Use of glycerophospholipid metabolites in alzheimer's disease

Abstract

The application provides application of glycerophospholipid metabolites in Alzheimer's disease. The application takes the fecal metabolite spectrum of a 5xFAD mouse after exercise remodeling as a starting point, and systematically analyzes the overall change characteristics of the metabolic network after exercise intervention. The results prove that four specific glycerophospholipid metabolites (PC(18:0 / 0:0), PC(18:1(11Z) / 0:0), PC(0:0 / 16:0) or PC(16:0 / 0:0)) are important metabolic signal molecules for exercise regulation of intestinal brain pathology, and have the potential to develop into an Alzheimer's disease diagnostic kit and an Alzheimer's disease treatment drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the diagnosis or treatment of Alzheimer's disease, specifically to the application of glycerophospholipid metabolites in Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline. With the accelerating aging of the global population, the number of AD patients is gradually increasing, leading to rising medical costs and a growing social burden, posing a significant challenge to public health systems. Current clinical treatment focuses primarily on improving symptoms and slowing disease progression. Although immunotherapy targeting β-amyloid protein has made some progress in recent years, its overall efficacy still suffers from significant individual variability and limited applicability.

[0003] In recent years, the interaction between gut microbiota and central nervous system function has received widespread attention, and the gut-brain axis theory has provided a new perspective for AD research. Studies have found that alterations in gut microbiota structure are common in AD patients and animal models, manifested as decreased microbiota diversity, reduced beneficial bacteria, and increased potentially pro-inflammatory bacteria, accompanied by impaired intestinal barrier integrity. Following intestinal barrier disruption, inflammatory molecules such as lipopolysaccharide (LPS) can enter the circulatory system, inducing peripheral immune activation and promoting intracranial inflammatory responses, thereby exacerbating the pro-inflammatory phenotype of microglia and ultimately leading to synaptic damage and cognitive impairment. Therefore, gut microbiota imbalance may promote the pathological progression of AD.

[0004] Exercise intervention, as a safe and effective non-pharmacological intervention, has significant potential in delaying the progression of Alzheimer's disease (AD). Previous studies have shown that regular exercise can improve learning and memory abilities, delay cognitive decline, and to some extent reduce Aβ deposition, improve synaptic plasticity, and inhibit neuroinflammatory responses (Dougherty RJ, Wang J, Tian Q, et al. Rising energetic cost of walking predicts cognitive impairment [J]. Alzheimers Dement, 2026, 22(2): e71193.; Chai GS, Gao TL, Bi SG, et al. Aerobic exercise facilitates p300 nuclear translocation via ADRB2-AMPKα signaling, leading to enhanced histone acetylation and mitigation of cognitive decline in APP / PS1 mice [J]. Alzheimers Res Ther, 2026.). However, the mechanism by which exercise improves AD pathology is not fully understood. Elucidating the mechanism by which regular exercise improves AD pathology is of great significance for the development of AD diagnostic kits and new drugs for the treatment of AD. Summary of the Invention

[0005] In order to address the problems in the prior art, the present invention aims to provide the application of glycerophospholipid metabolites in Alzheimer's disease.

[0006] The present invention adopts the following technical solution: Application of glycerophospholipid metabolites in Alzheimer's disease.

[0007] Specifically, the application of glycerophospholipid metabolites in the preparation of Alzheimer's disease diagnostic kits.

[0008] The glycerophospholipid metabolites described in this invention are selected from PC(18:0 / 0:0), PC(18:1(11Z) / 0:0), PC(0:0 / 16:0), or PC(16:0 / 0:0). The structural formula is as follows: Figure 1 As shown, this is the LPC subtype.

[0009] PC(18:0 / 0:0) is stearoyl lysophosphatidylcholine, with 18:0 connected at the sn-1 position and empty at the sn-2 position, belonging to saturated LPC.

[0010] PC(18:1(11Z) / 0:0) is oleoyl lysophosphatidylcholine, with an 18:1 monounsaturated fatty acid linked at the sn-1 position and an empty sn-2 position.

