Use of cxcl12 or cxcr4 as a target in the preparation of a medicament for treating cerebral small vessel disease

Abstract

The application discloses application of CXCL12 or CXCR4 as a target point in preparation of a drug for treating a cerebral small vessel disease, and can predict the cerebral small vessel disease by detecting expression levels of the CXCL12 or the CXCR4; and further discloses application of the CXCL12 or the CXCR4 as a diagnostic marker of the cerebral small vessel disease, wherein the CXCL12 or the CXCR4 inhibitor comprises Plerixafor hydrochloride. The application discloses a new use of the CXCL12 or the CXCR4 as a cerebral small vessel disease marker and a treatment target point, and provides an important basis for clinical transformation of an inhibitor thereof.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of the CXCL12 or CXCR4 signaling pathway as a target in the preparation of drugs for the treatment or prevention of cerebral small vessel diseases. Background Technology

[0002] Cerebral small vessel disease (CSVD) is a series of clinical, imaging, and pathological syndromes caused by damage to small arteries and their distal branches, capillaries, venules, and microveins in the brain due to various etiologies. Risk factors for CSVD mainly include non-modifiable factors (such as age and genetic factors) and modifiable factors (such as hypertension, diabetes, sleep apnea syndrome, smoking, hyperlipidemia, and hyperhomocysteinemia). The risk of developing CSVD increases with age. Many people are asymptomatic, but studies have shown that the burden of CSVD in an increasing number of individuals is associated with worse cognitive impairment, dementia, depression, bowel and bladder dysfunction, and motor and gait function, and is also associated with stroke risk or worse stroke outcomes.

[0003] Among the imaging features of CSVD (Cognitive Impairment Disease), white matter hyperintensity (WMH) has received widespread attention due to its high incidence and strong correlation with cognitive impairment. Studies have found that 100% of subjects aged 80-90 years have subcortical WMH, and 95% have periventricular WMH. Its pathophysiological process mainly involves reduced cerebral blood flow, ischemia and hypoxia leading to neurovascular unit dysfunction in the corresponding blood-supply area, and blood-brain barrier disruption, ultimately resulting in demyelination, axonal loss, and glial proliferation. Clinically, it presents as high signal intensity on T2 or T2-FLAIR sequences and isointense or hypointense signal intensity on T1 sequences. Overall, WMH reflects localized ischemic white matter damage; however, its pathogenesis is still incomplete, and effective treatment and prevention methods are lacking.

[0004] Researchers have discovered elevated levels of the chemokine CXCL12 during peripheral blood and cerebrospinal fluid protein assays. These findings suggest CXCL12 may serve as a potential disease biomarker, as it is observed in acute cerebrovascular diseases and neurodegenerative diseases such as brain atrophy, Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). CXCL12, also known as stromal cell-derived factor, belongs to the CXC chemokine family and is widely expressed in various tissues, including bone marrow, lymph nodes, liver, and the nervous system. It plays a role in cell migration, neuroinflammation, and embryonic development by binding to its receptor CXCR4. Studies have found that the CXCL12 / CXCR4 axis is highly expressed in the brain tissue of mice and patients with Parkinson's disease (PD). Accumulated α-synuclein induces the expression of the CXCL12 / CXCR4 axis through the TL-R4 / IκB-α / NF-κB pathway, further activating the FAK / Src / Rac1 pathway, leading to sustained microglial activation and inducing activated microglia to migrate to the substantia nigra, excessively releasing inflammatory factors, resulting in neuronal loss and worsening of Parkinson's disease. Inhibiting the CXCL12 / CXCR4 pathway with the inhibitor AMD3100 can improve cognitive impairment, reduce neuroinflammation, and improve pathophysiological markers in AD mouse models. However, the pathophysiological mechanism of the CXCL12 / CXCR4 axis in white matter injury remains unclear.

[0005] For cerebral small vessel disease, a mouse model of bilateral common carotid artery stenosis (BCAS) has been established to simulate ischemia and hypoxia. This model reduces cerebral blood flow (CBF) to varying degrees by using microcoils to narrow the bilateral carotid arteries. It effectively reproduces white matter damage and significant microglial activation and astrocyte proliferation. Furthermore, researchers found that mice exhibited poorer cognitive and gait balance functions approximately 28 days after BCAS surgery. This indicates that BCAS is a reliable animal model of cerebral small vessel disease and has significant implications for the clinical translation of related mechanistic studies. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] In view of the above background, the present invention provides the application of CXCL12 or CXCR4 as targets in the preparation of drugs for treating cerebral small vessel diseases, which solves the problem that the pathophysiological mechanism of the CXCL12 / CXCR4 axis in brain white matter injury is still unclear, and there is a lack of effective therapeutic targets and treatment strategies for cerebral small vessel diseases, especially brain white matter injury.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] Application of CXCL12 detection reagent in the preparation of cerebral small vessel disease detection reagent.

[0011] Furthermore, the CXCL12 detection reagent is selected from one or more of the following: ELISA detection reagents, Luminex detection reagents, immunofluorescence detection reagents, and proteomics detection reagents.

[0012] The present invention provides a detection system for cerebral small vessel disease, the detection system comprising a CXCL12 detection component and a result determination system.

