Use of psgl-1 antibodies in the preparation of a medicament for delaying aging of the body

Abstract

The application belongs to the field of biological medicine, and specifically discloses application of a PSGL-1 antibody in preparation of a medicine for delaying aging of a body, and discloses that the PSGL-1 antibody can effectively relieve formation of MPAs in peripheral blood of an old mouse, and further improve tissue degenerative diseases of the old mouse, and can relieve cell aging and inflammation; intervention on the old mouse by using the PSGL-1 antibody can obviously relieve aging, which can be reflected from a blood system, an inflammation factor level and a movement ability, and it is proved that the PSGL-1 antibody can be used for preparation of a medicine for delaying aging.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of a PSGL-1 antibody in the preparation of drugs that delay aging. Background Technology

[0002] Aging is a complex biological process involving the progressive decline of function in multiple organ systems. One of its core characteristics is chronic low-grade inflammation, or inflammatory aging. This persistent inflammatory state not only exacerbates damage to tissue cells but also directly promotes the occurrence and development of various age-related diseases such as atherosclerosis, neurodegenerative diseases, sarcopenia, and metabolic syndrome. During aging, the hematopoietic system, as a key site for metabolism and signal exchange, experiences particularly significant functional decline. The core characteristics of hematopoietic system aging are primarily manifested in a compensatory increase in the number of hematopoietic stem cells, but a significant decrease in their self-renewal, homing, and engraftment capabilities, exhibiting a clear myeloid differentiation bias, leading to a sharp reduction in lymphoid production and triggering immunosenescence. Simultaneously, changes in the bone marrow microenvironment are unfavorable for stem cell maintenance; furthermore, insufficient erythropoiesis leads to senile anemia, and abnormal megakaryocytic lineage function increases the risk of thrombosis.

[0003] Platelet-monocyte aggregates (MPA) are important biomarkers reflecting the activation status of platelets in vivo and a key bridge connecting thrombosis and inflammatory responses. During aging, circulating MPA levels significantly increase due to vascular endothelial damage and persistent low-level platelet activation. MPA formation primarily depends on the specific binding of P-selectin (expressed on the surface of activated platelets) to its main ligand, P-selectin glycoprotein ligand-1 (PSGL-1, expressed on the surface of monocytes). Once MPA forms, monocytes are further activated, releasing large amounts of pro-inflammatory factors (such as IL-1β, TNF-α, and MCP-1), while simultaneously upregulating tissue factor expression, exacerbating oxidative stress damage to the vascular endothelium and local microthrombus formation. This MPA-mediated thrombotic-inflammatory cascade is considered one of the important mechanisms driving age-related pathological changes such as atherosclerotic plaque progression, myocardial fibrosis, and cognitive decline. Therefore, blocking MPA formation has become a key entry point for intervening in vascular aging and related diseases.

[0004] P-selectin glycoprotein ligand-1 (PSGL-1), a transmembrane mucoprotein expressed on the surface of monocytes, plays a crucial role in mediating the rolling, adhesion, and migration of leukocytes to sites of inflammation or injury. Recent studies have found that PSGL-1 directly mediates the formation of MPA (pro-inflammatory glycoprotein) by binding to P-selectin on the surface of activated platelets, thus tightly coupling platelet activation with the pro-inflammatory function of monocytes. In aging individuals, due to increased platelet reactivity and dynamic changes in PSGL-1 expression levels, MPA production significantly increases, further amplifying local vascular inflammatory damage.

[0005] Current research on the application of PSGL-1 antibodies is mainly in the field of tumor immunotherapy. PSGL-1 molecules on the surface of tumor cells can serve as novel phagocytic checkpoints, transmitting signals to macrophages and thus helping tumors achieve immune escape. This invention utilizes an antibody intervention strategy targeting PSGL-1, specifically blocking the binding of PSGL-1 to P-selectin, effectively inhibiting the formation of MPA and fundamentally cutting off the abnormal interaction between platelets and monocytes. Studies have shown that anti-PSGL-1 antibody treatment can significantly reduce the level of circulating MPA, decrease the activation state and tissue infiltration capacity of monocytes, thereby alleviating the inflammatory burden on blood vessel walls and target organs.

[0006] Currently, research on PSGL-1 antibodies delaying the aging process by inhibiting platelet-monocyte aggregate formation is still in its early stages. Existing studies mostly focus on evaluating their efficacy in tumor disease models, while systematic reports on the effects and mechanisms of PSGL-1 antibodies in systemically improving physiological function and delaying the aging process in elderly individuals are still lacking. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides the application of PSGL-1 antibody in the preparation of drugs that delay aging. By treating an aging model with PSGL-1 antibody, it is observed that the treated model exhibits a significant decrease in the level of inflammation in the blood system, a more youthful distribution ratio in the blood system cell population, and a substantial improvement in motor ability, thereby achieving the effect of delaying aging.

[0008] To achieve the above objectives, the present invention is implemented according to the following technical solution: One of the objectives of this invention is to provide the application of PSGL-1 antibody in the preparation of drugs that delay aging.

[0009] A second objective of this invention is to provide a drug for delaying aging, comprising the aforementioned PSGL-1 antibody.

[0010] Furthermore, the aforementioned drugs for delaying aging may also contain pharmaceutically acceptable carriers.

[0011] Compared with existing anti-aging intervention technologies, the present invention has the following beneficial effects: This invention reveals and verifies for the first time that PSGL-1 antibody blocks the aging-related thrombotic inflammatory cascade at its source by specifically inhibiting the formation of platelet-monocyte aggregates, a method distinct from traditional broad-spectrum anti-inflammatory drugs or single-target intervention strategies. Experimental data show that mice treated with PSGL-1 antibody not only exhibited a significantly reduced proportion of circulating MPA-positive cells, but also showed a simultaneous downregulation of key pro-inflammatory factors in their serum and tissues, confirming its effective control of chronic low-grade inflammation at the molecular and cellular levels, and providing a new, precise immune regulatory pathway for delaying aging.

