Use of scemc10 in treating and diagnosing cerebral vascular endothelial cell senescence

Abstract

The application provides an application of scEMC10 in treatment and diagnosis of brain vascular endothelial cell aging, and relates to the technical field of biomedicine. The inventor finds that scEMC10 deficiency leads to brain microvascular endothelial cell aging; brain microvascular endothelial cells treated by scEMC10 can down-regulate beta-galactosidase, SASP and classic aging markers related to aging; after old mice and 5XFAD model mice of Alzheimer's disease are supplemented with scEmc10, the movement coordination ability and spatial cognitive ability of the mice can be obviously improved, and the relative content of the Parkinson's disease marker, phosphorylated alpha-synuclein, of the old mice can be obviously reduced, and the content of the tyrosine hydroxylase phosphorylation activity form can be increased. Therefore, scEMC10 can be used for treatment of brain microvascular endothelial cell aging, improvement of Alzheimer's disease and Parkinson's disease, and detection of brain microvascular endothelial cell aging as a marker.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of scEMC10 in the treatment and diagnosis of cerebral vascular endothelial cell senescence. Background Technology

[0002] Two rapamycin derivatives used to treat age-related diseases have been deemed ineffective in phase III clinical trials. Senolytics also present potential problems: firstly, their ability to target and eliminate senescent cells remains imprecise, with most senolytics still damaging normal cells; secondly, premature use of senolytics can lead to stem cell depletion, while delayed use can affect efficacy; and thirdly, the byproducts left after a large number of senescent cells are killed are often not promptly eliminated, posing a potential health risk. Existing clinical data on metformin's life-extending effects do not prove that this drug can extend the lifespan of non-diabetic patients. Acarbose and 17α-estradiol show gender differences in their anti-aging effects. Most anti-aging substances are still in the experimental stage, and their effects in vivo remain to be proven.

[0003] Therefore, it is necessary to find a new drug that can effectively delay aging.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The primary objective of this invention is to provide the application of scEMC10 in the preparation of drugs for anti-cerebral vascular endothelial cell aging, in order to solve the aforementioned technical problems.

[0006] A second objective of this invention is to provide a drug that combats vascular endothelial cell aging.

[0007] A third objective of this invention is to provide the application of reagents for detecting scEMC10 in the preparation of products for diagnosing cerebral vascular endothelial cell senescence.

[0008] The fourth objective of this invention is to provide a kit for diagnosing senescence of cerebral vascular endothelial cells.

[0009] The fifth objective of this invention is to provide the use of scEMC10 in the preparation of medicaments for treating Alzheimer's disease.

[0010] The sixth objective of this invention is to provide the use of scEMC10 in the preparation of medicaments for treating Parkinson's disease.

[0011] To achieve the above objectives, the following technical solution is adopted:

[0012] In a first aspect, the present invention provides the application of scEMC10 in the preparation of drugs for anti-aging of cerebral vascular endothelial cells.

[0013] In a second aspect, the present invention provides a drug for combating cerebral vascular endothelial cell aging, the drug comprising scEMC10.

[0014] As a further technical solution, the drug also includes pharmaceutical excipients;

[0015] The dosage form of the drug includes injections.

[0016] Thirdly, the present invention provides the application of reagents for detecting scEMC10 in the preparation of products for diagnosing cerebral vascular endothelial cell senescence.

[0017] As a further technical solution, the product includes a reagent kit.

[0018] As a further technical solution, if the expression level of scEMC10 in brain vascular endothelial cells is low, then the brain vascular endothelial cells are senescent.

[0019] Fourthly, the present invention provides a kit for diagnosing senescence of cerebral vascular endothelial cells, comprising an anti-scEMC10 antibody.

[0020] As a further technical solution, the kit can diagnose the aging of cerebral vascular endothelial cells by detecting the expression of scEMC10.

[0021] Fifthly, the present invention provides the use of scEMC10 in the preparation of medicaments for treating Alzheimer's disease.

