Use of the loop CH type finger protein 8 as a biomarker in assessing or aiding in the assessment of vascular endothelial cell senescence

Abstract

The application discloses application of a CH type finger protein 8 as a biomarker in evaluation or auxiliary evaluation of vascular endothelial cell aging, and a MARCHF8 expression level in vascular endothelial cells and / or on a cell membrane is negatively correlated with a degree of aging of the vascular endothelial cells. The MARCHF8 expression level in the vascular endothelial cells and / or on the cell membrane is detected to evaluate or assist in evaluating the vascular endothelial cell aging. The application discloses that the MARCHF8 is used as a vascular endothelial cell surface marker for early diagnosis of vascular aging, monitoring and targeted treatment of related diseases by determining the role of the MARCHF8 in the vascular aging process. With gradual aggravation of the vascular aging and atherosclerosis, the MARCHF8 as a new biomarker and target point will provide important support for early prevention, clinical diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of cyclic CH finger protein 8 (MARCHF8) as a biomarker in assessing or assisting in the assessment of vascular endothelial cell senescence. Background Technology

[0002] The vascular system is an indispensable part of human organ function. Vascular aging constitutes the basic physiological and pathological basis of age-related changes in multiple organ systems, and is also a common pathogenic mechanism for various chronic diseases in the elderly. Vascular aging is particularly associated with endothelial dysfunction, which promotes the development of cardiovascular and metabolic diseases, including atherosclerosis. Endothelial aging leads to a series of functional impairments, such as vasodilation, angiogenesis, and barrier function disorders, which are considered the core mechanisms of vascular aging and cardiovascular and metabolic disorders. Therefore, early assessment and diagnosis of vascular aging are important measures for evaluating and preventing vascular aging and related diseases, and have significant medical and social value.

[0003] However, the development of cell surface biomarkers for vascular aging remains largely unexplored. While some molecules potentially associated with vascular aging have been identified, their clinical value and early diagnostic role still require validation. Therefore, discovering new and reliable cell surface biomarkers related to vascular aging, especially those reflecting vascular endothelial cell aging, has become a pressing technical challenge.

[0004] In recent years, increasing evidence in vascular endothelial cell senescence research has shown that endothelial cell senescence not only directly affects vascular function but also promotes the occurrence and development of diseases such as atherosclerosis through multiple mechanisms. The main characteristics of vascular endothelial cell senescence include cell cycle arrest, increased inflammatory response, and decreased cell function. These senescence changes are often accompanied by alterations in the expression of biological markers, providing potential targets for the early diagnosis and treatment of vascular senescence.

[0005] Therefore, finding biomarkers that can reflect the aging process of vascular endothelial cells is of great clinical significance. Summary of the Invention

[0006] The purpose of this invention is to provide the application of membrane-associated cyclic CH finger protein 8 (MARCHF8) as a biomarker in assessing or assisting in the assessment of vascular endothelial cell senescence. This invention discovers a novel cell surface biomarker—membrane-associated cyclic CH finger protein 8 (MARCHF8)—which is closely related to vascular endothelial cell senescence and vascular senescence-related diseases (such as atherosclerosis). Studies have found that with the senescence of vascular endothelial cells, the gene and protein levels of MARCHF8, as well as its membrane protein levels, significantly decrease, and its expression level is closely related to vascular endothelial cell senescence, decreased vascular endothelial cell function (reduced cell proliferation, migration, tube formation, and nitric oxide synthesis capacity), and the development of atherosclerosis. Therefore, MARCHF8 can not only serve as a biomarker for vascular endothelial cell senescence and vascular senescence, but also as a biological biomarker and interventional therapeutic target for atherosclerosis, providing new insights for the early diagnosis and treatment of related diseases.

[0007] Compared with current methods for assessing vascular aging, the MARCHF8 biomarker provided in this invention exhibits higher sensitivity and specificity, effectively reflecting early changes in vascular endothelial cell aging. Furthermore, experimental results in a mouse model of atherosclerosis show that increased MARCHF8 expression significantly inhibits the occurrence and development of atherosclerosis, further validating its potential as a diagnostic and therapeutic target for atherosclerosis. Therefore, MARCHF8 not only serves as a biomarker for vascular aging but also holds promise for wide clinical applications, including early screening, prevention, and targeted therapy.

[0008] This invention clarifies the role of MARCHF8 in the vascular aging process and proposes its use as a biomarker on the surface of vascular endothelial cells for the early diagnosis, monitoring, and targeted therapy of related diseases. With the gradual aggravation of vascular aging and atherosclerosis, MARCHF8, as a novel biomarker and target, will provide important support for early prevention, clinical diagnosis, and treatment.