[0011] PC(0:0 / 16:0) is 2-palmitoyllysolecithincholine, with 16:0 connected at the sn-2 position and no position at the sn-1 position, belonging to 2-acyl LPC.

[0012] PC(16:0 / 0:0) is 1-palmitoyllysolecithincholine, with 16:0 at the sn-1 position and no position at the sn-2 position, belonging to 1-acyl LPC.

[0013] PC(18:0 / 0:0), PC(18:1(11Z) / 0:0), PC(0:0 / 16:0), and PC(16:0 / 0:0) share the common characteristic of having only one fatty acid chain and one phosphocholine head group attached to the glycerol backbone. Among them, PC(18:0 / 0:0) and PC(16:0 / 0:0) indicate that the fatty acid is located at the sn-1 position, while PC(0:0 / 16:0) indicates that the fatty acid is located at the sn-2 position; PC(18:1(11Z) / 0:0) contains an 18-carbon monounsaturated fatty acid side chain.

[0014] In this invention, the kit is used to measure the content of glycerophospholipid metabolites PC (18:0 / 0:0), PC (18:1(11Z) / 0:0), PC (0:0 / 16:0), or PC (16:0 / 0:0) in a sample. The sample is derived from feces, serum, or brain tissue.

[0015] The above-mentioned diagnostic kit can be used to detect Alzheimer's disease (AD) patients after they have engaged in regular exercise. The application of the above-mentioned glycerophospholipid metabolites in the preparation of the Alzheimer's disease diagnostic kit is to evaluate the rehabilitation effect of AD patients after regular exercise; the preferred regular exercise is running.

[0016] The aforementioned glycerophospholipid metabolites can be used as indicators for evaluating the efficacy of AD treatment drugs.

[0017] The above-mentioned diagnostic kit can be used to detect Alzheimer's disease (AD) patients after they have taken gut microbiota-enhancing agents. The application of the above-mentioned glycerophospholipid metabolites in the preparation of Alzheimer's disease diagnostic kits is to evaluate the recovery effect of AD patients after taking gut microbiota-enhancing agents.

[0018] The present invention also provides the use of the above-mentioned glycerophospholipid metabolites in the preparation of drugs for the prevention or treatment of Alzheimer's disease.

[0019] Beneficial effects

[0020] This invention provides the application of glycerophospholipid metabolites in Alzheimer's disease.

[0021] Studies have shown that regular exercise can improve learning and memory abilities, delay cognitive decline, and to some extent reduce Aβ deposition, improve synaptic plasticity, and inhibit neuroinflammatory responses (Dougherty RJ, Wang J, Tian Q, et al. Rising energetic cost of walking predicts cognitive impairment [J]. Alzheimers Dement, 2026, 22(2): e71193.;Chai GS, Gao TL, Bi SG, et al. Aerobic exercise facilitates p300 nuclear translocation via ADRB2-AMPKα signaling, leading to enhanced histone acetylation and mitigation of cognitive decline in APP / PS1 mice [J]. Alzheimers Res Ther, 2026.). In recent years, the gut-brain axis has gradually become a hot topic in the study of AD pathogenesis. Gut microbiota imbalance can affect central nervous system homeostasis by regulating metabolites, inflammatory responses, and intestinal barrier function. Whether exercise can improve AD cognitive dysfunction by regulating gut microbiota remains unclear.

