Use of antisilencing function 1a inhibitor in atherosclerosis mediated by histone lactylation modification

Abstract

The application discloses application of an antisilencing function 1A inhibitor in atherosclerosis mediated by histone lactic acid modification. The application first explicitly regulates the mechanism of ASF1A to histone H3K18 lactic acid modification, effectively prevents histone H3K18 lactic acid modification and occurrence of EndMT and AS mediated thereby, provides a new prevention and treatment drug research approach and a drug action target for diagnosis and treatment of AS, has very important medicinal value, and substances for inhibiting or knocking out ASF1A expression are expected to be candidate drugs for treating AS.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of substances that inhibit the activity of anti-silencing function 1A (ASF1A) in the preparation of drugs for the prevention and treatment of atherosclerosis-related vascular diseases. Background Technology

[0002] With the aging population becoming increasingly severe, the prevalence, incidence, and mortality rates of cardiovascular diseases (CVDs) are also rising continuously, posing a huge health threat and economic burden to people worldwide. In recent years, among the people who die from diseases in my country each year, those who die from CVDs account for 40% of all deaths, making CVDs the leading cause of disease and death among Chinese residents.

[0003] Atherosclerosis (AS) is the main cause of CVDs. According to statistics, the leading causes of death from CVDs worldwide are coronary artery disease (atherosclerosis affecting the arteries that supply blood to the heart) and stroke (atherosclerosis affecting the arteries that supply blood to the brain), making atherosclerosis a leading cause of death worldwide.

[0004] Atherosclerosis (AS) is a typical chronic inflammatory vascular disease caused by multiple factors. The aorta is divided into the intima, media, and adventitia from the inside out. The intima, a monolayer of cells mainly composed of endothelial cells (ECs), forms the inner wall of the vessel and regulates the exchange of oxygen and nutrients between blood and tissues. Therefore, ECs play a crucial role in maintaining the vascular barrier function. It has long been recognized that endothelial cell dysfunction is the initial process of AS, characterized by the upregulation of chemokines and adhesion molecules, lesion penetration, recruitment of circulating monocytes into the intima to become foam cells, and ultimately plaque formation. Activation and damage to the endothelium in AS-prone areas are one of the main initiating factors in AS development, primarily occurring in vulnerable areas of the arteries. When endothelial function is disordered, vascular intimal permeability increases, causing leakage of the endothelial layer, leading to massive lipid infiltration and macrophage infiltration, thereby initiating the pathological process of atherosclerosis. Therefore, maintaining the integrity of ECs is the first line of defense for protecting vascular function and is key to developing new strategies for the prevention and treatment of AS.

[0005] Endothelial-to-mesenchymal transition (EndMT) is a unique process in which endocrine glands (ECs) transform under the influence of various stimuli, including high-fat diets. During this process, ECs transform into mesenchymal cells under various stimuli, gradually losing endothelial-specific markers and acquiring a mesenchymal phenotype, thus impairing both their barrier and secretory functions. Abnormal occurrence of EndMT, frequently occurring in atherosclerosis-prone areas, has been identified as inducing neointimal proliferation and endothelial cell dysfunction, thereby promoting endothelial dysfunction and atherosclerosis. Studies have shown that EndMT can promote atherosclerosis (AS) by reducing the expression of characteristic EC markers and increasing the expression of extracellular matrix components such as fibronectin and adhesion molecules. However, the specific molecular mechanisms by which EndMT in endothelial cells leads to AS remain unclear.