[0013] Furthermore, the detection component of the CXCL12 contains one or more of the following: ELISA detection reagent, Luminex detection reagent, immunofluorescence detection reagent, and proteomics detection reagent.

[0014] Furthermore, the result determination system is used to output the risk of cerebral small vessel disease based on the CXCL12 expression results detected by the detection system.

[0015] Furthermore, the disease risk is determined by comparing the CXCL12 expression levels of the test sample and the normal sample. When there is a significant or extremely significant difference in the CXCL12 protein expression levels between the test sample and the normal sample, the test sample is judged to have a high disease risk. A significant or extremely significant difference is defined as a P-value < 0.05 between the two groups.

[0016] This invention also provides the application of the CXCL12 or CXCR4 signaling pathway as a target in the preparation of drugs for the treatment or prevention of cerebral small vessel diseases. The chemokine CXCL12, also known as stromal cell-derived factor, belongs to the CXC chemokine family and is widely expressed in various tissues such as bone marrow, lymph nodes, liver, and nervous system. It plays a role in cell migration, neuroinflammation, and embryonic development by binding to the receptor CXCR4.

[0017] Furthermore, the use of CXCL12 or CXCR4 signaling pathway inhibitors in the preparation of drugs for the treatment or prevention of cerebral small vessel diseases, wherein the CXCL12 or CXCR4 signaling pathway inhibitors are selected from CXCR4 inhibitors or CXCL12 inhibitors, wherein the CXCL12 inhibitors are selected from substances that inhibit CXCL12 activity, or substances that degrade CXCL12, or substances that reduce CXCL12 levels; and the CXCR4 inhibitors are selected from substances that inhibit CXCR4 activity, or substances that degrade CXCR4, or substances that reduce CXCR4 levels.

[0018] Furthermore, the CXCL12 / CXCR4 signaling pathway inhibitor is selected from CXCR4 inhibitors, among which plerixafor hydrochloride (AMD 3100) is a selective CXCR4 antagonist (IC50 of 44 nM) and also a potent CXCL12-mediated chemokine inhibitor (IC50, 5.7 nM). Plerixafor hydrochloride can interfere with the interaction between CXCR4 and its natural ligand SDF-1 (CXCL12). The structural formula of plerixafor hydrochloride is as follows:

[0019] .

[0020] This invention also provides the application of Plerixafor hydrochloride, an inhibitor of the CXCL12 / CXCR4 signaling pathway, in the preparation of a drug for treating cerebral small vessel disease, wherein the drug is administered by injection, specifically intraperitoneal injection, once daily.

[0021] In this invention, "prevention / treatment" refers to reducing, alleviating, or improving the symptoms of a disease or condition, improving underlying metabolic symptoms, or inhibiting a disease or condition, such as preventing the development of a disease or condition, alleviating a disease or condition, causing the remission of a disease or condition, relieving the condition caused by a disease or condition, or preventing the symptoms of a disease or condition. In this invention, CXCL12 or CXCR4 refers to microglia CXCL12 or CXCR4.

[0022] (III) Beneficial Effects

[0023] The beneficial effects of this invention are:

[0024] (1) This invention discovers CXCL12 or CXCR4, diagnostic and therapeutic markers for small cerebral vessels disease. Small cerebral vessels disease can be treated by targeting CXCL12 / CXCR4. At the same time, a small cerebral vessels disease detection system is proposed, which can accurately identify small cerebral vessels disease, thereby providing a basis for individualized treatment.

[0025] (2) This invention found that inhibitors of the CXCL12 / CXCR4 signaling pathway, such as plerixafor hydrochloride, can significantly improve brain white matter damage. CXCL12 / CXCR4 can serve as a diagnostic and therapeutic target for cerebral small vessel diseases, guiding the research and development and screening of related drugs.

[0026] (3) The present invention found that CXCL12 / CXCR4 signaling pathway inhibitor plerixafor hydrochloride can effectively improve the treatment of cerebral small vessel disease, indicating that the development of drugs for cerebral small vessel disease based on this has important clinical application value and market potential. Attached Figure Description

[0027] Figure 1 Serum CXCL12 can serve as a predictive biomarker for patients with cerebral small vessel disease. The diagram includes: (A) Proteomics PCA plot; (B) Proteomics differential gene volcano plot; (C) Metabolomics PCA plot; (D) Metabolomics differential gene volcano plot; (E) Venn diagram and bubble chart showing the intersection of differentially expressed pathways in proteomics and metabolomics analyses; (F) Bubble chart showing significantly altered lysosomal pathways in proteomics; (G) NES enrichment fraction histograms of the above five significantly altered pathways; (H) GSEA plot showing that, compared with the healthy control group, the lysosomal pathway and actin cytoskeleton regulatory pathway were significantly downregulated in the cerebral small vessel disease group; (I) Ring enrichment heatmap of differentially expressed proteins; (J) Comparison of peripheral blood CXCL12 levels between healthy controls and patients with cerebral small vessel disease; (K) Correlation analysis of peripheral blood CXCL12 levels with clinical and neuroimaging characteristics of the subjects.