[0012] This invention provides the first comprehensive animal study demonstrating that the PSGL-1 antibody can systematically improve physiological function and hematopoietic homeostasis in aged individuals. After antibody treatment, aged mice showed significant improvements in motor function in rotarod and grip strength tests, indicating substantial improvement in musculoskeletal function and overall vitality. Simultaneously, the proportion of youthful cell populations in the bone marrow suggested a synergistic effect in delaying the aging process of the hematopoietic system. Furthermore, the PSGL-1 antibody described in this invention exhibits excellent anti-aging effects, providing an important foundation for subsequent combination with other anti-aging drugs and optimization of dosing strategies, and possesses broad clinical translational prospects and patent value. Attached Figure Description

[0013] Figure 1 The correlation between MPA and age is illustrated as follows: A: Peripheral blood sample patterns for individuals aged 18 to 80 years; B: Flow cytometry analysis of average MPA levels in peripheral blood samples from different age groups, with average MPA flow cytometry plots for young individuals (left) and older individuals (right); C: Correlation plot between different age groups and the proportion of MPA in peripheral blood; D: Flow cytometry analysis of average MPA levels in peripheral blood from mice of different ages, with average MPA flow cytometry plots for young mice (left) and older mice (right); E: MPA ratio in peripheral blood from mice of different ages. Example: violin plot, F: Flow cytometry detection of the average MPA level in bone marrow samples of mice of different ages, flow cytometry plot of MPA ratio in bone marrow of young and old mice (left), box plot of MPA ratio in bone marrow of young and old mice (right), G: Schematic diagram of in vivo animal experiment, old mice were randomly divided into groups and treated with PBS and PSGL-1 antibody respectively, H: Flow cytometry plot of MPA ratio in peripheral blood of PSGL-1 antibody treatment group and PBS treatment control group (left), box plot of effect of PSGL-1 antibody treatment on MPA ratio in peripheral blood of old mice (right).

[0014] Figure 2Behavioral experiments showing the improvement of motor function in aged mice after PSGL-1 antibody treatment: A: Diagrams of open field, new object recognition, suspension, tension, and rotational fatigue tests performed on mice in the PSGL-1 antibody treatment group and the PBS treatment control group, respectively. B: Movement trajectories of mice in the open field test (left figure), effect of PSGL-1 antibody treatment on the total distance of movement in aged mice in the open field (middle figure), effect of PSGL-1 antibody treatment on the ratio of movement time to rest time in aged mice in the open field (right figure). C: Effect of PSGL-1 antibody treatment on the total distance of movement in aged mice in the fatigue test. D: Effect of PSGL-1 antibody treatment on the endurance of aged mice in the suspension test. E: Effect of PSGL-1 antibody treatment on the preference of aged mice in new object recognition. F: Effect of PSGL-1 antibody treatment on the grip strength and holding force of aged mice in the tension test.

[0015] Figure 3 The effects of PSGL-1 antibody treatment on the reduction of inflammation levels in aged mice are illustrated in the following diagrams: A: Effect of PSGL-1 antibody treatment on F4 / 80-positive areas in the kidneys of aged mice in immunohistochemistry. Left image shows scanned sections of F4 / 80-stained kidneys from aged mice under both treatments, and a statistical diagram of the positive areas after F4 / 80 staining in the kidneys of aged mice under both treatments (right image). B: Effect of PSGL-1 antibody treatment on F4 / 80-positive areas in the liver of aged mice in immunohistochemistry. Left image shows scanned sections of F4 / 80-stained livers from aged mice under both treatments, and a statistical diagram of the positive areas after F4 / 80 staining in the livers of aged mice under both treatments (right image). C: Effect of PSGL-1 antibody treatment on F4 / 80-positive areas in the lungs of aged mice in immunohistochemistry. Left image shows scanned sections of F4 / 80-stained lungs from aged mice under both treatments, and a statistical diagram of the positive areas after F4 / 80 staining in the lungs of aged mice under both treatments (right image). D: Effect of PSGL-1 antibody treatment on iNOS-positive areas in the kidney immunohistochemistry of aged mice. Scanned images of iNOS-stained kidney sections of aged mice under two treatments (left image). Statistical graph of iNOS-positive areas in the kidneys of aged mice after iNOS staining after two treatments (right image). E: Effect of PSGL-1 antibody treatment on iNOS-positive areas in the liver immunohistochemistry of aged mice. Scanned images of iNOS-stained liver sections of aged mice under two treatments (left image). Statistical graph of iNOS-positive areas in the livers of aged mice after iNOS staining after two treatments (right image). F: Effect of PSGL-1 antibody treatment on iNOS-positive areas in the lung immunohistochemistry of aged mice. Scanned images of iNOS-stained lung sections of aged mice under two treatments (left image). Statistical graph of iNOS-positive areas in the lungs of aged mice after iNOS staining after two treatments (right image). G: Effect of PSGL-1 antibody treatment on the level of inflammatory factors in the bone marrow supernatant of aged mice.

[0016] Figure 4 The following diagrams illustrate how PSGL-1 antibody treatment can induce a more youthful hematopoietic cell population in aged mice: A: Effect of PSGL-1 antibody treatment on the proportion of immature T cells in aged mice, with flow cytometry plots of immature T cell proportions under two treatments (left) and statistical plots of immature T cell proportions under two treatments (right); B: Effect of PSGL-1 antibody treatment on the proportion of lymphoid progenitor cells in aged mice, with flow cytometry plots of lymphoid progenitor cells under two treatments (left) and statistical plots of lymphoid progenitor cells under two treatments (right); C: Effect of PSGL-1 antibody treatment on the proportion of myeloid-biased hematopoietic stem cells in aged mice, with flow cytometry plots of myeloid-biased hematopoietic stem cells under two treatments (left) and statistical plots of myeloid-biased hematopoietic stem cells under two treatments (right); D: Effect of PSGL-1 antibody treatment on the proportion of MPP4 cells in the bone marrow of aged mice; E: Effect of PSGL-1 antibody treatment on the proportion of MPP1 cells in the bone marrow of aged mice; F: Effect of PSGL-1 antibody treatment on the proportion of MPP2 / 3 cells in the bone marrow of aged mice.