[0022] In a sixth aspect, the present invention provides the use of scEMC10 in the preparation of medicaments for treating Parkinson's disease.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] Research by the inventors has revealed that scEMC10 deficiency leads to senescence of brain microvascular endothelial cells. Treatment of brain microvascular endothelial cells with scEMC10 downregulates senescence-related β-galactosidase, SASP, and classic senescence markers (such as p16, p21, and phosphorylated p53). Replenishment of scEmc10 in aged mice and Alzheimer's disease 5XFAD model mice significantly improves motor coordination and spatial cognition, and significantly reduces the relative content of phosphorylated α-synuclein (p-α-synuclein S129), a marker of Parkinson's disease, while increasing the content of phosphorylated tyrosine hydroxylase (TH). Therefore, scEMC10 can be used to treat senescence of brain microvascular endothelial cells, improve Alzheimer's and Parkinson's diseases, and serve as a marker for detecting senescence of brain microvascular endothelial cells. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1A. After treatment with scEMC10 (100ug / ml) for 48 h, mRNA sequencing results showed that the senescence-associated secretory phenotype (SASP) of the human brain microvascular endothelial cell line HCMEC was significantly downregulated (n=3); B and C. After treatment with scEMC10 (100ug / ml) for 48 h, mouse brain microvascular endothelial cell line bEnd.3 and human brain microvascular endothelial cell line HCMEC showed a decrease in senescence-associated β-galactosidase staining, scale bar = 10μm (bEnd.3 cells) / 20μm (HCMEC cells). Statistical analysis was performed using an unpaired t-test, *: p<0.05, **: p<0.01; D. After perfusion of the heart with FITC-glucan (10kD), senescence-associated β-galactosidase staining results showed... E. After perfusion of the heart with FITC-glucan (10 kDa), the aging signal in the cerebral cortex of Emc10-deficient mice was significantly higher than that in wild-type mice (represented by FITC-glucan) (red arrow), scale bar = 200 μm. Statistical analysis was performed using an unpaired t-test, **: p < 0.01; E. After perfusion of the heart with FITC-glucan (10 kDa), the aging-related β-galactosidase staining results showed that the aging signal in the cerebral cortex of Emc10 microglia knockout mice was significantly higher than that in wild-type mice (represented by FITC-glucan) (black arrow), scale bar = 200 μm. Statistical analysis was performed using an unpaired t-test, **: p < 0.01;

[0027] Figure 2 A. After treatment of mouse brain microvascular endothelial cell line bEnd.3 with scEMC10 (100ug / ml) for 48h, the expression of aging-related markers p21, p-p53 / p53, and γ-H2A.X was significantly downregulated. Statistical analysis was performed using an unpaired t-test. *: p<0.05, **: p<0.01, ***: p<0.001. B. After treatment of human brain microvascular endothelial cell line HCMEC with scEMC10 (100ug / ml) for 48h, the expression of aging-related markers p21, p-p53 / p53, and p16 was significantly downregulated. Statistical analysis was performed using an unpaired t-test. *: p<0.05, **: p<0.01, ***: p<0.001.

[0028] Figure 3A. Representative Western blotting images of cortical MBP protein expression in A.WT and KO mice. The right image represents the results of ImageJ quantitative analysis. Values ​​are plotted as mean ± SEM. Statistical analysis was performed using an unpaired t-test. **p<0.01. n=6 mice; B. Representative immunofluorescence images of MBP staining in B.WT and KO mice. Red: MBP; Blue: DAPI. Black and white images are the results of ImageJ analysis. Scale bar = 200 μm. A' and A”: central and central brain regions; B' and B”: cerebral cortex and cCB region; cing: cingulate gyrus; cCB: corpus callosum; alv: hippocampal nerve fibers; scwm: supra-corpus callosum white matter. The right image represents the results of ImageJ quantitative analysis. Values ​​are plotted as mean ± SEM and normalized to the WT group. Student's t-test showed p<0.05. n = 4 mice; Representative images of LFB staining in C.WT and KO mice, blue: LFB; purple: H&E staining, scale bar = 2500 μm. A' and A”: central and central brain regions; B' and B”: cerebral cortex and ccb region; cing: cingulate gyrus; cdb: corpus callosum; alv: hippocampal nerve fibers; scwm: supra-corpus callosum white matter. The right image is the result of ImageJ quantitative analysis. Values ​​are plotted as mean ± SEM and normalized with reference to the WT group. Student's t test showed p < 0.05, n = 4 mice;