[0009] To achieve the above objectives, this application adopts the following technical solution:

[0010] In a first aspect, the present invention provides the application of cyclic CH finger protein 8 (MARCHF8) as a biomarker in assessing or assisting in the assessment of vascular endothelial cell senescence.

[0011] The gene ID of MARCHF8 is 312656 (updated on 5 Mar-2024). Diseases associated with vascular endothelial cell aging include atherosclerosis, coronary heart disease, aortic dissection, vascular calcification, hypertension, kidney disease, and cerebrovascular disease.

[0012] In the above technical solutions, the expression level of MARCHF8 in vascular endothelial cells and / or on the cell membrane is negatively correlated with the senescence degree of the vascular endothelial cells. When evaluating or assisting in the evaluation of the senescence degree and cell function of different vascular endothelial cells, the senescence degree of the tested vascular endothelial cells with low MARCHF8 expression is higher than that of the tested vascular endothelial cells with high MARCHF8 expression, and the function (cell proliferation, migration, tube formation, nitric oxide synthesis capacity) of the tested vascular endothelial cells with low MARCHF8 expression is lower than that of the tested vascular endothelial cells with high MARCHF8 expression.

[0013] In the above technical solutions, the expression level of MARCHF8 in vascular endothelial cells and / or cell membranes is detected to assess or assist in the assessment of vascular endothelial cell senescence.

[0014] In a second aspect, the present invention provides the use of MARCHF8 in the preparation of products for assessing or assisting in the assessment of vascular endothelial cell aging or function, or the use of substances that specifically bind to MARCHF8 or its gene in the preparation of products for assessing or assisting in the assessment of vascular endothelial cell aging or function.

[0015] Products used to assess or assist in assessing vascular endothelial cell senescence or function (cell proliferation, migration, tube formation, nitric oxide synthesis capacity) may contain substances that specifically bind to MARCHF8 or its gene. Substances specifically binding to MARCHF8 may be monoclonal or polyclonal antibodies against MARCHF8, while substances specifically binding to the MARCHF8 gene may be specific primer pairs for PCR amplification of the MARCHF8 gene.

[0016] Thirdly, the present invention provides the use of MARCHF8 in the preparation of products that regulate vascular endothelial cell aging or function, or the use of substances that specifically bind to MARCHF8 or its gene in the preparation of products that regulate vascular endothelial cell aging or function.

[0017] Products that regulate vascular endothelial cell aging or function (cell proliferation, migration, tube formation, and nitric oxide synthesis) may contain substances that specifically bind to MARCHF8 or its gene. Substances specifically binding to MARCHF8 may be monoclonal or polyclonal antibodies against MARCHF8, while substances specifically binding to the MARCHF8 gene may be specific primer pairs for PCR amplification of the MARCHF8 gene.

[0018] Products that regulate vascular endothelial cell aging or function (cell proliferation, migration, tube formation, nitric oxide synthesis capacity) also include substances that inhibit MARCHF8 expression, such as small interfering RNAs that interfere with MARCHF8 expression. Specifically, the target sequence of small interfering RNAs that interfere with MARCHF8 expression is st-h-MARCHF8 (i.e., CCTTGTATGTGCTCATTGA); or substances that promote MARCHF8 expression, such as exogenous MARCHF8 and its analogues, and / or drug components that enhance the stability of MARCHF8 protein, and / or nucleic acid molecules and their carriers that can express MARCHF8.

[0019] Fourthly, the present invention provides the use of a probe capable of detecting the expression level of MARCHF8 in vascular endothelial cells and / or on the cell membrane in the preparation of a kit for assessing or assisting in the assessment of vascular endothelial cell senescence.

[0020] Fifthly, the present invention provides a kit for assessing or assisting in the assessment of vascular endothelial cell senescence, the kit comprising a probe and a hybridization solution, the probe being capable of detecting the expression level of MARCHF8 in vascular endothelial cells and / or on the cell membrane.

[0021] In a sixth aspect, the present invention provides the use of an active ingredient in the preparation of a medicament for delaying vascular aging and / or preventing and treating vascular aging-related diseases, said active ingredient comprising a reagent capable of increasing the expression level of MARCHF8.

[0022] In the above technical solutions, the reagent includes exogenous MARCHF8 or its analogues; and / or the reagent includes a pharmaceutical component that enhances the stability of MARCHF8; and / or the reagent includes a nucleic acid molecule capable of expressing MARCHF8 and its carrier.

[0023] In a seventh aspect, the present invention provides a medicament for delaying vascular aging and / or preventing and treating vascular aging-related diseases, the medicament comprising a reagent capable of increasing the expression level of MARCHF8, and a pharmaceutically acceptable carrier.

[0024] This invention investigates the role of MARCHF8 in vascular endothelial aging and atherosclerosis through cell experiments and animal models, and reveals its application as a potential biomarker and target.

