Application of atf3 as a target in preparation of drugs and / or detection reagents for treating blood vessel aging

Abstract

The application discloses application of ATF3 as a target to preparation of a blood vessel aging treatment drug and / or a detection reagent, and belongs to the technical field of medicines. It is found by the application that the expression amount of ATF3 is related to blood vessel aging. It is found by research that the expression amount of ATF3 in blood vessel aging cells decreases, and ATF3 can be used to develop a blood vessel aging detection kit or chip to determine whether the detected blood vessel is aging. It is found by research that the occurrence and development of blood vessel aging can be prevented by improving the expression of the protein ATF3 in cells or tissues and activating the autophagy process, and the protein ATF3 can be used as a new target for prevention and treatment of blood vessel aging.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of ATF3 as a target in the preparation of drugs for treating vascular aging and / or detection reagents. Background Technology

[0002] Increased vascular stiffness is one of the important characteristics of vascular aging. Increased vascular stiffness can damage organs with high-flow, low-resistance vascular beds, such as the myocardium, kidneys, brain, placenta, and testes (Chirinos JA, Segers P, Hughes T, Townsend R. Large-Artery Stiffness in Health and Disease: JACC State-of-the-Art Review. J Am Coll Cardiol. 2019; 74(9):1237-1263., Nowak KL, Rossman MJ, Chonchol M, Seals DR. Strategies for Achieving Healthy Vascular Aging. Hypertension. 2018; 71(3):389-402), leading to systolic hypertension, left ventricular hypertrophy, heart failure, pulse wave-related nephropathy, cognitive impairment, cognitive decline, occasional dementia, preeclampsia, intrauterine growth retardation, and testicular aging.

[0003] Transcription Activator Factor 3 (ATF3) is an important transcription factor belonging to the Activator Transcription Factor / Cyclic AMP Response Element (CREB) family. Members of this family all contain a highly conserved basic leucine zipper (bZIP) domain, which consists of a basic amino acid region and a leucine zipper region that forms a homodimer. ATF3 forms heterodimers or homodimers with other bZIP proteins through its bZIP domain and binds to specific cis-acting elements on DNA (such as CREs, cAMP response elements), thereby regulating the transcriptional activity of target genes. ATF3 is a multifunctional transcription factor that plays a crucial role in multiple biological processes, including cellular stress responses, gene transcription regulation, cell cycle progression, immune and inflammatory responses, development, and differentiation. Its complex biological functions make it an important subject for studying stress response mechanisms, disease development, and the search for novel therapeutic targets.

[0004] Researchers have further explored the mechanisms of action of ATF3 at the molecular, cellular, and systemic levels and its correlation with various diseases using tools such as recombinant ATF3 protein and ELISA kits. However, whether ATF3 is a key factor in the development and progression of vascular aging has not yet been reported. Currently, in clinical practice, there is a lack of targets or interventions that can delay or reverse the increase in vascular stiffness at an early stage. Therefore, elucidating the mechanism of increased vascular stiffness in the development and progression of vascular aging will help identify new therapeutic targets, and screening anti-vascular aging drugs targeting ATF3 is of great significance for the treatment of patients with vascular aging. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an application of ATF3 as a target in the preparation of vascular aging therapeutic drugs and / or detection reagents.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides the application of ATF3 as a target in the preparation of drugs for anti-vascular aging.

[0008] This invention provides the application of ATF3 as a target in the preparation of drugs that combat vascular smooth muscle cell aging.

[0009] Preferably, the drug can exert an anti-vascular aging effect by increasing ATF3 expression or activity, wherein the mechanism includes activating autophagy.

[0010] Preferably, the methods for increasing ATF3 expression include at least one of transfection plasmid, viral vector, gene editing, transcriptional level, protein expression level, and post-translational modification; increasing ATF3 activity includes at least one of increasing ATF3 transcriptional activity and increasing ATF3 protein activity.