[0029] Figure 4A. Representative images of sa-β-gal in cortical microvessels of WT, KO, and KO supplemented scEMC10 mice, blue: sa-β-gal (senescent cells), scale bar = 250 μm; B. Representative Western blotting images of Aβ1-42 protein expression in WT, KO, and KO supplemented scEMC10 mice; C. Statistical results of the number of senescent cells on 100 μm microvessels, plotted as mean ± SEM, and normalized to the control group, **p < 0.01; ***Student's t-test p < 0.001, n = 3 mice; D. Quantitative analysis of the (B) western blotting results using ImageJ software, plotted as mean ± SEM, according to Student's t-test. t-test, ns: no statistical significance, *p<0.05, **p<0.01, n=4 mice; Representative immunofluorescence images of MBP staining in E.WT, KO, and KO supplemented scEMC10 mice, red: MBP; blue: DAPI; Black and white images were analyzed using ImageJ, scale bar = 1000μm, A', A”, A”': cingulate gyrus of the corpus callosum; B', B”, and B”': cerebral cortex and ccb area; F.WT, Representative images of LFB staining in scEMC10 mice, supplemented by KO and KO. Blue: local feedback; purple: he staining. Scale bar = 2500 μm. A', A”, A”': cingulate gyrus of the corpus callosum; B', B”, and B”': cerebral cortex and ccb area. G. Quantitative analysis of (E) myelin density and (F) LFB density using ImageJ. Values ​​are plotted as mean ± SEM and normalized to the WT+PBS group. Student's t-test showed no statistical significance (ns: *p<0.05). N = 4 mice. H. Representative western blotting images of MBP protein expression in scEMC10 mice, supplemented by WT, KO, and KO. The right image shows the quantitative analysis of the results using ImageJ. Values ​​are plotted as mean ± SEM. Student's t-test showed no statistical significance (ns: *p<0.05, **p<0.01). n = 6 mice.

[0030] Figure 5A. The left figure shows the movement trajectory of 90-week-old mice after supplementation with scEMC10 in the open field experiment. The right figure shows the movement speed of the mice and the percentage of the total movement time in the central region. The values ​​are plotted as mean ± SEM and normalized with the PBS group. According to Student's t test, ns: no statistical significance, *p<0.05. A. Quantitative analysis of spontaneous alternation behavior in the Y-maze test of 90-week-old mice after scEMC10 supplementation. Values ​​are plotted as mean ± SEM and normalized with the PBS group. *p<0.05. Student's t-test was performed. n=6 mice. B. Quantitative analysis of spontaneous alternation behavior in the Y-maze test of 90-week-old mice after scEMC10 supplementation. Values ​​are plotted as mean ± SEM and normalized with the PBS group. *p<0.05. Student's t-test was performed. n=6 mice. C. Quantitative analysis of grip strength in 90-week-old mice after scEMC10 supplementation. Values ​​are plotted as mean ± SEM and normalized with the PBS group. *p<0.05. Student's t-test was performed. n=6 mice. D. Representative images of sa-β-gal in cortical microvessels in 90-week-old mice after scEMC10 supplementation. Blue: sa-β-gal (senescent cells). Scale bar = 50 μm. E. Quantitative analysis of the number of sa-β-gal positive points per 100 μm microvessels and the density of sa-β-gal in the cerebral cortex. Values ​​are plotted as mean ± SEM and normalized with the PBS group. Student's t-test was performed. F. Western blotting images of mouse cortical aging markers. Protein expression levels were quantitatively analyzed using ImageJ. Values ​​were plotted as mean ± SEM and normalized to the PBS group. ns: Student's t test, *p<0.05, n=6 mice; G. Representative immunofluorescence images of MBP staining in 90-week-old mice after scEMC10 supplementation. Red: MBP; Blue: DAPI. Images were analyzed using ImageJ. Scale bar = 1000 μm. A', A”': Cingulate gyrus of the corpus callosum; B', B”: Cerebral cortex and ccb area; H. Quantitative analysis of myelin density (G) using ImageJ. Values ​​were plotted as mean ± SEM and normalized to the PBS group. Student's t test, ns: no statistical significance, *p<0.05.n = 6 mice; I. Representative images of LFB staining in 90-week-old mice after supplementation with scEmc10, blue: local feedback; purple: he staining, scale bar = 2500 μm, A', A”: cingulate gyrus of corpus callosum; B', B”: cerebral cortex and ccb area; J. Quantitative analysis of LFB density in (I) using ImageJ, values ​​are plotted as mean ± SEM and normalized with the PBS group, and analyzed by Student's t test, ns: no statistical significance, *p<0.05, n = 6 mice; K. Representative images of Aβ1-42 immunofluorescence staining in mice, A', B', A', B' are magnified images of specific locations, red: Aβ1-42; blue: DAPI, scale bar = 100 μm; Lp-Tau Representative images of T205 immunohistochemistry and H&E staining, A', B', A', B' are magnified images of specific locations, scale bar = 2500 μm; M. Quantification of Aβ1-42 density in (K) indicator region and p-Tau density in (L) indicator region, values ​​are plotted as mean ± SEM and normalized to PBS group, according to Student's t test, *p<0.05, **p<0.01, n = 5 mice;

