01). Compared with the individual drug administration groups, the combined gavage intervention of IBA and ILA had a more significant effect on improving nesting behavior, while also reducing the weight of unbuilt material ( p <0.05) and increase nest building score ( p <0.01). Intraperitoneal injection also showed a trend of action consistent with that of gavage.

[0100] (2) Open field experiment like Figure 2 As shown in Figure b, the open field experiment trajectories revealed that the NC group mice moved throughout all areas of the open field, while the HFD group mice primarily moved in the peripheral areas, with a significant reduction in their activity in the central area. Compared to the NC group, the HFD group mice exhibited a significantly shorter total distance in the open field. p<0.05, and the distance incenter also decreased significantly. p <0.01, suggesting that a high-fat diet can reduce spontaneous activity in mice and induce anxiety-like behavior. Oral administration of ILA alone partially reversed the above changes induced by HFD, as evidenced by a significant increase in total movement distance (…). p <0.05%. Compared with the individual drug administration groups, the combined intragastric administration of IBA and ILA (IBLA) was more effective in improving HFD-induced spontaneous activity reduction and anxiety-like behavior, and the total motor distance and central area motor distance ( p All levels (<0.05) further increased. Furthermore, in the open field experiment, intraperitoneal injection of IBA+ILA showed better improvement than gavage administration. However, CH-223191 intervention completely eliminated the improvement effect of IBLA.

[0101] (3) Elevated cross maze experiment like Figure 3 As shown, compared with the NC group, the HFD group mice showed a decreasing trend in both time spent in open arms and distance traveled in open arms, but the differences were not statistically significant. Oral administration of ILA alone significantly prolonged the time spent in open arms and increased the distance traveled in open arms in HFD mice. p <0.01). Compared with ILA intervention alone, combined IBA and ILA gavage (IBLA) showed a more significant improvement in the above indicators, with a further increase in open arm dwell time and movement distance. p <0.01). Mice in the intraperitoneal injection group of IBA and ILA showed better behavioral performance than mice in the gavage group. CH-223191 treatment completely eliminated the improving effect of IBA+ILA, and the open-arm dwell time and movement distance of the mice were significantly lower than those in the NC group ( p <0.05). The above results suggest that the protective effect of IBA combined with ILA on anxiety-like behavior mainly depends on the activation of the AhR pathway.

[0102] The combination of IBA and ILA reduced inflammatory damage in the hippocampus of obese mice and protected the blood-brain barrier. like Figure 4 As shown, compared with the NC group, the HFD group mice had higher levels of pro-inflammatory factors in their hippocampal tissue. Tnf-α , Il-1β , Il-6 and Tlr4 The mRNA expression levels were significantly upregulated. p <0.05 or p <0.01 indicates that a high-fat diet successfully induced an inflammatory response in the hippocampus. ILA alone (p <0.05) or combined administration of IBA and ILA ( p <0.01) can significantly reverse HFD-induced [conditions]. Tnf-α and Il-6 mRNA expression was upregulated. The anti-inflammatory effect of intraperitoneal injection was slightly weaker than that of gavage administration. Furthermore, after administration of the AhR inhibitor CH-223191, the inhibitory effects of ILA and IBA+ILA on the expression of the aforementioned pro-inflammatory genes were completely offset, and the levels of various inflammatory factors returned to levels comparable to those in the HFD group. p <0.05 or p <0.01). This result indicates that the anti-inflammatory effect of combined IBA and ILA intervention depends on the activation of the AhR pathway.

[0103] Furthermore, compared to the NC group, the HFD group mice showed higher levels of tight junction protein genes in their hippocampal tissue. ZO-1 and Claudin4 The mRNA expression of all of them was significantly downregulated ( p <0.01). IBA alone did not significantly improve its expression, while ILA alone significantly upregulated it. ZO-1 and Claudin4 level of expression ( p <0.05). Combined administration of IBA and ILA significantly upregulated [the condition]. ZO-1 ( p <0.01) and Claudin4 ( p The expression level of IBA and ILA was <0.05, and there was no significant difference in effect between intraperitoneal injection and gavage. CH-223191 treatment completely eliminated the upregulation effect of IBA+ILA on tight junction protein gene expression, and its expression level returned to the level of the HFD group, further confirming that the protective effect of IBA and ILA on blood-brain barrier integrity also depends on the AhR signaling pathway.

