Application of hypoglycosylated apolipoprotein D in preparation of medicine for repairing blood brain barrier

Abstract

The invention belongs to the technical field of medicine preparation, and particularly relates to application of hypoglycosylated apolipoprotein D in preparation of a medicine for repairing a blood brain barrier. Experiments in the invention show that in the molecular level, the binding affinity of low glycosylated apolipoprotein D (ApoD) and CD36 is increased by about 9 times compared with that of high glycosylated apolipoprotein D, and a key mechanism foundation is laid for function exertion; in an MCAO (middle cerebral artery occlusion) stroke model (middle cerebral artery occlusion model), the hypoglycosylated apolipoprotein D can remarkably repair a capillary structure, reduce an infarct area, improve neurological dysfunction, reduce blood-brain barrier permeability and enhance capillary perfusion, and the treatment efficiency is far superior to that of the hyperglycosylated apolipoprotein D; aiming at an elderly stroke model, the hypoglycosylated ApoD can improve the survival rate of mice, repair disordered and expanded capillaries, inhibit pathological endothelial cell proliferation and strengthen the integrity of a blood-brain barrier, and the hypoglycosylated ApoD can still continuously improve neurological functions and vascular phenotypes 30 days after stroke.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to the application of low-glycosylated apolipoprotein D in the preparation of drugs for blood-brain barrier repair. Background Technology

[0002] The blood-brain barrier (BBB) ​​is a core structural barrier maintaining the physiological homeostasis of the central nervous system (CNS). Its integrity is not only fundamental to ensuring a stable brain microenvironment and normal neural function, but also a crucial prerequisite for the recovery of neural function under pathological conditions. In the progression of various neurological diseases such as stroke, Alzheimer's disease, glioblastoma, and multiple sclerosis, BBB disruption is a hallmark pathological event, often accompanied by endothelial dysfunction and parietal cell dysregulation, leading to a series of malignant chain reactions such as exacerbated neuroinflammation, cerebral edema, and neuronal damage, directly and closely related to poor patient prognosis. Although the harmfulness of BBB dysfunction is widely recognized in clinical and research fields, its specific regulatory mechanisms remain incompletely understood. Currently, targeted therapeutic strategies for BBB integrity repair are still in the exploratory stage, and effective means for precise intervention in BBB damage are lacking in clinical practice.

[0003] Endothelial cells and parietal cells (including pericytes and vascular smooth muscle cells) are the core functional components constituting the microvascular structure of the blood-brain barrier. They form a highly coordinated intercellular communication network through direct contact and sharing the basement membrane, jointly maintaining vascular structural stability and barrier functional integrity. Compared to peripheral tissues, the proportion of parietal cells to endothelial cells is significantly higher in the brain. This specialized cell ratio is crucial for the precise regulation of the blood-brain barrier. When brain diseases occur, this intercellular coordination mechanism is disrupted, leading to endothelial dysfunction, parietal cell loss, and ultimately, loss of blood-brain barrier stability. However, the specific regulatory mechanisms of parietal-endothelial cell communication and their role in blood-brain barrier repair remain largely unexplored, hindering the development of targeted blood-brain barrier repair therapies.

[0004] Apolipoprotein D (ApoD), an important member of the lipid transporter family, has been shown to participate in various neuroprotective processes, exhibiting neuroprotective effects in disease models such as Alzheimer's disease and traumatic brain injury. Its upregulation is closely related to the repair process after nerve injury. However, to date, the specific functions and targets of ApoD in maintaining blood-brain barrier homeostasis, as well as the impact of post-translational modifications such as glycosylation on its function, have not been systematically and thoroughly studied. This current research status limits our understanding of the mechanisms by which ApoD regulates the blood-brain barrier and hinders its development as a therapeutic target for blood-brain barrier repair, necessitating further exploration and elucidation. Summary of the Invention

[0005] The purpose of this invention is to provide an application of low-glycosylated apolipoprotein D in the preparation of drugs for blood-brain barrier repair.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides the application of low-glycosylated apolipoprotein D in the preparation of drugs for blood-brain barrier repair.

[0007] Preferably, in the above applications, the low-glycosylated apolipoprotein D is obtained by treating the high-glycosylated apolipoprotein D with PNGase F.

[0008] More preferably, in the above applications, the method for preparing highly glycosylated apolipoprotein D includes: overexpressing the apolipoprotein D gene in engineered cells, and then purifying the cell culture supernatant to obtain highly glycosylated apolipoprotein D.

[0009] More preferably, in the above applications, the engineered cells are 293T cells.

[0010] Preferably, in the above applications, the drug has the effect of treating ischemic stroke.

[0011] Preferably, in the above applications, the subjects with ischemic stroke are elderly patients.

[0012] Preferably, in the above applications, the drug has one or more of the following effects: the effect of repairing microvascular structures; the effect of reducing infarct area; the effect of improving neurological deficits; the effect of promoting the reduction of pathological proliferation of endothelial cells; the effect of reducing blood-brain barrier permeability; and the effect of enhancing microvascular perfusion.