[0017] Figure 5 To assess the safety of PSGL-1 antibody treatment without affecting platelet function: A: Effect of PSGL-1 antibody treatment on the percentage of white blood cells in aged mice; B: Effect of PSGL-1 antibody treatment on the percentage of red blood cells in aged mice; C: Effect of PSGL-1 antibody treatment on the percentage of platelets in aged mice; D: Effect of PSGL-1 antibody treatment on the percentage of BMT cells in the peripheral blood of aged mice; E: Effect of PSGL-1 antibody treatment on the percentage of BMT cells in the bone marrow of aged mice; F: Effect of PSGL-1 antibody treatment on the platelet clot retraction assay in aged mice, including images of clot retraction time points (left) and a statistical graph of clot size changes over time (right); G: Effect of PSGL-1 antibody treatment on ATP release in aged mice; H: Effect of PSGL-1 antibody treatment on SELP levels in platelets of aged mice. + Effect of cell ratio, I: PSGL-1 antibody treatment on Jon / A ratio in platelets of aged mice + Effects on cell proportions: J: Effect of PSGL-1 antibody treatment for one month on the proportion of BMT cells in the bone marrow of aged mice; K: Effect of PSGL-1 antibody treatment for one month on the proportion of MPA cells in the peripheral blood of aged mice; L: Effect of PSGL-1 antibody treatment for one month on the proportion of various cell types in the bone marrow of aged mice. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0019] All chemical materials used in the following examples were purchased from the following companies: PSGL-1 antibody was purchased from BioXcell (catalog number BE0186-5MG); PBS was purchased from ELGBIO (catalog number EH80256); isoflurane was purchased from RWD (catalog number R510); PE Rat Anti-Mouse IL-6 was purchased from BD Pharmingen™ (catalog number 562050); Anti-APC Beads were purchased from Miltenyi (catalog number 130-090-855); APC-ckit was purchased from Thermo Fisher Scientific (catalog number 17-1171-83 / 2B8); APC-B220 was purchased from Biolegend (catalog number 103212 / RA3-). 6B2; PE-Cy7-B220 was purchased from Biolegend, catalog number 103221; APC-CD11b was purchased from Biolegend, catalog number 101212 / M1 / 70; APC-Cy7 SA was purchased from Thermo Fisher Scientific, catalog number 47-4317-82; BV605 CD150 was purchased from BD Biosciences, catalog number 115927 / TC15-12F12.2; FITC-CD34 was purchased from BD Biosciences. Biosciences, catalog number 553733 / RAM34; FITC-B220 purchased from Biolegend, catalog number 103206 / RA3-6B2; FITC-CD4 purchased from Biolegend, catalog number 100510 / RM4-5; FITC-CD8a purchased from Biolegend, catalog number 100706 / 53-6.7; APCSELP purchased from Invitrogen, catalog number 17-0626-82; PE Jon / A purchased from Biolegend, catalog number 101212 / M1 / 70; FITC BrdU purchased from Beyotime, catalog number ST1056-2g; APC CD41 was purchased from Biolegend (catalog number: 133913); AF700-CD16 / 32 was purchased from Thermo Fisher Scientific (catalog number: 56-0161-82 / 93); AF700-CD34 was purchased from BD Biosciences (catalog number: 560518 / RAM34); BV605-CD150 was purchased from BD Biosciences (catalog number: 115927 / TC15-12F12.2); Human FITC-CD11b was purchased from Biolegend (catalog number: 301330); Human PE-cy7 CD14 was purchased from Biolegend (catalog number: 367112); Human PE-CD41 was purchased from Biolegend (catalog number: 303706); and the enhanced ATP assay kit was purchased from Beyotime (catalog number: S0027). Unless otherwise specified, all other reagents used are commercially available pharmaceuticals, and all methods involved are known methods unless otherwise specified.

[0020] Example 1: The proportion of MPA in human peripheral blood increases with age. Healthy volunteers were recruited according to a protocol approved by the ethics committee, selecting individuals aged 18 to 80 and dividing them into youth and elderly groups, with a sample size of approximately 30-40 cases in each group. Figure 1As shown in section A, collect 2-3 mL of fasting peripheral venous blood using a sodium citrate anticoagulant tube. Immediately after collection, gently invert and mix 5-8 times to avoid platelet activation. All samples should be processed within one hour of collection. If immediate testing is not possible, whole blood can be stored at room temperature, but centrifugation and washing are strictly prohibited before antibody labeling to prevent artificial activation in vitro. Record donor information such as age, sex, blood pressure, and medication history, excluding individuals who have recently taken antiplatelet drugs or have inflammatory diseases or malignant tumors.

[0021] Take flow cytometry sample tubes and add 50 μL of thoroughly mixed whole blood to each tube. Then add fluorescently labeled antibodies sequentially: CD14-PE-Cy7 (labeled monocytes), CD41-PE (labeled platelets), and CD11b-FITC (labeled myeloid cells) to the detection tubes. Vortex to mix and incubate at room temperature in the dark for 20 min. After incubation, add 1 mL of 1× erythrocyte lysis buffer to each tube, vortex to mix, and lyse at room temperature in the dark for 10 min (note that the lysis time should not be too long to avoid damaging leukocytes). After lysis, centrifuge at 300 g for 5 min, discard the supernatant, wash once with 2 mL of PBS, centrifuge at 300 g for 5 min, discard the supernatant, and finally resuspend the cells in 300 μL of PBS. Immediately analyze using a flow cytometer. The P1 gate was set up to differentiate cell size and granularity using FSC-A / SSC-A, enriching nucleated cells and excluding debris and other impurities. The P2 gate used FSC-H / FSC-A to remove adherent cell clusters, obtaining a pure single-cell population. The P3 gate, combined with the PerCP-Cy5-5 channel, further delineated the target cell population. The Mono gate used CD11b (FITC channel) and CD14 (PE-Cy7 channel) as markers to separate CD11b+CD14+ monocytes from the P3 gate. The MPA gate, based on monocytes, excluded platelet contamination using CD41 (PE channel), precisely identifying the target MPA cell subset. Finally, the Meyloid PA population was additionally delineated for further myeloid-related analysis. Differences in MPA proportions, platelet activation levels, and the distribution of various monocyte subsets were compared among different age groups. Results are as follows: Figure 1 As shown, Figure 1 The B-flow cytometry plot shows that the average MPA level in the older population is significantly higher than that in the younger population. Figure 1 The C-value in the statistical correlation plot shows that MPA levels increase with age. In conclusion, this demonstrates a necessary link between the aging process and MPA levels.