[0006] Epigenomics and epigenetics research has provided new insights into various diseases caused by non-resolved inflammation, such as cancer, pulmonary fibrosis, and atherosclerosis. Growing evidence suggests that epigenetics plays a crucial role in cardiovascular disease, including DNA methylation, histone post-translational modifications, chromatin remodeling, and non-coding RNA. Histones are the core structure of nucleosomes and can influence chromatin accessibility in multiple ways. The tail of histones is restricted by various post-translational modifications, such as methylation, acetylation, phosphorylation, glycosylation, SUMOylation, and ubiquitination. These histone modifications can significantly regulate chromatin structure and gene expression by altering the chromatin microenvironment, playing a vital role in the development and progression of many diseases, including atherosclerosis. New research has found that lactate produced by glycolysis can serve as a precursor, adding lactyl groups to lysine residues of histones as a post-translational modification that regulates gene transcription, a process known as histone lactation. This histone modification affects gene expression by altering the physical accessibility of DNA molecules to proteins involved in DNA transcription. Furthermore, recent studies have shown that histone lactation is closely related to the occurrence and development of various diseases, such as tumors, neurodegenerative diseases, and cardiovascular diseases.

[0007] Mounting evidence suggests that aerobic glycolysis can stimulate histone lactation in macrophages. This energy-supply mechanism, which involves glucose consumption and lactate production even in the presence of oxygen, is known as the Warburg effect. While the Warburg effect has been described as a tumor-associated metabolic feature, it is now considered a physiological phenomenon. Endocrine disruptors (ECs) exhibit similar metabolic patterns, continuing aerobic glycolysis even under aerobic conditions. Glycolysis is the primary energy metabolism pathway for ECs under normal physiological conditions, meeting 85% of their ATP requirements. Glycolysis ensures normal metabolism and proliferation of ECs in atherosclerotic (AS)-prone areas, maintains EC homeostasis and vascular endothelial integrity, protects blood vessels, and reduces the development of AS. However, some studies indicate that while glycolysis is a protective mechanism for ECs, excessive glycolysis can exacerbate endothelial dysfunction. Numerous studies show that atherosclerotic plaques commonly occur at arterial openings, bifurcations, and tortuous areas, and low shear stress in blood flow is closely related to the occurrence of atherosclerosis. Endothelial cells exposed to atherosclerotic (AS)-prone areas with disturbed blood flow are subjected to laminar shear stress, leading to increased glycolysis and abnormally elevated levels of lactate and histone lactation. Excessive glycolysis induces excessive proliferation of endocrine disruptors (ECs), triggers endothelial permeability, damages the endothelial monolayer barrier, and thus accelerates AS progression, upregulating the inflammatory response. Currently, increased aerobic glycolysis by endothelial cells in atherosclerotic-prone areas is considered a cause of inflammation and atherosclerotic lesions. Previous studies have shown that the Warburg effect is also involved in end-mode thrombosis (EndMT) in pulmonary hypertension. However, the impact of aerobic glycolysis on EndMT in atherosclerosis remains unclear.

[0008] HAT P300, a histone acetyltransferase, is a potential "writer" protein for histone lactation. P300 catalyzes the transfer of lactate acyl groups from lactyl-CoA to histones. Whether P300-dependent histone lactation requires a cofactor to precisely regulate specific target genes remains unknown. ASF1A, an evolutionarily well-preserved histone H3 / H4 chaperone, is also an important regulator of gene transcription. In humans, the histone chaperone ASF1A regulates CBP-mediated histone H3K56 acetylation in cells. ASF1A has been reported to interact with P300 in Drosophila and HeLa cells, forming a complex that regulates histone modifications. Previous studies have shown that ASF1A plays a crucial role in tumorigenesis and development, replication-coupled ribosome assembly in anemia, and accelerating the development of chronic myeloid leukemia by activating Notch signaling, but its role in the cardiovascular field has not yet been reported. Histone epigenetic modifications are closely related to the occurrence and development of various diseases, but whether ASF1A-mediated epigenetic modifications are involved in the EndMT process is unknown.

[0009] Currently, there are no studies or reports on the role and mechanism of histone lactation in atherosclerosis. Whether the histone chaperone ASF1A and histone lactation modification are involved in EndMT and atherosclerosis also needs further investigation. Summary of the Invention

[0010] To address the aforementioned issues, this application provides a pharmaceutical use of a histone chaperone ASF1A inhibitor based on inhibiting the production of histone lactation H3K18la.

