Application of 2 '-deoxycytidine in preparation of medicine for treating atherosclerosis

Abstract

The invention discloses an application of 2 '-deoxycytidine or a pharmaceutical salt thereof in preparation of a medicine for preventing or treating atherosclerosis. The invention develops a new application of the 2 '-deoxycytidine, and also provides a new thought for prevention and treatment of atherosclerosis related cardiovascular diseases. Researches find that 2 '-deoxycytidine can significantly inhibit macrophage lipid accumulation and reduce ApoE <- / -> mouse atherosclerotic plaque formation. What's more important is that the inhibition effect of 2 '-deoxycytidine on Dax1 is stronger than that of decitabine.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to an application of 2'-deoxycytidine in the preparation of a drug for treating atherosclerosis. Background Art

[0002] The nucleoside family includes a variety of natural nucleosides and nucleoside analogs [Mikhailopulo IA, et al. New trends in nucleoside biotechnology. Acta Naturae. 2010; 2 (2): 36-59.]. The chemical structure of nucleoside analogs is similar to that of natural nucleosides, but the base or sugar group is modified. They compete with normal nucleosides to interfere with cell DNA synthesis and inhibit the binding of related polymerases, thereby blocking tumor cell proliferation and viral replication [Pawlowska R, et al. Nucleoside and Nucleotide Analogues as Potential Therapeutics. Curr Med Chem. 2023; 30 (11): 1207-1208.]. Studies have reported that various nucleoside analogs play an important role in the treatment of tumors and viral infection-related diseases [Guinan M, et al. Recent Advances in the Chemical Synthesis and Evaluation of Anticancer Nucleoside Analogues. Molecules. 2020; 25(9): 2050. Geraghty RJ, et al. Broad-Spectrum Antiviral Strategies and Nucleoside Analogues. Viruses. 2021; 13(4): 667.].Among them, decitabine is widely used in the treatment of tumor-related diseases such as acute myeloid leukemia (AML) and myelodysplastic syndrome [Pollyea DA, et al. Venetoclax with azacitidine or decitabine in patients with newly diagnosed acute myeloidleukemia: Long term follow-up from a phase 1b study. Am J Hematol. 2021; 96(2): 208-217. Xu K, Hansen E. Novel agents for myelodysplastic syndromes. J Oncol Pharm Pract. 2021; 27(8): 1982-1992. Yamada Y, et al. Targeting DNA methylation and B7-H3 in RB1-deficient and neuroendocrine prostate cancer. Sci Transl Med. 2023Nov 15; 15(722): eadf6732.]. Recent studies have found that decitabine can also play a protective role in cardiovascular diseases, especially atherosclerosis. Dunn et al. reported that decitabine treatment reduced ApoE by inhibiting endothelial inflammation in mice. - / -aortic disease (Dunn J, et al. Flow-dependent epigenetic DNA methylation regulates endothelial gene expression and atherosclerosis. J Clin Invest. 2014; 124(7): 3187–3199.). In addition, decitabine can also demethylate FOXP3, leading to an increase in the number of regulatory T cells [Lal G, et al. Epigenetic regulation of Foxp3 expression in regulatory T cells by DNA methylation. J Immunol. 2009; 182(1): 259–273. LüCX, et al. FOXP3 demethylation as a means of identifying quantitative defects in regulatory T cells in acute coronary syndrome. Atherosclerosis. 2013; 229(1): 263–270.), thereby reducing atherosclerosis [Sasaki N, et al. Regulatory T cells in atherogenesis. JA theroscler Thromb. 2012; 19(6): 503–515.). Cao et al. reported that decitabine regulates methylation in two key events involved in the development of atherosclerosis and plaque vulnerability, thereby reducing atherosclerosis in Ldlr. - / - It has an anti-atherosclerotic effect in mice [Cao Q, et al. Inhibiting DNA Methylation by 5-Aza-2'-deoxycytidine ameliorates atherosclerosis through suppressing macrophage inflammation. Endocrinology. 2014; 155(12): 4925-38]. These evidences suggest that nucleoside drugs are expected to be used in the treatment of cardiovascular diseases related to atherosclerosis.

[0003] The chemical structure of nucleoside analogs is similar to that of natural nucleosides. So are there natural nucleosides that have a similar structure to decitabine and can exert anti-atherosclerotic effects?

[0004] Atherosclerosis is a chronic inflammatory disease associated with lipid metabolism disorders, characterized by the formation of lipid-rich atherosclerotic plaques on the vascular wall, leading to vascular stenosis or obstruction, and triggering acute cardiovascular events such as myocardial infarction and stroke [Kong P, et al. Inflammation and atherosclerosis: signaling pathways and therapeutic intervention. Signal Transduct Target Ther. 2022; 7(1): 131.]. Among them, macrophages are the core participants in the development of atherosclerosis, from recruitment and migration to the subendothelium to induce local inflammation, phagocytosis of oxidized low-density lipoprotein to form foam cells, to lipid plaque formation and late thrombosis, throughout the disease [Barrett TJ. Macrophages in Atherosclerosis Regression. Arterioscler Thromb Vasc Biol. 2020; 40(1): 20–33]. Understanding the biological process of macrophage dysfunction remains an important area to be studied and is also the basis for future treatment of diseases. In recent years, macrophage autophagy has attracted much attention. It is a lysosome-dependent catabolic pathway that helps maintain cellular homeostasis. In the early stage of atherosclerosis, macrophage autophagy promotes lipid degradation and transport, reduces inflammation, and plays an anti-atherosclerotic role [Zhang Y, et al. Inhibition of Hedgehog signaling ameliorates foam cell formation by promoting autophagy in early atherosclerosis. Cell Death Dis. 2023; 14(11): 740]. As the disease progresses, atherosclerotic plaques are characterized by macrophage autophagy-lysosome dysfunction, manifested as reduced macrophage lipid clearance ability, increased inflammation and increased cell death [Maiuri MC, et al. Macrophage autophagy in atherosclerosis. Mediators Inflamm. 2013; 2013: 584715.]. These evidences suggest that autophagy is essential for regulating macrophage lipid homeostasis and reducing the formation of foam cells and lipid plaques.

[0005] Currently, the benefits of inhibiting the progression of atherosclerosis by giving intensive lipid-lowering therapy (such as statins and PCSK9 inhibitors) are still limited [Pierno S, et al. Pharmacotherapy of the Lipid-Lowering Drugs: Update on Efficacy and Risk. Int J Mol Sci. 2023, 24 (2). Fan J, et al. Atherosclerosis: Known and unknown. Pathol Int. 2022; 72 (3): 151-160. Arnold N, Koenig W. Lipid Lowering Drugs in Acute Coronary Syndromes (ACS). Curr Atheroscler Rep. 2023; 25 (12): 939-946.]. In addition, according to existing literature and diagnosis and treatment guidelines, other drugs used to treat atherosclerosis include: blood pressure control drugs (ACE inhibitors such as enalapril, calcium channel blockers such as amlodipine), antiplatelet drugs (such as aspirin and clopidogrel).

[0006] Patent applications with publication numbers WO2020146263A1, US20210330734A1, and EP3908374A4 disclose a composition and method for diagnosing and treating neurodegenerative diseases, provide compositions including small molecule biochemicals and their salts and derivatives, and methods for detecting, treating or preventing neurodegenerative diseases, including methods for detecting and treating conditions that cause neurodegenerative diseases, including peripheral and central inflammation, amyloid-β deposition in the brain, neuronal iron deposition, and hyperglycemia.

[0007] So far, there is no literature reporting that 2'-deoxycytidine has the effect of treating atherosclerosis. Summary of the invention

[0008] The purpose of the present invention is to provide an application of 2'-deoxycytidine in preparing a drug for preventing or treating atherosclerosis.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides a use of 2'-deoxycytidine or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating atherosclerosis.