[0031] Figure 6A. Quantitative analysis of grip strength in 24-week-old 5xFAD mice supplemented with scEmc10. Values ​​are plotted as mean ± SEM and normalized with the PBS group. *p<0.05. Student's t-test was performed. n=6 mice. B. The left figure shows the movement trajectory of 24-week-old 5xFAD mice after scEmc10 supplementation in the water maze test. The right figure shows the movement distance, time to first arrival at the platform, movement time in the platform area, and percentage of total movement time in the target quadrant. Values ​​are plotted as mean ± SEM and normalized with the PBS group. Student's t-test was performed. ns: no statistical significance, *p<0.05. n=6 mice. C. The left figure shows the movement trajectory of 24-week-old 5xFAD mice after scEmc10 supplementation in the open field test. The right figure shows the movement speed and percentage of total movement time in the central area. Values ​​are plotted as mean ± SEM and normalized with the PBS group. Student's t-test was performed. t-test, ns: no statistical significance, *p<0.05, n=6 mice; D. The upper image is a representative image of sa-β-gal in the cortical microvessels of 24-week-old 5xFAD mice after supplementation with scEmc10. Blue: sa-β-gal (senescent cells), scale bar = 50μm. The lower image shows the quantitative data of the number of sa-β-gal positive points per 100μm microvessels and the density of sa-β-gal in the cerebral cortex. The values ​​are plotted as mean ± SEM and normalized with the PBS group. Student's t-test, p<0.05, n=6 mice; E. Western blotting images of mouse cortical aging markers. The right and lower images are the results of quantitative analysis of protein expression levels using ImageJ. The values ​​are plotted as mean ± SEM and normalized with the PBS group. ns: no statistical significance, *p<0.05, n=6 mice; Student's t-test, p<0.05, n=6 mice; t-test, *p<0.05, n=6 mice; Representative western blotting images of Aβ1-42 protein expression in F. 24-week-old 5xFAD mice after scEMC10 supplementation. The right image is the ImageJ quantitative analysis of the results. The values ​​are plotted as mean ± SEM. Student's t-test, *p<0.05, n=6 mice;

[0032] Figure 7A. Representative immunofluorescence images of MBP staining in 24-week-old 5xFAD mice supplemented with scEMC10. Red: MBP; Blue: DAPI. Images were analyzed using ImageJ. Scale bar = 1000 μm. A', A”': Cingulate gyrus of the corpus callosum; B', B”': Cerebral cortex and ccb region. The following figure shows the quantitative analysis of myelin density using ImageJ. Values ​​are plotted as mean ± SEM and normalized with the PBS group. (Student's...) t-test, ns: no statistical significance, *p<0.05, n=6 mice; B. Representative images of LFB staining in 24-week-old 5xFAD mice after scEMC10 supplementation, blue: local feedback; purple: he staining, scale bar = 2500μm, A', A”: cingulate gyrus of the corpus callosum; B', B”: cerebral cortex and ccb region. The figure below shows the quantitative analysis of LFB density using ImageJ. The values ​​are plotted as mean ± SEM and normalized with the PBS group. t-test, ns: no statistical significance, *p<0.05, n=6 mice; C. Representative images of mouse Aβ1-42 immunofluorescence staining, A', B', A', B' are magnified images of specific locations, red: Aβ1-42; blue: DAPI, scale bar=100μm. The following figure shows the quantification of Aβ1-42 density in the indicator region using ImageJ. The values ​​are plotted as mean ± SEM and normalized to the PBS group. Student's t-test, *p<0.05, n=6 mice; Representative images of Dp-Tau T205 immunohistochemistry and H&E staining, A', B', A', B' are magnified images of specific locations, scale bar=2500μm. The following figure shows the quantification of p-Tau density in the indicator region using ImageJ. The values ​​are plotted as mean ± SEM and normalized to the PBS group. Student's t-test, *p<0.05, n=6 mice;

[0033] Figure 8 A. Western blot images showing the levels of phosphorylated α-synuclein (Ser129; p-α-Syn), total α-synuclein (α-Syn), phosphorylated tyrosine hydroxylase (Ser40; p-TH), and total tyrosine hydroxylase (TH) in the cortex of 90-week-old mice; B. Quantitative analysis of (A) results, numerical values ​​are plotted as mean ± SEM and normalized to the PBS group. Student's t-test showed *p<0.05, n=5 mice. Detailed Implementation

[0034] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0035] In a first aspect, the present invention provides the application of scEMC10 in the preparation of drugs for anti-aging of cerebral vascular endothelial cells.