[0011] Specifically, this application first provides the application of ASF1A inhibitors in the preparation of drugs for the prevention and treatment of cardiovascular diseases; particularly in the preparation of drugs for the prevention and treatment of atherosclerosis.

[0012] Furthermore, the aforementioned ASF1A inhibitors can serve as key agents affecting histone H3K18 lactation modification.

[0013] The objective of this invention can be achieved through the following technical solutions:

[0014] Application of ASF1A inhibitors in the preparation of drugs for the treatment and / or prevention of atherosclerosis.

[0015] As a preferred embodiment of the present invention, the ASF1A inhibitor is a substance that inhibits or knocks out ASF1A expression, selected from ASF1A siRNA, gene editing systems that specifically knock out ASF1A, or other small molecule compounds that can specifically inhibit ASF1A.

[0016] As a preferred embodiment of the present invention, the siRNA sequence of ASF1A is as follows:

[0017] 5'→3'GAGCAGUAAUCCAAAUCUATT (SEQ ID NO.1),

[0018] 3'→5'UAGAUUUGGAUUACUGCUCTT (SEQ ID NO. 2).

[0019] Application of reagents for detecting ASF1A in the preparation of auxiliary diagnostic kits for atherosclerosis.

[0020] Application of ASF1A as a detection target in screening drugs for the treatment of atherosclerosis.

[0021] A method for screening drugs for the treatment of atherosclerosis involves detecting the ASF1A level in plasma before and after drug administration, and assessing the efficacy of candidate drugs for the treatment of atherosclerosis by the degree of reduction in ASF1A level.

[0022] Endothelial cell-specific knockout of Asf1a ApoeKO mouse (Apoe) KO Asf1a ECKO The construction of ) and its application in the preparation of treatments for atherosclerosis.

[0023] The beneficial effects of this invention are:

[0024] The inventors discovered Apoe through experiments such as Western blotting. KO Histone lactation modification and EndMT activation by oxidized low-density lipoprotein (ox-LDL) are observed in mouse aortic vascular tissue, mouse aortic endothelial cells (MAECs), and human coronary artery endothelial cells (HCAECs). Under pathogenic stimuli, abnormally elevated glycolysis produces abnormally elevated lactate, and the resulting lactation modification may be associated with ankylosing spondylitis (AS) lesions and participate in the formation of AS. This invention clarifies for the first time the regulatory mechanism of ASF1A on histone H3K18 lactation modification, effectively preventing histone H3K18 lactation modification and its mediated EndMT and AS occurrence. This provides a new avenue for drug development and a drug target for the diagnosis and treatment of AS, possessing significant pharmaceutical value. Substances that inhibit or knock out ASF1A expression hold promise as candidate drugs for the treatment of AS. Attached Figure Description

[0025] Figure 1 The expression level of ASF1A in HCAECs after ox-LDL induction: (e.g.) Figure 1 As shown, an AS disease model was established by treating HCAECs with 50 μg / mL ox-LDL to induce damage. Cells were collected after 24 h, and the expression level of ASF1A protein was detected by Western Blot. (n = 3; * p < 0.05)

[0026] Figure 2To investigate the effect of ASF1A knockout on the expression levels of Pan Kla, H3K18la, H3K9la, and Snail proteins in HCAECs induced by ox-LDL: Small interfering RNA (siRNA) was transfected into HCAECs to knock out ASF1A, followed by treatment with 50 μg / mL ox-LDL to induce damage. Cells were collected after 24 h, and total HCAEC proteins were extracted. Western blot analysis was used to detect the expression levels of Pan Kla, H3K18la, H3K9la, and Snail proteins. (n = 3; **p < 0.01, ***p < 0.001; ns, no significance)