[0022] This invention uses the fecal metabolic profile of 5×FAD mice remodeled by exercise as a starting point to systematically analyze the overall changes in the metabolic network after exercise intervention. The results showed that the glycerophospholipid metabolic pathway exhibited a consistent upregulation trend across tissues after exercise, and four specific glycerophospholipid metabolites showed relatively consistent increases in feces, serum, and brain tissue. The inventors of this invention discovered that the cross-tissue synergistic changes in the glycerophospholipid metabolic pathway and the levels of the four specific glycerophospholipid metabolites suggest that exercise may participate in the regulation of AD-related pathological processes by remodeling the lipid metabolism network. Further metabolomics analysis of hippocampal tissue from FMT recipient mice revealed that differentially expressed metabolites were mainly enriched in pathways related to lipid metabolism, phagocytosis, and neurotransmitter regulation, and their overall levels showed an upward trend. This result supports the possibility, from a causal chain perspective, that exercise-remodeled gut microbiota can alter the central metabolic environment through metabolites, and further suggests a potential link between lipid metabolism and phagocytic function. This study observed that exercise intervention can upregulate the levels of four specific glycerophospholipid metabolites in 5×FAD mice, and these metabolites are closely related to changes in microglial inflammation and phagocytic function, thus providing new experimental evidence for the potential protective role of glycerophospholipid metabolites in AD.

[0023] This invention, from metabolomics screening to in vitro functional verification, systematically proposes and confirms that four specific glycerophospholipid metabolites are important metabolic signaling molecules for the regulation of enterocytosis by exercise. Their mechanism of action may involve microglial cell remodeling, manifested as enhanced Aβ phagocytosis and inhibition of inflammatory response, providing new directions for the development of AD diagnostic kits and new AD drugs. Attached Figure Description

[0024] Figure 1 These are the structural formulas of four specific glycerophospholipid metabolites.

[0025] Figure 2 This study investigated the effects of exercise on reducing Aβ deposition in the brains of 5×FAD mice and inhibiting microglial overactivation. (A) Representative immunofluorescence images show the expression of Aβ plaques (6E10, red), microglia (Iba1, green), and cell nuclei (DAPI, blue) in the cortical brain tissue of WT, 5×FAD, and 5×FAD-EX mice. Merge images are three-channel composites with a scale bar of 100 μM. (B) Quantitative analysis of the number of 6E10 positive plaques (cells / mm²). (C) Quantitative analysis of the percentage of 6E10 positive area. (D) Quantitative analysis of the percentage of Iba1 positive area in microglia. All data are expressed as Mean ± SEM. The sample size for each group was n = 5. Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among three groups. *P < 0.05, **P < 0.01.

[0026] Figure 3 This image shows the results of exercise-induced reduction of intestinal barrier damage in WT mice induced by fecal microbiota transplantation in 5×FAD mice. (A) Alixin blue staining shows the distribution of goblet cells in the colon tissues of WT, WT-AS, and WT-AE mice. Blue staining represents goblet cells that secrete acidic mucopolysaccharides. (B) Quantitative analysis (relative value) of the number of goblet cells in the colon unit crypts of the three groups of mice (WT, WT-AS, and WT-AE), with a scale bar of 100 μM. All experimental results are expressed as Mean ± SEM. The sample size for each group is n = 5. One-way ANOVA was used for comparison among the three groups. *P < 0.05, **P < 0.01.

[0027] Figure 4This is a graph showing the results of exercise-induced learning and memory dysfunction in WT mice induced by fecal microbiota transplantation in 5×FAD mice. (A) The escape latency curves of each group of mice finding the platform during the water maze hidden platform training phase (Acquisition); (B) The percentage of each group of mice swimming in the target quadrant during the water maze navigation test; (C) The percentage of each group of mice staying in the target quadrant during the water maze navigation test. All data are expressed as Mean ± SEM. The sample size for each group is n = 10. Two-way repeated measures ANOVA was used for the escape latency in the water maze, and one-way ANOVA was used for other comparisons among multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significant difference.

[0028] Figure 5 This is a heatmap of the expression of four common and differentially expressed LPC subtypes in feces, brain tissue, and serum.

[0029] Figure 6 This is a graph showing the potential metabolic mediator between the gut microbiota and the inflammatory response of microglia by LPC. (A) is a Pearson correlation heatmap between LPC and key differentially expressed bacterial genera; (B) is a Pearson correlation heatmap between LPC and inflammatory factors and microglia activation levels. Red represents a positive correlation, and blue represents a negative correlation. The intensity of the color reflects the strength of the correlation, and the numbers in the squares are the correlation coefficients (r values).