[0025] A study on the role of MARCHF8 in vascular endothelial cell senescence: By investigating the expression and function of MARCHF8 in vascular endothelial cells, it was found that MARCHF8 plays a crucial role in endothelial cell senescence. Using PCR primers and specific antibodies for MARCHF8, qPCR and Western blotting (WB) were performed on young and aged endothelial cells. The results showed that the expression levels of both the MARCHF8 gene and protein were significantly reduced in aged vascular endothelial cells compared to young endothelial cells.

[0026] Discovery of MARCHF8 in Atherosclerosis, a Disease Related to Vascular Aging: This invention investigated the role of MARCHF8 in atherosclerosis by establishing a high-fat diet-induced mouse model. Adeno-associated virus (AAV) was administered via tail vein injection to induce MARCHF8 overexpression in the mouse atherosclerosis model. Western blotting (WB) results showed a significant increase in MARCHF8 expression in the overexpression group, supported by both immunofluorescence and WB findings. Furthermore, Oil Red O staining of the arteries indicated that the atherosclerotic plaque area was reduced and lipid deposition was inhibited in mice overexpressing MARCHF8.

[0027] The role of MARCHF8 in the mechanisms of vascular aging and atherosclerosis:

[0028] Cell Model: Vascular endothelial cells (HUVECs) were infected with a packaged empty vector plasmid (Ctrl) and a lentivirus containing a MARCHF8 overexpression plasmid (M8-OE). Control Ctrl cell lines and MARCHF8-OE cell lines were obtained after selection with puromycin. Simultaneously, a MARCHF8 knockdown cell line (si-MARCHF8) and its siRNA control group (si-NC) were constructed using siRNA technology. The effects of MARCHF8 on endothelial cell growth and proliferation were examined using cell proliferation assays (EDU), cell migration assays (scratch healing assay), and endothelial cell function assays (nitric oxide (NO) secretion assay). The results showed that MARCHF8 knockdown inhibited the growth and proliferation of young endothelial cells and disrupted endothelial cell function, while accelerating the aging process. Overexpression of MARCHF8 promoted endothelial cell growth and proliferation, restored the function of senescent endothelial cells, and delayed the aging process.

[0029] Mouse model: Adeno-associated virus (AAV) containing packaged empty vector plasmid (CD-CV) and MARCHF8 overexpression plasmid (HFD-M8) was injected via tail vein into a mouse model of atherosclerosis, and the development of atherosclerosis was recorded at regular intervals. Four weeks after implantation, vascular stiffness and elasticity were assessed using ultrasound angiography, and lipid deposition in the vascular wall was assessed using Oil Red staining. The results showed that mice in the HFD-M8 group had significantly improved vascular stiffness and elasticity, as well as lipid deposition, compared to the HFD-CV group, suggesting that increased MARCHF8 expression can delay vascular aging and the onset of atherosclerosis.

[0030] The experimental results of this invention show that:

[0031] MARCHF8 plays a key role in the senescence of vascular endothelial cells and regulates senescence-related proteins.

[0032] Overexpression of MARCHF8 can significantly reduce the area of ​​atherosclerotic plaques in mice and inhibit lipid deposition;

[0033] Overexpression of MARCHF8 can improve the stiffness and elasticity of blood vessels in mice and delay the occurrence of atherosclerosis.

[0034] MARCHF8 participates in the development of vascular aging and atherosclerosis by regulating the growth, proliferation, and function of vascular endothelial cells.

[0035] The MARCHF8 protein of this invention can serve as an early diagnostic marker for vascular aging and atherosclerosis, and can also be used to prepare new vascular protective drugs targeting MARCHF8, especially for delaying the vascular aging process and improving the symptoms of atherosclerosis.

[0036] The beneficial effects of this invention are as follows: The biomarker MARCHF8 plays an important role in the screening and identification of vascular endothelial cells, can assess vascular aging and vascular aging-related diseases including atherosclerosis, and can also detect human vascular function and evaluate the degree of human vascular aging, providing important molecular indicators for the research and intervention of vascular aging-related diseases, especially in the assessment of human cardiovascular function and disease diagnosis, and has important clinical application value. Attached Figure Description

[0037] Figure 1 MARCHF8 mRNA levels are downregulated during vascular endothelial cell senescence, accompanied by an increase in the senescence marker P21 mRNA.

[0038] Figure 2 The expression of MARCHF8 protein in cells and on the cell membrane is downregulated with the aging of vascular endothelial cells.

[0039] Figure 3 Knockdown of MARCHF8 in vascular endothelial cells can promote cell senescence.

[0040] Figure 4 Knockdown of MARCHF8 in vascular endothelial cells leads to a decrease in MARCHF8 mRNA levels, accompanied by an increase in cellular inflammatory markers including IL6 and IL8 mRNA.