[0027] Figure 3 To investigate the effect of ASF1A knockout on the expression levels of EndMT-related biomarkers after ox-LDL-induced HCAECs: (e.g.) Figure 3 As shown, HCAECs were treated with siRNA to knock out ASF1A, followed by stimulation with 50 μg / mL ox-LDL to induce damage. RNA samples were collected, and the expression levels of EndMT-related biomarkers were detected by quantitative real-time polymerase chain reaction (RT-qPCR). The results are presented in heatmap and bar chart format, with blue (low values) and red (high values). The color of each box in the heatmap corresponds to the mean of the corresponding three independent experimental data. (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001)

[0028] Figure 4 The effect of endothelial cell-specific knockout of Asf1a on AS formation: such as Figure 4 As shown, Apoe KO Asf1a WT and Apoe KO Asf1a ECKO Mice were fed a normal chow diet (NC; maintenance diet for laboratory mice, Synergy Biotech) or a high-fat diet (HFD; atherosclerosis model diet, 21% fat and 1.25% cholesterol, Synergy Biotech) for 12 weeks. Aortic vessels were isolated, and Oil Red O staining was used to detect aortic plaque formation. (n = 6 mice per group; ***p < 0.001)

[0029] Figure 5 The effects of endothelial cell-specific knockout of Asf1a on AS formation and EndMT: such as Figure 5 As shown, Apoe KOAsf1a WT and Apoe KO Asf1a ECKO Mice were fed either non-nuclear (NC) or high-frequency (HFD) diets for 12 weeks. Aortic vessels were isolated, and the aortic root was frozen-embedded for serial sectioning. Endothelial metaplasia (EndMT) was detected in mouse aortic vessel sections using dual immunofluorescence staining with the endothelial marker platelet-endothelial cell adhesion molecule-1 (CD31) (green) and the mesenchymal marker alpha-smooth muscle actin (α-SMA) (red). (n = 6 mice per group)

[0030] Figure 6 The effect of endothelial cell-specific knockout of Asf1a on EndMT: such as Figure 6 As shown, Apoe KO Asf1a WT and Apoe KO Asf1a ECKO Mice were either non-nuclear (NC) or high-temperature-dissipated (HFD) for 12 weeks. Aortic vessels were isolated, and mammary artery endothelial cells (MAECs) were extracted. RNA was extracted from the samples, and EndMT marker expression was detected by RT-qPCR. Results are presented in heatmap and bar chart format, with blue (low values) and red (high values). The color of each box in the heatmap corresponds to the mean of the 10 independent experimental data. (n = 10 mice per group; **p < 0.01, ***p < 0.001)

[0031] Figure 7 Effects of endothelial cell-specific knockout of Asf1a on the morphology of EndMT: such as Figure 7 As shown, Apoe KO Asf1a WT and Apoe KO Asf1a ECKO Mice were fed either non-nuclear (NC) or high-frequency (HFD) diets for 12 weeks. Aortic vessels were isolated, and metastatic endothelial cells (MAECs) were extracted. Apoe were observed under an electron microscope in NC or HFD mice. KO Asf1a WT and Apoe KO Asf1a ECKO Morphology of aortic endothelial cells in mice. (n = 6 mice per group; ***p < 0.001)

[0032] Figure 8 The effect of endothelial cell-specific knockout of Asf1a on histone lactation modification: such as Figure 8 As shown, Apoe KO Asf1a WTand Apoe KO Asf1a ECKO Mice were fed either non-nuclear (NC) or high-frequency (HFD) diets for 12 weeks. Aortic vessels were isolated, and metastatic endothelial cells (MAECs) were extracted. Total cellular protein was extracted, and Western blot analysis was used to detect the expression levels of PanKla, H3K18la, and H3K9la proteins. (n = 6 mice per group; ***p < 0.001; ns, no significance) Detailed Implementation

[0033] The following embodiments are intended to enable those skilled in the art to fully understand the present invention, but do not limit the invention in any way.