[0013] Preferably, in the above applications, the dosage form of the drug is an injection solution or a powder.

[0014] The beneficial effects of this invention include at least the following: Experiments have shown that, at the molecular level, low-glycosylated apolipoprotein D (ApoD) has an affinity for CD36 that is approximately 9 times higher than that of high-glycosylated apolipoprotein D, laying a key mechanistic foundation for its functional performance; in the MCAO stroke model (middle cerebral artery occlusion model), low-glycosylated apolipoprotein D can significantly repair microvascular structure, reduce infarct area, improve neurological deficits, reduce blood-brain barrier permeability, and enhance microvascular perfusion, with therapeutic efficacy far superior to high-glycosylated ApoD; in elderly stroke models, low-glycosylated ApoD can improve mouse survival rate, repair disordered and dilated microvessels, inhibit pathological endothelial cell proliferation, strengthen blood-brain barrier integrity, and continue to improve neurological function and vascular phenotype 30 days after stroke. Attached Figure Description

[0015] Figure 1Western blot analysis was used to assess the level of ApoD glycosylation secreted by brain parietal cells. Figure 2 Micro-thermophoresis (MST) was used to analyze the affinity between low-glycosylated ApoD and high-glycosylated ApoD and the extracellular domain of CD36 protein; Figure 3 Biolayer interferometry (BLI) sensor images show the difference in affinity between low-glycosylated ApoD and high-glycosylated ApoD for the extracellular domain of CD36; Figure 4 A timeline and subsequent evaluation diagram of the experimental administration of recombinant apolipoprotein D (rApoD) via the tail vein after MCAO. Figure 5 To observe the distribution of ApoD protein in brain parenchyma using immunofluorescence staining (anti-ApoD antibody); Figure 6 Representative images of brain microvessels in ApoD- / - mice after treatment with hyperglycosylated ApoD, and quantitative analysis of vessel area, vessel diameter and parietal cell coverage (n=6). Figure 7 Representative images of brain microvessels in ApoD- / - mice after treatment with hypoglyco-ApoD, and quantitative analysis of vessel area, vessel diameter and parietal cell coverage (n=6). Figure 8 Immunostaining with MAP2 reveals infarcted areas (marked by dashed lines) in brain slices from ApoD- / - mice treated with low-glycosylation ApoD, with a scale bar of 1000 μm, and quantitative analysis of infarct area (n=6). Figure 9 Statistical analysis of neurological deficit scores in ApoD- / - mice treated with low-glycosylated ApoD (n=8). Figure 10 The pole climbing test (PCT) showed the iTurn and TD (n=8) of mice in the low-glycated ApoD treatment group and the control group after stroke. Figure 11 To show the latency of mice treated with low-glycosylated ApoD falling off the rotundus in the rotundus assay (n=8). Figure 12 Immunostaining for CD31, ERG and EdU showed the proliferation of mouse endothelial cells treated with low-glycosylation ApoD. Scale bar: 40 μm (n=6). Figure 13To assess the microvascular permeability of brain slices on day 5 after stroke using dextran-2000kDa and 70kDa (scale bar 40μm) and to perform quantitative analysis of vascular permeability (n=6). Figure 14 To determine the lectin perfusion in the microvessels of the peri-infarct area, scale bar: 50 μm, n=6; Figure 15 Immunofluorescence images show the interaction between parietal cells and microvessels in the brains of 2-month-old and 16-month-old mice; Figure 16 The results of ApoD protein blotting in adult and aged mice in the sham-operated and ischemic groups, n=3 mice, M is the molecular weight marker; Figure 17 CD31 immunostaining and quantitative analysis of vascular area in brain slices from 2-month-old and 16-month-old mice on day 5 after MCAO surgery (n=6). Figure 18 Representative images of microvascular permeability to dextran-2000kDa and 70kDa in 2-month-old and 16-month-old mice after stroke, as well as quantification of vascular permeability (n=6). Figure 19 A schematic diagram of the experimental timeline for tail vein hypoglycemic ApoD injection, MCAO surgery, and functional analysis; Figure 20 Statistical analysis of MAP2 immunostaining to show infarcted areas (indicated by yellow dashed lines) and infarct size (n=7); Figure 21 Image showing the neurological deficit measurement results on the 5th day after MCAO (n=8); Figure 22 Kaplan-Meier survival curves of hypoglycemic ApoD-treated mice and control mice undergoing MCAO surgery (n=15). Figure 23 The results of immunostaining of CD31 and CD13 in brain slices, as well as quantitative analysis of vascular area, average vascular diameter, vascular wall cell coverage, and vascular wall cell area (n=6). Figure 24 The results of immunostaining of CD31, ERG and EdU and quantitative analysis of endothelial cell proliferation in brain slices of mice in the low-glucose ApoD treatment group and the control group were presented (n=6). Figure 25 Representative images of microvascular permeability to Dextran-70 and vascular permeability (n=6). Figure 26Representative images of microvascular permeability to Dextran-2000 and vascular permeability (n=6); Figure 27 To determine the perfusion of lectins into the microvessels of the infarcted area (n=6); Figure 28 The representative LSCI plots show the CBF fluorescence display and content analysis of the two groups of mice before and after occlusion (n=6). Figure 29 Immunostaining of CD31 and tight junction protein ZO1 in brain slices and expression of tight junction protein (n=6). Figure 30 Immunostaining of CD31 and tight junction protein Claudin5 in brain slices and expression of tight junction protein (n=6). Figure 31 Immunostaining of CD31 and tight junction protein (Occludin) in brain slices and expression of tight junction protein (n=6). Figure 32 Immunostaining of CD31 and basement membrane protein collagen 4 in brain slices and expression of collagen 4 (n=6). Figure 33 Immunostaining of CD31 and basal laminin in brain slices and expression of laminin (n=6). Figure 34 The representative LSCI plot shows the CBF of the two groups of mice before and after occlusion; Figure 35 For the case of quantitative analysis of CBF (n=6); Figure 36 Immunostaining with MAP2 reveals the infarcted areas in brain slices from two groups of mice on day 30 post-stroke. Figure 37 The infarct area in brain slices of two groups of mice on day 30 after stroke (n=6); Figure 38 To assess the motor function of two groups of mice on day 30 after stroke using a correction wheel test (n=8); Figure 39 To assess motor function in two groups of mice on day 30 after stroke using PCT (n=8); Figure 40 Double immunostaining for CD31 and CD13 was used to show the microvessels and parietal cells in the periinfarct area on day 30 post-stroke (n=4). Figure 41 To assess the vascular permeability of the periinfarct region in two groups of mice by intravenous injection of dextran Dextran-2000 (n=4); Figure 42 To assess vascular permeability in the periinfarct region of two groups of mice by intravenous injection of Dextran-70 (n=4); Figure 43 Immunostaining and expression of the tight junction protein Claudin5 in microvessels surrounding the infarct area (n=4). Figure 44 Immunostaining and expression of the tight junction protein Occludin in microvessels surrounding the infarct area (n=4). Figure 45 Immunostaining and expression of the tight junction protein ZO1 in microvessels surrounding the infarct area (n=4). Detailed Implementation