[0011] The CAS number of the 2'-deoxycytidine is 951-77-9, and the structural formula is as follows (National Center for Biotechnology Information. "PubChem Compound Summary for CID 13711, 2'-Deoxycytidine" PubChem, https: / / pubchem.ncbi.nlm.nih.gov / compound / 2_-Deoxycytidine.):

[0012]

[0013] The invention provides an application of 2'-deoxycytidine or a pharmaceutically acceptable salt thereof in preparing a drug for preventing or treating atherosclerosis, wherein 2'-deoxycytidine is used as the only active ingredient.

[0014] The present invention also provides a pharmaceutical preparation, which is prepared from 2'-deoxycytidine or a pharmaceutically acceptable salt thereof and medically acceptable auxiliary materials.

[0015] The dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation, nano preparation and the like.

[0016] The pharmaceutically acceptable salt refers to a salt formed by the reaction of 2'-deoxycytidine with a pharmaceutically acceptable inorganic acid or organic acid; wherein: the inorganic acid is at least one of hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid or sulfuric acid; the organic acid is at least one of formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, asiatic acid, carbenoxolone acid, glycyrrhetinic acid, oleanolic acid, hawthorn acid, ursolic acid, corosolic acid, betulinic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid or amino acid.

[0017] In previous in vivo animal experiments, the results of Oil Red O staining of aorta macroscopic and aortic root sections proved that 2'-deoxycytidine can significantly reduce the area of ​​atherosclerotic plaques in mice and increase plaque stability without significantly changing the content of blood lipids (cholesterol and triglycerides). At the same time, it has no significant effect on the morphology of liver, kidney, and spleen tissues. Further mechanism studies have found that 2'-deoxycytidine inhibits the expression of nuclear receptor Dax1, promotes macrophage cholesterol transport, and enhances autophagy, thereby inhibiting lipid accumulation and foam cell formation in macrophages.

[0018] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0019] The present invention has developed a new use for 2'-deoxycytidine and also provides a new idea for the prevention and treatment of cardiovascular diseases related to atherosclerosis. Studies have found that 2'-deoxycytidine significantly inhibits macrophage lipid accumulation and reduces ApoE – / – Importantly, Dax1 can inhibit the function of multiple protective nuclear receptors, and both 2'-deoxycytidine and decitabine can inhibit the expression of Dax1 in a concentration-dependent manner, and the inhibitory effect of 2'-deoxycytidine on Dax1 is stronger than that of decitabine (IC 50 112.5 μM and 292.4 μM, respectively), suggesting that 2'-deoxycytidine may exert its anti-atherosclerotic effect by inhibiting the expression of Dax1. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 2'-deoxycytidine inhibits ApoE – / – Schematic representation of the results of atherosclerosis in mice.

[0021] Figure 2 2'-deoxycytidine is a – / – Schematic diagram of the effects of the basal state of mice.

[0022] Figure 3 This is a schematic diagram showing the results that 2'-deoxycytidine inhibits the expression of nuclear receptor Dax1 in a concentration-dependent manner.

[0023] Figure 4 Macrophage-specific Dax1 knockout alleviates ApoE – / – Schematic representation of the results of atherosclerosis in mice.

[0024] Figure 5 Schematic diagram of the results of Dax1 inhibiting cholesterol transport and autophagy in macrophages.

[0025] Figure 6 This is a schematic diagram of the result that Dax1 inhibits autophagy by binding to the transcription factor TFEB.

[0026] Figure 7 Schematic diagram of the results that 2'-deoxycytidine enhances autophagy and reduces lipid accumulation by inhibiting Dax1. DETAILED DESCRIPTION

[0027] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0028] Example 1

[0029] Effect and mechanism of 2'-deoxycytidine in inhibiting atherosclerosis

[0030] 1. Experimental materials:

[0031] 2'-Deoxycytidine (Cat. No. T4758) and decitabine (Cat. No. T1508) were purchased from Shanghai Taoshu Biotechnology Co., Ltd. Physiological saline or PBS was used for dilution in animal experiments and cell experiments, respectively.

[0032] 2. Experimental methods and results

[0033] 1.2'-Deoxycytidine reduces ApoE – / – Atherosclerosis in mice

[0034] Animal experiment: 8-week-old male ApoE knockout (ApoE – / – ) Mice were purchased from Saiye Biotechnology Co., Ltd. 30 mice were randomly divided into 3 groups, 10 mice in each group, namely, normal saline group, 0.25 mg / kg 2'-deoxycytidine group and 0.5 mg / kg 2'-deoxycytidine group. The specific operation is as follows:

[0035] Normal saline group: ApoE – / – Mice were intraperitoneally injected with normal saline at a volume of 100 μL / mice / time, once every other day.

[0036] 0.25mg / kg 2'-deoxycytidine group: ApoE was administered at a dose of 0.25mg / kg body weight – / – The mice were intraperitoneally injected with 2'-deoxycytidine at a volume of 100 μL / mouse / time, once every other day.

[0037] 0.5mg / kg 2'-deoxycytidine group: ApoE was administered at a dose of 0.5mg / kg body weight – / – The mice were intraperitoneally injected with 2'-deoxycytidine at a volume of 100 μL / mouse / time, once every other day.

[0038] From the beginning of the experiment, all mice were changed to a high-fat diet (containing 40% fat and 1.25% cholesterol, product number D12109C, purchased from Research Diets, USA) for 12 weeks to establish an atherosclerosis model. After 12 weeks of high-fat feeding, blood samples were collected to measure blood lipid levels, and the mice were euthanized, and the heart and aorta were collected for histological evaluation and analysis to clarify the formation of atherosclerosis.

[0039] 1) Blood lipid detection: The serum of mice was collected after fasting for 12 hours, and the serum total cholesterol and triglyceride levels were detected by colorimetric method.

[0040] 2) Aortic pathology detection: The mouse heart was exposed, PBS was perfused through the left ventricle, and the heart and aorta were carefully separated and fixed with paraformaldehyde.

[0041] 3) Aortic gross plaque detection: The fixed aorta was cut longitudinally along the aortic arch-thoracic aorta-abdominal aorta-iliac artery bifurcation, immersed in Oil Red O working solution for staining at room temperature in the dark for 15 minutes, rinsed with 60% isopropanol for 1 minute, and washed twice with deionized water, 1 minute each time. The gross plaque area of ​​the aorta was analyzed after taking pictures.

[0042] 4) Staining of frozen sections of aortic root:

[0043] (1) Oil Red O staining to detect plaque area: Frozen sections were rewarmed, fixed with paraformaldehyde for 15 minutes, and then washed with deionized water. Stained in Oil Red O working solution at room temperature in the dark for 15 minutes, rinsed with 60% isopropanol for 1 minute, and washed twice with deionized water, 1 minute each time. Stained in hematoxylin solution for 1 minute, rinsed with tap water, differentiated in 1% hydrochloric acid alcohol for 1 to 3 seconds, soaked in tap water for 5 to 10 minutes to turn blue, and sealed with glycerol gelatin and photographed under a microscope.

[0044] (2) Masson staining to detect plaque stability: Frozen sections were rewarmed, fixed with paraformaldehyde for 15 minutes, and then washed with deionized water. Stained with Weigert's iron serotonin solution for 10 minutes, washed with 1% hydrochloric acid alcohol for about 5 minutes, and stained with Biebrich Scarlet-Acid Fuchsin staining solution for 10 minutes. Differentiated with 1% phosphomolybdic acid solution, then stained with aniline blue for 5 minutes, washed with 0.2% glacial acetic acid aqueous solution, gently rinsed with PBS, and mounted with glycerol gelatin and photographed under a microscope.