[0036] The inventors discovered that treatment with scEMC10 can downregulate aging-related β-galactosidase, SASP, and classic aging markers (such as p16, p21, and phosphorylated p53) in brain microvascular endothelial cells. Replenishment of scEMC10 in middle-aged and aged mice significantly improved their motor coordination and spatial cognition. Therefore, scEMC10 can be used to treat aging of brain microvascular endothelial cells.

[0037] In a second aspect, the present invention provides a drug for combating cerebral vascular endothelial cell aging, the drug comprising scEMC10.

[0038] This drug can alleviate the aging of cerebral vascular endothelial cells.

[0039] In some alternative embodiments, the drug may further include pharmaceutical excipients.

[0040] This invention does not impose specific restrictions on pharmaceutical excipients; they can be selected as needed.

[0041] In some alternative embodiments, the dosage form of the drug includes, but is not limited to, injections, or other dosage forms well known to those skilled in the art.

[0042] Thirdly, the present invention provides the application of reagents for detecting scEMC10 in the preparation of products for diagnosing cerebral vascular endothelial cell senescence.

[0043] The inventors discovered that a deficiency in scEMC10 leads to senescence of brain microvascular endothelial cells. Therefore, scEMC10 can be used as a biomarker for the detection of senescence in brain microvascular endothelial cells.

[0044] In some alternative implementations, the product includes a reagent kit.

[0045] Fourthly, the present invention provides a kit for diagnosing senescence of cerebral vascular endothelial cells, comprising an anti-scEMC10 antibody.

[0046] The kit of the present invention diagnoses the aging of brain vascular endothelial cells by detecting the expression of scEMC10.

[0047] Fifthly, the present invention provides the use of scEMC10 in the preparation of medicaments for treating Alzheimer's disease.

[0048] In a sixth aspect, the present invention provides the use of scEMC10 in the preparation of medicaments for treating Parkinson's disease.

[0049] Research by the inventors has shown that rehydration with scEmc10 in Alzheimer's disease 5XFAD model mice significantly improves motor coordination and spatial cognition. Furthermore, it significantly reduces the relative content of phosphorylated α-synuclein (p-α-synuclein S129), a marker of Parkinson's disease, while increasing the content of phosphorylated tyrosine hydroxylase (TH). Therefore, scEmc10 has a beneficial effect on improving the characteristics of Alzheimer's and Parkinson's diseases.

[0050] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0051] Example 1

[0052] 1. Experimental Methods

[0053] 1.1 Cell Transcriptome Sequencing

[0054] RNA was extracted from the samples using Trizol, followed by RNA extraction according to the instructions of the EZ-press RNA Purification Kit (Ezbioscience, B0004D). RNA integrity and concentration were assessed using a bioanalyzer (NanoDrop ONE, ThermoFisher). Sequencing was performed by Suzhou Genewiz Biotechnology Co., Ltd. Sequencing data quality was assessed using FastQC software (v0.10.1). Library construction was performed using the ScriptSeq Complete Gold Kit (Epicentre, Illumina), with samples multi-coded and sequenced at 50 bp paired ends on an Illumina HiSeq2500. Eight samples were used per lane, and each sample was sequenced twice at a depth of 50 million reads.

[0055] 1.2 Staining of senescent β-galactosidase

[0056] For cultured cells, remove the culture medium from the culture medium and wash the cells once with PBS. For FITC-D perfused tissues, wash three times with PBS (0.01M) for 5 minutes each at room temperature. Add 1 ml of sa-β-galactosidase staining fixative to the cells or sections and incubate at room temperature for 15 minutes. Remove the fixative and wash the samples three times with PBS for 3 minutes each. Add 1 ml of staining working solution to each sample. Incubate at 37°C for 4 hours and detect sa-β-galactosidase under a light microscope.

[0057] 1.3 Mouse water maze test

[0058] (1) Experimental setup: A circular white pool with a diameter of 1m and a height of 0.5m was used. In the water maze software, two long line segments were used to divide the circular pool into four quadrants in a counterclockwise direction. Different colored and shaped stickers were used to mark the pool walls in each quadrant. A circular platform with a height of 30cm and a diameter of 6cm was placed in the center of the third quadrant. Water was poured into the pool until it was 2cm above the platform. Before the experiment, titanium dioxide powder was added to the water until the water turned white and the platform was covered.