[0016] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0017] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0018] In this invention, the term "hyperglycosylated apolipoprotein D (hyperglyco-ApoD)" (produced by 293T cells) is similar to the glycosylation level of serum ApoD; "hypoglycosylated apolipoprotein D (hypoglyco-ApoD)" refers to recombinant apolipoprotein D with reduced glycosylation level after deglycosylation treatment with PNGase F by hyperglycosylated apolipoprotein D.

[0019] This invention provides an application of low-glycosylated apolipoprotein D (low-glycosylated ApoD) in the preparation of a drug for blood-brain barrier repair.

[0020] It should be noted that the blood-brain barrier is a key structure for maintaining the stability of the brain tissue microenvironment. Damage to the blood-brain barrier is a core pathological link in central nervous system diseases such as ischemic stroke and traumatic brain injury, which can lead to cerebral edema, inflammatory infiltration, and neurological deficits, seriously affecting prognosis. Low-glycosylated apolipoprotein D has been experimentally verified to specifically target microvascular endothelial cells of the blood-brain barrier. By inhibiting the pathological proliferation of endothelial cells, it repairs the integrity of the barrier structure, providing a new target and drug raw material for the treatment of diseases related to blood-brain barrier damage.

[0021] In some specific examples, in the above applications, low-glycosylated apolipoprotein D is obtained by treating high-glycosylated apolipoprotein D with PNGase F.

[0022] It should be noted that PNGase F is a glycosidase that specifically removes N-linked glycans, exhibiting high catalytic efficiency and specificity. It can precisely remove the glycosylation modification of highly glycosylated apolipoprotein D. Apolipoprotein D expressed in eukaryotic cells of 293T cells is in a highly glycosylated state, and the glycan structure may affect the binding activity of ApoD protein to target proteins. The low-glycosylated form obtained after treatment with PNGase F is more likely to bind to CD36 protein, and its barrier repair effect is significantly better than that of the highly glycosylated form.

[0023] In some specific examples, the preparation method of highly glycosylated apolipoprotein D in the above applications includes: overexpressing the apolipoprotein D gene in engineered cells, and then purifying the cell culture supernatant to obtain highly glycosylated apolipoprotein D.

[0024] It should be noted that by overexpressing the apolipoprotein D gene in engineered cells, the protein can be produced on a large scale. Compared with natural extraction methods, the yield is higher and the purity is easier to control. Apolipoprotein D is a secreted protein, which can be released into the cell culture supernatant after expression, facilitating subsequent separation and purification and avoiding structural damage caused by intracellular protein extraction. The purification process can use conventional techniques in this field such as affinity chromatography and gel filtration, which can obtain highly glycosylated apolipoprotein D with a purity of ≥95%, meeting the quality requirements for subsequent deglycosylation treatment and drug preparation.