[0011] Preferably, the method of administration of the drug includes at least one of oral administration, intravenous administration, and intraperitoneal administration.

[0012] Preferably, the drug is any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions, and solutions.

[0013] This invention also provides the application of ATF3 as a target in the preparation of reagents for detecting vascular aging, wherein the reagents can detect the expression level of ATF3 in tissue cells or body fluids. The degree of vascular aging can be reflected by the ATF3 level.

[0014] The present invention also provides the application of ATF3 as a target in the preparation of a reagent for detecting senescence of vascular smooth muscle cells, wherein the reagent is capable of detecting the expression level of ATF3 in vascular smooth muscle cells.

[0015] Preferably, the tissue cells include at least one of blood vessels, liver, brain, heart, and kidney. The body fluids include at least one of serum, urine, cerebrospinal fluid, and sweat.

[0016] The detection reagent is one of the following: a chip or a reagent kit.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention discovers a correlation between ATF3 expression and vascular aging. Studies have shown that ATF3 expression decreases in aging vascular cells. ATF3 can be used to develop vascular aging detection kits or chips to determine whether vascular aging has occurred. Research has also found that increasing the expression of the protein ATF3 in cells or tissues can activate autophagy, thereby inhibiting the development of vascular aging. This suggests that ATF3 could serve as a novel target for the prevention and treatment of vascular aging. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Serum ATF3 expression levels and other baseline indicators were measured in patients with and without vascular stiffness; among them... Figure 1 A represents the serum ATF3 content in patients with arteriosclerosis; Figure 1 B represents the relationship between pulse wave velocity and serum ATF3 levels. Figure 1 C-1H represent the relationship between serum ATF3 levels and body weight, triglycerides, total cholesterol, high-density lipoprotein, low-density lipoprotein, and blood glucose, respectively.

[0021] Figure 2 The expression levels of ATF3 mRNA in different mouse models of vascular aging; among them, Figure 2 A represents the ATF3 mRNA content in the blood vessels of naturally aging mice. Figure 2 B represents the ATF3 mRNA content in the blood vessels of atherosclerotic mice. Figure 2 C represents the ATF3 mRNA content in the blood vessels of mice with accelerated aging.

[0022] Figure 3 The expression levels of ATF3 protein in different mouse models of vascular aging; among them... Figure 3 A represents the immunoblot of ATF3 protein in the blood vessels of naturally aging mice. Figure 3 B represents a semi-quantitative analysis of ATF3 protein content. Figure 3 C represents the immunoblot of ATF3 protein in the blood vessels of atherosclerotic mice. Figure 3 D represents a semi-quantitative analysis of ATF3 protein content. Figure 3 E is an immunoblot of ATF3 protein in the blood vessels of mice with accelerated aging. Figure 3 F represents a semi-quantitative analysis of ATF3 protein content.

[0023] Figure 4 The expression levels of ATF3 mRNA in replicative and induced aging vascular smooth muscle cells; among which, Figure 4 A represents the ATF3 mRNA content in replicating senescent vascular smooth muscle cells. Figure 4 B represents the ATF3 mRNA content in induced aging vascular smooth muscle cells.

[0024] Figure 5 The expression levels of ATF3 protein in replicative and induced aging vascular smooth muscle cells; among which, Figure 5 A represents the immunoblot of ATF3 protein in replicating senescent vascular smooth muscle cells. Figure 5 B represents a semi-quantitative analysis of ATF3 protein content. Figure 5 C represents the immunoblot of ATF3 protein in induced senescent vascular smooth muscle cells. Figure 5 D represents a semi-quantitative analysis of ATF3 protein content.