[0022] Example 2: The proportion of MPA in the peripheral blood of mice increases with age. Specific pathogen-free (SPF) grade C57BL / 6 male mice of different ages (purchased from Guangdong Kaisheng Biotechnology Co., Ltd.) were anesthetized with isoflurane reagent and placed on a test plate. The skin and flesh were cut open above the sternum to expose the sternum. The diaphragm was carefully cut open to expose the heart. The thin membrane on the surface of the heart was gently pulled apart with forceps, and a blood collection syringe (sodium citrate was added to the syringe to prevent blood agglutination) was inserted into the left ventricle to draw about 1 mL of blood, completing the sample collection process. When using flow cytometry to detect platelet-monocyte aggregate (MPA) content, the experimental procedure should focus on timely sample processing and appropriate antibody selection to avoid result deviations caused by in vitro artificial activation. Peripheral blood was collected using sodium citrate anticoagulant tubes. Whole blood samples should be processed as soon as possible. If immediate testing is not possible, they should be fixed with 1% paraformaldehyde and stored at low temperature. However, centrifugation of whole blood before antibody labeling should be absolutely avoided, as it will significantly increase the false positive rate of MPA. Take an appropriate amount of whole blood, add a combination of fluorescently labeled antibodies (such as anti-CD11b labeled myeloid cells, anti-CD41 labeled platelets, anti-Ly6G antibody negative and anti-Ly6C antibody positive labeled monocytes), incubate at room temperature in the dark for 15-30 minutes, then add hemolysin to lyse the red blood cells, wash and resuspend for later use.

[0023] Flow cytometry detection and data analysis: A P1 gate was set up to exclude dead cells and retain viable cells using DAPI; a P2 gate was set up to exclude debris and erythrocytes based on FSC-A / SSC-A to enrich nucleated cells; a P3 gate was set up to remove aggregated cell clusters using FSC-H / FSC-A to obtain a pure single-cell population; a CD11b gate was set up to identify myeloid cells from the single cells using CD11b as a marker; a Mono gate was set up to further separate monocytes from CD11b+ cells based on Ly6C expression; finally, an MPA gate was set up to exclude neutrophils and platelet contamination based on Ly6G and CD41, thus obtaining the percentage and absolute count of MPA from each subset. In addition, the expression rate of the platelet activation marker CD62P could be detected simultaneously to assess the correlation between MPA formation and platelet activation. Results are as follows: Figure 1 As shown, Figure 1 The D-flow cytometry results showed that the mean MPA level in the peripheral blood of aged mice was significantly higher than that in young mice. Figure 1 The statistical violin plot shows that MPA levels in peripheral blood increase with age in mice, indicating a necessary link between aging and MPA levels in mice. Figure 1 The F-flow cytometry plots show that the mean MPA level in the bone marrow of mice is significantly higher than that in young mice (left figure), and the box plots statistically show that the MPA level in the bone marrow of aged mice is extremely significantly higher than that in young mice. In conclusion, this demonstrates that MPA levels increase with aging in mice.

[0024] Example 3, In vivo animal experiments: Modeling of mice injected with PBS and those injected with PSGL-1 antibody. This embodiment aims to establish a mouse model of in vivo intervention with PSGL-1 antibody. By detecting the formation level of monocyte-platelet aggregates in peripheral blood, it verifies the effectiveness of PSGL-1 antibody in blocking P-selectin / PSGL-1 interaction, providing a basis for successful modeling in subsequent behavioral experiments.

[0025] Experimental animals: Middle-aged mice (14 months old) with C57BL / 6J background were selected and randomly divided into PBS control group and PSGL-1 antibody treatment group. The PBS group had n=17 and the PSGL-1 antibody group had n=21.

[0026] Main reagents: PSGL-1 functional blocking monoclonal antibody, phosphate-buffered saline (PBS, sterile), Staining Medium (PBS + 2% FBS + 1% penicillin-streptomycin, filtered through a 0.22 μm filter membrane), erythrocyte lysis buffer (ACK Lysis Buffer), flow cytometry antibodies: anti-CD11b labeled myeloid cells, anti-CD41 labeled platelets, anti-Ly6G antibody negative and anti-Ly6C antibody positive labeled monocytes.

[0027] PBS control group: Each mouse was injected intraperitoneally with 200 μL of sterile PBS. PSGL-1 antibody group: Each mouse was injected intraperitoneally with PSGL-1 monoclonal antibody (diluted in sterile PBS) at a dose of 200 μg / mouse and an injection volume of 200 μL.

[0028] Injection frequency: such as Figure 1 As shown in G, referring to the in vivo administration protocol of PSGL-1 antibody in existing studies, this embodiment uses an injection frequency of once every four days, for a total of ten injections. Samples are collected the day after the last injection.

[0029] The day after the last administration, mice were anesthetized by isoflurane inhalation. Blood was collected via cardiac puncture: the mice were fixed, and a 1 mL sterile syringe (pre-wetted with a small amount of anticoagulant) was inserted into the heart to slowly draw approximately 0.5-1 mL of whole blood. The collected blood was quickly transferred to a tube containing 3.8% sodium citrate (anticoagulant, 1:9 blood volume ratio) or EDTA anticoagulant, and gently inverted to mix to prevent clotting. Note: To avoid in vitro platelet activation affecting MPA test results, all procedures should be performed under mild conditions, avoiding violent shaking or low-temperature stimulation.