[0045] (3) Immunofluorescence staining to detect macrophage infiltration: frozen sections were rewarmed, fixed with paraformaldehyde for 15 minutes, washed twice with PBS, 5 minutes each time, permeabilized (0.2% Triton X-100 / PBS) for 15 minutes, blocked (10% goat serum) for 30 minutes, and incubated with primary antibody (anti-MOMA-2) at 4 degrees overnight. After washing with PBST, fluorescently labeled secondary antibody was added and incubated at room temperature for 1 hour. After washing with PBST, DAPI was added to stain the cell nucleus. After sealing with antifade sealing agent, the fluorescence microscope was used to take pictures.

[0046] 5) Hematoxylin-eosin (H&E) staining:

[0047] Mouse liver, kidney and spleen were fixed with paraformaldehyde and then paraffin sectioned. After the paraffin sections were baked at 70°C for 30 minutes, they were dewaxed in environmentally friendly dewaxing solution twice, 5 minutes each time. The sections were placed in 100%, 90%, 80%, and 70% ethanol for 5 minutes each for gradient hydration. The sections were stained with hematoxylin solution for 1 minute, rinsed with tap water, differentiated in 1% hydrochloric acid alcohol for 1 to 3 seconds, and soaked in tap water for 5 to 10 minutes to return to blue. The sections were stained in eosin solution for 15 seconds, dehydrated in 80%, 90%, 95% and 100% ethanol gradients for 5 minutes each, and transparentized with environmentally friendly dewaxing solution twice, 5 minutes each time. After sealing with neutral gum, the sections were photographed under a microscope.

[0048] The results are as follows Figure 1 As shown, Figure 1 2'-deoxycytidine inhibits ApoE – / – Schematic diagram of the results of atherosclerosis in mice. A is a schematic diagram of the experimental process of establishing an atherosclerosis model: 8-week-old ApoE – / – Mice were intraperitoneally injected with saline or 2'-deoxycytidine (2'-dC) (0.25 or 0.5 mg / kg, once every other day) and fed a high-fat diet for 12 weeks to induce atherosclerotic plaque formation (n=10 per group). – / – Representative images of mouse aorta stained with Oil Red O (n=10). C is ApoE in B. – / – Schematic diagram of statistical analysis of mouse aortic plaque area (n=10). Compared with the saline group, the aortic plaque area of ​​the 0.25mg / kg and 0.5mg / kg 2'-deoxycytidine treatment groups was significantly reduced. D is ApoE – / – Schematic representation of Oil Red O staining of the aortic root of mice (n=10). E is ApoE in D – / – Statistical analysis of Oil Red O staining of aortic root plaques in mice (n=10) is shown in Figure 1. Consistent with the results of gross staining of aorta, ApoE was significantly increased in mice treated with 2'-deoxycytidine (0.25 mg / kg and 0.5 mg / kg). – / – The plaque area in the aortic root of the mice was also significantly reduced. – / – Masson staining of mouse aorta root (magnification: 4x and 20x) represents schematic diagram (n=10), reflecting the stability of plaques. G is ApoE – / – Statistical analysis of collagen content in mouse aortic root (n=10) Schematic diagram. Compared with the saline group, the collagen content in the aortic root of the 0.25mg / kg and 0.5mg / kg 2'-deoxycytidine-treated groups increased significantly, indicating that the plaques were more stable. H is ApoE – / –Statistical analysis of the diameter of the necrotic core in the aortic root of mice (n=10) shows that the diameter of the necrotic core in the aortic root of the 0.25mg / kg and 0.5mg / kg 2'-deoxycytidine-treated groups was significantly reduced compared with the saline group, indicating an increase in plaque stability. I is ApoE – / – Representative schematic diagram of co-staining of macrophage marker protein (MOMA-2) and cell nucleus (DAPI) in mouse aortic root (n=10), reflecting the infiltration of macrophages in plaques. J is ApoE – / – Statistical analysis of MOMA-2 positive area in mouse aortic root (n=10) shows that compared with the saline group, the macrophage infiltration in the aortic root of the 0.5 mg / kg 2'-deoxycytidine treatment group was significantly reduced, indicating that the plaque inflammation response was reduced. The statistical significance of multiple group comparisons was tested by One-Way ANOVA. Data are expressed as mean ± standard error. *p<0.05; **p<0.01; ns indicates no significance.

[0049] Oil red O staining results showed that compared with the saline group, administration of 2'-deoxycytidine (0.25 mg / kg and 0.5 mg / kg) could significantly reduce ApoE – / – Aortic plaque in mice ( Figure 1 In addition, the collagen content increased and the necrotic core decreased in aortic root plaques of mice treated with 2'-deoxycytidine ( Figure 1 F to H). The decrease in macrophage infiltration was observed by MOMA-2 immunofluorescence staining ( Figure 1 This part of the results shows that 2'-deoxycytidine inhibits ApoE from reducing plaque formation, increasing plaque stability and reducing inflammation. – / – Atherosclerosis in mice, suggesting that 2'-deoxycytidine may have the effect of treating atherosclerosis.

[0050] ApoE was administered at doses of 0.25 mg / kg and 0.5 mg / kg body weight. – / – Mice were injected intraperitoneally with 2'-deoxycytidine (2'-dC) every other day and fed a high-fat diet for 12 weeks. After 12 weeks, blood samples were collected to measure lipid levels, and mice were euthanized and the heart and aorta were harvested for histological evaluation and analysis. Figure 2 2'-deoxycytidine is a – / – Schematic diagram of the effect of basal status of mice. – / – There is no significant effect on the basic state of mice. – / – Schematic diagram of the results of mouse body weight (n=10). B is ApoE – / –Schematic diagram of the spleen weight of mice (n=10). C is ApoE – / – Schematic diagram of mouse plasma total cholesterol level (n=10). D is ApoE – / – Schematic diagram of the results of mouse plasma triglyceride levels (n=10). E is ApoE – / – Representative schematic diagram of HE staining of mouse liver, kidney and spleen. Statistical significance of multiple group comparisons was tested by One-Way ANOVA. Data are expressed as mean ± standard error. ns: not significant.

[0051] 2'-deoxycytidine-treated ApoE – / – Body weight of mice and control mice ( Figure 2 (shown in A), spleen weight ( Figure 2 B), total cholesterol in plasma ( Figure 2 C) and triglycerides ( Figure 2 There was no difference in the levels of CKD (Fig. 5D), nor in the morphological observations of the liver, kidney, and spleen among the three groups ( Figure 2 E), indicating that administration of 2'-deoxycytidine has no obvious toxicity and side effects.

[0052] 2'-Deoxycytidine reduces the expression of nuclear receptor Dax1, while Dax1 expression is increased in macrophages of atherosclerotic plaques

[0053] 1) Cell experiment: Peritoneal macrophages of C57BL / 6J mice were isolated and cultured, and treated with gradient concentrations (0, 5, 10, 20, 40, 80, 160 μM of decitabine and 2'-deoxycytidine for 12 hours.

[0054] (1) Immunoblotting was used to detect the expression level of Dax1 and calculate the half inhibitory concentration (IC) of 2'-deoxycytidine 50 For subsequent experiments.

[0055] (2) Macrophages were treated with 2'-deoxycytidine and then immunofluorescence staining was performed. The expression and localization of Dax1 in macrophages were observed using a confocal microscope.

[0056] 2) Immunofluorescence detection of human vascular tissue: Dax1 expression in atherosclerotic and non-atherosclerotic vessels and its co-localization with macrophages (CD68), endothelial cells (CD31) and smooth muscle cells (α-SMA).

[0057] 3) Peripheral blood mononuclear cells (PBMCs) were isolated from healthy subjects and patients with coronary artery disease (CAD), and the expression level of Dax1 was detected by immunoblotting.

