Application of 4-octyl itaconic acid in preparation of medicine for preventing and treating AS

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of 4-octyl itaconic acid in preparation of a medicine for preventing and treating AS. In-vivo and in-vitro model research finds that 4-octyl itaconic acid further promotes ABCA1 expression by activating an NRF2-HO-1 / NQO1 antioxidant network, promoting formation of an NQO1-p53 compound, blocking ubiquitination degradation of p53 and improving p53 stability, so that cholesterol excretion of VSMCs is enhanced, formation of foam cells is reduced, and AS plaque lipid deposition is remarkably relieved. The invention systematically reveals the action mechanism of 4-OI in AS lesion for the first time, and provides a new treatment strategy and theoretical basis for early intervention of AS.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of 4-octyl itaconic acid in preparation of a medicine for preventing and treating AS. BACKGROUND

[0002] Atherosclerosis (AS) is a chronic inflammatory vascular disease characterized by lipid metabolism disorder, and the transformation of vascular smooth muscle cells into foam cells is an important link to promote the progression of the disease. The disease often appears in middle age or old age, and can involve the brain, heart, kidneys, other important organs and medium and large arteries of the legs. The causes of AS formation are complex, and dyslipidemia, hypertension, diabetes, genetic factors, etc. are the main risk factors. With the improvement of people's living standards and the change of eating habits, cardiovascular and cerebrovascular diseases caused by AS have become the main cause of global population death.

[0003] 4-octyl itaconate (4-OI) is a membrane permeable derivative of itaconic acid, which can activate Nrf2 by alkylation of KEAP1, and has strong anti-inflammatory, antioxidant, antiviral and cell protection effects. The structural formula of 4-octyl itaconate is shown as formula I, and the CAS number is 3133-16-2.

[0004]

[0005] I.

[0006] 4-OI has been proved to have anti-inflammatory, antiviral, metabolic regulation, and tissue damage protection effects in various cells and disease models. For example, CN120585811A patent reports that 4-OI can effectively improve pulmonary arterial hypertension, and in vivo experiments show that 4-OI can significantly reduce the thickening of pulmonary vascular wall and collagen deposition in PAH rats, and improve PAH and vascular remodeling; further in vitro experiments reveal that 4-OI can inhibit ERS through the PERK-eIF2a pathway, block the SM22-dependent smooth muscle cell phenotype transformation and EndMT process, and ultimately inhibit vascular remodeling, thereby playing a role in treating PAH. For another example, CN115400113A patent reports that 4-OI has the effects of inhibiting the activation of peritoneal macrophages and inhibiting the expression of pro-inflammatory factors, and can be used for prevention or treatment of ovarian cancer. For another example, CN112402446A patent reports that 4-OI can reduce inflammation and oxidative stress damage in sepsis acute lung injury caused by MRSA.

[0007] At present, the mechanism of 4-OI in AS lesions has not been elucidated. SUMMARY

[0008] Therefore, one of the purposes of the present application is to provide an application of 4-octyl itaconic acid in preparing a medicine for preventing and / or treating atherosclerosis. The present application has found that 4-octyl itaconic acid has the effect of improving atherosclerotic lesions.

[0009] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0010] The application of 4-octyl itaconic acid in preparing a medicine for preventing and / or treating atherosclerosis.

[0011] Preferably, the atherosclerosis includes atherosclerosis induced by hyperlipidemia.

[0012] Preferably, the medicine prevents and / or treats atherosclerosis by reducing the formation of VSMC-derived foam cells.

[0013] Preferably, the application of 4-octyl itaconic acid in preparing a medicine has any one of the following effects 1) to 5):

[0014] 1) inhibiting the progression of atherosclerotic lesions;

[0015] 2) inhibiting lipid accumulation in atherosclerotic plaques;

[0016] 3) reducing the area and distribution range of aortic plaque;

[0017] 4) reducing plaque vulnerability;

[0018] 5) reducing serum lipid levels;

[0019] 6) promoting cholesterol efflux.