[0025] Figure 6 The results of experiments on the aging behavior of ATF3 overexpression in senescent vascular smooth muscle cells; among which, Figure 6 A-6B represent the expression of ATF3 in vascular smooth muscle cells at p53 and p21 mRNA levels, respectively. Figure 6 C represents the immunoblotting of P53 and P21 proteins in smooth muscle cells. Figure 6 D represents a semi-quantitative analysis of P53 protein content. Figure 6 E represents a semi-quantitative analysis of P21 protein content.

[0026] Figure 7 The results of the phenotypic transformation experiment in senescent vascular smooth muscle cells by overexpression of ATF3; among which, Figure 7 A represents the immunoblot of SM22α and OPN proteins in smooth muscle cells. Figure 7 B represents a semi-quantitative analysis of SM22α protein content. Figure 7 C represents a semi-quantitative analysis of OPN protein content. Figure 7D represents immunofluorescence labels SM22α and OPN.

[0027] Figure 8 The experimental results show that ATF3 induces transcriptional activation of the autophagy pathway by ATG7; among them, Figure 8 A is the ATF3 sequence recognition map. Figure 8 B represents the dual-luciferase assay. Figure 8 C represents the immunoblotting of ATG7 and LC3 proteins in smooth muscle cells. Figure 8 D represents the ratio of LC3Ⅱ to LC3Ⅰ. Figure 8 E represents a semi-quantitative analysis of ATG7 protein content.

[0028] Figure 9 The experimental results showed that knocking down ATF3 in primary mouse smooth muscle cells could reveal the number of autophagosomes; among them, Figure 9 A represents fluorescently labeled autophagosomes and autolysosomes in smooth muscle cells; 9B represents a statistical analysis of the number of autophagosomes and autolysosomes. Figure 9 C represents the transmission electron microscopy observation of the number of autophagosomes in smooth muscle cells.

[0029] Figure 10 The experimental results show that inhibiting the autophagy pathway blocks the anti-aging effect of ATF3; among them, Figure 10 A represents the immunoblot of P53 and P21 proteins in smooth muscle cells. Figure 10 B represents a semi-quantitative analysis of P53 protein content. Figure 10 C represents a semi-quantitative analysis of P21 protein content. Figure 10 D represents the immunoblot of P53 and P21 proteins in smooth muscle cells. Figure 10 E represents a semi-quantitative analysis of P53 protein content. Figure 10 F represents a semi-quantitative analysis of P21 protein content.

[0030] Figure 11 The results of the phenotypic transformation experiment to knock down ATG7 in primary mouse vascular smooth muscle cells; among them, Figure 11 A represents the immunoblot of SM22α and OPN proteins in smooth muscle cells. Figure 11 B represents a semi-quantitative analysis of SM22α protein content. Figure 11 C represents a semi-quantitative analysis of OPN protein content. Figure 11 D represents immunofluorescence labels SM22α and OPN.

[0031] Figure 12 The results of an experiment to inhibit LC3 phenotypic transformation in primary mouse vascular smooth muscle cells; Figure 12 A represents the immunoblot of SM22α and OPN proteins in smooth muscle cells. Figure 12 B represents a semi-quantitative analysis of SM22α protein content. Figure 12C represents a semi-quantitative analysis of OPN protein content. Figure 12 D represents immunofluorescence labels SM22α and OPN. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0033] 1. Collection and testing of serum samples from patients with and without arteriosclerosis

[0034] All human specimen acquisition protocols involved in this study were approved by the Medical Ethics Committee of Tongji Hospital, Huazhong University of Science and Technology.

[0035] This study enrolled patients aged 40 to 70 years who had abnormally elevated pulse wave velocity (PWV > 1400 m / s), exceeding the normal reference value, had not experienced cardiovascular events including coronary heart disease, aortic aneurysm, or stroke, had normal liver and kidney function, no hematological diseases, and had signed informed consent forms.

[0036] This study excluded: patients who need or are taking lipid-lowering or hypoglycemic drugs due to abnormal blood sugar or blood lipids; patients with angina pectoris, cardiomyopathy, valvular coronary artery disease, or other problems; patients with malignant tumors; patients taking drugs that affect coagulation function and platelet function; and patients participating in other clinical trials.