[0025] In some specific examples, the engineered cells in the above applications are 293T cells.

[0026] It should be noted that 293T cells are a commonly used engineered cell line for recombinant protein expression, with advantages such as high transfection efficiency, high protein expression levels, and mild culture conditions. They can efficiently express the apolipoprotein D gene and undergo natural glycosylation modification. This cell line has a clear genetic background, is non-pathogenic, and the expressed protein is highly safe, meeting the compliance requirements for biopharmaceutical production and ensuring the structural correctness and biocompatibility of highly glycosylated apolipoprotein D.

[0027] In some specific examples, the drugs described above are used to treat ischemic stroke.

[0028] It should be noted that after ischemic stroke, cerebral ischemia and hypoxia lead to damage to the endothelial cells of the blood-brain barrier and increased vascular permeability, resulting in secondary cerebral edema and neuronal death, which are key factors in the progression of the disease. Low-glycosylated apolipoprotein D can block the above-mentioned pathological processes by repairing the blood-brain barrier. Validated in a mouse model of ischemic stroke, this drug can significantly improve cerebral blood flow perfusion and reduce neurological function damage in the model animals, providing a new targeted treatment for ischemic stroke, especially suitable for patients in the acute and recovery phases.

[0029] In some specific examples, the subjects of ischemic stroke in the above applications were elderly patients.

[0030] It should be noted that elderly patients (usually ≥60 years old) have decreased vascular elasticity and endothelial function, resulting in more severe blood-brain barrier damage after ischemic stroke and weaker repair capabilities, leading to a poorer clinical prognosis. The drug of this invention targets the pathological characteristics of elderly patients, can effectively repair the damaged blood-brain barrier, and has the advantages of low toxicity and good tolerability. Validated in elderly animal models, its therapeutic effect and safety are superior to traditional drugs, filling the gap in targeted therapy for ischemic stroke in the elderly.

[0031] In some specific examples, in the above applications, the drug has one or more of the following effects: the ability to repair microvascular structures; the ability to reduce infarct area; the ability to improve neurological deficits; the ability to promote the reduction of pathological proliferation of endothelial cells; the ability to reduce blood-brain barrier permeability; and the ability to enhance microvascular perfusion.

[0032] It should be noted that the above-mentioned efficacy has been verified by in vitro and in vivo experiments: repairing microvascular structure is achieved by stabilizing tight junctions between endothelial cells and the integrity of the basement membrane; reducing infarct area is due to barrier repair reducing damage to surrounding normal brain tissue caused by cerebral edema and inflammatory factor infiltration; improving neurological deficits (such as motor and cognitive impairment) is achieved by protecting the microenvironment for nerve cell survival and promoting neural circuit repair; inhibiting pathological proliferation of endothelial cells can prevent abnormal vascular remodeling and maintain normal microvascular function; reducing blood-brain barrier permeability reduces plasma leakage and alleviates cerebral edema; enhancing microvascular perfusion can improve blood oxygen supply to ischemic areas and promote tissue repair. The synergistic effect of these various efficacy measures comprehensively improves the treatment effect of ischemic stroke.

[0033] In some specific examples, the dosage form of the drug in the above applications is an injection or a powder.

[0034] It should be noted that the injectable formulation can be administered intravenously to directly enter the bloodstream, quickly reaching the site of blood-brain barrier damage to exert its effects. It is suitable for emergency intervention in the acute phase of ischemic stroke and can rapidly control the progression of the disease. The powder is a lyophilized formulation, which removes moisture through freeze-drying technology, effectively protecting the biological activity of low-glycosylated apolipoprotein D and avoiding protein denaturation caused by temperature and humidity changes during storage and transportation. It also has a long shelf life, is easy to carry, and can be reconstituted with sterile water for injection before administration. It is suitable for medication needs in different scenarios such as clinical emergency, hospitalization, and home rehabilitation. The excipients for both formulations are selected from pharmaceutically acceptable components (such as phosphate buffer for the injectable formulation and mannitol for the powder), which are non-allergenic and have high safety.

[0035] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0036] In the following examples, to explore the potential translational value of the findings, an in-depth analysis was conducted to explain why elevated serum apolipoprotein D (ApoD) has little effect on maintaining the integrity of the blood-brain barrier (BBB), while parietal cell-derived ApoD is crucial for this function. This invention, through Western blot experiments, revealed that the glycosylation level of ApoD secreted by brain parietal cells is lower than that of plasma ApoD (…). Figure 1 Furthermore, the glycosylation level of ApoD derived from parietal cells closely matches that of ApoD from brain tissue. This difference in glycosylation patterns suggests that plasma-derived and brain parietal cell-derived ApoD may have unique biological functions.