[0104] The combination of IBA and ILA protects hippocampal neurons and synaptic structures in obese mice. like Figure 5 a and Figure 5 As shown in Figure c, compared with the NC group, the number of Nissl positive neurons in the hippocampus of mice in the HFD group was significantly reduced. ImageJ semi-quantitative analysis showed that the number of Nissl positive cells in the hippocampus of mice in both the HFD and CH-223191 groups was significantly lower than that in the NC group. p <0.01, suggesting that a high-fat diet can induce hippocampal neuronal damage or loss. Oral administration of IBA or ILA alone significantly increased the number of Nissl-positive neurons ( p <0.01), and the combined intervention of the two (IBLA) also showed a similar protective effect. Comparison of administration routes showed that intraperitoneal injection of IBA+ILA had a weaker protective effect on neurons than gavage.

[0105] Observation of hippocampal synaptic ultrastructure under transmission electron microscopy Figure 5 (b, d, and e) The results showed that compared with the NC group, the length of the hippocampal active zone in the HFD group mice was significantly shortened. p <0.01, the width of the synaptic cleft showed an increasing trend, suggesting that a high-fat diet can induce synaptic structural abnormalities, which may affect synaptic transmission efficiency and lead to cognitive dysfunction. Administration of IBA or ILA alone significantly improved the above structural abnormalities, manifested as a significant increase in the length of the active zone and a significant decrease in the width of the synaptic cleft. p <0.05 or p <0.01). The improvement effect on synaptic structure in the IBA and ILA combined administration group was similar to that in the ILA or IBA alone intervention group, while the improvement effect in the IBA_IP group was slightly weaker than that in the IBA+ILA gavage group. After administration of the AhR inhibitor CH-223191, the protective effect of IBA+ILA on synaptic ultrastructure almost completely disappeared, further confirming that the synaptic protective effect of the combined intervention depends on the activation of the AhR pathway. In conclusion, the combined intervention of IBA and ILA can effectively alleviate hippocampal synaptic structural abnormalities caused by a high-fat diet and plays an important role in protecting synaptic function and promoting nerve signal transmission.

[0106] The combined use of IBA and ILA regulates the activation state of hippocampal microglia and astrocytes in obese mice. Microglia: such as Figure 6 As shown in the figure, compared with the NC group, the HFD group mice had a higher concentration of ion-calcium-binding adapter molecule 1 in their hippocampal tissue. Iba-1 mRNA expression levels were significantly upregulated. p <0.05, significantly enhanced immunofluorescence intensity ( p <0.05). Skeletal analysis showed that resting microglia were branched, with small cell bodies, long and thin processes, and a highly branched structure. Microglia in the HFD group exhibited a typical hyperactivated morphology, characterized by enlarged cell bodies and short, thick processes. Compared to the HFD group, ILA administration alone significantly inhibited... Iba-1 mRNA expression level ( p <0.01) and the fluorescence intensity of Iba-1 ( p<0.05), and caused morphological reversal in activated microglia, manifested as shortening (or retraction) of primary processes and further shrinkage of the cell body. The inhibitory effect of combined administration of IBA and ILA (i.e., the IBLA group) was basically consistent with the effect of ILA intervention alone. In addition, the inhibitory effect of intraperitoneal injection of IBA+ILA (IBLA_IP) was comparable to that of intragastric administration of IBA+ILA. It is worth noting that pretreatment with the AhR antagonist CH-223191 can weaken the inhibitory effect of combined IBA and ILA on excessive microglia activation.