[0028] Figure 2 In the middle, (A) forest plot shows the results of Mendelian randomization analysis between cerebral small vessel disease and peripheral blood CXCL12 levels. (a) Dots represent the magnitude of the standardized effect, and horizontal lines represent the corresponding 95% confidence intervals (CarcelMarquez-2024 study n=198048; TraylorM-2021 study n=232596; MalikR-2018 study n=5816); (b) Statistical graphs of baseline age, sex, baseline white matter high signal volume, and baseline CXCL12 content in the community population; (c) Correlation analysis of baseline CXCL12 concentration and ΔWMH; (d) Correlation analysis of baseline CXCL12 concentration and ΔMoCA score; (e) Correlation analysis of baseline CXCL12 concentration and TUG time; (f) Forest plot comparison of standardized coefficients of the three multiple linear regression models (dots represent β estimates, horizontal lines represent 95% CI. Vertical dashed line β=0 indicates no effect. Asterisks indicate significance levels: *P<0.05, **P<0.01, ***P<0.001.)

[0029] Figure 3 The study investigated the successful establishment of the cerebral cerebral arthroscopy (BCAS) model and the changes in CXCL12 levels after BCAS surgery. The results included: (A) a schematic diagram of cerebral blood flow changes before and after BCAS modeling; (B) a statistical graph of cerebral blood flow changes after BCAS surgery; (C) ELISA quantitative analysis of CXCL12 levels in peripheral blood of mice (n=10); and (D) ELISA quantitative analysis of CXCL12 levels in the corpus callosum region (n=5; data are expressed as mean ± standard error, analyzed using one-way ANOVA and Bonferroni post-hoc test); and (E) correlation analysis between peripheral blood and CXCL12 levels in the corpus callosum region (dashed lines represent the regression line and 95% confidence interval. Partial correlation coefficients (r) and p-values ​​are shown in the figure). (*: vs sham; #: vs vehicle; *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001)

[0030] Figure 4 Inhibition of the CXCR4 / CXCL12 pathway effectively improved cognitive and gait function in mice. Specifically, (A) Spatial learning and memory abilities of mice were assessed using a water maze test. Red circles represent platform locations, and green curves represent swimming paths; (B) Statistical graphs of water maze latency, target quadrant exploration time, and platform traversal counts; (C) Assessment of mice's new object recognition and memory abilities. Blue and red objects represent old and new objects, respectively, and green curves depict exploration trajectories; (D) Statistical graphs of new object preference index in the new object recognition experiment; (E) Partial least squares discriminant analysis plots showing overall gait changes in mice from different treatment groups; (F) Heatmaps presenting gait parameters showing differences between groups. (n=10 per group, data are expressed as mean ± standard error, one-way ANOVA and Bonferroni post-hoc test were used; * : vs sham; # : vs vehicle; *p < 0.05, **p < 0.01, ***p < 0.005)

[0031] Figure 5Inhibition of the CXCR4 / CXCL12 pathway effectively improved myelin damage in the corpus callosum region of mice. (A) Representative images assessing myelin formation status using multiple techniques, including: immunofluorescence staining of myelin-associated glycoprotein (MAG) (scale bar = 100 μm); myelin LFB staining (scale bar = 100 μm); and electron micrographs showing ultrastructural details of the myelin sheath (scale bar = 1 μm). (B) Quantitative analysis of corpus callosum myelin sheath integrity, including: statistical analysis of MAG immunofluorescence intensity; statistical scoring of LFB staining; and quantitative analysis of G-ratio and its correlation with axonal diameter. (C) Representative immunofluorescence micrographs of oligodendrocyte lineage cells in the corpus callosum. Includes: single staining of CC1, a marker of mature oligodendrocytes (scale bar = 20 μm); single staining of oligodendrocyte precursor cells and GST-π, a marker of mature cells (scale bar = 20 μm); co-staining of NG2, a marker of oligodendrocyte precursor cells, and Ki67, a proliferation marker (scale bar = 20 μm). (D) Statistical graphs of the number of CC1+ mature oligodendrocytes, GST-π+ oligodendrocytes, and NG2+Ki67+ proliferating oligodendrocyte precursor cells in the corpus callosum. (n=5 per group, data are expressed as mean ± standard error, one-way ANOVA and Bonferroni post-hoc test were used; * : vs sham; # : vs vehicle; *p < 0.05, **p < 0.01)

[0032] Figure 6AMD3100 administration reduced microglial overactivation in the corpus callosum of mice after BCAS. (A) CXCL12 was mainly expressed on microglia in the corpus callosum region after BCAS. (B) Includes: representative immunofluorescence images (scale bar = 20 μm) and Imaris 3D reconstructed images (scale bar = 5 μm) showing Iba1⁺CXCL12⁺ colocalization; quantitative analysis of Iba1⁺CXCL12⁺ cell percentage, Iba1⁺ cell density, and microglial morphological parameters (surface area and volume). (C) Representative immunofluorescence images (scale bar = 20 μm) and magnified views (scale bar = 5 μm) showing Iba1⁺CXCR4⁺ colocalization, along with quantitative analysis. (D) Representative images and corresponding quantitative analysis of Iba1 and CD68 colocalization (scale bar = 20 μm). (E) Representative images of co-localization of Iba1 and LC3B and corresponding quantitative analysis (scale bar = 20 μm). (F) Representative images of co-localization of Iba1 and Lamp1 and corresponding quantitative analysis (scale bar = 20 μm). (G) Correlation analysis of mouse peripheral blood CXCL12 levels, including cognitive function (water maze: latency, target quadrant time); gait (regularity, speed); demyelination (MAG intensity, LFB score); and microglia activation (Iba1⁺ cell density, morphology [area, volume], and percentage of Iba1⁺CXCL12⁺ cells) (dashed lines represent regression lines and 95% confidence intervals. Partial correlation coefficients (r) and P-values ​​are shown in the figure). (Each group n=5, data are expressed as mean ± standard error, one-way ANOVA and Bonferroni post-hoc test were used; * : vs sham; # : vs vehicle; *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001)