[0037] In the following example, the preparation process of glycosylated ApoD is as follows: (1) Obtaining hyperglycosylated ApoD (1-1) Preparation of recombinant plasmid and host cell: The mouse ApoD plasmid with His tag (pMKH-His-mApoD) was used as the expression vector, and 293T cells were selected as the eukaryotic expression host cells.

[0038] (1-2) Cell culture and protein expression: 293T cells were seeded in a culture system containing special medium (MCE, catalog number HY-P7S0082). When the cells grew to the logarithmic growth phase, recombinant plasmids were transfected to overexpress His-mApoD protein in 293T cells. ApoD is a secreted protein and was released into the cell culture supernatant after expression.

[0039] (1-3) Protein purification: Collect the culture supernatant of transfected 293T cells and perform affinity purification using a HisPur™ Ni-NTA column (Thermo Fisher Scientific, catalog number 88226). Refer to the kit instructions for specific steps: first, equilibrate the column with equilibration buffer, then load the culture supernatant to allow the His tag to bind with the Ni-NTA protein. 2+ After removing impurities with washing buffer, the target protein is eluted with elution buffer to obtain high-purity His-mApoD.

[0040] (1-4) Verification of hyperglycosylation: The recombinant protein was derived from the 293T eukaryotic expression system. Its glycosylation level was similar to that of serum ApoD, which is hyperglycosylated ApoD.

[0041] (2) Obtaining hypoglycosylated ApoD (2-1) Deglycosylation treatment: Take the purified high-glycosylated ApoD and add PNGase F (Beyotime, catalog number P2330S) for deglycosylation reaction (use 1 μL of PNGase F (500U / μL) per 100μg of glycoprotein and react at 37℃ for 1 hour); PNGase F can specifically cleave the oligosaccharide chain linked by asparagine, thereby reducing the glycosylation level of ApoD.

[0042] (2-2) Post-treatment purification: After the deglycosylation reaction is completed, the free PNGaseF and the cleaved sugar chains are removed by centrifugation and dialysis to obtain hypoglycosylated ApoD (hypoglyco-ApoD).

[0043] In the following examples, all data are expressed as mean ± standard error; statistical analysis was performed using GraphPadPrism 9.5 software. One-way ANOVA was used to compare differences between groups, and Tukey's HSD test was used for post-hoc pairwise comparisons; Student's t test was used to compare differences between groups; a p-value less than 0.05 was considered statistically significant.

[0044] In the examples below, all animal experimental protocols were approved by the Animal Ethics Committee of the Army Medical University and comply with the U.S. Department of Public Health's policy on the use of laboratory animals.

[0045] Example 1: Binding affinity test of high / low glycosylated ApoD to CD36 (1) Microscale thermophoresis (MST) to detect binding affinity (1-1) Preparation of experimental materials: Recombinant ApoD proteins include hyperglycosylated ApoD and hypoglycosylated ApoD, wherein hypoglycosylated ApoD is obtained by treating hyperglycosylated ApoD with PNGase F (PNGase F is purchased from Beyotime, catalog number P2330S).

[0046] Recombinant CD36 extracellular domain protein: obtained by purification using a prokaryotic expression system (E. coli BL21) or a eukaryotic expression system (293T cells), and is tagged with His for easy purification.

[0047] Analysis buffer: Buffer containing 10 mM HEPES (pH 7.5) and 150 mM sodium chloride.

[0048] Experimental instrument: Monolith NT.115 affinity analyzer from Nanotemper GmbH, Germany.

[0049] (1-2) Experimental Procedure 1) Dilute high-glycosylated ApoD and low-glycosylated ApoD to 400 nM concentration and dissolve them in 100 μL of analysis buffer to prepare ligand solutions.

[0050] 2) The experiment was set up in two groups (high sugar group and low sugar group), with 16 test tubes in each group, numbered 1 to 16. Add 10 μL of analysis buffer to each of the test tubes numbered 2 to 16.

[0051] 3) Add 20 μL of the corresponding 400 nM ligand solution to the first tube of each group, and then transfer 10 μL of the solution to tubes 2 to 16 for a 2-fold serial dilution to obtain a series of ligand solutions with different concentrations.

[0052] 4) Add 10 μL of fluorescently labeled CD36 extracellular domain protein to each test tube (refer to the instrument manual for labeling method) and mix thoroughly.

[0053] 5) After incubating the sample at room temperature for 15 minutes, transfer it to a sample tray and place it in a Monolith NT.115 affinity analyzer for detection, recording the changes in the thermophoretic signal.

[0054] 6) Use the instrument's software to fit the data and calculate the dissociation constant (KD) value.

[0055] (1-3) Results Micro-thermophoresis (MST) showed that the dissociation constant (KD) of highly glycosylated ApoD with CD36 was 3.03 μM, while that of low-glycosylated ApoD with CD36 was 0.33 μM. Figure 2This means that the affinity of low-glycosylated ApoD for CD36 is about 9 times higher than that of high-glycosylated ApoD for CD36.