[0107] Astrocytes: such as Figure 7 As shown in the figure, compared with the NC group, the HFD group mice had higher levels of glial fibrillary acidic protein (GFI) in their hippocampal tissue. Gfap The mRNA expression level of ) was also significantly upregulated ( p <0.01), significantly enhanced immunofluorescence intensity ( p <0.01). Skeletal analysis showed that, compared with the NC group, astrocytes in the HFD group exhibited typical hyperactivated morphology, characterized by cell hypertrophy, thickened processes, and increased branching density. Compared with the HFD group, administration of IBA or ILA alone significantly inhibited the mRNA expression level and fluorescence intensity of Gfap. p <0.05), causing morphological reversal in activated microglia, manifested as cell shrinkage and shortening (or retraction) of primary processes. When IBA and ILA were administered in combination (i.e., the IBLA group), the inhibitory effect was significantly more pronounced than that of the ILA-only intervention group. Gfap The downregulation of mRNA expression levels was largely consistent with the downregulation of protein levels. Similarly, the inhibitory effect of IBLA_IP was comparable to that of oral IBA+ILA. CH-223191 also weakened the regulatory effect of IBA+ILA combination on astrocyte activation.

[0108] Molecular mechanisms by which IBA and ILA synergistically improve neuroinflammation (2) Detection of mRNA expression of AhR and downstream signaling pathway-related genes in mouse hippocampus KEGG enrichment analysis of proteomics in cell experiments ( Figure 8Studies (ab) showed that AhR's downstream target proteins were mainly enriched in the Toll-like receptor signaling pathway and neurodegenerative disease-related pathways. In the Toll-like receptor signaling pathway, key inflammation-related proteins such as Jun, Il1b, Cd14, and Stat1 were enriched, suggesting that AhR may participate in the regulation of inflammatory responses through these proteins. In the neurodegenerative disease pathway, proteins related to neuronal damage and oxidative stress, such as Il1a, Nos2, Map2k6, and Ndufs1, were significantly enriched, suggesting that AhR may participate in the molecular mechanisms of neurodegenerative diseases. Further drug intervention experiments showed that, compared with LPS treatment alone, IBA, ILA, and the combination of both (IBLA) could significantly regulate the expression levels of key proteins (Stat1, Nos2, etc.) in the above pathways; among them, the regulatory effect of IBA and ILA combined was significantly stronger than that of single drug administration, suggesting a synergistic effect between the two in regulating inflammation and neurodegenerative-related pathways.

[0109] Figure 9 Image a shows the mRNA expression levels of AhR and its downstream key genes in the mouse hippocampus. Compared with the NC group, the expression levels of AhR and its downstream key genes in the hippocampus of mice in the HFD and CH-223191 groups were significantly lower. Ahr The mRNA expression level was significantly reduced ( p <0.05), its pathway-related core genes Stat1 ( p <0.05), Cxcl9 ( p <0.01) Cxcl10 ( p <0.01) Cxcl11 ( p <0.01) and Nos2 ( p The expression levels of mRNAs with a concentration <0.05 were significantly upregulated, suggesting that a high-fat diet can activate the AhR pathway and downstream inflammatory responses in the hippocampus. Administration of either IBA or ILA alone significantly reduced... Cxcl9 , Cxcl10 , Cxcl11 , Nos2 mRNA expression level ( p <0.05 or p <0.01); When IBA and ILA were administered in combination, the effects on Stat1 and ILA were significantly downregulated compared to interventions alone. Cxcl9 , Cxcl10 , Cxcl11 , Nos2 The mRNA expression levels were measured. The regulatory effect of IBLA_IP was consistent with the trend of IBLA administration via gavage. CH-223191 weakened the regulatory effect of IBA+ILA on the above genes.

[0110] The results are as follows Figure 9 As shown in bc, compared with the NC group, the HFD group ( p =0.082) and CH-223191 group ( p <0.05) AhR protein expression was downregulated in mouse brain tissue, while p-Stat1 expression was significantly increased. p <0.01). Administration of IBA or ILA alone significantly increased AhR protein expression levels and inhibited Stat1 phosphorylation levels ( ). p <0.05 or p <0.01). When IBA and ILA were administered in combination, their regulatory effects on AhR and p-Stat1 were slightly enhanced compared to IBA or ILA alone, as evidenced by a further increase in AhR protein expression and a further decrease in p-Stat1 phosphorylation.