[0033] Figure 7 AMD3100 promotes the clearance of myelin fragments by modulating the functional state of microglia. (A) Representative in vivo two-photon microscopy images (scale bar = 10 μm) showing myelin fragment dynamics, microglia morphology, and cell motility. (B) Quantitative analysis of myelin fragment fluorescence intensity, microglia density, and microglia surface area. (C) Pathways enriched between the Vehicle group and the AMD3100-treated group by RNA sequencing analysis. (D) Heatmap of differentially expressed genes in pathways related to cell migration, Fcγ-mediated phagocytosis, lipid droplet organization, fatty acid oxidation, phospholipase D signaling, and inflammatory responses. (n=5 per group, data are expressed as mean ± standard error, two-way ANOVA and Bonferroni post-hoc test were used; *: vssham; #: vs vehicle; *p < 0.05, **p < 0.01) Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] Example

[0036] (a) Community population data collection

[0037] This study, based on the "Tongji Cerebral Small Vessel Disease and Aging Cohort" research platform, has received funding. The study has been approved by the ethics review committee, and the informed consent of the enrolled population was fully respected during data collection (ethics approval number: 2019-S105).

[0038] This study included 100 eligible community participants in the cross-sectional cohort, assessed using the Fazekas score for white matter hyperintensity. The white matter hyperintensity group consisted of 50 participants with moderate to severe white matter hyperintensity (Fazekas total score ≥ 3), while the healthy control group consisted of 50 participants without white matter hyperintensity (Fazekas total score = 0). The longitudinal cohort also included 100 participants. All participants underwent cranial MRI scans at baseline and approximately 2 years later. Based on the change in total white matter hyperintensity volume (ΔWMH) over two years, participants were divided into a "slow progression group" (ΔWMH ≤ 0.01 mL, n=50) and a "rapid progression group" (ΔWMH > 0.2 mL, n=50). There were no significant differences between the two groups in baseline age, sex, body mass index (BMI), and baseline white matter hyperintensity volume.

[0039] In this study, physicians with professional training inquired about basic information of the participants, such as age, gender, height, weight, and years of education. They also recorded the presence of vascular-related risk factors, including BMI, systolic blood pressure, diastolic blood pressure, current smoking and drinking history, hypertension, diabetes, hyperlipidemia, history of coronary heart disease, and history of transient ischemic attack.

[0040] In this study, radiologists used a United Imaging uMR 780 MRI scanner to acquire four sequences from patients: T1WI, T2WI, T2FLAIR, and SWI. Researchers then uniformly assessed the volume of high signal in the brain white matter.

[0041] Five mL of fasting blood was collected from the included population and the serum was obtained by centrifugation at 3000 rpm for 10 min. The serum was stored at -80℃ for further proteomics and metabolomics analysis.

[0042] The participants underwent assessments of overall cognitive function and multidimensional cognitive function (attention, learning and memory ability, visuospatial ability, language function, and executive function).

[0043] This study used the Hamilton Anxiety Scale (HAMA) and the Hamilton Depression Scale (HAMD) to assess the patients' mental and emotional state.

[0044] This study used a gait analyzer (APDM Wearable Technologies Inc, Portland, OR, USA) to assess the gait function of the population, mainly evaluating gait speed, stride length (the distance from the heel of one foot to the heel of the opposite foot during walking), and swing time (the time from when the foot leaves the ground to when it re-consumes the ground during the gait cycle).

[0045] This study performed Mendelian randomization analysis based on publicly available GWAS data. Significantly associated SNPs with CXCL12 levels were extracted as instrumental variables from a plasma protein QTL study (GCST90088430), and corresponding outcome association data were obtained from three large white matter hypersignaling GWAS studies (n=198048 in CarcelMarquez-2024; n=232596 in TraylorM-2021; n=5816 in MalikR-2018) to explore the potential causal effects of CXCL12 on white matter hypersignaling.

[0046] (II) Animal Model Construction

[0047] Wild-type C57BL / 6 mice, 8-12 weeks old and weighing 20-25 g, were purchased from Shanghai Southern Model Biotechnology Co., Ltd. This research protocol has been reviewed and approved by the Experimental Animal Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Approval No.: T1-2024-07-006).