[0058] It has been reported that the anti-tumor drug decitabine (5-aza-2'-deoxycytidine) affects the methylation of Dax1 by recognizing the CpG site in the promoter region of the nuclear receptor Dax1, thereby reducing its expression [Liu J, et al. DNA methylation affects cell proliferation, cortisol secretion and steroidogenic gene expression in human adrenocortical NCI-H295R cells. J Mol Endocrinol. 2004; 33(3): 651–662.32].According to the literature, Dax1 is a corepressor of estrogen receptor (ER), NR4A1, peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptor α (LXRα) [Zhang H, et al. DAX-1 functions as an LXXLL-containing corepressor for activated estrogen receptors. J Biol Chem. 2000; 275(51): 39855-39859. Song KH, et al. The atypical orphan nuclear receptor DAX-1 interacts with orphan nuclear receptor Nur77 and represses its transactivation. Mol Endocrinol. 2004; 18(8): 1929-1940. Kim GS, et al. The orphan nuclear receptor DAX-1 acts as a novel transcriptional corepressor of PPARgamma. Biochem Biophys Res Commun.2008;370(2):264-268.Nedumaran B,et al.Orphan nuclear receptor DAX-1acts as a novel corepressor of liver X receptor alpha and inhibits hepatic lipogenesis.J Biol Chem.2010;285(12):9221-9232.16-19], thereby inhibiting the activity of these anti-atherosclerotic nuclear receptors. Therefore, Dax1 may cause these vascular protective factors to be weakened, suggesting that Dax1 may be related to atherosclerosis.

[0059] The results are as follows Figure 3 As shown, Figure 3The figure is a schematic diagram of the results that 2'-deoxycytidine inhibits the expression of nuclear receptor Dax1 in a concentration-dependent manner. A is a representative immunoblot image of Dax1 protein expression in peritoneal macrophages of wild-type mice treated with 0, 5, 10, 20, 40, 80, and 160 μM 5-aza-2'-deoxycytidine (5-Aza-dC) and 2'-deoxycytidine (2'-dC) (three independent repeated experiments). B is a schematic diagram of the statistical analysis of A, and the results show that the half-maximal inhibitory concentration (IC 50 ) is 112.5 μM, and the IC of decitabine 50 is 292.4 μM. C is a schematic diagram of confocal images of Dax1 immunofluorescence staining of peritoneal macrophages of wild-type mice treated with 2'-dC (112.5 μM) or PBS for 12 hours. D is a schematic diagram of the statistical analysis of Dax1 fluorescence intensity in peritoneal macrophages of wild-type mice treated with 2'-dC (112.5 μM) or PBS for 12 hours. E is a schematic diagram of the co-localization images and statistical analysis of Dax1 and macrophages (CD68) in non-atherosclerotic and atherosclerotic blood vessels detected by immunofluorescence staining. F is a schematic diagram of the co-localization images and statistical analysis of Dax1 and endothelial cells (CD31) in non-atherosclerotic and atherosclerotic blood vessels detected by immunofluorescence staining. G is a schematic diagram of the co-localization images and statistical analysis of Dax1 and smooth muscle cells (α-SMA) in non-atherosclerotic and atherosclerotic blood vessels detected by immunofluorescence staining. H is a representative figure of the protein expression of Dax1 in peripheral blood mononuclear cells (PBMC) of healthy subjects (n=16) and patients with coronary artery disease (CAD) (n=19) detected by immunoblotting. I is a representative figure of the protein expression of Dax1 in peripheral blood mononuclear cells (PBMC) of male and female CAD patients (n=18 in each group) detected by immunoblotting. J is a schematic diagram of the statistical analysis of Figure H. K is a schematic diagram of the statistical analysis of Figure I. The statistical significance of the comparison between the two groups was tested by Student's t test. Data are expressed as mean ± standard error. ***p<0.001; ns: not significant.

[0060] The present invention uses 2'-deoxycytidine, which is an analog of decitabine. It is worth noting that both decitabine and 2'-deoxycytidine reduce the level of Dax1 in macrophages in a concentration-dependent manner (0, 5, 10, 20, 40, 80, 160 μM), and the inhibitory effect of 2'-deoxycytidine is more obvious, with a half-maximal inhibitory concentration (IC 50 ) was 112.5 μM, while the IC 50 292.4μM( Figure 3 In addition, using confocal microscopy, it was observed that 2'-deoxycytidine significantly reduced the expression of Dax1 in the nucleus ( Figure 3 (as shown in C and D in Figure 1). This suggests that 2'-deoxycytidine may affect atherosclerosis by regulating the expression of Dax1. However, whether Dax1 is involved in atherosclerosis has not been studied. Therefore, the present invention first studies the relationship between Dax1 and atherosclerosis. The expression and localization of Dax1 in atherosclerotic blood vessels were determined by immunofluorescence staining. It is worth noting that compared with control blood vessels, the expression of Dax1 was significantly increased and was mainly located in macrophages in atherosclerotic plaques ( Figure 3 In addition, the level of Dax1 in peripheral blood mononuclear cells (PBMCs) of patients with coronary artery disease (CAD) was significantly higher than that in healthy controls ( Figure 3 As shown in I and J in Figure 1, the gene encoding Dax1, NR0B1, is expressed on chromosome X, the present invention further compares the Dax1 levels in PBMCs of different genders. There was no significant difference in the Dax1 levels between the two genders ( Figure 3 H and K in Figure 3). These results indicate that 2'-deoxycytidine can reduce the expression of nuclear receptor Dax1, while Dax1 expression is increased in macrophages of atherosclerotic plaques, suggesting that Dax1 may be associated with the development of atherosclerosis and that 2'-deoxycytidine may affect the progression of atherosclerosis by regulating Dax1 expression.

[0061] 3. Macrophage-specific Dax1 knockout inhibits atherosclerosis

[0062] In order to further determine whether Dax1 in macrophages mediates atherosclerosis, the present invention constructed macrophage-specific Dax1 knockout mice (Dax1 ΔMac / ApoE – / – ) and studied the effect of macrophage Dax1 deficiency on plaque formation in mice, taking into account gender factors. A model of atherosclerosis was established by high-fat diet for 12 weeks ( Figure 4 (shown in A).

[0063] Animal experimentation: Dax1 flox / flox and Lyz2-Cre mice were purchased from Jackson Lab and bred to generate macrophage-specific Dax1 knockout (Dax1 ΔMac ) mice, and then ApoE – / – Crossbreeding of mice to generate Dax1 ΔMac / ApoE – / – Mice, littermate controls are Dax1 WT / ApoE – / – Mice. 8-week-old Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / –Mice, 10 male and female mice in each group, were fed a high-fat diet for 12 weeks to establish an atherosclerosis model.

[0064] 1) Sampling: The mouse heart was exposed, PBS was perfused through the left ventricle, the heart and aorta were carefully separated, and then fixed with paraformaldehyde.

[0065] 2) Aortic gross plaque detection: The fixed aorta was cut longitudinally along the aortic arch-thoracic aorta-abdominal aorta-iliac artery bifurcation, immersed in Oil Red O working solution for staining at room temperature in the dark for 15 minutes, rinsed with 60% isopropanol for 1 minute, and washed with deionized water twice, 1 minute each time. The gross plaque area of ​​the aorta was analyzed after taking pictures.

[0066] 3) Staining of frozen sections of aortic root:

[0067] (1) Oil Red O staining to detect plaque area: Frozen sections were rewarmed, fixed with paraformaldehyde for 15 minutes, and then washed with deionized water. Stained in Oil Red O working solution at room temperature in the dark for 15 minutes, rinsed with 60% isopropanol for 1 minute, and washed twice with deionized water, 1 minute each time. Stained in hematoxylin solution for 1 minute, rinsed with tap water, differentiated in 1% hydrochloric acid alcohol for 1 to 3 seconds, soaked in tap water for 5 to 10 minutes to turn blue, and sealed with glycerol gelatin and photographed under a microscope.

[0068] (2) Masson staining to detect plaque stability: Frozen sections were rewarmed, fixed with paraformaldehyde for 15 minutes, and then washed with deionized water. Stained with Weigert's iron serotonin solution for 10 minutes, washed with 1% hydrochloric acid alcohol for about 5 minutes, and stained with Biebrich Scarlet-Acid Fuchsin staining solution for 10 minutes. Differentiated with 1% phosphomolybdic acid solution, then stained with aniline blue for 5 minutes, washed with 0.2% glacial acetic acid aqueous solution, gently rinsed with PBS, and mounted with glycerol gelatin and photographed under a microscope.