[0030] Figure 7 LPC promotes the absorption of Aβ by BV2 microglia. 42 The phagocytic results are shown in the figure, where (A) the proportion of FITC-positive cells was quantitatively analyzed by flow cytometry; and (B) the phagocytic response of BV2 cells to FITC-Aβ was detected by immunofluorescence. 42 Phagocytosis, Iba1 (red) marking microglia, FITC-Aβ 42 (Green) labeling of phagocytic substrate, DAPI (blue) labeling of cell nuclei, scale bar = 20 μM; (C) Immunofluorescence quantitative analysis of the proportion of FITC-positive phagocytic cells. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA or independent samples t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed Implementation

[0031] To further illustrate the purpose, technical solution, and advantages of this invention, the technical solution of this invention will be further described below. All reagents used in this invention are commercially available products. Unless otherwise specified, all percentages mentioned in this invention are mass percentages. Example 1: Exercise reduces Aβ deposition in the brain of 5×FAD mice and inhibits excessive microglial activation.

[0032] All animals involved in this experiment were purchased from Shanghai Southern Model Organisms Center Co., Ltd., including 5×FAD transgenic mice and age-matched wild-type (WT) mice, with a background of C57BL / 6J. The mice were bred and housed in a specific pathogen-free (SPF) environment at the Experimental Animal Center of the Second Affiliated Hospital of Chongqing Medical University. The light and dark conditions were maintained at a 12-hour light-dark cycle, and water and food were provided in abundance. All animal experiments were approved by the Experimental Animal Management and Use Committee of the Second Affiliated Hospital of Chongqing Medical University (Approval No.: IACUC-SAHCQMU-2023-0039). Animal groups: (1) Control group (WT): C57BL / 6J mice; (2) AD model group (5×FAD): 5×FAD transgenic mice; (3) Exercise intervention group (5×FAD-EX): 5×FAD transgenic mice underwent high-intensity intermittent training.

[0033] Mice underwent HIIT (High-Intensity Intervention) on a treadmill. An adaptation phase was established before the experiment: mice were placed on an electric treadmill and trained at gradually increasing speeds, starting at 5 m / min and gradually increasing to 15 m / min, for 30 minutes daily for 5 consecutive days to reduce exercise stress. The formal training phase lasted 8 weeks. The initial speed in week 1 was 15 m / min, increasing by 1 m / min each week thereafter, reaching a maximum speed of 22 m / min in week 8. Training was conducted 5 days a week, with each session lasting 60 minutes. Each training session consisted of 15 high-intensity running phases, each lasting 2 minutes, interspersed with 2-minute recovery phases. The mice's movement was monitored throughout the training process, with brief adjustments made as needed to avoid over-fatigue. A sedentary control group of mice was placed on the same type of treadmill but did not undergo exercise training to eliminate environmental interference. Fecal samples were collected on the second day after the 8-week training period ended. During sampling, gently hold the mouse by its back and tail to position it in a supine position. Place a sterile cryopreservation tube near the mouse's anus and collect 2-3 fresh rectal feces after the mouse defecates naturally. Avoid contact between feces and urine or other contaminants during sampling. Immediately immerse the collected fecal samples in liquid nitrogen for flash freezing, then transfer them to a -80°C freezer for long-term storage for subsequent gut microbiota gene sequencing and metabolomics analysis.

[0034] 5×FAD transgenic mice are a commonly used AD mouse model. Aβ plaques appear in the brain between 2 and 4 months of age, triggering a strong microglial response and inflammatory process, as well as synaptic and neuronal loss. Therefore, 4 months of age is generally considered a crucial stage in the progression of early pathology to more pronounced pathological changes in this model. Based on this, this study selected 4 months of age as the starting point for exercise intervention, implementing HIIT for 8 weeks in 5×FAD transgenic mice to investigate the effect of exercise intervention on AD-related pathological changes. After the exercise intervention, the Aβ plaque burden was assessed by immunofluorescence staining of brain tissue. The results showed that, compared with the model group, exercise intervention significantly reduced the number and deposition area of ​​Aβ plaques in the cerebral cortex of 5×FAD mice, suggesting that exercise can effectively alleviate the core pathological changes in AD.