[0041] Figure 5 Knockdown of MARCHF8 in vascular endothelial cells leads to a decrease in MARCHF8 protein levels, accompanied by an increase in the aging marker P21 protein.

[0042] Figure 6 Overexpression of MARCHF8 in vascular endothelial cells can inhibit cell senescence.

[0043] Figure 7 Overexpression of MARCHF8 in vascular endothelial cells leads to an increase in MARCHF8 mRNA levels, accompanied by an increase in cellular inflammatory markers, including a decrease in the mRNA levels of IL6 and IL8.

[0044] Figure 8 Overexpression of MARCHF8 in vascular endothelial cells leads to an increase in MARCHF8 protein levels, accompanied by a decrease in the aging marker P21 protein.

[0045] Figure 9 MARCHF8 can improve the proliferation, migration, tube formation, and NO synthesis of endothelial cells. Young HUVECs were transfected with a siRNA targeting human MARCHF8 (siM8) and a negative control siRNA (siNC), while senescent HUVECs were infected with a control lentiviral vector (Ctrl) or a lentivirus overexpressing MARCHF8 (M8-OE). (A) Cell proliferation was detected by an Edu binding assay. (B) Cell migration was detected by a wound healing assay. Images were taken at 0, 16, and 24 hours. Gradients, 50 μm. (C) Tube formation was observed in 96-well plates coated with Matrigel medium. Representative images of network morphological changes are shown. Total tube length and branch number were quantified using ImageJ. (D) Representative images and histograms of intracellular nitric oxide (NO) production in HUVECs. Scale bar: 100 μm (si) / 500 μm (OE). Quantitative data are expressed as mean ± SEM of three independent experiments (n = 3). * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

[0046] Figure 10 ApoE - / -Overexpression of MARCHF8 in mice led to a significant increase in MARCHF8 protein levels in the aorta of the HFD-M8 group mice. ApoE - / - Mice were randomly divided into three groups (n=10 per group): 1) Ctrl: control diet (CD) + AAV empty control vector (Control, Ctrl) group; 2) HFD-Ctrl: high-fat diet (HFD) + Ctrl group; 3) HFD-M8: HFD + AAV MARCHF8 overexpression group (M8) group. Western blot analysis of MARCHF8 protein levels in the aorta was performed in the three groups, with GAPDH serving as an internal control (n=6).

[0047] Figure 11 ApoE - / - Overexpression of MARCHF8 in mice significantly delayed atherosclerosis. The effect was assessed using aortic oil red O analysis. Figure 10 The degree of atherosclerosis in the three groups of mice after 12 weeks of feeding (n=10). (A) Lipid deposition in atherosclerotic mice was analyzed using the entire aorta. Three representative aortas were used from each group of mice. Scale line, 20 μm. The relative area of ​​atherosclerotic lesions was quantitatively analyzed as a percentage of the total aortic area (n=3). (B) Representative examples and quantitative comparisons of atherosclerotic lesions stained with Oil Red O at the aortic root (n=3). Scale line, 50 μm.

[0048] Figure 12 ApoE - / - Overexpression of MARCHF8 in mice significantly reduced HFD-induced plasma IL6 levels and decreased systemic inflammation levels.

[0049] Figure 13 ApoE - / - Overexpression of MARCHF8 in mice can reduce pulse wave velocity (PWV) in a mouse model of HFD-induced atherosclerosis and reduce vascular stiffness in mice. Detailed Implementation

[0050] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims. The abbreviations used in this invention are shown in Table 1 below.

[0051] Table 1 List of Abbreviations

[0052]

[0053] The materials and equipment used in the experiments of this invention are shown in Table 2 below.

[0054] Table 2 Main Reagents

[0055]

[0056]

[0057] Cell Culture: The use of human umbilical cords was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. As previously described, HUVECs were extracted from three different human umbilical cords and cultured in EGM2 medium (Lonza) at 37°C and 5% CO2 with an EGM-2 supplement kit. HUVECs were passaged every 2 or 3 days with 0.05% trypsin-EDTA (Gibco, USA), and the population doubling level (PDL) of the cells was measured. Replicative senescence of HUVECs was induced sequentially by young cells until their proliferation nearly ceased. Young cells (PDL5-13) and senescent cells (PDL25-33) were used in the experiments. This study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and written informed consent was obtained from the donor fathers (Agreement No.: TJ-IRB20230711).