[0069] (3) Immunofluorescence staining to detect macrophage infiltration: frozen sections were rewarmed, fixed with paraformaldehyde for 15 minutes, washed twice with PBS, 5 minutes each time, permeabilized (0.2% Triton X-100 / PBS) for 15 minutes, blocked (10% goat serum) for 30 minutes, and incubated with primary antibody (anti-MOMA-2) at 4 degrees overnight. After washing with PBST, fluorescently labeled secondary antibody was added and incubated at room temperature for 1 hour. After washing with PBST, DAPI was added to stain the cell nucleus. After sealing with antifade sealing agent, the fluorescence microscope was used to take pictures.

[0070] The results are as follows Figure 4 As shown, Figure 4 Macrophage-specific Dax1 knockout alleviates ApoE – / –Schematic diagram of the results of atherosclerosis in mice. A is a schematic diagram of the process of establishing an atherosclerosis model: 8-week-old Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Mice, 10 male and female mice per group, were fed a high-fat diet for 12 weeks. B is a male Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic representation of Oil Red O staining of mouse aorta (n=10). C is a diagram of Dax1 in male mice shown in B. WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic diagram of statistical analysis of mouse aortic plaques (n=10), and Dax1 WT / ApoE – / – Compared with mice, Dax1 ΔMac / ApoE – / – The aortic plaque area of ​​mice was significantly reduced. D is male Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic representation of Oil Red O staining of the aortic root of mice (n=10). E is the Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Statistical analysis of aortic root plaque area in mice (n=10) and Dax1 WT / ApoE – / – Compared with mice, Dax1 ΔMac / ApoE – / – The aortic root plaque area of ​​mice was significantly reduced. F is a male Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic representation of Masson staining of mouse aortic root (magnification: 4x). G is the male Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic diagram of the statistical analysis of collagen content in mouse aortic root and Dax1 WT / ApoE – / – Compared with mice, Dax1 ΔMac / ApoE – / –Increased collagen content in aortic root plaques in mice. H is male Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic representation of Masson staining of mouse aortic root (magnification: 20 times). I is the male Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Statistical analysis of the diameter of the necrotic core in the mouse aortic root (n=10), and Dax1 WT / ApoE – / – Compared with mice, Dax1 ΔMac / ApoE – / – The diameter of the necrotic core of aortic root plaques in mice was significantly reduced. J is a male Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Representative schematic diagram of co-staining of macrophage marker protein (MOMA-2) and cell nucleus (DAPI) in mouse aortic root, reflecting the infiltration of macrophages in plaques. K is the male Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Statistical analysis of the MOMA-2 positive area in the mouse aortic root (n=10), and Dax1 WT / ApoE – / – Compared with mice, Dax1 ΔMac / ApoE – / – The infiltration of macrophages in the aortic root plaques of mice was significantly reduced, indicating a reduced inflammatory response. L is a female Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic representation of Oil Red O staining of mouse aorta (n=10). M is a female Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic diagram of statistical analysis of mouse aortic plaques (n=10), and Dax1 WT / ApoE – / – Compared with mice, Dax1 ΔMac / ApoE – / – The aortic plaque area of ​​mice was significantly reduced. N is female Dax1 WT / ApoE – / –and Dax1 ΔMac / ApoE – / – Schematic representation of Oil Red O staining of the aortic root of mice. O is the female Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Statistical analysis of aortic root plaque area in mice (n=10) and Dax1 WT / ApoE – / – Compared with mice, Dax1 ΔMac / ApoE – / – The aortic root plaque area of ​​mice was significantly reduced. P is female Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic representation of Masson staining of mouse aortic root (magnification: 4x). Q is the female Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic diagram of the statistical analysis of collagen content in mouse aortic root and Dax1 WT / ApoE – / – Compared with mice, Dax1 ΔMac / ApoE – / – Increased collagen content in aortic root plaques in mice. R is female Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Schematic representation of Masson staining of mouse aortic root (magnification: 20 times). S is the female Dax1 in R. WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Statistical analysis of the diameter of the necrotic core in the mouse aortic root (n=10), and Dax1 WT / ApoE – / – Compared with mice, Dax1 ΔMac / ApoE – / – The diameter of the necrotic core of aortic root plaques was significantly reduced in mice. T is female Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / –Representative schematic diagram of co-staining of macrophage marker protein (MOMA-2) and cell nucleus (DAPI) in mouse aortic root, reflecting the infiltration of macrophages in plaques. U is the female Dax1 WT / ApoE – / – and Dax1 ΔMac / ApoE – / – Statistical analysis of the MOMA-2 positive area in the mouse aortic root (n=10), and Dax1 WT / ApoE – / – Compared with mice, Dax1 ΔMac / ApoE – / – The infiltration of macrophages in the aortic root plaques of mice was significantly reduced, indicating that the inflammatory response was alleviated. The statistical significance of the two groups was compared by Student's t test. Data are expressed as mean ± standard error. *p<0.05; **p<0.01.

[0071] With Dax1 WT / ApoE – / – Compared with mice, male and female Dax1 ΔMac / ApoE – / – The atherosclerosis of mice was significantly reduced (p<0.05) ( Figure 4 Pathological examination of the aortic root also confirmed that knockout of macrophage Dax1 significantly reduced plaque area ( Figure 4 At the same time, the plaques of macrophage Dax1 knockout mice showed a significant increase in collagen content and a decrease in necrotic core ( Figure 4 F to I, P to S), macrophage infiltration (MOMA-2 positive area) was significantly decreased ( Figure 4 The results of the study suggest that the plaque stability was increased and the inflammatory response was reduced. These effects were consistent in male and female mice. The above results suggest that the knockout of Dax1 in macrophages significantly reduces atherosclerosis.

[0072] 4.Dax1 inhibits macrophage cholesterol transport and autophagy

[0073] 1) RNA sequencing: extraction of Dax1 WT and Dax1 ΔMac RNA was extracted from primary peritoneal macrophages of mice using TRIzol. After RNA quality inspection (purity, concentration and integrity), the library was constructed and sequenced using the Illumina Novasek 6000 platform (Shanghai Ouyi Biomedical Technology Co., Ltd.).

[0074] 2) Cell Oil Red O staining: Dax1 was treated with ox-LDL (80 μg / mL) WT and Dax1ΔMac Primary peritoneal macrophages of mice for 24 hours. Fix with paraformaldehyde for 20 minutes, wash with PBS 3 times, 1 minute each time. Add Oil Red O working solution and stain for 30 minutes at room temperature in the dark. Wash with deionized water 3 times, 1 minute each time. Counterstain the nucleus with hematoxylin for 1 minute, wash with running water for 5 minutes to turn blue, seal with glycerol gelatin and take pictures with a microscope.

[0075] 3) Detection of Dax1 by immunoblotting WT and Dax1 ΔMac The expression levels of cholesterol transport-related protein LXRα and its target molecules ATP-binding cassette transporter A1 (ABCA1), ABCG1 and autophagy-related proteins Beclin1 and LC3 in primary peritoneal macrophages of mice.

[0076] 4) Immunoprecipitation: HEK 293 cells were co-transfected with Dax1 overexpression plasmid (His-Dax1) and LXRα overexpression plasmid (Flag-LXRα). After 36 hours, the cells were lysed. Immunoprecipitation experiments were performed using anti-Flag or anti-His antibodies.

[0077] 5) Promoter dual luciferase reporter gene experiment: HEK 293 cells were co-transfected with ABCA1 promoter reporter plasmid, His-Dax1 and Renilla luciferase plasmids, and treated with LXRα agonist T0901317 (20 μM) at the same time. Cells were collected 36 hours later and dual luciferase reporter experiments were performed.