[0048] Construction of the BCAS animal model: Male C57BL / 6 mice were selected, anesthetized, and fixed. A midline incision was made in the neck, and the common carotid artery on one side was isolated and exposed. A spring was used to wrap around the artery to create a model on one side, and the same method was used on the other side. In the sham-operated group, only the blood vessel was exposed without placing the spring. After modeling, the wound was sutured, and the mice were kept warm until they regained consciousness. A separate drug treatment group was established, which received plerixafor hydrochloride (AMD3100) in addition to the modeling treatment.

[0049] Administration of Plexafor AMD3100 hydrochloride: AMD3100 powder was dissolved in sterile PBS to a 6.25 mg / mL stock solution, aliquoted into centrifuge tubes, and stored at -80°C. One week prior to surgery, mice in the BCAS group received an intraperitoneal injection of 200 μL of sterile PBS as a drug control; mice in the BCAS + AMD3100 group received an intraperitoneal injection of 200 μL of 0.625 mg / mL working solution (preparation: 180 μL sterile PBS + 20 μL AMD3100 stock solution), with consistent daily injection times.

[0050] Behavioral testing: 28 days after modeling, mice were assessed for cognitive function using water maze and new object recognition experiments, and for gait function using a gait analyzer.

[0051] Quantitative analysis of mouse CXCL12 by ELISA: The CXCL12-specific ELISA kit was used, and the operation was strictly followed according to the instructions to detect the CXCL12 levels in mouse peripheral blood and corpus callosum tissue. A standard curve was established by serially diluting the standard, and quantitative analysis was performed.

[0052] Immunofluorescence: Brain sections and cell slides were fixed, perforated, and blocked, then incubated overnight at 4°C with the corresponding primary antibody. After washing, the samples were incubated at room temperature with the secondary antibody for 1 hour. Imaging was performed using a fluorescence microscope and a laser confocal microscope (Olympus FV1000). Images were analyzed using ImageJ software, and three-dimensional reconstruction and colocalization analysis were performed using Imaris software.

[0053] Laugh blue staining (LFB): After dehydration with a gradient of ethanol (75%, 95%, 100%), the sections were immersed in preheated LFB staining solution and stained at 60°C for 6-8 hours. Subsequently, they were destained with 0.05% lithium carbonate and differentiated with 70% ethanol. After cooling to room temperature, the sections were observed and images were acquired using an optical microscope (BX51; Olympus). The severity of white matter lesions was assessed using the LFB score.

[0054] Transmission electron microscopy: Transmission electron microscopy was used to observe the ultrastructure of myelin sheath. After euthanasia, 1×1×1 mm³ corpus callosum tissue was collected, fixed, dehydrated, embedded, sectioned, stained, and observed. The integrity of myelin sheath and axon was quantitatively analyzed by the G-ratio value (axon diameter / total diameter of axon and myelin sheath).

[0055] Two-photon microscopy: In vivo two-photon imaging was performed using CX3CR1-GFP mice and PKH26-labeled myelin. Mice in the experimental group received intraperitoneal injections of AMD3100 daily for 7 consecutive days prior to surgery, while mice in the control group received PBS. On the day of surgery, 0.5 μL of PKH26-labeled myelin was stereotactically injected into the target brain region (anterior fontanelle coordinates: AP: 0 mm; lateral margin: ML: 1.00 mm; depth: DV: 0.3 mm), followed by cranial fenestration at the same location. Dynamic imaging was performed using a two-photon microscope system at 1, 2, 3, 4, 5, and 24 hours post-fenestration, continuously capturing fluorescence signals. Monitoring included myelin fluorescence intensity, microglia morphology, cell number, and migration.

[0056] Flow cytometry cell sorting: First, a single-cell suspension of corpus callosum tissue was prepared using a tissue dissociation kit. The suspension was pre-incubated for 15 minutes with an Fc blocking agent (anti-CD16 / 32 antibody). Subsequently, surface antibody staining was performed: cells were incubated with CD45 and CD11b antibodies at 4°C in the dark for 30 minutes. Using a standardized gating strategy on a BD FACSAria™ flow cytometer, the CD45lowCD11bhigh cell population was directly sorted into lysis buffer and stored at -80°C for subsequent omics analysis.

[0057] (III) Specific experimental results:

[0058] In serum proteomics, principal component analysis showed that the samples from the healthy control group and the CSVD group were completely separated in the multivariate space defined by the first two principal components, strongly confirming that the pathophysiological process of cerebral small vessel disease involved systematic and global changes in proteins (see...). Figure 1 A). To identify specific differentially expressed proteins, we performed differential analysis and plotted a volcano plot, identifying a total of 91 differentially expressed proteins. Among them, 41 molecules were significantly upregulated in the CSVD group, and 50 molecules were significantly downregulated (see A). Figure 1 B).

[0059] In serum metabolomics, principal component analysis showed that the samples from the healthy control group and the CSVD group were completely separated in the multivariate space defined by the first two principal components, strongly confirming that the pathophysiological process of cerebral small vessel disease involved systematic and global changes in metabolites (see...). Figure 1 C). To identify specific differentially expressed metabolites, we performed differential analysis and generated a volcano plot, identifying a total of 185 differentially expressed metabolites. Among them, 82 molecules were significantly upregulated in the CSVD group, and 103 molecules were significantly downregulated (see [link to relevant documentation]). Figure 1 D).