[0111] The results are as follows Figure 9 As shown in Figure d, compared with the NC group, the protein levels of chemokines CXCL9, CXCL10, CXCL11 and inflammatory factor NOS2 in the brain tissue of mice in the HFD group were significantly increased. p <0.01), CXCL9, CXCL10, and CXCL11 are mainly produced by glial cells in the nervous system (such as astrocytes and microglia) under the stimulation of inflammatory signals (especially interferon-γ and IFNγ). The simultaneous upregulation of CXCL9, CXCL10, CXCL11, and NOS2 in brain tissue is a clear signal of a strong inflammatory response in the central nervous system (CNS). Administration of IBA or ILA alone can significantly reduce the levels of these inflammatory factors. p <0.01). Combined gavage administration of IBA and ILA further downregulated the expression of these proteins compared to IBA or ILA treatment alone. The anti-inflammatory effect in the IBLA_IP group was largely consistent with that in the IBLA group. Treatment with CH-223191 completely eliminated the inhibitory effect of IBA+ILA on inflammatory factors.

[0112] Figure 9 As shown in Figure e, the core mechanism of this invention is as follows: IBA and ILA act as ligands for AhR, activating the AhR signaling pathway; the activated AhR inhibits the phosphorylation activation of STAT1 through protein-protein interactions or transcriptional regulation mechanisms, thereby downregulating the expression of STAT1-dependent chemokines (CXCL9 / CXCL10 / CXCL11) and inflammatory mediators (NOS2), ultimately alleviating neuroinflammation and protecting cognitive function.

[0113] The combined use of IBA and ILA reduced liver and colon inflammation and decreased serum LPS levels in obese mice. like Figure 10As shown in Figure a, compared with NC, HFD mice exhibited significant steatosis and inflammatory cell infiltration in their liver tissue. IBA or ILA alone partially improved the steatosis and inflammatory infiltration in the liver tissue, while combined IBA and ILA intervention showed significantly better improvement than either treatment alone, resulting in liver morphology closer to normal. The improvement effect of intraperitoneal injection of IBA+ILA was comparable to that of gavage administration of IBA+ILA. The AhR inhibitor CH-223191 significantly inhibited the protective effect of IBA+ILA on liver tissue morphology.

[0114] like Figure 10 As shown in bg, compared with the NC group, the HFD group and the CH-223191 group of mice had significantly higher levels of inflammatory factors in their liver tissues. Tnf- α , Il-1β , Il-6 , Tlr4 and Cd14 The mRNA expression levels were significantly upregulated. p <0.01 or p <0.05), indicating that a high-fat diet can induce a significant inflammatory response in the liver. IBA or ILA intervention alone showed limited effects in reducing liver inflammation; IBA intervention only reduced liver inflammation compared to the HFD group. Tnf-α mRNA expression ( p <0.05); When IBA and ILA were administered in combination, their anti-inflammatory effect was stronger than that of IBA or ILA alone, as evidenced by the fact that combined intervention with IBA and ILA significantly reduced the inflammation. Tnf-α(p <0.01 ） and Il-1β ( p <0.05) mRNA expression level, the effect of intraperitoneal injection is comparable to that of gavage, and overall, the combination of IBA and ILA also plays a certain role in improving the level of liver inflammation in obese mice.