[0056] (2) Further verification by biolayer interferometry (BLI) (2-1) Preparation of experimental materials: Recombinant CD36 extracellular domain protein, high-glycosylated ApoD, low-glycosylated ApoD (same as MST detection).

[0057] Biotinylation reagent: EZ-Link NHS-LC-LC-Biotin.

[0058] Streptavidin (SA) biosensor (18-5019, Fortebio).

[0059] Buffer: PBS (containing 0.005% Tween 20); 72.5 μM oleic acid (OA) needs to be added in the competition experiment.

[0060] Competing ligands: 10 nM human oxidized low-density lipoprotein (ox-LDL; IOI300, Solarbio), 72.5 μM palmitic acid (PA).

[0061] Experimental apparatus: Octet R8 biolayer interferometer (Sartorius, Göttingen, Germany), 96-well black substrate (3915, Corning, China).

[0062] (2-2) Experimental steps: 1) Biotinylation of CD36 protein: CD36 protein was dissolved in PBS at a concentration of 100 μg / ml, and an appropriate amount of EZ-Link NHS-LC-LC-Biotin reagent was added. The mixture was incubated at room temperature for 30 minutes, and unbound biotin was removed by dialysis.

[0063] 2) Sensor pretreatment: The streptavidin (SA) biosensor was pre-wetted with PBS to establish baseline readings.

[0064] 3) Protein immobilization: Biotinylated CD36 protein was immobilized on the SA biosensor at a flow rate of 20 μL / min for 600 seconds to ensure stable immobilization.

[0065] 4) Gradient binding experiment: High-glycosylated ApoD and low-glycosylated ApoD were diluted in PBS (containing 0.005% Tween 20) to a series of concentrations of 2.5 nM, 5 nM, 10 nM, 20 nM and 40 nM, respectively, with a final volume of 200 μL per well. A control group containing only buffer was also set up.

[0066] 5) Detection process: The process involves sequentially performing the following steps: sample loading for 300 seconds, baseline measurement for 60 seconds, binding for 180 seconds, and dissociation for 180 seconds. The changes in the interference signal are recorded using Octet R8 data acquisition and analysis software.

[0067] 6) Competition experiment: After binding the CD36-immobilized sensor to 4 μM ApoD (high sugar or low sugar) for 180 seconds, it was exposed to buffer containing 10 nM ox-LDL (supplemented with 72.5 μM OA) or 72.5 μM PA to evaluate the competitive binding.

[0068] 7) Data processing: The KD value is calculated by fitting the curve using a 1:1 model.

[0069] (2-3) Results The KD value of highly glycosylated ApoD with CD36 was 38.34 nM, while the KD value of low-glycosylated ApoD with CD36 was 2.65 nM, further confirming that the binding affinity of low-glycosylated ApoD to CD36 was significantly higher than that of highly glycosylated ApoD. Figure 3 ).

[0070] Example 2: Effect of Glycosylation on ApoD's Blood-Brain Barrier (BBB) ​​Repair Function (1) Laboratory animals and grouping (1-1) Laboratory animals ApoD knockout (ApoD- / -) mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were half male and half female and were housed in a specific pathogen-free environment with a standard 12-hour light-dark cycle, with free access to food and water.

[0071] (1-2) Grouping situation Control group: 6 ApoD- / - mice were injected with saline via the tail vein; Highly glycosylated ApoD treatment group: divided into dose subgroups of 50 μg / kg, 100 μg / kg, 200 μg / kg and 400 μg / kg, with 6 ApoD- / - mice in each group; Low-glycosylated ApoD treatment group: divided into subgroups with doses of 50 μg / kg, 100 μg / kg, 200 μg / kg, and 400 μg / kg, with 5 ApoD- / - mice in each subgroup.

[0072] (2) Construction of MCAO model (middle cerebral artery occlusion model) (2-1) Preoperative preparation Mice were anesthetized by intraperitoneal injection of pentobarbital (75 mg / kg body weight), fixed supine on the operating table, and their necks were prepared and disinfected.

[0073] (2-2) Surgical procedure 1) Make an incision along the midline of the neck and separate the left common carotid artery (CCA), left external carotid artery (ECA), and left pterygopalatine artery under an operating microscope.

[0074] 2) Use a small vascular clamp to clamp the peripheral area at the bifurcation of the internal carotid artery (ICA) and the pterygopalatine artery.

[0075] 3) Insert a silicone-coated filament (Jialing, Shanghai, China, 1800) through the ICA until slight resistance is felt, ensuring that the distance from the tip of the filament to the bifurcation of the ICA-pterygopalatine artery and the bifurcation of the ICA-ECA is approximately 6 mm and 9 mm respectively, to achieve occlusion of the middle cerebral artery.