[0020] Preferably, the reduction of serum lipid levels includes reducing the levels of serum total cholesterol, triglycerides and / or LDL.

[0021] Preferably, the reduction of plaque vulnerability specifically refers to down-regulating the expression level of matrix metalloproteinase 9 protein.

[0022] Preferably, the promotion of cholesterol efflux is to enhance ABCA1-mediated cholesterol efflux.

[0023] The second purpose of the present application is to provide an application of 4-octyl itaconic acid in preparing a foam cell formation inhibitor.

[0024] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0025] The application of 4-octyl itaconic acid in preparing a foam cell formation inhibitor.

[0026] Preferably, the foam cells are derived from vascular smooth muscle cells.

[0027] A third object of the present application is to provide a use of 4-octyl itaconic acid in the preparation of a medicament for preventing and / or treating atherosclerosis by inhibiting the formation of foam cells derived from VSMCs.

[0028] To achieve the above object, the present application adopts the following technical solutions:

[0029] 4-octyl itaconic acid in the preparation of a medicament for preventing and / or treating atherosclerosis by inhibiting the formation of foam cells derived from VSMCs. The present application researches and finds that 4-octyl itaconic acid can improve atherosclerotic lesions by inhibiting the formation of foam cells derived from VSMCs.

[0030] Preferably, the 4-octyl itaconic acid can improve atherosclerotic lesions by reducing the formation of foam cells derived from VSMCs.

[0031] Preferably, the 4-octyl itaconic acid can improve atherosclerotic lesions by activating the NRF2-HO-1 / NQO1 antioxidant network, promoting the formation of NQO1-p53 complex, inhibiting the ubiquitination degradation of p53, improving the stability of p53, further promoting the expression of ABCA1, and inhibiting the foam of vascular smooth muscle cells.

[0032] The present application researches and confirms that 4-octyl itaconic acid can reduce the formation of muscle-derived foam cells in AS plaques by enhancing ABCA1-mediated cholesterol efflux and reducing lipid accumulation in VSMCs. Specifically, 4-OI can reduce CD68 expression, up-regulate ACTA2 expression, and promote ABCA1 expression after treatment. Further researches find that 4-octyl itaconic acid can enhance ABCA1-mediated cholesterol efflux by up-regulating p53. Specifically, 4-OI can stabilize p53 / ABCA1 by activating the NRF2-HO-1 / NQO1 antioxidant pathway to inhibit the foam of VSMCs, including up-regulating the expression of NRF2 downstream antioxidant-related genes (such as HO-1, NQO1 and Gsta2), and enhancing the nuclear localization of NRF2, up-regulating the downstream antioxidant proteins HO-1 and NQO1. In addition, 4-OI can also promote NQO1-p53 interaction to inhibit p53 ubiquitination and stabilize the p53 / ABCA1 pathway.

[0033] A fourth object of the present application is to provide a pharmaceutical composition for preventing and / or treating atherosclerosis.

[0034] To achieve the above object, the present application adopts the following technical solutions:

[0035] A pharmaceutical composition for preventing and / or treating atherosclerosis, the pharmaceutical composition including 4-octyl itaconic acid and a pharmaceutically acceptable carrier or excipient.

[0036] As preferred, the dosage form of the medicine includes any one or more of tablets, capsules, granules, powders, oral liquids, pills, injections.

[0037] The present application has the beneficial effects in that:

[0038] 1、The present application combines in vivo and in vitro models to first explore the effect of 4-OI on the progression of AS and the formation of VSMC-derived foam cells. The experimental results show that 4-OI up-regulates NQO1 through the NRF2 signaling pathway, promotes the formation of NQO1-p53 complex, inhibits the ubiquitination and degradation of p53, and thus stabilizes the p53 / ABCA1 pathway, promotes the cholesterol efflux of VSMCs, reduces the formation of myogenic foam cells, and significantly reduces the lipid deposition of AS plaques. The present application first systematically reveals a new mechanism of 4-OI for inhibiting the foaming of VSMCs by activating the NRF2-HO-1 / NQO1 axis, stabilizing p53 and enhancing the ABCA1 expression mediated by p53, which not only deepens the understanding of the role of the p53 / ABCA1 pathway in the progression of AS, but also provides a theoretical basis for taking 4-OI and its regulated pathway as a target for early intervention of AS.