[0037] Blood samples are collected from patients fasting in the morning via venous blood collection. The collected blood samples are placed in appropriate test tubes or centrifuge tubes and centrifuged to separate the serum. The separated plasma or serum samples are stored in appropriate refrigeration equipment, typically at -20°C or lower, to ensure sample stability and preservation.

[0038] The ATF3 content in serum was determined by enzyme-linked immunosorbent assay (ELISA). ATF3 antibody was added to the ELISA plate and incubated at 37°C for 30 minutes. The serum sample to be tested and a series of standards of known concentrations were added to the corresponding wells. Each sample and standard was repeated in duplicate to obtain reliable results. Incubation was performed at 37°C for 1 hour to allow the antigen or antibody in the sample to bind to the antigen or antibody on the coating. Finally, horseradish peroxidase-HRP-conjugated secondary antibody was added, followed by washing to remove unbound detection antibody. The absorbance of each well was measured within 10 minutes after the addition of substrate staining agent and reaction stop solution. The concentration of the target molecule in the sample was determined by comparison with a standard curve.

[0039] Simultaneously, biochemical analysis was performed on blood samples to obtain blood lipid and blood glucose information, and the patient's height and weight were recorded. T-tests and Pearson correlation analysis were used to draw relevant conclusions and results.

[0040] 2. Construction of a mouse model of vascular aging and detection of ATF3 content

[0041] Ten-month-old male accelerated aging mice (SAMP8) were used to simulate a natural aging model; APOE- / - mice were used in combination with a high-fat diet (HFD) to construct an atherosclerosis model, which was fed the high-fat diet for 3 months; each group consisted of 10 mice, with 12 hours of light per day, temperature maintained between 20-26℃, and humidity maintained between 40-60%.

[0042] In vivo vascular function was assessed by measuring blood pressure, pulse wave velocity (PWV), and vascular ring tension. Blood pressure in mice was measured using a Coda 21.0 blood pressure system. PWV in mice was measured using Doppler ultrasound (Mouse Doppler, Indus Instruments). Mice anesthetized with isoflurane were placed supine on a heated platform at a constant temperature of 38°C. The limbs of the mice were fixed to ECG electrode plates, and conductive paste was applied to the fingertips. Electrocardiograms were continuously recorded. Pressure waveforms were detected in the descending aorta and abdominal aorta using a 20MHz probe. The arrival and transit times over five cardiac cycles were calculated, and the distance between the descending aorta and abdominal aorta was measured using a ruler. Mouse PWV (m / s) = propagation distance / propagation time.

[0043] Vascular specimens were extracted for smooth muscle cell function and aging marker detection: Mice were anesthetized intraperitoneally with 3% pentobarbital, and blood was collected from the eyeballs. The collected blood was centrifuged at 2000 rpm for 10 minutes, and the serum was then stored at -80°C. After dissection, the mice were perfused with pre-chilled sterile PBS to isolate the entire aorta, including the aortic arch, thoracic aorta, and abdominal aorta.

[0044] Detection of ATF3 protein expression levels: Animal tissue specimens were minced, and a suitable amount of lysis buffer was added before homogenization using a grinder. Protein concentration was determined using a BCA kit, and the concentration of the sample was calculated based on a standard curve. Proteins of different molecular weights were separated by SDS-PAGE gel electrophoresis, and the proteins were then transferred to a PVDF membrane. Non-specific regions on the PVDF membrane were blocked with 5% BSA at room temperature, and incubated overnight at 4°C using a specific primary antibody dilution. Then, a suitable secondary antibody dilution was added, and incubation was performed at room temperature for 1 hour. The PVDF membrane was developed with ECL developing solution and exposed using a high-sensitivity gel imaging system.