[0058] Laboratory animals: Animal experiments were conducted in accordance with the regulations of the Institutional Animal Use and Care Committee of Huazhong University of Science and Technology (HUST) (IACUC no. 3141). Five-week-old male ApoE - / - Mice were obtained from Beijing HFK Bioscience Co., Ltd. MARCHF8 was overexpressed using bGlobin-MCS-AAV-EGFP-3-FLAG-WPRE-hGHpolyA carrying the CMV promoter. MARCHF8 was purchased from Jikai Gene Co., Ltd. (Shanghai, China). The specific sequence is shown in Table 2-2. A blank vector (bGlobin-MCS-EGFP-3FLAG-WPRE-hGH polyA) containing the CMV promoter and enhanced green fluorescent protein was used as a control vector. Mice were randomly divided into three groups (n=10 per group): Group 1, control group (CD) fed + AAV blank control vector group (CV); Group 2, high-fat diet (HFD) fed + CV; Group 3, HFD + AAV MARCHF8 overexpression group (M8). During the first two weeks of HFD (Cat.#XT108C, Xitong, Jiangsu, China), ApoE... - / - Mice were intravenously injected with MARCHF8 or an AAV vector overexpressing AAV, followed by a 12-week high-fat diet (HFD). After 12 weeks of HFD treatment, mice in all three groups were fasted overnight and euthanized, and blood and tissues were collected. The remaining tissues were collected and frozen for future use.

[0059] Example 1: MARCHF8 expression levels are downregulated with endothelial cell senescence.

[0060] Detection of intracellular MARCHF8 and p21 mRNA levels: Total RNA was extracted from young and senescent endothelial cells using an RNA purification kit (Guangzhou Meiji Biotechnology Co., Ltd.), and analyzed using ReverTra... Reverse transcription was performed using an RT kit (Takara, Japan). The mRNA levels of the gene were determined using Green Realtime PCR Master Mix (TOYOBO, Japan) and primers on an ABI Step One Plus (Applied Biosystems, USA). GAPDH and β-TUBULIN were used as endogenous normalization controls. Two [tests were conducted]. -ΔΔCt The relative changes in mRNA levels were calculated using the method. Each assay was performed at least three times independently. Primer sequences are shown in Table 3. Quantitative results of intracellular MARCHF8 and p21 mRNA levels are shown below. Figure 1 As shown, the results indicate that compared with young endothelial cells, the expression level of MARCHF8 mRNA in senescent endothelial cells was significantly reduced, while the expression level of p21 mRNA was significantly increased.

[0061] Detection of intracellular MARCHF8 protein levels: Western blotting (WB) was performed using standard methods. Cells were lysed with RIPA buffer (Biotech, China), with a protease inhibitor mixture (Boster Biological, China) added. Protein lysates were separated by polyacrylamide-sodium dodecyl sulfate gel electrophoresis and then transferred to polyacrylamide difluoride membranes (Millipore, USA). After electrophoresis, the membranes were blocked with 5% skim milk and then incubated with primary antibodies. The membranes were subsequently washed and incubated with appropriate secondary antibodies, and finally developed using a chemiluminescence assay kit (Beyotime, China). Membrane and cytoplasmic proteins were separated using a membrane extraction kit (KGI Biotech, China). The antibodies used in this study are listed in the main reagents table. The WB results and quantification results of intracellular MARCHF8 protein levels are as follows: Figure 2 As shown, the results indicate that, compared with young endothelial cells, the total cellular protein level and membrane protein level of MARCHF8 in aged endothelial cells are significantly reduced.

[0062] Example 2: MARCHF8 knockdown and overexpression experiments

[0063] MARCHF8 gene interference: Small interfering RNAs (siRNAs) targeting MARCHF8, ADAM10, and their negative control (NC) were designed and synthesized (RiboBio, China). HUVECs were inoculated in complete culture medium and grown to 70-80% confluence, then treated with Lipofectamine premixed with siRNA. TM siRNA was transfected with 3000 μL (Thermo Fisher Scientific, USA) at a concentration of 50 nM. Six hours later, the medium containing siRNA and Lipofectamine was replaced with complete culture medium. TM 3000 culture medium.

[0064] Table 3 MRAF H8 Target Information

[0065] ID Target sequence information st-h-MARCHF8 CCTTGTATGTGCTCATTGA(SEQ ID NO.1)

[0066] st-h-MARCHF8 is referred to as si-MARCHF8 below.

[0067] Interference gene infection of HUVEC cells and verification

[0068] Overexpression of MARCHF8 viral packaging:

[0069] ① Resuscitate 293T cells to package lentiviruses, culture them in DMEM complete medium, and use cells from earlier passages.

[0070] ②Day 1: The day before transfection, digest with trypsin at 293T for 1 min, count the cells, and then inoculate into 10cm dishes, ensuring that each dish contains at least 6.5 × 10⁻⁶ cells. 6 One cell, with a culture medium volume of 8.5 mL, was used for transfection.

[0071] ③Day 2: Take a 2mL EP tube, add 1.5mL of OPTI-MEM, then add 7.5μg of lentiviral packaging helper plasmid psPAX2, 2.5μg of pMD2.G, and 10μg of the target plasmid. Mix well, then add 30μL of HG-Trans293. TM Mix the transfection reagent thoroughly, let it stand at room temperature for 30 minutes, and then evenly add it dropwise to the 293T cells seeded the day before, being careful not to blow the cells away. Replace with DMEM complete medium after 8-12 hours.