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

[0035] Example 1

[0036] 1.1 Induction of human coronary artery endothelial cells:

[0037] Human coronary artery endothelial cells were stimulated with 50 μg / mL ox-LDL for 24 h to induce injury and establish an AS endothelial injury cell model.

[0038] To investigate changes in the histone chaperone ASF1A in ankylosing spondylitis (AS), the applicant purchased human coronary artery endothelial cells (HCAECs) from the ScienCell research laboratory and cultured them in ECM medium containing 10% fetal bovine serum. After reaching 80%-90% confluence, the cells were passaged. An AS model was established by stimulating HCAECs with 50 μg / mL ox-LDL for 24 h, with an equal amount of PBS added as a control group. Total cell protein was then extracted, and the expression level of ASF1A protein was detected using Western blotting. The results are as follows: Figure 1 As shown, the expression level of ASF1A protein in HCAECs was significantly increased after ox-LDL stimulation-induced injury. Figure 1 The above experimental results suggest that ASF1A is upregulated in endothelial cells during the AS process.

[0039] Example 2

[0040] 2.1 Small interfering RNA (siRNA) transfection of human coronary artery endothelial cells

[0041] (1) HCAECs were seeded into small dishes for transfection. The transfection system is as follows:

[0042] A: ECM 125 μL + siRNA 6.25 μL

[0043] B: ECM 125 μL + lipo3000 3 μL

[0044] (2) Prepare solutions A and B above, mix them gently with a pipette, and let them stand at room temperature for 5 min. Mix the two solutions together, mix them gently with a pipette, let them stand at room temperature for 15-20 min, and then immediately transfect.

[0045] (3) Before transfection, replace the cells with preheated fresh complete culture medium, add the mixture from step (2) into the wells, and gently shake the plate to distribute the complex evenly.

[0046] (4) Incubate the cells at 37°C for 4-6 hours, then replace with a new preheated complete culture medium.

[0047] In this invention, siRNA was designed based on the ASF1A gene sequence. The siRNA sequence is: 5'→3'GAGCAGUAAUCCAAAUCUATT.

[0048] 3'→5'UAGAUUUGGAUUACUGCUCTT.

[0049] 2.2 Administer HCAECs ox-LDL stimulation

[0050] HCAECs were stimulated with 50 μg / mL ox-LDL for 24 h to induce lesions.

[0051] 2.3 RT-qPCR

[0052] (1) Collect the processed samples and extract total RNA according to the instructions in the Trizol kit. Wear a mask to avoid RNase to prevent RNA degradation, use an RNase-free pipette tip, and DEPC water;

[0053] (2) Wash the cells in the six-well plate with pre-cooled PBS and add 1 mL of Trizol to each well;

[0054] (3) After 10 s, transfer the cell lysis buffer to an EP tube and incubate on ice for 10 min for lysis;

[0055] (4) Add 200 μL of chloroform to each tube, mix by inverting, and place on ice for 10 min to lyse;

[0056] (5) Centrifuge at 4 ℃, 12000 rpm for 15 min;

[0057] (6) Carefully aspirate the supernatant into a new EP tube, add an equal volume of isopropanol, invert to mix, and place on ice for 10 min;

[0058] (7) Centrifuge at 4 ℃, 12000 rpm for 15 min, and discard the supernatant;

[0059] (8) Invert the paper to absorb the remaining liquid. A white feather-like precipitate will be visible. Add 75% ethanol diluted with DEPC water and mix by inverting.

[0060] (9) Centrifuge at 4 ℃, 12000 rpm for 15 min, and discard the supernatant ethanol;

[0061] (10) Remove residual ethanol and place the cell culture chamber in the ventilation hole of the clean bench for 5-10 minutes to air dry.

[0062] (11) Add 20 μL of DEPC water to dissolve the RNA, measure the RNA concentration with NanoDrop, and store in a -80 ℃ refrigerator for later use.