[0060] We then performed further differential gene pathway enrichment analysis on the differentially expressed genes from proteomics and metabolomics, and found four differential pathways by taking the intersection of the enriched pathways: Fcγ receptor-mediated phagocytosis, phospholipase D signaling pathway, regulation of the actin cytoskeleton, and calcium ion signaling pathway (see...). Figure 1 E), in which the proteomics pathway enrichment results showed that the lysosomal pathway had significant differential gene enrichment between the two groups (see E). Figure 1 F).

[0061] We then further refined the GSEA analysis. Compared with the healthy control group, the regulation of the actin cytoskeleton, lysosomal pathway, phospholipase D signaling pathway, and Fcγ receptor-mediated phagocytosis were significantly downregulated in the CSVD group, while the calcium ion signaling pathway showed positive enrichment in CSVD. This result indicates that the experimental intervention synergistically activated a series of interrelated biological processes involving autophagy and degradation, cell morphology and motility, immune phagocytosis, and cell signal transduction (see...). Figure 1 GH).

[0062] The proteomic differential protein ring heatmap results between the CSVD and HC groups showed that CXCL12 was highly expressed in CSVD patients compared to healthy controls (see...). Figure 1 I).

[0063] Serum CXCL12 levels in the community population were positively correlated with age, negatively correlated with average gait speed and average stride length, and positively correlated with white matter hyperintensity score and total cerebral small vessel disease burden (see [link to relevant data]). Figure 1 JK).

[0064] The Mendelian randomization analysis included one CXCL12 QTL study and three GWAS studies on cerebral small vessel disease (n=198048 in CarcelMarquez-2024; n=232596 in TraylorM-2021; n=5816 in MalikR-2018). The results showed an association between higher CXCL12 levels and an increased risk of white matter hyperintensity (see Mendelian randomization analysis). Figure 2 A).

[0065] Based on this, we conducted a two-year longitudinal follow-up of the community population, including a total of 100 patients who were divided into a slow-progression group (ΔWMH_V < 0.01 ml, n=50) and a rapid-progression group (ΔWMH_V > 0.2 ml, n=50). We followed up on the white matter high-signal imaging, cognition, and gait of the included subjects. The results showed that the two groups were comparable at baseline, but the serum CXCL12 concentration at baseline was significantly higher in the rapid-progression group than in the slow-progression group (ΔWMH_V > 0.2 ml, n=50). Figure 2 B). Baseline serum CXCL12 concentration was significantly positively correlated with ΔWMH (r = 0.3, P = 0.002). Figure 2 C). After adjusting for age, sex, baseline WMH volume, years of education, and major vascular risk factors (including blood pressure, blood glucose, and lipid levels), multiple linear regression analysis confirmed that CXCL12 concentration remained an independent predictor of ΔWMH (β = 0.287, 95% CI: 0.095–0.479, P < 0.004). Figure 2 The F indicates that higher baseline CXCL12 levels are associated with faster progression of white matter damage over the next two years. In addition to imaging indicators, baseline CXCL12 levels are also closely related to cognitive decline. CXCL12 concentration was significantly negatively correlated with a decrease in the Montreal Cognitive Assessment (MoCA) total score (ΔMoCA) (r = -0.248, P = 0.015). Figure 2 D), and this negative association remained significant in the fully corrected model (β = -0.207, 95% CI: -0.408 ~ -0.006, P = 0.047). Figure 2 F). Regarding motor function, baseline CXCL12 concentration was significantly positively correlated with prolonged standing-walking time (TUG) (r = 0.226, P = 0.043). Figure 2 E). After adjusting for confounding factors, CXCL12 still independently predicted the increase in TUG time (β = 0.256, 95% CI: 0.021–0.491, P = 0.036). Figure 2The results (F) indicate that high CXCL12 levels are associated with a decline in dynamic balance and mobility. Although CXCL12 showed a negative correlation with changes in gait speed and stride length, this correlation did not reach statistical significance. All of the above longitudinal data suggest that baseline serum CXCL12 levels are not only an independent predictor of the radiographic progression of white matter injury, but also significantly associated with cognitive decline and gait impairment.

[0066] Studies show that the BCAS model was successfully constructed, and the blood flow in the corresponding blood supply area decreased significantly, with statistically significant differences (see [link to study]). Figure 3 AB).

[0067] The study used ELISA to detect CXCL12 concentration. Results showed high expression of CXCL12 in the peripheral blood and white matter region of the corpus callosum in the BCAS mouse model, and a correlation was found between peripheral blood CXCL12 levels and corpus callosum tissue CXCL12 levels (see [link to study].) Figure 3 CE).

[0068] Studies have shown that mice after BCAS surgery exhibited increased latency, reduced target quadrant exploration time, and fewer platform crossings during the water maze exploration phase. Intraperitoneal administration of AMD3100 can improve cognitive function in mice to some extent (see...). Figure 4 AB).

[0069] Studies have shown that mice exhibited reduced exploration in novel object recognition tests after BCAS surgery, while intraperitoneal administration of AMD3100 improved their exploration ability (see...). Figure 4 CD). The above results indicate that, under chronic cerebral hypoperfusion in mice, administration of AMD3100 to inhibit CXCL12 / CXCR4 improved spatial learning and memory abilities to some extent and enhanced the mice's ability to explore and remember new things.