[0035] Since Aβ deposition is not only a pathological outcome but also a significant stimuli inducing abnormal microglial activation, and persistent overactivation of microglia can further exacerbate neuroinflammation and nerve damage, this study further analyzed the microglial state using immunofluorescence. The results are as follows... Figure 2 As shown, exercise intervention significantly improved the overactivation of microglia in the brains of 5×FAD mice. Example 2: Does exercise intervention reduce the pathogenicity of gut microbiota in 5×FAD mice?

[0036] The inventors transplanted the gut microbiota collected from 5×FAD mice in Example 1 into WT recipient mice to simulate the effects of exogenous gut microbiota imbalance on the host. This serves two purposes: firstly, to verify whether the gut microbiota of 5×FAD mice is sufficient to induce AD-like pathological changes and related neuroinflammatory responses in WT mice; and secondly, to further evaluate whether exercise intervention can alleviate the damaging effects of the gut microbiota of 5×FAD mice on WT mice.

[0037] The animal model used in this experiment was the wild-type C57BL / 6J mouse, which was purchased from Shanghai Southern Model Organisms Center Co., Ltd. The animals were divided into three groups: (1) control group (WT); (2) group transplanted with fecal microbiota from 5×FAD mice (WT-AS); and (3) group transplanted with fecal microbiota from 5×FAD mice with exercise intervention (WT-AE).

[0038] Before the gut microbiota transplantation, the WT-AS and WT-AE groups first received a one-week oral administration of an antibiotic cocktail (ABX) (metronidazole 30 mg / mL, vancomycin 15 mg / mL, neomycin 30 mg / mL, ampicillin 30 mg / mL) to clear or significantly reduce the original gut microbiota, thereby creating conditions for the subsequent colonization of exogenous microbiota.

[0039] Fecal samples stored at -80°C were removed and thawed on ice. A bacterial suspension was prepared by adding 1 g of feces to 10 mL of sterile PBS. After soaking at 4°C for 20 min, the mixture was thoroughly shaken to ensure even dispersion of the feces in the PBS. The suspension was then centrifuged at 4°C and 2000 rpm for 5 min. The supernatant was collected and aliquoted (3 mL / tube). The prepared bacterial suspension was immediately placed in a -80°C freezer and used immediately before gavage.

[0040] On day 1 after antibiotic pretreatment, mice in the WT-AS and WT-AE groups began receiving bacterial suspension via gavage. Initially, gavage was administered once daily for one month, with each gavage volume of 100 μL, five times a week. Subsequently, the gavage frequency was adjusted to three times a week for two months. Mice were fasted and deprived of water for 4 hours before each gavage, and all gavage syringes were sterilized and dried. The mice's mental state and behavior were closely monitored during gavage; if significant adverse reactions occurred, gavage was immediately stopped, and fluid replacement and subsequent treatment were administered as needed.

[0041] This invention first pretreated recipient WT mice with ABX to eliminate their original gut microbiota, followed by transplantation of fecal microbiota from 5×FAD and 5×FAD-EX mice. After transplantation, fecal samples were collected, behavioral tests were performed, and tissue samples were collected and relevant experimental analyses were conducted after week 14. Following the intervention, morphological observation of the colonic tissue of the recipient mice was performed using Alixin Blue staining. The results showed that, compared with normal WT mice, the number of goblet cells in the colon of WT mice receiving 5×FAD microbiota transplantation was significantly reduced (P < 0.01). Figure 3 This suggests that the gut microbiota of 5×FAD mice can disrupt the intestinal mucus barrier structure. However, after transplantation of microbiota from exercise-interventional 5×FAD mice, the number of goblet cells in WT recipient mice was restored compared to the group receiving untreated 5×FAD microbiota (P < 0.05). Figure 3 This indicates that the gut microbiota remodeled by exercise can partially improve intestinal mucosal barrier damage.