[0063] (12) Reverse transcription was performed using the Hifair® Ⅱ 1st Strand cDNA Synthesis Kit. The total reaction volume was 20 μL, and the specific composition is shown below:

[0064] Total RNA 1 μg 5×Hifair® Ⅱ Buffer plus 4 μL

[0065] (13) After mixing thoroughly, reverse transcription was performed using a PCR instrument:

[0066] 42 ℃ 30 min 85℃ 5 min

[0067] (14) After reverse transcription, the cDNA is diluted in 80 μL of DEPC water at a ratio of 1:4 and stored at -20 ℃ for later use.

[0068] (15) The target gene was relatively quantified using Hieff® qPCR SYBR Green Master Mix. The PCR reaction system is as follows:

[0069] Hieff® qPCR SYBR Green Master Mix 10 µL Upstream primer, 10 µM 0.4 µL Downstream primer, 10 µM 0.4 µL Sterile ultrapure water to 20

[0070] (16) Divide into groups and calculate the system (add cDNA at the end);

[0071] (17) Seal the plate, centrifuge the 384-well plate, and then place it in a real-time PCR instrument;

[0072] (18) The reaction was performed on a Bio-Rad 480II quantitative PCR instrument.

[0073] The primer sequences are as follows:

[0074] siASF1A Forward: GAGCAGUAAUCCAAAUCUATT

[0075] Reverse: UAGAUUUGGAUUACUGCUCTT

[0076] To further confirm the effect of histone chaperone ASF1A on histone lactation in HCAECs, the applicant cultured HCAECs and transfected them with ASF1A small interfering RNA for 48 h during the logarithmic growth phase. Cells were then stimulated with 50 μg / mL ox-LDL, and harvested 24 h later. Western blotting was used to detect the protein expression levels of Pan Kla, H3K18la, H3K9la, and the classical EndMT transcription factor Snail. Results are as follows: Figure 2 As shown, the deficiency of ASF1A significantly inhibited the increase in H3K18la and Snail protein levels induced by ox-LDL, as well as the EndMT process. Figure 2 ).

[0077] To further clarify the role of ASF1A in EndMT and AS, the applicant cultured HCAECs, transfected the cells with small interfering RNA of ASF1A during the logarithmic growth phase, and then stimulated them with 50 μg / mL ox-LDL. After 24 h, the cells were collected, and the expression level of EndMT markers was detected by RT-qPCR. During EndMT, the expression levels of endothelial markers platelet-endothelial cell adhesion molecule-1 (CD31), vascular endothelial cadherin (VE-cadherin), endothelial nitric oxide synthases (eNOS), and ocludin decreased, while the expression levels of VSMCs or mesenchymal stem cell-like markers such as neural cadherin (N-cadherin), calmodulin, alpha-smooth muscle actin (α-SMA), fibronectin 1, type I collagen 1A (Collagen 1A), and vimentin increased in endothelial cells. Results are as follows: Figure 3As shown, the lack of ASF1A significantly inhibited the decrease in the expression level of ox-LDL-induced endothelial cell-specific markers, while also inhibiting the increase in the expression level of ox-LDL-induced mesenchymal markers. Figure 3 The above results indicate that ASF1A deficiency significantly inhibits the ox-LDL-induced EndMT process.

[0078] Example 3

[0079] 3.1 Construction of Apoe cells with Asf1a-specific knockout in endothelial cells KO mouse (Apoe) KO Asf1a ECKO )