[0059] (2) Start of the experiment: The water temperature was maintained at 24±3℃ during the experiment, and the environment was quiet and undisturbed. Mouse training for 4 days: Mice were placed at the edge of the pool corresponding to the first, second, third and fourth quadrants respectively. The time and trajectory of the mice finding the platform and staying there for 20 seconds were recorded. If the mice did not find the platform within 60 seconds, the experimenters placed the mice on the platform for 20 seconds for training.

[0060] (3) Formal experiment: Remove the platform and place the mouse at the edge of the pool corresponding to the first quadrant, and record the mouse’s movement trajectory within 60 seconds.

[0061] 1.4 Spontaneous Alternation Experiment in Mouse Y-Maze

[0062] Spatial working memory was evaluated using a Y-maze spontaneous alternation task. Mice were placed at the end of one arm of a Y-shaped maze (the three arms were at a 120° angle) and allowed to explore freely for 8 minutes. Alternation was defined as consecutive entry into all three arms without repetition. The percentage of spontaneous alternations was calculated as the ratio of actual alternations to possible alternations: (number of alternations) / (total arm entry - 2) × 100%. The total number of arm entryes was recorded to control motor activity. Video tracking and analysis of behavior were performed using SuperMaze V2.0 software (Shanghai Xinruan, China).

[0063] 1.5 Mouse gripping force test

[0064] Forelimb strength was measured using a grip strength meter (BIOSEB, EB Instruments). Mice were allowed to grasp a metal grid with their forepaws while their tails were gently pulled horizontally until they released their grip. Each mouse underwent three trials, and force and grip time were recorded using SuperGSM software (Shanghai Xinruan, China).

[0065] 1.6 Open Field Experiment

[0066] Before the experiment, the animals were allowed to acclimatize to the testing environment for 30 minutes. The animals were placed in the center of the testing chamber in the same location and orientation. Testing began immediately after placement and lasted for 5 minutes. Parameters were set in the VisuTrack software, and the animal's number, date, and status information were recorded.

[0067] 1.7 Protein extraction, SDS-PAGE and Western blotting

[0068] Mouse cerebral cortex tissue was sonicated in RIPA buffer (pH 7.4, with added protease and serine / threonine and tyrosine phosphatase inhibitors). The homogeneous mixture was centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was retained for protein analysis. Sample quantification was performed using the BCA protein assay. Samples (25 μg / lane) were mixed with reducing sample buffer containing SDS and DTT and heated to 95°C for 5 minutes. Proteins of different molecular weights were separated by SDS-page on an appropriately concentrated polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked in 5% skim milk for 1 hour and then diluted in blocking buffer at 4°C with primary antibodies (Emc10, Abcam, 1:1000; β-Actin, CST, 1:5000). After thorough washing, the membrane was incubated with peroxidase-conjugated secondary antibody at room temperature for 2 hours, thoroughly washed, and rinsed with a chemiluminescent substrate (Thermo Scientific). Density assays were performed using ImageJ (National Institutes of Health) to quantify the proteins expressed in each channel.

[0069] 2. Experimental Results

[0070] Endothelial cells are more prone to aging due to their structural characteristics and endothelial cell turnover rate. Tissues with high endothelial cell content exhibit the highest concentrations of senescent cells, and endothelial cells are among the earliest cell types to age. Many arterial beds in vivo demonstrate endothelial cell aging, including the brain. Mechanistically, senescent endothelial cells increase adhesion to the basement membrane, hindering proper alignment of blood flow. This suggests that endothelial cell aging plays a role in various disease processes associated with vascular dysfunction.

[0071] Senescent cells are characterized by a senescence-associated secretory phenotype (SASP), which is heterogeneous and determined by cell type and senescence mechanism.

[0072] Cultured human brain microvascular endothelial cell line HCMEC was treated with scEMC10 (100 μg / ml) for 48 h, followed by mRNA sequencing. An untreated cell line was used as a control group. RNA sequencing results showed that scEMC10 stimulation of HCMEC significantly downregulated SASP-related genes. Figure 1 In Figure A, con_1, con_2, and con_3 are the control groups, and scEMC10_1, scEMC10_2, and scEMC10_3 are the experimental groups.