[0045] Detection of ATF3 mRNA expression levels: Carotid artery RNA was extracted using Trizol reagent. The total mRNA amount from all samples was adjusted to 2 μg, and reverse transcription was performed. The reaction was carried out at 37℃ for 15 minutes followed by enzyme inactivation at 98℃ for 5 minutes, yielding cDNA samples. Quantitative real-time PCR was performed using a TOYOBO SYBR Green instrument, and the relative expression level of RNA was calculated using the 2-ΔΔCt method.

[0046] Figure 1 Serum ATF3 expression levels and other baseline indicators were measured in patients with and without vascular stiffness; among them... Figure 1 A indicates a decrease in serum ATF3 levels in patients with arteriosclerosis; Figure 1 B indicates a certain correlation between pulse wave conduction velocity and serum ATF3 levels. Figure 1 C-1H indicates that serum ATF3 levels are not correlated with body weight, triglycerides, total cholesterol, high-density lipoprotein, low-density lipoprotein, or blood glucose. Therefore, in clinical samples, serum ATF3 levels are decreased in individuals with high vascular stiffness, and this is independent of other baseline indicators.

[0047] Figure 2 The expression levels of ATF3 mRNA in different mouse models of vascular aging; Figure 2 A indicates a decrease in ATF3 mRNA levels in the blood vessels of naturally aging mice. Figure 2 B indicates a decrease in ATF3 mRNA levels in the blood vessels of atherosclerotic mice. Figure 2 C indicates a decrease in ATF3 mRNA levels in the blood vessels of mice with accelerated aging. This confirms that a decrease in ATF3 mRNA expression levels was observed in the aortic tissues of prematurely aging mice, naturally aging mice, and mice with arteriosclerosis.

[0048] Figure 3 The expression levels of ATF3 protein in different mouse models of vascular aging; Figure 3 A-3B indicates a decrease in ATF3 protein levels in the blood vessels of naturally aging mice. Figure 3 C-3D indicates a decrease in ATF3 protein levels in the blood vessels of atherosclerotic mice. Figure 3 E-3F indicates a decrease in ATF3 protein content in the blood vessels of mice with accelerated aging. This confirms that a decrease in ATF3 protein expression was observed in the aortic tissues of prematurely aging mice, naturally aging mice, and mice with arteriosclerosis.

[0049] All mice used in this study were purchased from Vital River Pharmaceuticals Ltd. (Beijing) and housed at the SPF-grade Animal Laboratory Center of Huazhong University of Science and Technology. All animal experimental protocols used in this study were approved by the Animal Experiment Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and all followed the International Association for the Study of Pain's guidelines for animal research and standard biosafety and institutional safety procedures.

[0050] 3. Culture of replicative / induced senescent vascular smooth muscle cells and detection of ATF3 content

[0051] Primary mouse smooth muscle cells were isolated using an enzymatic digestion method. A replicative aging model was constructed using a continuous passage method: 7-week-old male C57BL / 6 mice were anesthetized with 2% sodium pentobarbital and fixed on a dissecting board. The thoracic and abdominal aortas of the mice were isolated using high-pressure instruments. Excess fat and connective tissue outside the blood vessels were removed under a stereomicroscope. The isolated blood vessels were placed in a laminar flow hood and rinsed repeatedly with pre-cooled sterile PBS 3-5 times. Then, 2 mL of type II collagenase (1 mg / mL) was added and digested at 37°C for 20 minutes. After 20 minutes, the collagenase was aspirated, rinsed with sterile PBS, and placed under a stereomicroscope. The adventitia of the blood vessels was dissected using sterile instruments, the blood vessels were longitudinally incised, and the endothelial tissue was scraped away. The blood vessels were then placed in a laminar flow hood and rinsed repeatedly with sterile PBS 3-5 times. Use sterile scissors to cut the vascular tissue into small pieces. Spread the cut vascular tissue evenly in a T75 culture flask, add ordinary high-glucose medium containing 20% ​​fetal bovine serum, and place it in an incubator. Observe the cell growth after 5-7 days and change the medium according to the cell condition.