[0076] 4) After 90 minutes of occlusion, remove the silicone-coated filaments, restore blood flow perfusion, suture the incision layer by layer, and keep the patient warm and recover postoperatively.

[0077] (2-3) Model Validation The model was successfully constructed by detecting ipsilateral cortical cerebral blood flow (CBF) using laser speckle contrast imaging (LSCI) and a CBF reduction of more than 50%.

[0078] (2-4) Setup of sham surgery group The artery was isolated without occlusion, and the remaining steps were the same (for baseline control).

[0079] (3) Drug administration Timing of administration: Administration was given on day 2 and day 4 post-stroke (corresponding to the peak of natural ApoD expression) (see [link to relevant documentation]). Figure 4 ).

[0080] Administration method: The high-glycosylated ApoD treatment group and the low-glycosylated ApoD treatment group were injected with the corresponding dose of protein solution (solvent is physiological saline) via tail vein injection, while the blank group was injected with an equal volume of physiological saline. The volume of administration was adjusted according to the body weight of the mice (0.1 mL per 10 g body weight).

[0081] (4) Detection indicators, methods and results (4-1) Detection of ApoD distribution in the brain After drug administration, the distribution of ApoD protein in the brain parenchyma was observed by immunofluorescence staining (anti-ApoD antibody). The results showed that ApoD protein successfully entered the brain parenchyma. Figure 5 ).

[0082] (4-2) Assessment of microvascular structure On the 5th day after stroke, mouse brain tissue was collected, frozen sections were prepared, and CD31 (endothelial cell marker) and CD13 / PDGFRβ (parietal cell marker) immunofluorescence staining were performed. The vascular area, vascular diameter and parietal cell coverage were quantitatively analyzed using ImageJ software.

[0083] The results showed that low doses of highly glycosylated ApoD (50 μg / kg, 100 μg / kg, 200 μg / kg) failed to improve the poor microvascular phenotype in ApoD- / - mice, while high doses (400 μg / kg) achieved moderate microvascular repair, including a reduction in vessel area and diameter and a slight improvement in parietal cell coverage. Figure 6 In contrast, low-glycosylated ApoD at doses of 200 μg / kg and 400 μg / kg significantly repaired microvascular structure, as evidenced by a marked reduction in vessel area and diameter, and an increase in parietal cell coverage. Figure 7 ).

[0084] (4-3) Measurement of infarct area MAP2 immunofluorescence staining (a neuron-specific marker) was used, and the percentage of infarct volume to total brain tissue volume was calculated using ImageJ software.

[0085] The results showed that hypoglycosylated ApoD significantly reduced the infarct area (see...). Figure 8 ).

[0086] (4-4) Neurological function assessment 1) Neurological deficit score: According to the literature: Validity and Reliability of NeurologicalScores in Mice Exposed to Middle Cerebral Artery Occlusion. Stroke. 2019 Oct;50(10):2875-2882., the score range is 0-5 points (0 points means no deficit, 5 points means death).

[0087] 2) Rotating bar test: to assess motor coordination and balance ability, and to record the latency of mice falling from a rotating beam with gradually increasing speed (5 rpm to 45 rpm).

[0088] 3) Pole Climbing Test (PCT): Record the mouse's turning time (iTurn) and total climbing time (TD) on the stick.

[0089] The results showed that low-glycosylated ApoD significantly improved neurological deficits and promoted stroke recovery in apolipoprotein D knockout mice. Figure 9 to Figure 11 ).

[0090] (4-5) Endothelial cell proliferation detection After stroke, patients were given intraperitoneal injections of EdU (50 mg / kg body weight) for 5 consecutive days. On the 5th day, brain tissue sections were taken for EdU, ERG (endothelial cell nuclear marker), and CD31 immunofluorescence staining, and the number of EdU+ / ERG+ proliferating endothelial cells was counted.

[0091] The results showed that the treated mice exhibited a pathological reduction in endothelial cell proliferation (see...). Figure 12 ).

[0092] (4-6) Blood-brain barrier permeability test Mice were injected with dextran-FITC (70 kDa and 2000 kDa) via the tail vein. Two minutes later, the mice were sacrificed, and brain tissue sections were collected. The extravasation of blood vessels was quantitatively analyzed by fluorescence intensity (permeability index = extravasated FITC fluorescence intensity / total FITC fluorescence intensity × 100).

[0093] The results showed that the treated mice exhibited decreased blood-brain barrier permeability (see...). Figure 13 ).

[0094] (4-7) Microvascular perfusion detection Mice were injected with Lectin-DyLight 594 via the tail vein, and sacrificed 15 minutes later. Brain tissue sections were collected, stained with CD31, and the ratio of perfused vascular area (CD31 / lectin double-positive area) to total vascular area (CD31 positive area) was calculated.

[0095] The results showed that the treated mice exhibited enhanced microvascular perfusion (see...) Figure 14 ).

[0096] These results indicate that hypoglycosylation significantly affects the interaction between apolipoprotein D (ApoD) and CD36, and greatly enhances the functional efficacy of ApoD in blood-brain barrier (BBB) ​​repair and stroke recovery.