[0073] For Emc10 - / - Single-cell transcriptome sequencing analysis of the cerebral cortex of mice (Emc10 gene knockout mice) revealed that scEMC10 in the brain is secreted by microglia (macrophages in the brain). Therefore, Emc10 gene knockout mice (Emc10 gene knockout mice) were constructed. - / - KO mice and two microglia Emc10 gene selective knockout mice (Flox mice) + / + Tmem119 Cre+ Mice and Flox + / + Cx3cr1 Cre+ Mice (cKO mice). β-galactosidase (β-gal) is a glycosidase that accumulates primarily in the lysosomes of irreversibly arrested cells. This marker is detectable by histochemical staining in most cell cycle-arrested cells, but is generally absent in quiescent or immortal cells. Cultured mouse brain microvascular endothelial cell line bEnd.3 and human brain microvascular endothelial cell line HCMEC were treated with scEMC10 (100 μg / ml) for 48 h, with an untreated cell line serving as a control. Staining results showed that, compared to the control group, scEMC10 treatment reduced the senescence phenotype (…). Figure 1 (B and C in the text). Furthermore, after perfusion of the heart with FITC-glucan (10kD), aging-related β-galactosidase staining results showed that the aging signal in the cerebral cortex vessels of Emc10-deficient mice was significantly higher than that of wild-type mice, and the aging signal in the cerebral cortex vessels of Emc10 microglia knockout mice was significantly higher than that of wild-type mice. Figure 1 (C and D in the text).

[0074] Tumor suppressor factors in mouse brain microvascular endothelial cell line bEnd.3 and human brain microvascular endothelial cell line HCMEC were detected after treatment with scEMC10 (100 μg / ml) for 48 h. The results showed that two key tumor suppressor factors, cyclin-dependent kinase inhibitor 1A (CDKN2A / p21) and cyclin-dependent kinase inhibitor 2A (CDKN2A / p16), were upregulated in responses to various senescence-inducing stressors. Furthermore, the phosphorylated form of p53 upstream of p21 is also commonly used as a marker of aging. Another sensitive molecular marker, γH2AX, is associated with DNA damage and repair. Our experimental results indicate that treatment of HCMEC / bEnd.3 cells with recombinant scEmc10 downregulated the protein levels of senescence markers p16, p21, p-p53 / p53, and γ-h2AX. Figure 2 (A and B in the original text). In summary, our study demonstrates that scEMC10 has potential anti-aging effects on brain vascular endothelial cells in vitro. The study also includes in vivo observations of signs of vascular endothelial cell senescence when microglia-derived scEMC10 is absent.

[0075] To investigate brain damage caused by Emc10 deficiency, we systematically examined the coronal plane covering the temporal cortex of WT and KO mice using histological methods, observing changes in the expression of axonal and myelin protein markers. Western blotting analysis showed that Emc10 deficiency led to the loss of MBP protein in the brains of KO mice. Figure 3 (A) Myelin basic protein (MBP) is a marker of myelin fibers. Immunofluorescence also shows that scEMC10 deficiency in the cingulate tract and supracallosal white matter (scwm) region induces demyelination. LFB staining results also confirm white matter damage in the cing and scwm regions of KO mice. Figure 3 (B and C in the middle).

[0076] We injected scEMC10 into KO mice aged 12-18 weeks via tail vein for 14 days. Sa-β-gal staining of mouse brain sections showed that scEMC10 supplementation restored normal cerebral cortical blood vessels. Figure 4 The protein level of amyloid-beta (Aβ1-42), a marker of the neurodegenerative disease Alzheimer's, was significantly increased in the cerebral cortex of KO mice, and returned to normal levels after scEMC10 supplementation. Figure 4 (B in the text). Furthermore, the demyelination of white matter in KO mice was alleviated after scEMC10 rehydration ( Figure 4 The CE in the brain of KO mice). Western blotting analysis also confirmed that scEMC10 supplementation could alleviate the loss of MBP protein in the brain of KO mice. Figure 4 (F in the text)

[0077] To verify the intervention effect of scEMC10 protein on brain injury in 90-week-old mice, we injected recombinant scEMC10 protein into mice via tail vein for 14 days, followed by a series of experiments. Given the close relationship between scEMC10 and cognitive decline, we used three cognitive assessment methods to evaluate behavioral changes in 90-week-old mice after scEMC10 supplementation, including the open field test, the Y-maze spontaneous alternation test, and the grasp test. Behavioral analysis in the open field test confirmed that scEMC10 supplementation helped reduce anxiety in mice. However, improvements in motor skills were not significant. Figure 5 (A) In the Y-maze spontaneous alternation experiment, scEMC10 supplementation significantly enhanced the memory ability of mice to recognize new environments. Figure 5 (B in the text). Furthermore, grip strength tests showed that scEMC10 supplementation significantly improved muscle strength in mice. Figure 5 (C in the text). Supplementation with scEMC10 in aged mice also reduced the degree of aging of cortical blood vessels. Figure 5 The levels of D and E in the brain cortex were reduced, as were the aging markers p21 and p53 and their phosphorylation forms. Figure 5 (F in the text). To verify the recovery of white matter damage in 90-week-old mice after scEMC10 supplementation, myelin was stained using LFB and MBP immunofluorescence staining to determine MBP protein levels. The results showed that scEMC10 supplementation could promote the recovery of white matter damage (F in the text). Figure 5 The immunofluorescence staining of amyloid-beta (Aβ1-42), a biomarker of Alzheimer's disease, and the immunohistochemical and H&E staining results of p-Tau showed that supplementation with scEMC10 in 90-week-old mice effectively reduced the accumulation of Aβ1-42 and p-Tau in the cortex and hippocampus. Figure 5 (KM in the middle).