[0052] A stress-induced aging model was constructed by intervening with doxorubicin (100 nM) for 48 h: 10% fetal bovine serum (FBS) was added to DMEM (Dulbecco's Modified Eagle Medium), and cells were cultured in a cell culture incubator with a CO2 concentration of 5% under constant temperature conditions of 37°C.

[0053] Smooth muscle cell function (scratch assay, etc.) and aging markers (SA-β-gal, P53 / P21) were detected using the Beyotime β-galactosidase kit. Primary mouse vascular smooth muscle cells were seeded into 12-well plates at a specific ratio and treated with different culture media after adhesion. After treatment, the culture medium was aspirated, and the cells were washed once with PBS. 200 μL of paraformaldehyde fixative was added to each well, and the cells were fixed at room temperature for 20 minutes. The fixative was removed, and the cells were washed three times with PBS. β-galactosidase working solution was prepared. 500 μL of working solution was added to each well, and the cells were incubated overnight at 37°C. The next day, the working solution was aspirated, and the cells were washed three times with PBS to remove residual working solution. Microscopic images were taken to observe and count the cells, and the results were analyzed. Simultaneously, the mRNA and protein levels of phenotypic transformation markers such as ATF3, α-SMA, SM22α, and OPN were detected.

[0054] Figure 4 The expression level of ATF3 mRNA in vascular smooth muscle cells of replicative and induced aging; Figure 4 A indicates a decrease in ATF3 mRNA levels in replicative aging vascular smooth muscle cells. Figure 4 B indicates a decrease in ATF3 mRNA levels in induced senescent vascular smooth muscle cells. This suggests that a decrease in ATF3 mRNA expression levels was observed in both replicative senescent and induced senescent smooth muscle cells.

[0055] Figure 5 The expression level of ATF3 protein in vascular smooth muscle cells of replicative and induced aging; Figure 5 A-5B indicates a decrease in ATF3 protein content in replicative senescent vascular smooth muscle cells. Figure 5 C-5D indicates a decrease in ATF3 protein content in induced senescent vascular smooth muscle cells. This suggests that a decrease in ATF3 protein expression was observed in both replicative senescent and induced senescent smooth muscle cells.

[0056] 4. Knockdown and overexpression of ATF3 were used to detect its regulatory effect on smooth muscle cell senescence.

[0057] To assess the role of ATF3 in regulating smooth muscle cell senescence, ATF3 was silenced using siRNA. Primary mouse smooth muscle cells were seeded in 6-well plates, and after 24 hours, the cell density was maintained at 40%–45%. siRNA-ATF3 solutions were prepared by dissolving siRNA in enzyme-free water to create different concentration gradients. For cell transfection, 0.5 mL of serum-free medium was added, along with transfection reagents Lipo3000 and Opti-MEM, and the mixture was repeatedly pipetted until homogeneous. After 8 hours of incubation, the transfection reagents were replaced with complete medium. RNA was collected after 36 hours, or protein was extracted after 72 hours to determine the knockdown efficiency of the target gene.

[0058] ATF3 was overexpressed using adenovirus to assess the role of ATF3 in regulating smooth muscle cell senescence by detecting smooth muscle cell function (scratch marks) and senescence markers (SA-β-gal, P53 / P21). Primary mouse smooth muscle cells were cultured to an appropriate state and transfected at a cell density of 50-60%. The constructed ATF3-adenovirus mixture was added to the cell culture medium, along with a certain proportion of transfection enhancement reagent, and co-cultured with the target cells for 12 hours before changing the medium. After 48 hours, successfully transfected cells were screened based on the intensity of fluorescence expression.