[0039] 2、The present application researches and confirms that 4-OI can not only improve atherosclerotic lesions and reduce plaque vulnerability, but also effectively reduce the formation of foam cells by regulating the foaming process of VSMCs, which has important significance for understanding the early occurrence and pathological process of AS, and provides a new direction for clinical treatment.

[0040] 3、The present application deeply explores the role and mechanism of the p53 / ABCA1 signaling pathway in the formation of VSMC-derived foam cells, deepens the understanding of the role of this pathway in the progression of AS, and provides a new theoretical basis for early intervention of AS. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The experimental results of 4-OI for improving atherosclerotic lesions and reducing plaque vulnerability, wherein, Figure 1 A is the detection result of IRG1 level in the aortic tissue of ApoE - / - mice fed with a regular diet (RD) or a high-fat diet (HFD), with GADPH as an internal reference; Figure 1 B is the result of Figure 1 A experimental results are quantified (***p<0.001); Figure 1-C represents the results of serological testing of serum total cholesterol (T-CHO) and triglyceride (TG) levels to evaluate the efficacy of 4-OI treatment (*p<0.05, **p<0.01, ***p<0.001). Figure 1 -D is a result image of gross Oil Red O staining of AS plaques in the aortic arch to verify the modeling and treatment effects; Figure 1 -E is for pairs Figure 1 -D experimental results quantified (****p<0.0001, ***p<0.001). Figure 1 -F is an image of an oil red O staining of a frozen section of the aortic root; Figure 1 -G is a result of HE staining, Sirius red staining and Masson staining of aortic root sections. HE staining of aortic root sections shows the area of ​​AS plaques, and Sirius red staining of aortic root sections shows the collagen content in AS plaques. Figure 1 -H is for pairs Figure 1 - The result of quantifying Masson staining in G (****p<0.0001); Figure 1 -I is for pairs Figure 1 - The result of quantifying the Sirius red staining results in G (****p<0.0001); Figure 1 -J is the result of WB detection of matrix metalloproteinase 9 (MMP9) level in tissue (with GADPH as internal control); Figure 1 -K represents pairs Figure 1 - The results of the J experiment were quantified (****p<0.0001, ***p<0.001).

[0042] Figure 2 The figure shows the results of Western blot and immunofluorescence detection of changes in the expression of foam cell-related markers. Figure 2 -A is the result of WB detection of CD68 and ACTA2 expression levels in tissue (with GADPH as an internal reference), which shows the degree of myogenic foamy transformation; Figure 2 -B indicates a pair Figure 2 -The results of quantifying the expression level of ACTA2 in A (**p<0.01); Figure 2 -C is for... Figure 2 -The results of quantifying CD68 expression levels in A (**p<0.01, ***p<0.001); Figure 2 -D is an immunofluorescence staining image of ACTA2 (green) and CD68 (red) on a section of aortic root; Figure 2 -E is for pairs Figure 2 -D The result of quantifying the relative positive area of ​​ACTA2 (*p<0.05, **p<0.01). Figure 2 -F indicates that -F is for...Figure 2 - Results plot for quantification of CD68 relative positive area in D (***p<0.001).

[0043] Figure 3 t-SNE plot for selecting human atherosclerotic plaque for dimensionality reduction clustering analysis, which shows 5 VSMCs subpopulations in human AS plaque (SMC1-5).