[0070] The study, using PLS-DA results, showed that the gait data of the Sham and Vehicle groups were completely separated, while the AMD3100 group fell in between, indicating overall comparability among the three groups. Further visualization analysis of the differential gait data revealed expression differences among the three groups, suggesting that chronic cerebral hypoperfusion has a certain impact on gait function in mice. Administration of AMD3100 to inhibit CXCL12 / CXCR4 can improve gait function in mice to some extent (see...). Figure 4 EF).

[0071] The results showed that compared with the Sham group, the MAG fluorescence intensity in the corpus callosum region of mice after BCAS was significantly decreased, and LFB staining revealed disordered myelin sheath arrangement and obvious demyelination areas in the corpus callosum region. Transmission electron microscopy also showed that the myelin sheath thickness was significantly thinner after BCAS, exhibiting pathological morphology of myelin sheath layering and separation. However, after intraperitoneal injection of AMD3100 to inhibit CXCL12 / CXCR4, compared with the Vehicle group, the AMD3100 group showed increased MAG fluorescence intensity, improved disordered white matter fiber arrangement in LFB staining, and reduced myelin sheath thinning and myelin sheath layering, with statistically significant differences (see [link to study]. Figure 5 AB).

[0072] Immunofluorescence staining revealed a significant decrease in CC1-positive mature oligodendrocytes after BCAS treatment, while an increase in GST-π and NG2+Ki67+ double-positive cells, which reflect oligodendrocyte proliferative capacity. Treatment with AMD3100 significantly increased the number of CC1-positive mature oligodendrocytes, and their proliferative capacity was higher than that of the BCAS group. These results indicate that AMD3100 treatment can promote oligodendrocyte maturation and differentiation to a certain extent (see...). Figure 5 CD).

[0073] To further investigate the mechanism of action of CXCL12, we performed immunofluorescence staining on frozen sections of mouse brain tissue and found that CXCL12 was mainly expressed on microglia in the corpus callosum region of mice after BCAS surgery (see...). Figure 6 A).

[0074] Further staining of microglia revealed that, compared to the Sham group, CXCL12 expression was upregulated in microglia of the corpus callosum region after BCAS surgery, and the proportion of IBA1+CXCL12+ double-positive cells was significantly reduced after AMD3100 treatment. Compared to the Vehicle group, the number of microglia in the corpus callosum region was reduced in the AMD3100 group, with a statistically significant difference. To further assess the degree of microglia activation, morphological analysis of microglia in the corpus callosum region was performed using Imaris. The results showed that, compared to the Vehicle group, the surface area and volume of microglia were significantly reduced after AMD3100 treatment, while there was no significant difference in sphericity between the two groups. These results indicate that the brain hypoperfusion model induces amoeboid changes in microglia morphology, and that AMD3100 treatment effectively reduces the degree and number of microglia by inhibiting CXCL12 / CXCR4 (see [link to relevant documentation]). Figure 6 B).

[0075] Simultaneously, co-labeling of the CXCL12 receptor CXCR4 with microglia revealed a significant increase in CXCR4 expression in corpus callosum microglia after BCAS surgery, while the co-labeling ratio of CXCR4 and IBA1 significantly decreased after administration of the pathway antagonist AMD3100 (see...). Figure 6 C).

[0076] To investigate changes in microglia phenotype and function, we used immunofluorescence staining to count the percentages of IBA1+LC3B+ double-positive cells, Iba1+Lamp1+ double-positive cells, and Iba1+CD68+ double-positive cells, observing whether AMD3100 administration affected the proportion of microglia in the corpus callosum region after BCAS surgery. The results showed that compared to the Vehicle group, the proportion of LC3B and Iba1 co-labeled cells was significantly reduced in the AMD3100 group; and through Lamp1 and CD68 co-labeling staining, we found that phagocytic activation of microglia was significantly decreased after AMD3100 administration (see [link to AMD3100]). Figure 6 DF).

[0077] The results showed that the level of CXCL12 in peripheral blood of mice was correlated with indicators such as cognitive function, gait, myelin damage, and microglia activation. Specifically, CXCL12 levels were positively correlated with the latency of the water maze and negatively correlated with the time spent exploring the target quadrant; negatively correlated with the proportion of normal gait and walking speed; negatively correlated with MAG fluorescence intensity and positively correlated with LFB score; and positively correlated with the number of microglia, the proportion of Iba1+CXCL12+ double-positive cells, and the surface area and volume of microglia (see...). Figure 6 G).

[0078] We then performed in vivo two-photon imaging to assess the dynamic changes in myelin fragment fluorescence intensity, microglia morphology, and cell motility. The results showed that compared to the PBS group, the AMD3100 group exhibited a significant decrease in myelin fluorescence intensity and a smaller surface area of ​​microglia surrounding the myelin sheath, while the number of microglia did not differ statistically between the two groups. This indicates that the AMD3100 group experienced reduced microglia activation and increased myelin clearance rate (see...). Figure 7 AB).