[0059] Figure 6 To improve smooth muscle cell senescence by overexpressing ATF3 in senescent vascular smooth muscle cells; among which, Figure 6 A-6B indicates that overexpression of ATF3 can reduce the expression of P53 and P21 mRNA in smooth muscle cells. Figure 6 C-6E indicates that overexpression of ATF3 can reduce the expression of P53 and P21 proteins in smooth muscle cells. Therefore, it can be concluded that overexpression of ATF3 can improve smooth muscle cell senescence in induced senescence.

[0060] Figure 7 To reverse the phenotypic transformation of smooth muscle cells by overexpressing ATF3 in senescent vascular smooth muscle cells; among which, Figure 7 A-7C indicates that overexpression of ATF3 increases the expression level of the synthetic phenotypic marker SM22α protein in smooth muscle cells, while decreasing the expression level of the secretory phenotypic marker OPN protein in smooth muscle cells. Figure 7 D represents the immunofluorescence label SM22α, OPN. This indicates that overexpression of ATF3 can reverse the phenotypic transformation of smooth muscle cells in induced senescence.

[0061] 5. Validation of the mechanism by which ATF3 improves aging by promoting autophagy

[0062] (1) ATF3 acts as a transcription factor to regulate the transcription of ATG7: The relevant sites in the ATG7 promoter region were predicted using JASPAR software; ATG7 promoter region mutants were constructed; the binding status was detected by luciferase reporter assay to identify the mutation sites.

[0063] The ATG7 promoter luciferase reporter vector plasmid and the internal reference gene TK vector were co-transfected into HEK293T cells. Transfection can be performed when the cell density is 50-60%. Each cell group was overexpressed with ATF3 or as a control, depending on experimental requirements. Thirty-six hours after transfection, cells were lysed with cell lysis buffer and centrifuged to remove cell debris. The cell lysates were mixed with the luciferase substrate, and the fluorescence signals of the two luciferases were measured using a fluorescence microplate reader. The luciferase activity of the target gene was calculated, and the fluorescence intensity of the target gene was divided by the fluorescence intensity of the internal reference gene to eliminate differences in transfection efficiency and cell number.

[0064] (2) Knockdown of ATF3 using siRNA to detect ATG7 protein expression and the LC3BⅡ / LC3BⅠ ratio in smooth muscle cells: Cell or tissue samples were observed using transmission electron microscopy. Autophagosomes or autolysosomes are vesicle structures enclosed by a single or multiple membrane. Immunoblot analysis was performed using antibodies against autophagy-labeled proteins (LC3B). LC3B in autophagosomes is recognized by the antibody and displayed as a specific protein band. The presence and activity of autophagosomes can be inferred by detecting the conversion and expression levels of LC3B protein. Cells were transfected with exogenous autophagy viruses to express LC3B protein with red or green fluorescence. The green fluorescent probe interacts with the acidic environment within the lysosome, and the degree of autophagy activation is determined based on the ratio of red to green fluorescence.

[0065] Figure 8 ATF3 activates the autophagy pathway by promoting ATG7 transcription; among which, Figure 8 A-8B indicates that ATF3 can bind to the ATG7 promoter to promote transcription. Figure 8 C-8E indicates that knocking down ATF3 reduces ATG7 protein expression and the LC3Ⅱ / LC3Ⅰ ratio in smooth muscle cells. This suggests that ATF3's role as a key protein in aging regulation is achieved by influencing the autophagy pathway.

[0066] Figure 9 Knocking down ATF3 in primary mouse smooth muscle cells resulted in a reduced number of autophagosomes; among which, Figure 9 A-9B indicates that smooth muscle cells were transfected with GFP-mCherry-LC3 virus, and the autophagosomes and autolysosomes showed red and green fluorescence. Figure 9 C represents the number of autophagosomes in smooth muscle cells observed using transmission electron microscopy. This indicates that knocking down ATF3 in primary mouse smooth muscle cells results in a reduced number of autophagosomes.