[0097] Example 3: An experiment on the improvement of blood-brain barrier integrity and neurological function in aged stroke mice by low-glycosylated ApoD (1) Experimental animals and model construction (1-1) Laboratory animals Sixteen-month-old wild-type (WT) mice (aged group) and two-month-old WT mice (young control group) were purchased from the Animal Center of Daping Hospital (Chongqing, China) and were housed under the same conditions as in Example 2.

[0098] (1-2) Validation of characteristics of aged mouse model 1) Detect the interaction between vascular wall cells and endothelial cells (CD31 and PDGFRβ colocalization) by immunofluorescence staining.

[0099] 2) Detect the intrinsic ApoD level in the brain microvascular system (ELISA and Western blotting).

[0100] 3) Assess microvascular morphology (vessel area, diameter) and blood-brain barrier permeability (dextran-FITC tracing) to verify the characteristics of aggravated microvascular abnormalities and increased blood-brain barrier permeability in aged mice. Figure 15 , Figure 16 , Figure 17 and Figure 18 ).

[0101] (1-3) Construction of MCAO model The surgical procedures and model validation standards are the same as in Example 2.

[0102] (2) Drug administration (2-1) Experimental Grouping Aged WT mice were divided into a treatment group and a control group, with 6 mice in each group; young WT mice served as an additional control.

[0103] (2-2) Dosing regimen The treatment group received a tail vein injection of low-glycosylated ApoD (200 μg / kg / 48h) after MCAO surgery, while the control group received an equal volume of normal saline. Administration continued until the detection time point. Figure 19 ).

[0104] (3) Detection indicators, methods and results Infarct area measurement, neurological function assessment (neurological deficit score, rotarod test, pole climbing test), microvascular structure assessment, endothelial cell proliferation detection, blood-brain barrier permeability detection, and microvascular perfusion detection (same as Example 2).

[0105] Results showed that on day 5 post-stroke: the infarct area in the treatment group was significantly reduced, neurological function prognosis was improved, and survival rate was increased. Figure 20 , Figure 21 and Figure 22 ).

[0106] In addition, the microvascular morphology has been restored (the vessels are thin and uniform, the area is normal, and the coverage of parietal cells has improved). Figure 23 ).

[0107] In addition, pathological endothelial cell proliferation is inhibited ( Figure 24 ); In addition, improved microvascular perfusion reduces blood-brain barrier permeability. Figure 25 , Figure 26 and Figure 27 ); In addition, the tight junction proteins and basement membrane components were detected: the arrangement and expression levels of occludin, claudin-5, ZO-1 (tight junction proteins), laminin, and collagen 4 (basement membrane components) were examined by immunofluorescence staining. The results showed that the tight junction proteins and basement membrane components were more orderly arranged. Figure 28 to Figure 33 ).

[0108] In addition, long-term rehabilitation assessment: The above test indicators were repeated 30 days after stroke to assess the long-term therapeutic effect of low-glycosylated ApoD. Results showed that the treatment group maintained a smaller infarct area, better neurological function, and improved vascular phenotype, highlighting the long-term rehabilitation effect. Figure 34 to Figure 45 ).

[0109] The data above indicate that Hypoglyco-ApoD provides an effective strategy for improving the rehabilitation of elderly stroke patients by inhibiting pathological endothelial proliferation, promoting parietal cell-endothelial interaction, and enhancing BBB integrity.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of hypoglycosylated Apolipoprotein D in the preparation of a medicament for repairing blood brain barrier.

2. Use according to claim 1, characterized in that, The hypoglycosylated Apolipoprotein D is obtained by treating the hyperglycosylated Apolipoprotein D with PNGase F.

3. Use according to claim 2, characterized in that, The method for preparing the hyperglycosylated Apolipoprotein D comprises: overexpressing the Apolipoprotein D gene in an engineered cell, and obtaining the hyperglycosylated Apolipoprotein D from the cell culture supernatant, and then purifying.

4. Use according to claim 3, characterized in that, The engineered cell is a 293T cell.

5. Use according to any one of claims 1 to 4, characterized in that, The medicament has the efficacy of treating ischemic stroke.

6. Use according to claim 5, characterized in that, The subject of ischemic stroke is an elderly patient.

7. Use according to claim 5, characterized in that, The medicament has one or more of the following effects: has the effect of repairing the structure of microvessels; has the effect of reducing the infarction area; has the effect of improving neurological impairment; has the effect of promoting the reduction of pathological proliferation of endothelial cells; has the effect of reducing the permeability of the blood brain barrier; has the effect of enhancing microvascular perfusion.

8. Use according to claim 1, 2, 3, 4, 6 or 7, characterized in that, The dosage form of the medicament is an injection liquid or powder.

9. Use according to claim 5, characterized in that, The dosage form of the medicament is an injection liquid or powder.

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