[0030] Referring to the hematopoietic stem cell (HSPC) detection protocol and the whole blood flow cytometry method reported in the literature, the MPA detection protocol is established as follows: (1) Whole blood staining: Take 100 μL of anticoagulated whole blood and transfer it to a flow cytometry tube or a 96-well U-plate. Add the flow cytometry antibody mixture to each tube and stain on ice for 30 minutes in the dark. The antibody combination is as follows: anti-CD11b labeled myeloid cells, anti-CD41 labeled platelets, anti-Ly6G antibody negative and anti-Ly6C antibody positive labeled monocytes.

[0031] (2) Lysis of red blood cells After staining, add 1-2 mL of 1× erythrocyte lysis buffer to each tube and incubate at room temperature in the dark for 8-10 minutes, gently vortexing once during incubation to promote lysis. After lysis, add 2 mL of staining medium to terminate the lysis reaction. Centrifuge at 1200 rpm / min, 4°C for 5 minutes, and discard the supernatant.

[0032] (3) Washing and resuspension Add 2 mL of staining medium to each tube to resuspend the cells, centrifuge and wash again (under the same conditions as above), and discard the supernatant. Add 300-400 μL of staining medium to each tube to resuspend the cells and transfer them to flow cytometry tubes. Before flow cytometry: Add DAPI (final concentration 1 μg / mL) to each tube to distinguish dead cells, store in an ice box protected from light, and immediately perform flow cytometry analysis.

[0033] (3) Flow cytometry analysis strategy The system employs a P1 gate to exclude dead cells and retain live cells using DAPI. A P2 gate, based on FSC-A / SSC-A, excludes debris and erythrocytes to enrich nucleated cells. A P3 gate, using FSC-H / FSC-A, removes aggregated cell clusters to obtain a pure monocyte population. A CD11b gate, using CD11b as a marker, identifies myeloid cells from the monocyte population. A Mono gate further separates monocytes from CD11b+ cells using Ly6C expression. Finally, an MPA gate, combining Ly6G and CD41, excludes neutrophil and platelet contamination, thus obtaining the percentage and absolute count of MPA from each subset. This percentage represents the percentage of circulating monocyte-platelet aggregates (MPA) in the total number of monocytes. A comparison of the MPA ratio in the peripheral blood of mice in the PBS control group and the PSGL-1 antibody treatment group was conducted: PBS control group: A certain baseline level of MPA was present in the peripheral blood of mice. PSGL-1 antibody treatment group: Due to the blockade of PSGL-1 function, P-selectin on the platelet surface cannot effectively bind to PSGL-1 on the monocyte surface, resulting in the inhibition of MPA formation. Figure 1As shown in H, the proportion of MPA in the PSGL-1 antibody-treated group in peripheral blood was significantly lower than that in the control group (P < 0.05), indicating that the PSGL-1 antibody successfully blocked the platelet-monocyte interaction in vivo, and the model was successfully established.

[0034] Example 4: The motor ability of mice was significantly improved after the application of PSGL-1 antibody. Muscle strength and motor coordination tests, such as Figure 2 As shown in A, it includes: 1. Pull test: Place the mouse's forepaws on the metal grid or lever of the pull tester. Once the mouse has a firm grip, the experimenter pulls the mouse's tail backward at a constant speed until the mouse's forepaws release the grid. The instrument automatically records the maximum gripping force. Each mouse is measured twice, and the average value is taken as its forepaw muscle strength index.

[0035] 2: Wire mesh suspension experiment: Set the wire mesh angle to 60°, place the mouse in the center of the wire mesh, and after the mouse has gripped the mesh with its limbs, hang the mouse upside down. Immediately record the time from the start of the upside-down position until the mouse falls, and record the time after the mouse falls. This is to assess the mouse's muscle endurance and balance ability.

[0036] 3. Fatigue Rotary Run Experiment: Mice were placed on a rotating rotundus for four consecutive days. The first three days were training days, with an initial blank rotation speed (10 revolutions / minute). After running for ten minutes, the speed was gradually increased to 40 revolutions / minute. Six mice were trained per round. On the fourth day, the initial blank rotation speed (10 revolutions / minute) was set for three minutes, and then gradually increased to 40 revolutions / minute. The time it took for the mice to continue moving on the rotundus until they fell, the rotation speed at the fall, and the total distance traveled were recorded. This was to evaluate the motor coordination and fatigue resistance of each group of mice.

[0037] Spontaneous activity and anxiety behavior assessment, open field test: Mice were placed from a fixed position in the center of an open field test chamber (an open rectangular chamber with the bottom equally divided into a central area and a peripheral area). A camera above the chamber automatically recorded the mice's spontaneous activity trajectory over 10 minutes. Analytical indicators included total movement distance (reflecting spontaneous activity level), time spent in the central area and number of entries (reflecting anxiety level), and number of times the mice stood up (reflecting exploratory behavior). After each experiment, feces were cleaned from the chamber, and the chamber walls and bottom were wiped with 75% alcohol to eliminate odor interference before the next round of experiments.

[0038] Learning and memory ability testing, new object recognition experiment: The experiment is divided into three phases: adaptation period, familiarization period, and testing period. Adaptation period: Mice are placed in the experimental environment for 30 minutes. Familiarization period: Two identical objects are placed symmetrically in the box, and the mice are placed inside, allowed to explore freely for 10 minutes. After 10 minutes of exploration, the mice are removed and allowed to rest outside the box for 20 minutes. Testing period: One of the objects is replaced with a new object (pink cylinder represents the old object, red cone represents the new object), and the mice are placed back in the box to explore for 5 minutes. The time spent exploring the old and new objects during these 5 minutes is recorded. The recognition index (time spent exploring the new object / total exploration time) or discrimination index is calculated to assess the mice's cognitive and memory functions.