[0078] 5xFAD transgenic mice are one of the most commonly used commercial Alzheimer's disease (AD) mouse models, derived from mutations in the genes encoding APP and presenilin (PSEN1 and PSEN2), leading to increased production of β-amyloid peptides, particularly the highly aggregated β42 subtype. Cognitive functions, including learning and memory abilities such as visual recognition, association, spatial work, reference learning, and memory, show signs of decline in mice aged 4–6 months. We investigated whether administering recombinant scEMC10 protein via tail vein injection to 24-week-old 5xFAD mice could alleviate Alzheimer's disease symptoms. Grasp strength tests and water maze experiments showed that scEMC10 protein supplementation not only improved muscle strength in mice (… Figure 6 A in the formula also enhanced the spatial cognitive ability of mice. Figure 6(B in the text). Furthermore, open field assays showed that supplementation with scEMC10 recombinant protein significantly enhanced exploratory behavior and curiosity about new environments in 5xFAD mice, while also reducing anxiety-like behaviors (B in the text). Figure 6 (C in the text). Supplementation with scEMC10 in 5xFAD transgenic mice also reduced the aging of vascular endothelium. Figure 6 (D in the middle), and the aging markers of the cerebral cortex, p21 and p53, and their phosphorylation forms were all reduced ( Figure 6 In addition, the protein level of amyloid-beta (Aβ1-42), a marker of Alzheimer's disease, decreased after scEMC10 supplementation. Figure 6 (F in the text). In 5xFAD mice, white matter density in the cerebral cortex increased in areas such as the cingulate gyrus and corpus callosum after the addition of scEMC10. Figure 7 (A and B in the text). Immunofluorescence staining of amyloid-beta (Aβ1-42), a biomarker of Alzheimer's disease, and immunohistochemical and H&E staining results of p-Tau showed that supplementation with scEMC10 in 5xFAD mice effectively reduced the accumulation of Aβ1-42 and p-Tau in the cortex and hippocampus. Figure 7 (C and D in the text).

[0079] Alpha-synuclein is a protein composed of 140 amino acids and is highly expressed in the brain. It is also a major component of pathogenic Lewy bodies and Lewy neurites. Studies have shown that phosphorylation at α-Synuclein S129 (p-α-Syn) is closely related to Parkinson's disease. Tyrosine hydroxylase (TH) is an enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to dihydroxyphenylalanine (DOPA). In Parkinson's disease (PD), characterized primarily by dysfunction of the substantia nigra-striatal dopamine system, the expression or active phosphorylation of TH undergoes abnormal changes. We examined the relative levels of p-α-Syn and p-TH in the cerebral cortex of 90-week-old wild-type mice after reintroduction with scEMC10. The results showed that the level of p-α-Syn significantly decreased after reintroduction, while the level of p-TH significantly increased. Figure 8 (A and B in the text).

[0080] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of scEMC10 in the preparation of drugs for anti-cerebral vascular endothelial cell senescence, characterized in that, The drug includes scEMC10.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutical excipients; The dosage form of the drug includes injections.

3. Application of reagents used to detect scEMC10 in the preparation of products for diagnosing cerebral vascular endothelial cell senescence; If the expression level of scEMC10 in brain vascular endothelial cells is low, it indicates senescence of brain vascular endothelial cells.

4. The application according to claim 3, characterized in that, The product includes a reagent kit.

5. The application according to claim 4, characterized in that, The kit includes an anti-scEMC10 antibody.

6. The application of scEMC10 in the preparation of drugs for treating Alzheimer's disease, characterized in that, The drug includes scEMC10.

7. The application of scEMC10 in the preparation of drugs for treating Parkinson's disease, characterized in that, The drug includes scEMC10.

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