[0067] 6. Inhibiting the autophagy pathway to detect the degree of cellular senescence

[0068] We used siRNA to silence ATG7 and LC3 inhibitors to inhibit autophagosome formation. We then used the results to assess the role of autophagy in regulating smooth muscle cell senescence by detecting smooth muscle cell function (scratch) and senescence markers (SA-β-gal, P53 / P21).

[0069] Primary mouse vascular smooth muscle cells were seeded in 6-well plates. When the cell density approached 90% and formed a continuous monolayer, a scratch assay was created. A straight line was drawn on the cell monolayer using a pipette tip. Cells were washed with serum-free culture medium or PBS to remove free cells and cell debris. Cell migration was observed at 0, 12, 24, and 48 hours until the scratch healed. The location and morphological changes of the scratch were recorded using an electron microscope. ImageJ image processing software was used to analyze the scratch assay images and calculate cell migration at different time points and for different groups.

[0070] DC-LC3-D5 blocks autophagy by inhibiting LC3B lipidation. When primary smooth muscle cells reached 70% confluence, they were incubated in serum-free medium for 12 hours and then starved. They were then treated with a 10 nM LC3 inhibitor for 6 hours, during which cell growth was monitored and the complete medium was replaced as needed. Subsequently, the cells were digested enzymatically, and proteins and mRNA were extracted for further analysis.

[0071] Figure 10 To inhibit the autophagy pathway and block the anti-aging effect of ATF3; among which, Figure 10 A-10C knocks down ATG7, promoting the expression of P53 and P21 proteins in smooth muscle cells. Figure 10 D-10F uses an LC3 inhibitor to promote the expression of P53 and P21 proteins in smooth muscle cells.

[0072] Figure 11 To promote phenotypic transformation of smooth muscle cells by knocking down ATG7 in primary mouse vascular smooth muscle cells; among which, Figure 11 A-11C knocks down ATG7, reducing the expression level of the synthetic phenotypic marker SM22α protein in smooth muscle cells and increasing the expression level of the secretory phenotypic marker OPN protein in smooth muscle cells. Figure 11 D represents the immunofluorescence label SM22α, OPN.

[0073] Figure 12 To inhibit LC3 in primary mouse vascular smooth muscle cells and promote phenotypic transformation of smooth muscle cells; among which, Figure 12 A-12C knocks down ATG7, reducing the expression level of the synthetic phenotypic marker SM22α protein in smooth muscle cells and increasing the expression level of the secretory phenotypic marker OPN protein in smooth muscle cells. Figure 12 D represents the immunofluorescence label SM22α, OPN.

[0074] Therefore, it can be seen that inhibiting the autophagy pathway can block the anti-aging effect of ATF3; knocking down ATG7 in primary mouse smooth muscle cells can promote smooth muscle cell phenotypic transformation; and inhibiting LC3 in primary mouse smooth muscle cells can promote smooth muscle cell phenotypic transformation.

[0075] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of ATF3 as a target in the preparation of drugs that combat vascular aging by promoting ATG7 transcriptional activation of the autophagy pathway, wherein the drugs can activate the autophagy process by increasing ATF3 expression levels to produce an anti-vascular aging effect, and the method of increasing ATF3 expression levels is by using an adenovirus vector.

2. The application of ATF3 as a target in the preparation of drugs that combat vascular smooth muscle cell senescence by promoting ATG7 transcriptional activation of the autophagy pathway, wherein the drugs can activate the autophagy process by increasing ATF3 expression levels to produce an anti-vascular senescence effect, and the method of increasing ATF3 expression levels is by using an adenovirus vector.

3. The application according to claim 1 or 2, characterized in that, The drug can be administered via at least one of intravenous injection and intraperitoneal injection.

4. The application according to claim 1 or 2, characterized in that, The drug is in a pharmaceutically acceptable dosage form, including at least one of injections, suspensions, and solutions.

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