[0039] The results are as follows Figure 2 As shown, Figure 2 In the open field experiment, B indicates that the movement trajectories of the two groups of aged mice were recorded ten minutes after entering the open field. Compared with the PBS group, the PSGL-1 antibody treatment group had a longer total movement distance and a longer movement time than the resting time, indicating that PSGL-1 treatment made the aged mice more inclined to move and improved their movement ability. Figure 2 The "C" indicates that in the mouse fatigue test using a rotating bar, mice treated with the PSGL-1 antibody were able to stay on the rotating bar for a longer time and run a longer total distance after training, showing significant improvement in physical strength and endurance. Figure 2 The "D" indicates that in the suspension experiment, mice in the PSGL-1 antibody treatment group had a longer suspension time and significantly improved arm strength and muscle strength. Figure 2 The "E" indicates that in the new object recognition experiment, mice treated with the PSGL-1 antibody were able to effectively distinguish between new and old objects and maintain a preference for novel objects, indicating that the object recognition memory ability of older mice was improved and they showed a preference for new objects similar to that of younger mice. Figure 2 The F in the figure indicates that the PSGL-1 antibody-treated mice had stronger grip strength in the tensile test, indicating that the upper limb muscle strength of the mice was significantly improved.

[0040] Example 5: Inflammation levels decreased in mice after administration of PSGL-1 antibody. Immunohistochemical detection of tissue inflammatory infiltration: After anesthetizing the mice, fix them supine on a dissecting board and disinfect the abdominal skin with 75% alcohol. Lift the skin along the midline of the abdomen with forceps and cut along the midline from bottom to top to expose the abdominal cavity. Gently lift the liver with forceps, cut off the hepatic lobe, and immediately rinse in pre-cooled PBS to remove blood. Locate the kidneys (bean-shaped) on both sides of the abdominal cavity, peel off the perirenal fat, cut the renal pedicle, remove the kidney, remove the capsule, and rinse with PBS. Cut the diaphragm along the costal arch, then cut the sternum along both sides of the ribs to expose the thoracic cavity. Carefully separate the lungs from the surrounding tissues, cut off the entire lung, and rinse with PBS to remove blood. Immediately fix the removed liver, kidney, and lung tissues in pre-cooled 4% paraformaldehyde. Dehydrate the fixed tissues with ethanol solution, then soak them in xylene until completely transparent. After transparency, soak the tissues in melted paraffin wax, then remove them for embedding and sectioning.

[0041] The prepared paraffin sections were sequentially immersed in environmentally friendly dewaxing solution (G1128) I for 10 min, followed by environmentally friendly dewaxing solution (G1128) II for 10 min, then environmentally friendly dewaxing solution (G1128) III for 10 min, then anhydrous ethanol I for 5 min, then anhydrous ethanol II for 5 min, then anhydrous ethanol III for 5 min, and finally washed with distilled water. After dewaxing to water, antigen retrieval was performed. During this process, excessive evaporation of the buffer solution should be prevented, and the slides should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time. After antigen retrieval, endogenous peroxidase blocking was performed. The sections were placed in 3% methanol-hydrogen peroxide solution and incubated at room temperature in the dark for 25 min. The slides were then placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time. The subsequent experimental steps were as follows: serum blocking: 3% BSA was added to the histochemistry zone to evenly cover the tissue, and the tissue was blocked at room temperature for 30 min. (For primary antibodies derived from goat, use rabbit serum for blocking; for other sources, use BSA for blocking.) Add primary antibody: Gently shake off the blocking solution, add the prepared primary antibody in PBS at a specific ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight. Add secondary antibody: Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker, 5 min each time. After slightly drying the slide, add the corresponding species of secondary antibody (HRP-labeled) to the circle to cover the tissue, and incubate at room temperature for 50 min. DAB staining: Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker, 5 min each time. After slightly drying the slide, add freshly prepared DAB staining solution to the circle, and control the staining time under a microscope. A positive result is brownish-yellow. Rinse the slide with tap water to stop staining. Counterstain cell nuclei: Counterstain with hematoxylin for about 3 min, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, use hematoxylin blue solution to return to blue, and rinse with running water. Dehydration and mounting: Immerse the sections sequentially in 75% ethanol for 5 min, 85% ethanol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and clear the sections. Remove the sections from the xylene and allow them to dry slightly before mounting with mounting adhesive. Interpret the results under a white light microscope.

[0042] The results are as follows Figure 3 As shown, in F4 / 80 staining, macrophages stained with F4 / 80 in the PSGL-1 antibody-treated group showed significant differences in the kidneys and livers. The active area of ​​macrophages in the PSGL-1 antibody-treated group was significantly smaller than that in the PBS-treated control group (see [link to original text]). Figure 3 A in Figure 3 In iNOS (nitric oxide synthase) staining, there were significant differences between the PSGL-1 antibody-treated group and the PBS-treated control group in the kidneys and liver. The area of ​​pro-inflammatory macrophage activity stained by iNOS in the PSGL-1 antibody-treated group was significantly smaller than that in the PBS-treated control group (see B). Figure 3 D in Figure 3 (E in the text). There was no significant difference between the two immunohistochemical staining methods in lung tissue (see [link]). Figure 3 C in Figure 3 (F in the text). In summary, the low proportion of macrophages and low proportion of nitric oxide synthase in the PSGL-1 antibody treatment group indicate that there are fewer pro-inflammatory macrophages and the inflammatory infiltration of the tissue has been improved.

[0043] Detection of inflammatory factors in the supernatant: This can be performed using plasma or bone marrow samples. Plasma samples: Collect whole blood from mice in an EDTA anticoagulant tube, gently invert to mix, centrifuge at 3000 rpm / min for 10 min at 4°C, carefully aspirate the upper pale yellow plasma layer (avoiding the leukocyte layer), transfer to a new 1.5 mL centrifuge tube, and freeze at -80°C for later use or use directly for detection. Bone marrow supernatant samples: When preparing the bone marrow single-cell suspension, carefully aspirate the supernatant obtained after the first centrifugation (1200 rpm / min, 4°C, 8 min), transfer to a new centrifuge tube, and freeze at -80°C for later use. Avoid repeated freeze-thaw cycles for all samples. Before detection, thaw slowly on ice and briefly centrifuge again to remove any precipitate.