[0078] 6) Transmission electron microscopy: Dax1 was incubated with 80 μg / mL oxidized low-density lipoprotein (ox-LDL) WT and Dax1 ΔMac Mouse peritoneal macrophages for 24 hours. Fixed in 2.5% glutaraldehyde at room temperature for 1 hour and then placed at 4°C overnight. The collected macrophages were treated with osmium tetroxide, embedded in epoxy resin, cut into thin sections and counterstained with lead citrate. Hitachi TEM (HT7800, Hitachi, Japan) was used for observation and photography. The number of lipid droplets was counted and the diameter was measured using Image Pro Plus 6.0 software.

[0079] 7) Immunofluorescence staining of mouse aortic root plaques: The frozen sections of the aortic root were rewarmed to room temperature, fixed with paraformaldehyde for 15 minutes, washed twice with PBS, 5 minutes each time, permeabilized (0.2% Triton X-100 / PBS) for 15 minutes, blocked (10% goat serum) for 30 minutes, and incubated with primary antibodies (anti-LC3, anti-LAMP1, anti-MOMA-2) at 4 degrees overnight. After washing with PBST, fluorescently labeled secondary antibodies were added and incubated at room temperature for 1 hour. After washing with PBST, DAPI was added to stain the cell nucleus. After sealing with antifade sealing agent, the fluorescence microscope was used to take pictures.

[0080] The results are as follows Figure 5 As shown, Figure 5 This is a schematic diagram of the results of Dax1 inhibiting cholesterol transport and autophagy in macrophages. A is the RNA sequencing analysis of Dax1 WT and Dax1 ΔMac Differentially expressed genes in mouse peritoneal macrophages and GO enrichment analysis (n=3 per group) Schematic diagram showing that cholesterol metabolism and autophagy pathways are enriched in Dax1 knockout macrophages. B is Dax1 treated with ox-LDL (80 μg / mL) for 24 hours. WT and Dax1 ΔMac Representative schematic diagram of Oil Red O staining in mouse peritoneal macrophages. C is a schematic diagram of the statistical analysis of Figure B (n = 100 cells per group), and the results show that the Oil Red O staining area in Dax1 knockout macrophages is reduced, indicating reduced lipid accumulation. D is Dax1 WT and Dax1 ΔMacRepresentative immunoblot images of cholesterol transport-related proteins (LXRα, ABCA1, and ABCG1) and proinflammatory factor TNF-α expression levels in mouse peritoneal macrophages. E is a schematic diagram of the statistical analysis of D (n = 6 per group), and the results show that the levels of cholesterol transport-related proteins LXRα, ABCA1, and ABCG1 in Dax1-knockout macrophages are increased, and the expression of proinflammatory factor TNF-α is reduced, indicating that the cholesterol transport capacity of macrophages is enhanced and inflammation is reduced. F is a schematic diagram of the results of immunoprecipitation experiments using anti-Flag or anti-His antibodies after co-transfection of Dax1 overexpression plasmid (His-Dax1) and LXRα overexpression plasmid (Flag-LXRα) into HEK 293 cells, indicating that Dax1 interacts with LXRα. G is a schematic diagram of the results of co-transfection of ABCA1 promoter reporter plasmid and Dax1 overexpression plasmid (His-Dax1) into HEK293 cells, and treatment with LXRα agonist T0901317 (20 μM) for 36 hours, followed by dual luciferase reporter gene assay (n=3 per group), indicating that Dax1 reduces the transcriptional activity of LXRα downstream target gene ABCA1 by interacting with LXRα. H is Dax1 WT and Dax1 ΔMac Representative immunoblot images of the levels of Beclin1, LC3I / II, and LAMP1, a lysosomal marker protein, in peritoneal macrophages of mice. I is a schematic diagram of the statistical analysis of H (n=6 per group), showing that autophagy in macrophages knocked out of Dax1 was enhanced. J is a transmission electron microscopy image of Dax1 after 24 hours of treatment with ox-LDL (80 μg / mL). WT and Dax1 ΔMac Representative images of autophagosomes, lysosomes, and lipid droplets in mouse macrophages. K and L are schematic diagrams of the number and diameter of lipid droplets in J (n=10 per group), respectively. The results show that the number and diameter of lipid droplets in Dax1 knockout macrophages are reduced. M is Dax1 WT and Dax1 ΔMac Schematic diagram of representative immunofluorescence images of LC3, LAMP1 and MOMA-2 in mouse aortic root plaques. N is the percentage of LC3 / LAMP1 double-positive macrophages in the total number of macrophages (n=10 per group). The results show that Dax1 ΔMac The number of LC3 / LAMP1 double-positive macrophages in mouse aortic root plaques increased, indicating enhanced autophagy. The statistical significance of the two-group comparison was tested by Student's t test. The statistical significance of multiple group comparisons was tested by One-Way ANOVA. Data are expressed as mean ± standard error. *p<0.05; **p<0.01; ns: not significant.

[0081] In order to clarify the mechanism by which Dax1 promotes atherosclerosis, the present invention investigates the expression of Dax1 in WT and Dax1 ΔMac RNA sequencing was performed on peritoneal macrophages of mice. GO enrichment analysis showed that the differentially expressed genes between the two macrophages were enriched in the autophagy and cholesterol metabolism (including cholesterol efflux) pathways ( Figure 5 A number of evidences have shown that cholesterol metabolism disorders are the key pathological basis of atherosclerosis [Groenen AG, et al. Cholesterol efflux pathways, inflammation, and atherosclerosis. Crit Rev Biochem Mol Biol. 2021; 56(4): 426–439. Skeyni A, et al. Cholesterol trafficking, lysosomal function, and atherosclerosis. Am J Physiol Cell Physiol. 2024; 326(2): C473-C486. Poznyak AV, et al. Cholesterol Transport Dysfunction and Its Involvement in Atherogenesis. Int J Mol Sci. 2022; 23(3): 1332. 28-30]. Compared with control cells, Dax1 ΔMac Lipid accumulation in mouse peritoneal macrophages was significantly reduced ( Figure 5 Western blot analysis showed that knocking out Dax1 significantly upregulated the expression of LXRα and its downstream target molecules ABCA1 and ABCG1 in macrophages, and reduced the level of TNF-α ( Figure 5 In addition, the present invention observed that this interaction affected the transcriptional activity of the LXRα downstream gene ABCA1 ( Figure 5 These data suggest that the interaction of Dax1 with LXRα represses transcription of downstream target genes, thereby inhibiting cholesterol transport and promoting lipid accumulation in macrophages.

[0082] Immunoblotting results showed that the levels of proteins related to autophagosome formation (such as Beclin1, LC3 I / II) and lysosomal marker proteins (such as LAMP1) increased in Dax1-knockout macrophages ( Figure 5 H and I). After macrophages were incubated with ox-LDL, Dax1 WT Compared with the mice group, Dax1 was observed by transmission electron microscopy ΔMacThe number of autophagosomes and lysosomes increased in mouse macrophages, while the size and number of lipid droplets decreased ( Figure 5 The results of immunofluorescence staining showed that the expression of Dax1 WT Compared with mice, Dax1 ΔMac Increased autophagosomes (LC3) and lysosomes (LAMP1) in macrophages of mouse plaques Figure 5 M and N in the figure). Given that macrophage autophagy promotes lipid droplet degradation and cholesterol transport [Skeyni A, et al. Cholesterol trafficking, lysosomal function, and atherosclerosis. Am JPhysiol Cell Physiol. 2024; 326(2): C473-C486. Laval T, et al. A role for lipophagyin atherosclerosis. Nat Rev Cardiol. 2023; 20(7): 431–432. 29, 31], increased autophagy helps to enhance lipid processing capacity, thereby reducing Dax1 ΔMac Lipid accumulation in macrophages.