[0042] To assess the impact of gut microbiota transplantation on the cognitive function of recipient mice, a water maze test was used to assess spatial learning and memory abilities. During the platform hiding training phase, WT recipient mice transplanted with 5×FAD mouse microbiota showed a significantly prolonged escape latency to the platform on day 5 of training, which was significantly lower than that of the WT control group, suggesting impaired spatial learning ability. Figure 4 WT recipient mice transplanted with the 5×FAD microbiota from mice with exercise intervention showed a significantly shorter escape latency compared to the 5×FAD microbiota transplantation group, indicating that the microbiota formed by exercise intervention can partially improve spatial learning impairment caused by the 5×FAD microbiota. Figure 4In the navigation test, compared with WT mice, recipient mice transplanted with 5×FAD mouse microbiota showed significantly reduced time spent in the target quadrant and swimming distance in the target quadrant. Recipient mice transplanted with exercise-interventional 5×FAD mouse microbiota showed increased scores in both of these indicators compared to the 5×FAD microbiota transplantation group, suggesting that exercise intervention can alleviate spatial memory impairment induced by 5×FAD microbiota transplantation. This indicates that the role of exercise in improving AD-like behavioral phenotypes may be closely related to its remodeling of the gut microbiota. Example 3: Non-targeted metabolomics analysis

[0043] To identify the key metabolites involved in the improvement of AD-like pathology by exercise through gut microbiota, this invention performed non-target metabolomics analysis on fecal samples from three groups of mice (WT, 5×FAD, and 5×FAD-EX, Example 1). The results showed significant separation in the metabolomics profiles of the three groups, suggesting that different genotypes and exercise interventions can significantly alter the composition of fecal metabolites. Differentially expressed metabolites were screened based on VIP > 2 and P < 0.05. The results showed that, compared to the WT group, the 5×FAD group had 91 significantly downregulated and 57 significantly upregulated metabolites; compared to the 5×FAD group, the 5×FAD-EX group had 94 significantly downregulated and 93 significantly upregulated metabolites. KEGG pathway enrichment analysis showed that, compared to the WT group, the overall trend of glycerophospholipid metabolism pathway-related metabolites was decreased in the 5×FAD group. Compared to the 5×FAD group, differentially regulated metabolites in the 5×FAD-EX group were also enriched in the glycerophospholipid metabolism pathway and showed an upregulation trend, suggesting that exercise intervention may reverse the abnormal glycerophospholipid metabolism in 5×FAD mice. To verify whether this pathway change has cross-tissue consistency, non-target metabolomics analysis was further performed on brain tissue and serum, and the results were consistent with the trend of changes in fecal metabolomics. Compared to the WT group, the downregulated differentially regulated metabolites in the 5×FAD group were enriched in the glycerophospholipid metabolism pathway, while in the 5×FAD-EX group compared to the 5×FAD group, the upregulated differentially regulated metabolites were also enriched in this pathway, suggesting that glycerophospholipid metabolism may be a key metabolic pathway for exercise intervention to exert a protective effect. Subsequently, an intersection analysis was performed on the metabolomic data from feces, brain tissue, and serum, identifying four differentially expressed metabolites with consistent changes across tissues: PC(18:0 / 0:0), PC(18:1(11Z) / 0:0), PC(0:0 / 16:0), and PC(16:0 / 0:0)[Rac]. Heatmap analysis showed that, compared to the 5×FAD group, the 5×FAD-EX group exhibited an increasing trend for all four LPCs in all three tissue types, with the most significant changes observed in fecal samples. Figure 5 These results suggest that LPC may be an important candidate metabolite for regulating the gut microbiota-metabolite axis and exerting neuroprotective effects through exercise.