[0072] ④Day 4: 48 hours after transfection, the supernatant was collected into a 50mL centrifuge tube, temporarily stored at 4℃, and 10mL of LDM complete medium was added again. The transfection efficiency was observed by taking pictures under a fluorescence microscope, and then incubated at 37℃ and 5% CO2.

[0073] ⑤Day 5: Collect the supernatant at 72h and combine it with the supernatant at 48h. Centrifuge at 1000g / min for 10min. Aliquot according to the specific usage and store at -80℃.

[0074] Viral infection of HUVEC cells:

[0075] Jikai synthesized two target plasmids, VC being an empty vector and M8-OE being a MARCHF8 overexpression plasmid. Both vectors packaged lentiviruses.

[0076] Table 4 LV-MARCHF8 Target Information

[0077]

[0078] HUVEC cells were seeded in six-well plates one day in advance, with 2 × 10⁶ cells per well. 6 Nine wells were prepared. Infection: 500 μL of VC virus and 1000 μL of M8-OE virus were added to each well, and the remaining medium was added to 2 mL with McCoy's 5A complete medium. 2 μL of polybrene, an auxiliary infection reagent, was added to each well, and the mixture was incubated. After 8 h, the medium was replaced with McCoy's 5A complete medium to infect HUVEC cells. Cell morphology and infection efficiency were observed under a fluorescence microscope after 24 h, 48 h, 72 h, and 96 h.

[0079] MARCHF8 knockdown, MARCHF8 overexpression RNA level verification, cell RNA extraction and PCR steps are the same as above.

[0080] MARCHF8 knockdown, validation of MARCHF8 overexpression protein levels:

[0081] Cells were collected 96 h after lentiviral infection (after trypsin digestion, neutralization with McCoy's 5A complete medium, centrifugation at 300 g / min, removal of medium, washing with PBS, centrifugation, and removal of PBS as much as possible), lysed by 2D-lysis, and lysed on ice for half an hour with shaking every 5 min, then centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was collected. The quantification steps were the same as above. 15 μg of immunoblot protein was loaded, and the Western blot experiment was performed in the same manner as above.

[0082] Example 3: MARCHF8 affects the expression of cellular senescence and cellular inflammation markers.

[0083] Cellular phenotype identification of senescent cells: SA-β-gal activity was measured using a senescence β-galactosidase staining kit (Beyotime, China). Cells were first treated with 1x fixation buffer for 6-7 minutes at room temperature, then stained overnight with a staining mixture at 3°C ​​without carbon dioxide. Subsequently, the number of blue-stained cells and the total cell count were observed under a light microscope and counted using ImageJ software (National Institutes of Health, USA). Compared with the control group, the proportion of blue-stained cells in the MARCHF8 knockdown group was significantly increased. Figure 3 As shown, compared with the control group, the proportion of blue-stained cells in the MARCHF8 overexpression group was significantly reduced. Figure 6 As shown in the figure; the results indicate that MARCHF8 can delay cell senescence.

[0084] Cellular senescence and cellular inflammation markers were detected using mRNA and Western blotting. The cellular inflammation markers used in this invention were interleukin-6 (IL6) and interleukin-8 (IL8), and the senescence marker was p21. Primer information is shown in Table 5, and antibody information is listed in the main reagents table; the cell RNA extraction and PCR steps, as well as the cell protein extraction and Western blotting procedures, were the same as above. Compared with the control group, the mRNA levels of IL6 and IL8 in the MARCHF8 knockdown group were significantly increased, such as... Figure 4 As shown; p21 protein levels are significantly increased, such as Figure 5 As shown; compared with the control group, the mRNA levels of IL6 and IL8 in the MARCHF8 overexpression group were significantly reduced, such as Figure 7 As shown, the protein level of p21 is significantly reduced, such as Figure 8 As shown, the results indicate that MARCHF8 can reduce cellular inflammation and delay cellular senescence at both the RNA and protein levels.

[0085] Table 5. PCR Primer Sequence List

[0086]

[0087]

[0088] Example 4: Effects of MARCHF8 on Cell Function

[0089] (1) Cell proliferation function - Edu proliferation experiment

[0090] 1) Prepare the constructed MARCHF8 knockout HUVECs (si-M8) and their control group HUVECs (si-NC) according to the above steps, and the MARCHF8 overexpressing HUVECs (MARCHF8-OE) and their control group HUVECs (VC). After digestion, count and prepare 5×10⁻⁶ HUVECs. 4A cell suspension of 100 μL / mL was evenly added to each well of a 96-well plate to maintain a cell count of 5 × 10⁶ cells / mL. 3 After complete adhesion to the wells, discard the culture medium and wash with PBS. Add 500 μL of fresh culture medium to each well. Dilute Edu working solution to 20 μM / L with cell culture medium, and add 500 μL of the diluted Edu working solution to each well, resulting in a final concentration of 10 μM / L. After addition, incubate the plates for 24 hours.