[0083] 5. Dax1 inhibits autophagy by binding to transcription factor EB (TFEB)

[0084] 1) Immunoprecipitation combined with mass spectrometry analysis of interacting proteins: HEK 293 cells were transfected with Dax1 overexpression plasmid (His-Dax1) or control empty vector plasmid, and the cells were lysed after 36 hours. Anti-His antibody was used for immunoprecipitation experiment, and the eluted protein was subjected to SDS-PAGE electrophoresis, stained with Coomassie brilliant blue, cut into gel for enzymatic digestion, and detected by mass spectrometry.

[0085] 2) Detection of Dax1 by immunoblotting WT and Dax1 ΔMac Expression levels of TFEB in primary peritoneal macrophages of mice.

[0086] 3) Immunoprecipitation: HEK 293 cells were co-transfected with Dax1 overexpression plasmid (His-Dax1) and LXRα overexpression plasmid (Flag-TFEB). After 36 hours, the cells were lysed. Immunoprecipitation experiments were performed using anti-Flag or anti-His antibodies.

[0087] 4) Promoter dual luciferase reporter gene experiment: HEK 293 cells were co-transfected with BECN1 (TFEB target gene) promoter reporter plasmid, His-Dax1 and Renilla luciferase plasmids, and TFEB agonist (1 μM) was given at the same time. Cells were collected 36 hours later and dual luciferase reporter experiments were performed.

[0088] The results are as follows Figure 6 As shown, Figure 6 Figure 1 is a schematic diagram of the results of Dax1 inhibiting autophagy by binding to the transcription factor TFEB. A is the experimental scheme: transfecting HEK 293 cells with His-Dax1 or control plasmid, performing immunoprecipitation experiments with anti-His antibodies, and then performing mass spectrometry analysis of proteins interacting with Dax1. B is a schematic diagram of the cellular localization of proteins interacting with Dax1. Red indicates nuclear proteins, dark blue indicates membrane proteins, and light blue indicates cytoplasmic proteins. C is a schematic diagram of the results of GO enrichment analysis of different nuclear proteins, indicating that nuclear proteins interacting with Dax1 are significantly enriched in autophagy regulation-related pathways. D is a schematic diagram of autophagy-related nuclear proteins (TFEB, PLEKHF1, MTMR9, TSPO, and SREBF1). E is a schematic diagram of the characteristic peptide spectrum of TFEB. F is a schematic diagram of the interaction strength of Dax1 with TFEB, PLEKHF1, MTMR9, TSPO, and SREBF1 predicted by binding affinity analysis, and the prediction results show that Dax1 has the strongest binding force with TFEB. G is a schematic diagram of the conformation of the complex formed by Dax1 and TFEB predicted by molecular docking. H is from Dax1 WT and Dax1 ΔMac Representative immunoblot images of TFEB protein expression in peritoneal macrophages of mice (n=6 per group). I is from Dax1 WT and Dax1 ΔMac Schematic diagram of statistical analysis of TFEB protein expression in peritoneal macrophages of mice (n=6 per group), showing that Dax1 does not alter TFEB expression. J is a schematic diagram of co-transfection of His-Dax1 and Flag-TFEB in HEK 293 cells for immunoprecipitation assays with anti-Flag or anti-His antibodies. K is a schematic diagram of the results of co-transfection of HEK 293 cells with BECN1 promoter reporter plasmid and His-Dax1, treatment with TFEB activator 1 (1 μM) for 36 hours, and then dual luciferase reporter gene assay (n=6 per group), indicating that the interaction of Dax1 with TFEB reduces the transcriptional activity of the TFEB target gene BECN1. The statistical significance of the comparison between the two groups was tested by Student's t test. The statistical significance of the comparison of multiple groups was tested by One-Way ANOVA. Data are expressed as mean ± standard error. ***p<0.001; ns: not significant.

[0089] In order to analyze the mechanism by which Dax1 affects autophagy, the present invention analyzes proteins interacting with Dax1 by immunoprecipitation and mass spectrometry. Figure 6 Among the 87 differentially identified proteins, 29 were nuclear proteins ( Figure 6 Further GO enrichment analysis results showed that nuclear proteins interacting with Dax1 were significantly enriched in autophagy regulation-related pathways ( Figure 6 C), including autophagy-related proteins TFEB, PLEKHF1, MTMR9, TSPO and SREBF1 ( Figure 6 Among them, mass spectrometry detected that TFEB has a characteristic peptide segment ( Figure 6 Binding force prediction analysis found that among the five autophagy-related nuclear proteins mentioned above, Dax1 has the strongest binding force with TFEB ( Figure 6 Molecular docking analysis showed that Dax1 formed a complex with TFEB through multiple hydrogen bonds ( Figure 6 However, Dax1 did not significantly regulate the expression of TFEB ( Figure 6 H and I). Through immunoprecipitation experiments, the present invention verifies the interaction between Dax1 and TFEB ( Figure 6 J), and clarified that the interaction between Dax1 and TFEB affects TFEB's transcriptional regulation of the downstream autophagy-related gene BECN1 ( Figure 6 K), thereby affecting the autophagic flow and reducing the ability of macrophages to process lipids, leading to lipid accumulation in macrophages.

[0090] 6.2'-Deoxycytidine promotes macrophage cholesterol transport and autophagy by inhibiting Dax1

[0091] 1) Peritoneal macrophages of C57BL / 6J mice were treated with 2'-deoxycytidine (112.5 μM) or PBS for 12 hours, and the levels of autophagy-related proteins (TFEB, Beclin1, etc.), cholesterol transport-related proteins (LXRα, ABCA1, etc.) and inflammatory factor interleukin-1β (IL-1β) were detected by immunoblotting.

[0092] 2) Cell Oil Red O staining: After pre-treating C57BL / 6J mouse peritoneal macrophages with 2'-deoxycytidine or PBS for 3 hours, add ox-LDL (80μg / mL) and continue incubation for 24 hours. Fix with paraformaldehyde for 20 minutes and wash with PBS 3 times, 1 minute each time. Add Oil Red O working solution and stain at room temperature in the dark for 30 minutes. Wash with deionized water 3 times, 1 minute each time. Counterstain the nucleus with hematoxylin for 1 minute, wash with running water for 5 minutes to turn blue, and take pictures with a microscope after sealing with glycerol gelatin.

[0093] 3) Autophagic flux detection: Macrophages were pretreated with 2'-deoxycytidine (112.5 μM) for 3 hours, then incubated with autophagosome and lysosome fluorescent probes DAPRed (0.1 μM) and DALGreen (1 μM) for 30 minutes, and cultured with ox-LDL (25 μg / mL) for 8 hours in the presence of 2'-deoxycytidine. Cellular autophagic flux was detected by laser scanning confocal microscopy (Leica TCS Sp8 STED, Leica Microsystems, Germany).

[0094] 4) Flow cytometry:

[0095] (1) Peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease (CAD) (n=31) were isolated using Ficoll-Paque PLUS density gradient medium.

[0096] (2) The isolated PBMCs were washed with HBSS, centrifuged, and suspended in RPMI 1640 medium containing 1% penicillin / streptomycin and 10% FBS. The PBMCs of each CAD patient were inoculated in two wells of a 12-well culture plate at a density of 2×10 6 cells / well.

[0097] (3) PBMCs from each CAD patient were pretreated with 2'-deoxycytidine (112.5 μM) or PBS for 3 h, incubated with ox-LDL (25 μg / mL) for 12 h, and stained with the lipid dye BODIPY (5 μM) for 30 min to visualize lipid droplets in the cells.

[0098] (4) Collect cells and wash with HBSS. Use fluorescently labeled antibodies anti-CD45-APC and anti-CD14-PE-cy7 to stain PBMC marker proteins. Use Beckman Coulter Cyto Flex S flow cytometer for flow cytometry, and use Flow Jo software to analyze BODIPY in PBMC. + The proportion of monocytes.