[0044] Building upon this foundation, to further explore the role of gut microbiota in the regulation of metabolic changes during exercise, this study conducted a precise metabolomics analysis of the hippocampus tissue of recipient mice after FMT. KEGG pathway enrichment analysis of the screened differentially regulated metabolites revealed that the upregulated metabolites were mainly enriched in phagocytosis-related pathways, cell signaling pathways, lipid metabolism pathways, and neurotransmitter-related pathways. Among the lipid metabolism-related pathways, the glycerophospholipid metabolism pathway and the linoleic acid metabolism pathway were significantly enriched, indicating that lipid metabolism reprogramming may play an important role in the neuroprotective effect of exercise intervention. Furthermore, the apoptotic cell clearance pathway was also significantly enriched, suggesting that phagocytosis-related processes may be regulated by metabolites derived from gut microbiota. Simultaneously, nervous system-related pathways such as the glutamatergic synaptic pathway and the long-term potentiation pathway were also significantly enriched, suggesting that exercise may participate in the regulation of cognitive function by influencing the neurotransmitter system and synaptic plasticity. Further heatmap analysis of LPC levels revealed that, compared with the WT-AS group, the overall LPC level in the hippocampus tissue of recipient mice transplanted with fecal microbiota from 5×FAD exercise mice showed an increasing trend. In summary, these results indicate that the gut microbiota remodeled by exercise can significantly alter the metabolic profile of the hippocampus in recipient mice. These changes mainly involve multiple biological processes, including lipid metabolism, phagocytosis-related processes, and neurotransmitter regulation, providing important clues for further analysis of the mechanisms of action of key metabolites in neuroprotection.

[0045] In the aforementioned non-target metabolomics analysis, this study screened four LPC subtypes that showed consistent variations in feces, serum, and brain tissue: PC(18:0 / 0:0), PC(18:1(11Z) / 0:0), PC(0:0 / 16:0), and PC(16:0 / 0:0)[Rac]. To further clarify their association with the gut microbiota, Pearson correlation analysis was performed on the above LPC subtypes and the major differentially expressed bacterial genera. The results showed that LPCs were significantly positively correlated with some probiotic genera, while they were significantly negatively correlated with some potentially pathogenic bacterial genera. Figure 6 A) suggests that changes in LPC levels may be closely related to an imbalance in the gut microbiota structure.

[0046] Given the important role of LPCs in neuroinflammatory regulation and immune homeostasis, this study further explored their potential association with intracranial inflammatory markers. Correlation analysis showed that the relative abundance of the four LPC subtypes was negatively correlated with the levels of inflammatory factors IL-1β, IL-6, and TNF-α, and also negatively correlated with the proportion of Iba1 positive signal area. Figure 6(B) This suggests that decreased LPC levels may be closely related to microglial overactivation and enhanced neuroinflammation. These results indicate that LPC may play a crucial mediating role between the gut microbiota and the microglial inflammatory response. Exercise intervention, by reshaping the gut microbiota structure and improving LPC levels, thereby alleviating microglial overactivation and reducing neuroinflammation, provides new research clues for elucidating the mechanism by which exercise improves AD-like pathology. Example 4

[0047] This invention uses the immortalized mouse microglial cell line BV2 for in vitro experiments. All cell-related operations were performed under aseptic conditions. Strict aseptic procedures were followed during cell culture, and all culture vessels and reagents used were sterilized. Cell grouping: (1) Control group: BV2 cells without any treatment; (2) Group Aβ: Using Aβ 42 Oligomer intervention in BV2 cells; (3) Aβ + LPC group: using Aβ 42 Oligomers and LPCs jointly intervene in BV2 cells.

[0048] Based on the correlation between LPC levels and microglia activation status in previous animal experiments, this study hypothesizes that LPC may play a key role in the functional regulation of microglia, particularly in the phagocytosis of Aβ by microglia. To further verify the direct impact of LPC on microglia function, this study used the mouse immortalized microglia line BV2 to construct an in vitro model for functional research.