[0091] 2) Cell fixation and permeabilization: Discard the culture medium and wash with PBS. Fix with 4% paraformaldehyde for 15 min at room temperature, then discard the paraformaldehyde. Wash with PBS containing 3% BSA. Then add 0.5% Triton X100 dissolved in PBS to the well plate for permeabilization and let stand at room temperature for 20 min.

[0092] 3) Edu detection: This experiment uses a 100μL iClick reaction solution system, and the system components are shown in Table 6 below.

[0093] Table 6 System Components

[0094] Added components volume iClick EdU reaction buffer 86μL CuSO4 4μL Andy Fluor 555azide 0.3μL 1x iclickEdU buffer additive 10μL Total volume 100μL

[0095] Discard the cell permeabilization buffer and wash again with PBS containing 3% BSA. After washing, add the prepared iClick reaction solution to each well of the plate. Prepare the solution fresh each time you use it. Incubate at room temperature for 30 minutes, protecting from light. Discard the reaction solution and wash again with PBS containing 3% BSA.

[0096] 4) Imaging: Add 200 μL of DAPI-containing anti-quenching agent to the well plate and then observe it under a microscope.

[0097] 5) Repeat the above experiment 3 times.

[0098] The results of Edu fluorescence detection and quantification are as follows: Figure 9 As shown in Figure A, the results showed that compared with the control group, the cell fluorescence level in the MARCHF8 knockdown group was significantly reduced, while the cell fluorescence level in the MARCHF8 overexpression group was significantly increased compared with the control group; the results indicate that MARCHF8 can promote the proliferation function of endothelial cells.

[0099] (2) Cell migration function - cell scratch assay

[0100] 1) The migration ability of HUVECs was assessed using a wound healing assay. Cells were cultured in 6-well plates until fully confluent, and the cell monolayer was scratched into a straight line. Images were taken at 0, 16, and 24 hours after the scratch.

[0101] 2) Repeat the above experiment 3 times.

[0102] The results and quantitative results of the cell scratch assay are as follows: Figure 9 As shown in Figure B, the results showed that the scratch repair rate was significantly lower in the MARCHF8 knockdown group compared with the control group, while the scratch repair rate was significantly higher in the MARCHF8 overexpression group compared with the control group. The results indicate that MARCHF8 can promote the migration function of endothelial cells.

[0103] (3) Cell tube formation function - tube formation experiment

[0104] 1) The tube-forming ability of HUVECs was assessed using a Matrigel tube formation assay. HUVECs were seeded at a density of 20,000 cells / well into 96-well plates pre-coated with growth factor-reducing Matrigel (Corning International, Inc.), and then cultured at 37°C and 5% CO2 for 6 hours. The tube formation process was photographed using an inverted optical microscope and quantified using the Angiogenesis Analyzer plugin in ImageJ.

[0105] 2) Repeat the above experiment 3 times.

[0106] The results and quantitative results of the tube formation test are as follows: Figure 9 As shown in Figure C, the results showed that compared with the control group, the MARCHF8 knockdown group had significantly reduced cell tube formation ability and tube length; while compared with the control group, the MARCHF8 overexpression group had significantly increased cell tube formation ability and tube length; the results indicate that MARCHF8 can promote the tube formation function of endothelial cells.

[0107] (4) Cellular NO synthesis function - NO synthesis experiment

[0108] 1) The production of NO in HUVEC cells was assessed using the specific NO probe DAF-FM DA (Beaudio, China). After removing the culture medium, cells were stained with 2.5 μmol / L DAF-FM DA in Hank's solution (pH 7.4) for 20 minutes at 37°C. After washing three times with Hank's solution to completely remove the probe, the cells were observed and photographed using a laser confocal microscope (Nikon C2+, Tokyo, Japan).

[0109] 2) Repeat the above experiment 3 times.

[0110] The results and quantitative results of NO synthesis experiments are as follows: Figure 9 As shown in Figure D, the results showed that compared with the control group, the NO synthesis function of the MARCHF8 knockdown group was significantly reduced; while compared with the control group, the NO synthesis function of the MARCHF8 overexpression group was significantly increased; the results indicate that MARCHF8 can promote NO synthesis in endothelial cells.

[0111] Example 5: MARCHF8 can delay atherosclerosis.