[0099] The results are as follows Figure 7 As shown, Figure 7Figure 1 is a schematic diagram of the results that 2'-deoxycytidine enhances autophagy and reduces lipid accumulation by inhibiting Dax1. Figure 2 is a schematic diagram of representative immunoblot images of wild-type mouse peritoneal macrophages treated with 2'-deoxycytidine (2'-dC, 112.5μM) or PBS for 12 hours to detect the expression of autophagy, cholesterol transport and inflammation-related proteins. Figure 3 is a schematic diagram of the statistical analysis of Figure 1 (n = 6 per group). The results showed that 2'-deoxycytidine did not affect the expression of TFEB, but it could increase the expression of proteins related to autophagy (Beclin1, LC3, etc.) and cholesterol transport (LXRα, ABCA1, etc.) and reduce the level of proinflammatory factor IL-1β. Figure 4 is a schematic diagram of representative images of lipid droplets stained with Oil Red O in peritoneal macrophages of wild-type mice pretreated with 2'-deoxycytidine (112.5μM) or PBS for 3 hours and then incubated with ox-LDL (80μg / mL) for 24 hours. Figure 5 is a schematic diagram of the statistical analysis of Figure 1 (n = 100 cells per group). E is a diagram of peritoneal macrophages of wild-type mice pretreated with 2'-deoxycytidine (112.5μM) or PBS for 12 hours, incubated with autophagosome and lysosome probes DAPRed (0.1μM) and DALGreen (1μM) for 30 minutes, and then treated with ox-LDL (25μg / mL) for another 8 hours. A schematic diagram of the colocalization of DAPRed and DALGreen in macrophages observed using a confocal microscope. F and G are schematic diagrams of the colocalization intensity tracks of DAPRed and DALGreen in macrophages treated with PBS and 2'-deoxycytidine (112.5μM) in Figure E, respectively. The results show that the colocalization intensity of DAPRed and DALGreen in macrophages treated with 2'-deoxycytidine increased, indicating enhanced autophagy. H is a schematic diagram of DAPRed in Figure E. + / DALGreen + The number of cells accounted for all DAPRed + Schematic diagram of statistical analysis of the percentage of cell numbers (n=8 per group), showing that DAPRed + / DALGreen +The increase in cell number indicates enhanced autophagy. I is a representative confocal image of autophagosomes (LC3), lysosomes (LAMP1), and lipid droplets (BODIPY) in wild-type mouse macrophages incubated with ox-LDL (25 μg / mL) for 24 hours after pretreatment with 2'-deoxycytidine (112.5 μM) or PBS for 3 hours. J is a schematic diagram of statistical analysis of the average fluorescence intensity of LC3, LAMP1, and BODIPY in Figure I (n = 100 cells per group). Compared with the PBS group, the fluorescence intensity of LC3 and LAMP1 in 2'-deoxycytidine-treated macrophages was enhanced, and the fluorescence intensity of BODIPY was weakened, indicating that autophagy was enhanced and intracellular lipid accumulation was reduced. K is the isolation and culture of peripheral blood mononuclear cells (PBMC) from patients with coronary artery disease (CAD), pretreated with 2'-deoxycytidine (112.5 μM) for 3 hours, then incubated with oxLDL (25 μg / mL) for 12 hours, stained with BODIPY (5 μM) for 30 minutes, and CD45 was detected by flow cytometry. + / CD14 + / BODIPY + L is a schematic diagram of the statistical analysis of Figure K, the results show that compared with the PBS group, the CD45 + / CD14 + / BODIPY + The decrease in cell proportions suggests that 2'-deoxycytidine significantly reduces lipid accumulation in PBMCs of CAD patients. The statistical significance of the two-group comparison was tested by Student's t test. The statistical significance of multiple group comparisons was tested by One-Way ANOVA. Data are expressed as mean ± standard error. **p<0.01; ***p<0.001; ns: not significant.

[0100] The above results clearly show that Dax1 promotes atherosclerosis by interacting with LXRα and TFEB, leading to abnormal cholesterol transport and autophagy in macrophages, which suggests that Dax1 may be a potential therapeutic target. In addition, the present invention has clearly shown that 2'-deoxycytidine significantly reduces the level of Dax1 in macrophages ( Figure 3 As shown in A to D, Figure 7 As shown in A and B in the figure, the present invention speculates that 2'-deoxycytidine can inhibit the expression of Dax1 in macrophages, improve cholesterol transport, and enhance autophagy, thereby alleviating atherosclerosis. The following is a related verification at the cellular level.

[0101] Immunoblotting results showed that 2'-deoxycytidine did not affect the expression of TFEB. However, it significantly increased the expression of TFEB downstream Beclin1, LC3 I / II, and LAMP1 in macrophages, while reducing the level of autophagy substrate P62, indicating that autophagy was enhanced after Dax1 inhibition. In addition, 2'-deoxycytidine promoted the expression of LXRα and its downstream ABCA1 and ABCG1, and reduced the level of IL-1β ( Figure 7 Oil red O staining revealed that 2'-deoxycytidine significantly reduced lipid accumulation in macrophages ( Figure 7 Using autophagosome and lysosome fluorescent probes (DAPRed and DALGreen), 2'-deoxycytidine increased autophagy in macrophages ( Figure 7 Immunofluorescence staining results showed that the formation of autophagosomes (LC3) and lysosomes (LAMP1) increased, while the number of lipid droplets (BODIPY) decreased in 2'-deoxycytidine-treated macrophages compared with control cells ( Figure 7 In addition, PBMCs were collected from CAD patients and pretreated with 2'-deoxycytidine for 3 hours before incubation with ox-LDL (at a concentration of 25 μg / mL) for 12 hours. Lipid droplets were stained with BODIPY and then subjected to flow cytometry. The results showed that lipid accumulation in PBMCs treated with 2'-deoxycytidine was significantly reduced compared with that in PBS-treated PBMCs ( Figure 7 Therefore, inhibition of Dax1 can increase lipid transport and autophagy in macrophages, which may be the key mechanism by which 2'-deoxycytidine reduces lipid accumulation in macrophages and thus exerts anti-atherosclerotic effects.

[0102] Results and Discussion:

[0103] Nuclear receptors are closely related to the pathophysiological process of cardiovascular diseases, among which Dax1 is a co-inhibitor of multiple anti-atherosclerotic nuclear receptors Nur77, PPARγ and LXRα. This effect leads to the weakening of vascular protective factors, suggesting that Dax1 may be related to atherosclerosis. The present invention shows that Dax1 promotes atherosclerosis. Further, the present invention clarifies the mechanism by which Dax1 promotes atherosclerosis, namely, by interacting with LXRα and TFEB, inhibiting lipid transport and autophagy, respectively. Thus, the present invention determines that 2'-deoxycytidine inhibits atherosclerosis and inhibits the expression of Dax1, while Dax1 promotes atherosclerosis, suggesting that 2'-deoxycytidine inhibiting Dax1 may be its way to exert anti-atherosclerotic effects, further verifying that 2'-deoxycytidine promotes macrophage lipid transport and autophagy, which may be an important mechanism for 2'-deoxycytidine to inhibit the formation of macrophage foam cells, thereby exerting anti-atherosclerotic effects.

[0104] Table 1 is a brief comparison of decitabine and 2'-deoxycytidine:

[0105] Table 1

[0106]

[0107] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. Use of 2'-deoxycytidine or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating atherosclerosis.

2. Use of 2'-deoxycytidine or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing or treating atherosclerosis, characterized in that: The CAS number of the 2'-deoxycytidine is 951-77-9, and the structural formula is as follows:

3. Use of 2'-deoxycytidine or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing or treating atherosclerosis, characterized in that: In the application, 2'-deoxycytidine is the only active ingredient.

4. A pharmaceutical preparation, prepared from the 2'-deoxycytidine or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 and a medically acceptable adjuvant.

5. The pharmaceutical preparation according to claim 4, characterized in that The dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation, and nano preparation.

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