[0049] First, assess Aβ 42 The effect of LPC on BV2 cell viability. CCK-8 results showed that, with the increase of Aβ... 42 With increasing Aβ concentration, BV2 cell viability gradually decreased, and the difference was statistically significant at concentrations ≥ 15 μM, suggesting that high concentrations of Aβ... 42 It exhibits some cytotoxicity. Therefore, subsequent experiments uniformly used 5 μM oligomer Aβ. 42 A stimulation model was constructed. The effects of different concentrations of LPC on BV2 cell viability were further investigated. The results showed that LPC significantly reduced cell viability at concentrations of 10 μM and above; however, no significant toxic effects were observed in the 0-5 μM range. Cell viability in the 2 μM group was slightly higher than that in the control group, indicating that low concentrations of LPC have good biosafety for BV2 cells under the experimental conditions.

[0050] After determining the safe concentration range, FITC-labeled Aβ was used. 42 (FITC-Aβ) 42A phagocytosis model was constructed. BV2 cells were pretreated with different concentrations of LPC (0, 1, 2, 3, 4, 5 μM) and then phagocytosed with FITC-Aβ. 42 After co-incubation for 3 hours, the proportion of FITC-positive cells was detected by flow cytometry. The results showed that, compared with the control group, LPC treatment significantly increased the FITC-Aβ responsiveness of BV2 cells. 42 The phagocytic capacity of the 3 μM LPC group was significantly enhanced, with the phagocytic enhancement effect being the most pronounced. Figure 7 To further visually verify the changes in phagocytic phenotype, immunofluorescence was used for observation. Results showed that, compared to the Aβ group, the Aβ + LPC group had significantly higher FITC-Aβ levels. 42 The green fluorescence signal was significantly enhanced, the proportion of FITC-positive cells increased significantly, and the cytoplasmic punctate distribution was more obvious.

[0051] In summary, the in vitro experimental results indicate that LPC can significantly enhance the response of BV2 cells to Aβ. 42 The phagocytic capacity suggests that LPC may be involved in the microglia-mediated Aβ phagocytosis process as a key metabolite after exercise intervention.

Claims

1. Application of glycerophospholipid metabolites in the preparation of Alzheimer's disease diagnostic kits; wherein the glycerophospholipid metabolites are selected from PC(18:0 / 0:0), PC(18:1(11Z) / 0:0), PC(0:0 / 16:0) or PC(16:0 / 0:0); PC(18:0 / 0:0) is stearoyl lysophosphatidylcholine; PC(18:1(11Z) / 0:0) is oleoyl lysophosphatidylcholine; PC(0:0 / 16:0) is 2-palmitoyl lysophosphatidylcholine; PC(16:0 / 0:0) is 1-palmitoyl lysophosphatidylcholine.

2. The application as described in claim 1, characterized in that, The kit is used to measure the content of glycerophospholipid metabolites PC (18:0 / 0:0), PC (18:1(11Z) / 0:0), PC (0:0 / 16:0), or PC (16:0 / 0:0) in a sample.

3. The application as described in claim 2, characterized in that, The samples were derived from feces, serum, or brain tissue.

4. The application as described in any one of claims 1-3, characterized in that, The glycerophospholipid metabolites are used as an evaluation indicator for the efficacy of AD treatment drugs.

5. The application as described in claim 4, characterized in that, To evaluate the recovery effect of intestinal flora preparations in Alzheimer's disease (AD) patients.

6. The application as described in any one of claims 1-3, characterized in that, The application is used to evaluate the rehabilitation effect of AD patients after regular exercise.

7. The application as described in claim 6, characterized in that, The exercise in question is running.

8. The use of glycerophospholipid metabolites in the preparation of drugs for the prevention or treatment of Alzheimer's disease; wherein the glycerophospholipid metabolites are selected from PC(18:0 / 0:0), PC(18:1(11Z) / 0:0), PC(0:0 / 16:0) or PC(16:0 / 0:0); PC(18:0 / 0:0) is stearoyl lysophosphatidylcholine; PC(18:1(11Z) / 0:0) is oleoyl lysophosphatidylcholine; PC(0:0 / 16:0) is 2-palmitoyl lysophosphatidylcholine; PC(16:0 / 0:0) is 1-palmitoyl lysophosphatidylcholine.

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