[0044] Figure 4 Results plot for analyzing CD68 and ABCAl gene expression levels in SMC5 cluster, wherein, Figure 4 - A is a smooth muscle cell marker gene TAGLN subtype plot; Figure 4 - B is a results plot for analyzing CD68 gene expression levels in SMC5 cluster; Figure 4 - C is a results plot for analyzing ABCAl gene expression levels in SMC5 cluster.

[0045] Figure 5 Results plot for 4-OI reducing foam cell formation by regulating ABCAl, wherein, Figure 5 - A is an immunofluorescence staining plot of ACTA2 (green), CD68 (red) and ABCAl (yellow) in aortic root section, which shows the existence of ABCAl+CD68+ VSMCs subpopulation in plaque area; Figure 5 - B is a results plot for WB detecting ABCAl expression levels in tissues (GADPH as internal reference); Figure 5 - C is a results plot for Figure 5 - B experimental results are quantified in the results plot (*p<0.05).

[0046] Figure 6 Results plot for Example 3, wherein, Figure 6 - A is a results plot for observing lipid droplet accumulation by BODIPY 493 / 503 staining; Figure 6 - B is a results plot for detecting CD68 and ACTA2 protein expression levels in cells, which shows the degree of myogenic foam-like transformation, with GADPH as internal reference; Figure 6 - C is a results plot for Figure 6 - B is a results plot for quantifying ACTA2 protein expression levels in B (*p<0.05,); Figure 6 - D is a results plot for quantifying CD68 protein expression levels in B (**p<0.01); Figure 6 - D is a results plot for quantifying CD68 protein expression levels in B (**p<0.01); Figure 6 - E is a results plot for WB detecting p53 expression levels in cells (GADPH as internal reference); Figure 6 - F is a results plot for Figure 6 - E experimental results are quantified in the results plot (**p<0.01); Figure 6-G is an immunofluorescence staining image of p53 (red) in cells; Figure 6 -H represents intracellular lipid droplets stained with Oil Red O to verify the modeling and the effect of 4-OI treatment. Figure 6 -I is for pairs Figure 6 -H The result of quantifying the relative positive area of ​​oil red O (*p<0.05); Figure 6 -J is the graph showing the validation results of siRNA knockout of p53 (protein expression level) (using GADPH as an internal control); Figure 6 -K represents pairs Figure 6 - The results of the J experiment were quantified (***p<0.001). Figure 6 -L is the result of WB detection of CD68 and ACTA2 protein expression levels under different group conditions (with GADPH as internal reference). Figure 6 -M is for pairs Figure 7 The results of quantifying the expression level of ACTA2 protein in -L (*p<0.05, ***p<0.001). Figure 7 -N represents pairs Figure 7 The results of quantifying CD68 protein expression in -L (ns indicates no statistical difference, ***p<0.001).

[0047] Figure 7 The figure shows the experimental results related to 4-OI upregulating p53 to enhance ABCA1-mediated cholesterol efflux. Figure 7 -A is the result of Oil Red O staining of intracellular lipid droplets to verify the effect of p53 knockout on the treatment effect of 4-OI; Figure 7 -B indicates a pair Figure 7 -Results of quantitative analysis of the relative positive area of ​​O in oil red in A (ns indicates no significant difference, **p<0.01); Figure 8 -C shows the results of WB detection of ABCA1 protein expression levels under different group conditions (with GADPH as an internal reference). Figure 9 -D indicates a pair Figure 10 - The results of the C experiment were quantified (ns indicates no significant difference, **p<0.01).

[0048] Figure 10 A heatmap showing the separation of gene expression profiles between the 4-OI group and the Ctrl group.

[0049] Figure 10 Volcano plot to detect the expression of p53 downstream target genes Hmox1, Cdkn1c and Pdgfrb in the 4-OI group.

[0050] Figure 11 The figure shows the results of GSEA analysis on the enrichment of porphyrin metabolic pathways and reactive oxygen species response pathways in the 4-OI group. Figure 11- A is the result graph of GSEA analysis of the enrichment of porphyrin metabolism pathway in the 4-OI group; Figure 11 - B is the result graph of GSEA analysis of the enrichment of reactive oxygen response pathway in the 4-OI group.