[0044] Quantitative detection of inflammatory factors was performed using a flow cytometry multifactor assay kit. First, standards were prepared and serially diluted. Then, 50 μL of mixed capture microspheres, 50 μL of standards or test sample (plasma or bone marrow supernatant) were added sequentially to flow cytometry tubes, and incubated at room temperature in the dark for 1 h. Next, 50 μL of PE-labeled detection antibody was added, and incubation continued at room temperature in the dark for another 1 h. 1 mL of wash buffer was added, and the tubes were centrifuged at 1200 rpm for 5 min, and the supernatant was carefully discarded. 300 μL of wash buffer was added to each tube to resuspend the microspheres, and the tubes were immediately analyzed by flow cytometry. Appropriate FSC / SSC thresholds were set to exclude debris, and fluorescence signals were collected in the PE channel (or the corresponding detection channel). After completion, the concentrations of each inflammatory factor (e.g., IL-6, TNF-α, CCL2, etc.) in the sample were calculated based on a standard curve. Throughout the process, it was crucial to avoid light exposure, ensure thorough mixing of the microspheres and sample, and resuspend each tube before analysis. Results are as follows: Figure 3 As shown in G, the levels of inflammatory factors CCL2 and IL-1β were significantly downregulated, indicating that the secretion of inflammatory factors in the bone marrow supernatant decreased after PSGL-1 antibody treatment.

[0045] Example 6: Applying PSGL-1 antibody to rejuvenate the hematopoietic cell population Mouse leg bones, iliac bones, and spine were collected and placed in 15 mL centrifuge tubes containing 4 mL of staining medium (PBS + 2% FBS + 1% p / s, filtered through a 0.22 μm filter). The bone and liquid were poured into a mortar and pestle, and the bone was pressed vertically until all the bone marrow was extracted (note: press vertically, do not rotate or grind). The mixture was then filtered through a 200-mesh filter into a new 15 mL centrifuge tube. The process was repeated, adding 4 mL of staining medium and pressing and filtering, then combining the suspensions. The cells were centrifuged at 1200 rpm / min at 4°C for 8 min, the supernatant was discarded, and the bone marrow cells were resuspended in 2 mL of staining medium. Approximately 1 × 10⁻⁶ cells were collected. 7 One cell line (approximately 100 μL) was placed in a 96-well U-type plate. 10 μL of Lin-mix antibody (containing Anti-Ter-119, Gr-1, Mac-1, B220, CD4, CD8, and Staining Medium) was added to each well. The plate was stained on ice in the dark for 30 min. After staining, 200 μL of Staining Medium was added to each well, and the plate was centrifuged at 1200 rpm / min at 4°C for 5 min. The supernatant was discarded.

[0046] After discarding the supernatant, add 25 μL of master mix (containing CD34, Flt3, IL7R, Sca-1, c-Kit, Lin / SA, CD16 / 32, CD150, and CD48) to each well and stain the samples at 4°C on an ice box. Add 200 μL of staining medium to each well, centrifuge at 1200 rpm / min at 4°C for 5 min, discard the supernatant, and absorb excess liquid with absorbent paper. Resuspend the cells in 400 μL of staining medium to each well, transfer to flow cytometry tubes, and store in an ice box protected from light until analysis. Add DAPI before analysis, with a final DAPI concentration of 1 μg / mL. Throughout the process, maintain the total secondary antibody staining volume at 25-30 μL to increase the CD34 antibody concentration. Maintain 4°C for all centrifugation steps and strictly avoid light exposure from the addition of the Lin-mix antibody. Results are as follows. Figure 4 In flow cytometry analysis of cell populations in the bone marrow hematopoietic system, PSGL-1 antibody treatment significantly improved the differentiation lineage of bone marrow hematopoietic stem cells and progenitor cells, reversing aging-related cell population abnormalities: specifically, it enhanced lymphoid differentiation potential—an increase in lymphoid progenitor cells (CLPs) and naive T cells (see [link to relevant documentation]). Figure 4 A in Figure 4 (B in the text) Inhibits pathological myeloid bias—reduction of myeloid-biased hematopoietic stem cells (My-HSCs) (see also B) Figure 4 (C in the text), and rebalance the MPP subpopulations—promoting a lymphoid bias in MPP1 / MPP4 (see C in the text), and rebalancing the MPP subpopulations—promoting a lymphoid bias in MPP1 / MPP4 (see C in the text). Figure 4 D in Figure 4E in the middle), reduce the bias towards myeloid / megakaryotype MPP2 / 3 (see Figure 4 (F in the text). This demonstrates that the PSGL-1 antibody can induce the remodeling of the bone marrow hematopoietic microenvironment towards a younger phenotype in aging mice, and holds promise for delaying or treating hematopoietic system aging and related immune function decline.

[0047] Example 7: The application of PSGL-1 antibody had no side effects on mice. This study aimed to evaluate the effect of continuous treatment with anti-PSGL-1 antibody on peripheral blood platelet function in mice. Given the important role of the P-selectin / PSGL-1 pathway in platelet-leukocyte interactions and thrombus formation, and previous studies have shown that intervention strategies targeting this pathway have the potential to maintain safety without affecting hemostasis, this study systematically assessed platelet count, activation markers, ATP release capacity, and in vitro clot retraction function to verify the hematological safety of long-term PSGL-1 antibody administration.

[0048] Middle-aged C57BL / 6J mice were randomly divided into a control group (injected with PBS) and an experimental group (anti-PSGL-1 monoclonal antibody, dose of 200 μg / mouse, administered via intraperitoneal injection). Blood samples were collected within 24 hours after the last administration and tested again one month after the last administration.