[0079] Next, we focused on microglia, using flow cytometry to extract microglia from the corpus callosum region for transcriptome analysis. Pathway enrichment analysis showed that the Vehicle group genes were enriched in pathways such as inflammatory responses, cell chemotaxis, responses to interleukin-1, immune system processes, and lipid droplet formation. In contrast, the AMD3100-treated group was mainly enriched in pathways such as cellular responses to calcium ions, calcium-regulated exocytosis, phospholipase D signaling pathway, exocytosis, cholesterol metabolism, fatty acid oxidation, ATP biosynthesis, lipid binding, Fcγ receptor-mediated phagocytosis, and cholesterol efflux (see...). Figure 7 C).

[0080] We then created a heatmap of differentially expressed genes (see...). Figure 7 (D), mainly involving cell migration, Fcγ-mediated phagocytosis, lipid droplet organization, fatty acid oxidation, phospholipase D signaling pathway and inflammatory response-related pathways. The above results show that the Vehicle group exhibits a typical pro-inflammatory immune phenotype, accompanied by reprogramming of lipid metabolism. Administration of AMD3100 is beneficial to improve lipid metabolism and promote phagocytosis, fatty acid oxidation, cholesterol efflux and other pathways.

[0081] Based on the above results, this study identified the key protein CXCL12 using high-throughput proteomics and clinical characteristics of a community population. Mendelian randomization and longitudinal follow-up revealed that baseline serum CXCL12 levels are not only an independent predictor of the radiographic progression of white matter injury, but also significantly correlated with cognitive decline and gait impairment, supporting CXCL12 as a potential blood biomarker for CSVD progression. Subsequently, by constructing a BCAS mouse model, it was found that under cerebral hypoperfusion, CXCL12 levels were positively correlated with microglial activation, neuroinflammation, and myelin damage, and negatively correlated with gait and cognitive function. Administration of the pathway inhibitor AMD3100 resulted in a transformation of the microglial phenotype to an anti-inflammatory phenotype, alleviating demyelinating damage in the corpus callosum region and improving cognitive and gait functions in mice to some extent. Furthermore, immunofluorescence and other methods revealed that CXCL12 and CXCR4 proteins play a role in BCAS progression. Postoperative expression was mainly found on microglia. Significant changes were also found in the lipophagy and lysosomal pathways of microglia between the two groups. Transcriptomics further showed that microglia after BCAS surgery transformed into a pro-inflammatory phenotype, accompanied by reprogramming of lipid metabolism. Administration of AMD3100 to inhibit CXCL12 / CXCR4 was beneficial to promoting lipid metabolism pathways such as fatty acid oxidation and cholesterol efflux.

[0082] Based on the above results, this invention proposes that the CXCL12 / CXCR4 axis may become a new clinical intervention target for cerebral small vessel disease, providing a new direction for the prevention and treatment of cerebral small vessel disease in the future.

[0083] Finally, it should be noted that the above embodiments are only for illustrating the present invention and not for limiting the scope of protection of the present invention. Furthermore, after reading the technical content of this invention, those skilled in the art can make various modifications, alterations, or variations to the present invention, and all such equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. Application of CXCL12 detection reagent in the preparation of cerebral small vessel disease detection reagent.

2. The application according to claim 1, characterized in that, The CXCL12 detection reagent is selected from one or more of the following: ELISA detection reagent, Luminex detection reagent, immunofluorescence detection reagent, and proteomics detection reagent.

3. A detection system for cerebral small vessel disease, characterized in that, The detection system includes the CXCL12 detection component and the result determination system.

4. The detection system according to claim 3, characterized in that, The detection component of the CXCL12 contains one or more of the following: ELISA detection reagent, Luminex detection reagent, immunofluorescence detection reagent, and proteomics detection reagent.

5. The detection system according to claim 3, characterized in that, The result determination system is used to output the risk of cerebral small vessel disease based on the CXCL12 expression results detected by the detection system.

6. The detection system according to claim 5, characterized in that, The disease risk mentioned is based on the comparison of CXCL12 expression levels between the test sample and the normal sample. When there is a significant or extremely significant difference in CXCL12 protein expression levels between the test sample and the normal sample, the test sample is judged to have a high disease risk. A significant or extremely significant difference is defined as a P value < 0.05 between the two groups.

7. Application of CXCL12 or CXCR4 signaling pathways as targets in the preparation of drugs for the treatment or prevention of cerebral small vessel diseases.

8. The use of CXCL12 or CXCR4 signaling pathway inhibitors in the preparation of drugs for the treatment or prevention of cerebral small vessel diseases, characterized in that, The CXCL12 or CXCR4 signaling pathway inhibitors are selected from CXCR4 inhibitors or CXCL12 inhibitors. The CXCL12 inhibitors are selected from substances that inhibit CXCL12 activity, degrade CXCL12, or reduce CXCL12 levels. The CXCR4 inhibitors are selected from substances that inhibit CXCR4 activity, degrade CXCR4, or reduce CXCR4 levels.

9. The inhibitor according to claim 8, characterized in that, The CXCL12 or CXCR4 signaling pathway inhibitor is plerixafor hydrochloride, which is a selective CXCR4 antagonist and also an effective CXCL12-mediated chemokine inhibitor.

10. The use of plerixafor hydrochloride in the preparation of drugs for treating cerebral small vessel diseases, characterized in that, The drug is administered by injection.

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