[0080] The Asf1a gene has six transcripts. Based on the structure of the Asf1a gene, exon 2 of Asf1a-201 (ENSMUST00000020004.8) is recommended as the gene knockout region. This region contains a 116 bp coding sequence. Knocking out this region will result in the disruption of gene function. In this project, we used CRISPR-Cas9 technology to modify the Asf1a gene. The brief process is as follows: The CRISPR-Cas9 system and Donor microinjection were performed into the fertilized eggs of C57BL / 6JGpt mice. After embryo transfer, positive F0 mice were obtained, and the genotype was confirmed by PCR and targeted amplicon sequencing. The positive F0 mice were mated with C57BL / 6JGpt mice to obtain a stable F1 mouse strain, and the required mutant alleles were confirmed by PCR and targeted amplicon sequencing. This yielded the Asf1a gene. flox / flox In mice, this gene constructs a flux structure by inserting loxp sequences flanking exon 2. In the cre-expressing state, cre, a nuclease, recognizes the flux structure upon entering the nucleus, circularizes it, and removes the sequence between the two loxp sequences, thus achieving exon knockout and frameshift mutation, preventing the normal transcription and translation of the target gene Asf1a. When mated with mice expressing Cre recombinase, the flux mice are knocked out, resulting in the loss of function of the target gene Asf1a in specific tissues and cell types. flox / flox Mice and endothelial cell-specific Cdh5-cre mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. C57BL / 6J and Apoe KO Mice were also purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and housed in a temperature-controlled SPF-grade animal facility. (The sentence fragment about Apoe appears unrelated and likely refers to a separate, incomplete thought.) KO Background: Asf1a floxed (Asf1a fl / flA mouse was obtained by crossing a mouse with a Cre recombinase system driven by the vascular endothelial adhesion protein (Cdh5) promoter (Cdh5-Cre) to obtain a mouse with a specific deletion of Asf1a in endothelial cells (Apoe). KO Asf1a ECKO ). (with Apoe from the same litter) KO Asf1a WT mouse (Apoe) KO Asf1a fl / fl As a control, mice were fed a normal chow (NC; maintenance diet for laboratory mice, Synergistic Bio) or a high-fat diet (HFD; atherosclerosis model diet, 21% fat and 1.25% cholesterol, Synergistic Bio) for 12 weeks to establish an AS mouse model and simulate the AS disease process.

[0081] 3.2 Gross Oil Red Staining

[0082] The AS mouse model established in 3.1 after 12 weeks of high-fat feeding was used to detect the area of ​​the overall plaque in the aorta of the mice by gross Oil Red O staining.

[0083] Gross Oil Red O staining procedure: The mouse aorta was dissected and removed. Perivascular adipose tissue was removed with forceps. The vessel was fixed with 4% paraformaldehyde for 10 min, followed by rinsing with PBS for 10 min. The dissected vessel was stained with Oil Red O working solution for 1 h, then removed and rinsed three times with 60% isopropanol. Fat plaques within the lumen appeared orange-red or bright red, while other areas were nearly colorless. Finally, the vessel was rinsed twice with distilled water. The stained gross tissue was spread and fixed on a glass slide and photographed under good lighting conditions.

[0084] Test results as follows Figure 4 As shown, Apoe fed with NC or HFD are compared. KO Asf1a ECKO and Apoe KO Asf1a WT Mice, Oil Red O staining showed that, relative to Apoe KO Asf1a ECKO Mouse, Apoe KO Asf1a WT Lipid burden in the mouse aorta was significantly reduced. Quantitative data from Oil Red O staining of mouse aortic vessels showed a reduction in plaque area after Asf1a knockdown in mouse endothelium. Figure 4 ).

[0085] 3.3 Angiographic Immunofluorescence

[0086] In addition, to further clarify the role of Asf1a in endothelial dysfunction and AS, the applicant isolated Apoe fed either NC or HFD. KO Asf1a WT and Apoe KO Asf1a ECKO Mouse aortic vessels were stained with CD31 (green) and α-SMA (red) double immunofluorescence on frozen sections of the aorta, and cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (blue).