[0115] like Figure 11 As shown in Figures a–f, compared to NC, pro-inflammatory genes were present in the colonic tissue of HFD mice. Tnf-a , Il-1β , Il-6 , Tlr4 and Cd14 The mRNA expression levels were significantly upregulated. p <0.05 or p <0.01, suggesting that a high-fat diet can induce a significant inflammatory response in colonic tissue. When IBA or ILA were administered alone, only Il-6 ( p <0.05) and Tlr4 ( pThe mRNA expression level was <0.01. When IBA and ILA were combined, the anti-inflammatory effect was superior to IBA or ILA alone, as evidenced by the IBLA group mice. Tnf-a ( p <0.05), Il-6 ( p <0.05) and Tlr4 ( p The mRNA expression levels of the group with a concentration <0.01 were significantly lower than those in the HFD group. The anti-inflammatory effect of intraperitoneal injection of IBA+ILA was comparable to that of intragastric administration of IBA+ILA, indicating that the route of administration had no significant impact on its efficacy. CH-223191 intervention weakened the inhibitory effect of the combined use of IBA and ILA on pro-inflammatory genes.

[0116] Figure 11 In the figure, g–i represents the mRNA expression level of tight junction-related genes in colonic tissue. Compared with the NC group, the expression levels of tight junction protein genes in the colonic tissue of mice in the HFD and CH-223191 groups were significantly higher. ZO-1 , Claudin4 and Occludin The mRNA expression levels were significantly downregulated. p <0.01 indicates that a high-fat diet can disrupt the integrity of the colonic mucosal barrier. The improvement effect of IBA or ILA alone is limited. IBA ( p <0.01) and ILA ( p <0.05) significantly improved compared to the HFD group Claudin4 The mRNA expression level of mice was observed. When IBA and ILA were administered together by gavage, the expression levels of these molecules were significantly reduced. ZO-1 ( p <0.01) and Claudin4 ( p The expression levels of <0.05 were significantly higher than those in the HFD group.

[0117] like Figure 12 As shown in the figure, compared with NC, the levels of LPS, TNF-α, IL-1β and IL-6 in the serum of HFD mice were significantly increased ( p <0.01, suggesting that a high-fat diet can induce a systemic inflammatory response. Compared with the HFD group, ILA alone ( p <0.01) or IBA ( p When the concentration of LPS was <0.05, it was significantly reduced; in addition, compared with the HFD group, administration of ILA alone reduced serum TNF-α ( p <0.05), IL-1β ( p <0.01) and IL-6 ( pAt levels <0.01), IBA alone can reduce the concentration of the aforementioned pro-inflammatory factors to some extent. Combined administration of IBA and ILA also showed a strong anti-inflammatory effect. Treatment with CH-223191 eliminated the ameliorative effect of IBA+ILA on inflammation in obese mice.

[0118] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of indolebutyric acid combined with indolelactic acid in the preparation of health food products that help improve memory.

2. A health food product that helps improve memory, characterized in that, The active ingredients include indolebutyric acid and indolelactic acid.

3. The health food according to claim 2, characterized in that, The mass ratio of indolebutyric acid to indolelactic acid in the health food is 1:5 to 5:

1.

4. The health food according to claim 2 or 3, characterized in that, The dosage forms of the health food include capsules, tablets, granules, or oral liquids.

5. The use of indolebutyric acid in combination with indolelactic acid in the preparation of medicaments for the prevention or improvement of complications arising from metabolic syndrome, wherein the complications include one or more of the following: 1) Cognitive impairment; 2) Neuroinflammation; 3) Damage to related tissues.

6. The application according to claim 5, characterized in that, The metabolic syndrome mentioned refers to metabolic syndrome caused by a high-fat, high-calorie diet.

7. The application according to claim 5 or 6, characterized in that, The related tissue damage includes one or more of the following: intestinal mucosal barrier damage, hepatic steatosis, and inflammatory damage to brain tissue.

8. The application according to claim 7, characterized in that, The brain tissue inflammation damage includes upregulation of the expression of brain tissue inflammation-related genes; the brain tissue inflammation-related genes include Cxcl9, Cxcl10, Cxcl11 and Nos2 One or more of them.

9. The application according to claim 5 or 6, characterized in that, Prevention or improvement of cognitive impairment caused by metabolic syndrome includes one or more of the following: reducing anxiety, restoring normal motor function, and improving learning and memory function.

10. The application according to claim 5 or 6, characterized in that, The neuroinflammation includes inflammatory damage caused by lipopolysaccharide pathway activation due to metabolic syndrome.

Images