[0049] 1. Cell population detection Whole blood was collected from mice, and the number of mature cells (platelets, white blood cells, and red blood cells) in peripheral blood was measured using an automated hematology analyzer or flow cytometry (combined with CD41 / CD61 staining). Differences were compared between the control group and the antibody-treated group. Results are as follows: Figure 5 After continuous treatment with PSGL-1 antibody, there was no significant difference in the number of platelets, white blood cells, and red blood cells in the peripheral blood of mice compared with the control group (see [link to study]). Figure 5 A in Figure 5 B in Figure 5 (C) This result indicates that continuous treatment with anti-PSGL-1 antibody does not cause a decrease in the three types of mature cells in peripheral blood, suggesting that it does not have a significant bone marrow suppression or peripheral blood platelet destruction effect. Combined with CD11b, CD4 / CD8, and B220 staining, the number of BMT (B for B cells, M for myeloid cells, and T for T cells) cell populations in peripheral blood and bone marrow was measured, and the results are as follows: Figure 5 D in Figure 5 As shown in E, PSGL-1 antibody treatment did not affect the number of BMT cell populations in peripheral blood and bone marrow, indicating that terminally differentiated cell populations were unaffected.

[0050] 2. In vitro clot retraction experiment PRP was prepared from whole blood collected from mice. Thrombin and calcium ions were added to initiate the coagulation process. The mixture was placed in a constant temperature environment of 37°C, and the clot volume or area was observed and measured at the same time points (every 20 minutes) to assess platelet-mediated clot retraction ability. In vitro clot retraction experiments showed no significant difference in platelet-mediated clot retraction volume between the PSGL-1 antibody treatment group and the control group within the same observation time window (see [link to study]). Figure 5 (F in the figure). This result further confirms that continuous treatment with PSGL-1 antibody does not affect platelet contractile function or its mediated coagulation cascade.

[0051] 3. ATP release experiment Washed platelets or platelet-rich plasma were prepared, and the supernatant was collected under resting and thrombin stimulation conditions. ATP content was measured using a chemiluminescence assay (luciferin-luciferase system) to reflect platelet dense granule release function. The ATP release assay results showed no significant difference in ATP content in the platelet secretion supernatant between the control group and the PSGL-1 antibody-treated group, regardless of whether under resting or thrombin stimulation (see [link to ATP release assay]). Figure 5 (G in the text). This result confirms that continuous treatment with PSGL-1 antibody does not affect the release of dense granules from platelets.

[0052] 4. Platelet activation status assessment Alpha granule release: Whole blood was collected, and the outward movement level of P-selectin on the platelet surface was detected by flow cytometry at rest and after agonist stimulation to evaluate alpha granule release function. Alpha granule release: At rest, the expression levels of P-selectin on the platelet surface in both groups were at baseline low. After thrombin stimulation, the outward movement level of platelets in both groups significantly increased, and there was no statistically significant difference between the treatment group and the control group (see [link to relevant documentation]). Figure 5 (H in the text). This indicates that PSGL-1 antibody treatment does not affect the α-granule release function of platelets.

[0053] Integrin activation: The activation epitopes of integrin αIIbβ3 were detected using specific antibodies to evaluate the ability of integrins to transition from the resting to the activated state. Integrin activation: The proportion of JON / A-positive platelets was low in both groups at rest. After thrombin stimulation, the integrin activation levels in both groups were significantly upregulated, and the response levels in the treatment and control groups were comparable (see [link to relevant documentation]). Figure 5 The I in the figure indicates that the ability of platelet integrins to transition from the resting state to the activated state was not affected by antibody treatment.

[0054] One month later, various cell populations were tested again. One month after the last dose, samples were taken again to detect the percentage of cell populations in bone marrow and peripheral blood.

[0055] Detection of BMT in bone marrow: Combining CD11b, CD4 / CD8, and B220 staining, the number of BMT cells in peripheral blood and bone marrow was determined. Results are as follows: Figure 5 As shown in J, after one month of PSGL-1 antibody treatment, there was still no significant difference in bone marrow BMT compared to the PBS control group, proving that PSGL-1 antibody treatment has no potential effect on terminally differentiated cells.

[0056] Detection of MPA in peripheral blood: A combination of fluorescently labeled antibodies (anti-CD11b-labeled myeloid cells, anti-CD41-labeled platelets, anti-Ly6G antibody-negative and anti-Ly6C antibody-positive labeled monocytes) was added to detect changes in the percentage of MPA in bone marrow. The results are as follows: Figure 5 As shown in K, the number of MPA in peripheral blood was not significantly different from that in the PBS control group after one month of PSGL-1 antibody treatment, proving that PSGL-1 antibody treatment has no long-term effect on peripheral blood MPA.

[0057] Detection of various cell populations in bone marrow: The staining protocol used was CD34, Flt3, IL7R, Sca-1, c-Kit, Lin / SA, CD16 / 32, CD150, and CD48. Changes in the percentage of various hematopoietic cell populations in the bone marrow were detected. The results are as follows: Figure 5 As shown in L, after one month of treatment with PSGL-1 antibody, the differences between various cell types in the bone marrow and the PBS control group disappeared, proving that PSGL-1 antibody has no long-term effect on various cell groups in the bone marrow hematopoietic system.

[0058] In summary, while continuous treatment with anti-PSGL-1 antibody significantly interfered with MPA formation in the peripheral blood and bone marrow of middle-aged mice, it had no significant effect on platelet count or various functions (including α-granule release, integrin activation, dense granule release, and clot retraction). This indicates that the antibody intervention strategy targeting the P-selectin / PSGL-1 pathway has good safety. This case provides important data support for the preclinical safety assessment of anti-PSGL-1 antibody, suggesting that this antibody does not interfere with normal platelet function during application.

[0059] In summary, the PSGL-1 antibody can effectively improve the excessive formation of MPA in aged mice, significantly alleviate various physiological changes caused by aging, and alleviate cellular senescence and inflammatory responses. Intervention with the PSGL-1 antibody in aged mice can significantly alleviate aging, as evidenced by changes in the blood system, inflammatory factor levels, and motor function. This indicates that the PSGL-1 antibody can be used to prepare drugs that delay aging in mice. Further human clinical trials are needed to further verify the anti-aging efficacy of the PSGL-1 antibody.

[0060] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Application of PSGL-1 antibody in the preparation of drugs to delay aging.

2. A drug for delaying aging, characterized in that, It contains the PSGL-1 antibody as described in claim 1.

3. The drug for delaying aging according to claim 2, characterized in that: It also includes pharmaceutically acceptable carriers.

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