[0087] The applicant first isolated the proximal portion of the ascending aorta from mice in different groups, and then embedded it with OCT frozen section embedding medium. PBS was pre-chilled at 4°C. The aorta was flash-frozen at -80°C and cut into 8 μm frozen sections. The frozen sections were baked at 55°C for 15 min, then fixed in 4% paraformaldehyde for 20 min, followed by inoculation with 0.1% Triton X-100 for 20 min to rupture the membrane. Subsequently, the samples were blocked with 10% BSA for 1 hour and incubated overnight at 4°C with a specific primary antibody. The next day, the samples were washed with PBS and then incubated with a secondary antibody at 37°C for 1 h. Finally, the cell nuclei were stained with DAPI at a ratio of 1:1000 for 10 min. Images were obtained using a laser confocal scanning microscope.

[0088] The results are as follows Figure 5 As shown, Apoe fed with HFD KO Asf1a WT In mice, the endothelial cell marker CD31 was significantly downregulated in the vascular intima and plaques, which was observed in Apoe KO Asf1a ECKO The effect was reversed in mice; the mesenchymal transition cell marker α-SMA showed the opposite phenomenon, suggesting that Asf1a may be related to the occurrence of endothelial dysfunction-induced EndMT. Figure 5 ).

[0089] Example 4

[0090] To confirm whether Asf1a affects the occurrence of EndMT in endothelial dysfunction, the applicant extracted samples from Apoe fed either NC or HFD. KO Asf1a WT and Apoe KO Asf1a ECKO The expression levels of EndMT-related markers in mouse aortic endothelial cells were detected using RT-qPCR. Results are as follows: Figure 6As shown, endothelial cell-specific knockout of Asf1a significantly inhibits the EndMT process. These findings further confirm the crucial role of endothelial cell Asf1a in histone lactation and the EndMT phenotype. Figure 6 ).

[0091] To determine the effects of endothelial cell-specific Asf1a knockout at the cellular level, the applicant extracted and cultured Apoe cells from NC-fed or HFD-fed cells. KO Asf1a WT and Apoe KO Asf1a ECKO MAECs in mice. Results are as follows: Figure 7 As shown, under high-fat feeding, compared with Apoe KO Asf1a ECKO Compared to mice, the Apoe KO Asf1a WT The cell morphology of mouse ECs was significantly altered, changing from oval to elongated spindle-shaped, exhibiting the classic EndMT cell morphology phenotype. Figure 7 ).

[0092] To verify the effect of endothelial cell-specific Asf1a knockout on histone lactation, the applicant extracted and cultured Apoe cells from NC-fed or HFD-fed cells. KO Asf1a WT and Apoe KO Asf1a ECKO MAECs in mice. The expression levels of PanKla, H3K18la, and H3K9la proteins were detected by Western blotting. Results are as follows: Figure 8 As shown, with Apoe KO Asf1a WT Compared to mice, the Apoe KO Asf1a ECKO The expression levels of Pan Kla and H3K18la proteins in mouse ECs were significantly increased. Figure 8 ).

[0093] The above experimental results suggest that Asf1a is involved in histone H3K18 lactation-mediated EndMT, thereby promoting the transformation of mouse endothelial cells to the EndMT phenotype.

[0094] The above experimental results fully demonstrate that ASF1A-dependent H3K18 lactation modification is involved in the progression of AS. By inhibiting H3K18la production through ASF1A inhibitors and downregulating histone H3K18 lactation modification, the occurrence and development of AS can be effectively suppressed. Therefore, we believe that ASF1A can serve as a new and important clinical target for the treatment of AS, and has potential clinical application value in the prevention and treatment of AS.

[0095] The above specific examples provide a detailed description of the present invention, but are merely for illustrating the technical concept and features of the invention. The aim is to enable those skilled in the art to understand and implement the invention, and should not be construed as limiting the scope of protection of the invention. The present invention is not limited to the above examples, and variations can be made without departing from the spirit and essence of the invention. All equivalent transformations or modifications made in accordance with the spirit of the invention should be covered within the scope of protection of the invention.

Claims

1. The use of ASF1A inhibitors in the preparation of drugs for the prevention and / or treatment of atherosclerosis, characterized in that, The ASF1A inhibitor is a small interfering RNA that inhibits or knocks out ASF1A expression, with sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

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