[0112] A mouse model of atherosclerosis was established by feeding mice a high-fat diet (HFD) for 12 weeks. The lipid deposition in the aorta was detected by Oil Red O staining to confirm successful model establishment. After euthanasia, the mice were perfused with ice-cold PBS for 5 minutes, followed by perfusion with 4% buffered paraformaldehyde containing 2 mM EDTA and 2% glucose.

[0113] (1) Obtaining and validating MARCHF8 differentially expressed protein in an animal atherosclerosis model

[0114] Western blot (WB) experiments validated the upregulation of MARCHF8 expression in a mouse model of atherosclerosis overexpressing MARCHF8. Aortic tissue from mice in three groups were subjected to WB experiments: 1) CD-CV: control diet (CD) + AAV empty control vector (CV); 2) HFD-CV: high-fat diet (HFD) + CV; and 3) HFD-M8: HFD + AAV MARCHF8 overexpression (M8) groups. Six samples were collected from each group (n=6). WB analysis of MARCHF8 protein levels in the aorta was performed using GAPDH as an internal control. Western blot (WB) was performed using standard WB methods. Cells were lysed using RIPA buffer (Boster Biological, China) supplemented with protease inhibitors. Protein lysates were separated by electrophoresis on SDS-PAGE gels and then transferred to PVDF membranes (Millipore, USA). After electrophoresis, cell membranes were blocked with 5% skim milk and incubated with primary antibody. Cell membranes were then washed with appropriate secondary antibodies and incubated, followed by visualization using a chemiluminescence kit (Beyotime, China). MARCHF8 expression was upregulated in the aortic tissue of mice in the HFD-M8 group, such as... Figure 10 As shown.

[0115] (2) MARCHF8 reduced lipid deposition in a mouse model of atherosclerosis. The entire aorta was carefully dissected, connective tissue and fat removed, and longitudinal sections were prepared on paraffin-coated culture dishes. The aorta was incubated with Oil Red O in propylene glycol for 2 hours at room temperature. After staining, the aorta was destained three times with 60% ethanol. The aorta, then immersed in PBS, was photographed against a black background, and the images were analyzed using ImageJ to assess the percentage of Oil Red O stained areas. Figure 11 As shown, compared with the control group, the area of ​​Oil Red staining in the aorta was significantly reduced in the MARCHF8 overexpression group, indicating that MARCHF8 can reduce lipid deposition in the mouse atherosclerosis model.

[0116] (3) MARCHF8 can reduce systemic inflammatory response. In this invention, the indicator used to reflect the systemic inflammatory response is the plasma IL-6 level. (BD) TM The Flow Cytometry Magnetic Bead Array (CBA) Mouse IL6 Detection Kit (#558301, BD Biosciences, USA) was used to determine IL6 levels in mouse plasma. Data were acquired and analyzed using BD FACSDiva software on a FACS CantoII flow cytometer (BD Biosciences, USA). Figure 12 As shown, compared with the control group, the plasma IL6 level in the MARCHF8 overexpression group was significantly reduced, indicating that MARCHF8 can reduce the systemic inflammatory response in a mouse atherosclerosis model.

[0117] (4) MARCHF8 can reduce PWV in a mouse model of atherosclerosis. For measuring pulse wave velocity (PWV) in mice, mice were placed in a closed air-anesthesia chamber and inhaled 3% isoflurane for approximately 15 seconds. After checking the degree of anesthesia by pinching the toes, the mice were fixed on a sensor plate, and the isoflurane concentration was adjusted to a maintenance concentration of 1.0%-1.5%. After hair removal, an ultrasound coupling agent was applied. The mouse's electrocardiogram (ECG) was first observed using a sensor. Once the ECG stabilized, the ultrasound sensor probe was slowly slid upwards along the mouse's sternum until the thoracic aorta waveform was found and photographed, while simultaneously recording the position. Then, the probe was slowly slid upwards along the umbilicus until the abdominal aorta waveform was found, and the position was photographed and recorded. The distance between the thoracic and abdominal aorta waveforms was measured, and the conduction time was analyzed based on the waveform. Pulse wave velocity was calculated as the separation distance (mm) between the two measurement points divided by the pulse transmission time (ms). Figure 13 As shown, compared with the control group, the plasma PWV of the MARCHF8 overexpression group was significantly reduced, indicating that MARCHF8 can reduce PWV in a mouse atherosclerosis model.

[0118] In the embodiments of the present invention, the obtained data were analyzed using SPSS 22.0 statistical software. The experiment was repeated 3 times, and P < 0.05 was considered statistically significant.

[0119] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The use of an active ingredient in the preparation of a medicament for delaying and / or preventing vascular aging-related diseases, characterized in that: The active ingredient includes a reagent that can increase the expression level of MARCHF8 protein, the reagent including a nucleic acid molecule and its carrier that can express MARCHF8 protein, and the vascular aging-related disease is atherosclerosis.

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