[0051] Figure 12 is the result graph of GO enrichment analysis, wherein, Figure 12 - A is the column chart of Top 30 GO terms of GO enrichment analysis of up-regulation; Figure 12 - B is the column chart of Top 30 GO terms of GO enrichment analysis of down-regulation; the results show that N-acetyllactosamine synthase activity and glutathione transferase activity are up-regulated, and cell chemotaxis and inflammatory response are down-regulated.

[0052] Figure 12 is the result graph of NRF2-HO-1 / NQO1 pathway related detection, wherein, Figure 12 - A is the result graph of cluster analysis showing the expression of Hmox1, NQO1 and Gsta2 in the 4-OI group; Figure 12 - B is the result graph of WB detection of the levels of NRF2, NQO1 and HO-1 in cells (GADPH as internal reference); Figure 12 - C is the result graph of quantification of HO-1 detection results in B (**p<0.01); Figure 12 - B is the result graph of quantification of HO-1 detection results in B (**p<0.01); Figure 12 - D is the result graph of quantification of NQO1 detection results in B (***p<0.001); Figure 13 - B is the result graph of quantification of NQO1 detection results in B (***p<0.001); Figure 13 - E is the result graph of quantification of NRF2 detection results in B (****p<0.0001). Figure 13 - B is the result graph of quantification of NRF2 detection results in B (****p<0.0001).

[0053] Figure 13 is the result graph of Example 5, wherein, Figure 13 - A is the result graph of WB detection of the expression levels of NQO1, p53 and ABCA1 in VSMCs under ox-LDL and dicoumarol (di) treatment (GADPH as internal reference); Figure 13 - B is the result graph of quantification of NQO1 detection results in A (***p<0.001); Figure 13 - B is the result graph of quantification of NQO1 detection results in A (***p<0.001); Figure 13 - C is the result graph of quantification of p53 detection results in A (****p<0.0001); Figure 13 - C is the result graph of quantification of p53 detection results in A (****p<0.0001);Figure 13 -D is para Figure 13 -A is a result graph of quantification of ABCA1 detection results in B; Figure 13 -E is a result graph of oil red O staining to detect the effect of di on the treatment effect of 4-OI; Figure 13 -F is a structure diagram of STRING interaction analysis of the functional correlation between NQO1 and p53; Figure 13 -G is a result graph of immunofluorescence staining to detect the expression content of NQO1 (red) and p53 (green) in VSMCs under ox-LDL and 4-OI treatment; Figure 14 -H is a result graph of Co-IP experiment for detecting specific bands of NQO1 in protein complexes pulled down by anti-p53 antibody (HA-p53); Figure 1 -I is a result graph of Co-IP experiment for detecting specific bands of p53 in protein complexes pulled down by anti-NQO1 antibody (FLAG-NQO1); Figure 1 -J is a result graph of WB for detecting the expression level of protein after IP combined with anti-ubiquitin antibody.

[0054] Figure 1 is a mechanism diagram of NRF2-HO1 / NQO1 and p53 / ABCA1 synergistic action. DETAILED DESCRIPTION

[0055] The technical solutions of the present application will be further clearly and completely described below in combination with specific examples. Obviously, the described examples are only some of the embodiments of the present application, not all the embodiments. Therefore, all other embodiments obtained by those skilled in the art based on the examples in the present application without creative labor are within the protection scope of the present application.

[0056] In the embodiments of the present application, the animal experiments and grouping are as follows:

[0057] 8-week-old male ApoE- / -mice (C57BL / 6J) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were raised in standard cages in an SPF environment, with a light / dark cycle of 12 hours, and could freely access food and water. All animal experiments were approved by the Animal Care and Use Committee of Chongqing Medical University, and all procedures were in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0058] Mice were randomly divided into three groups: regular diet group (RD), high-fat diet group (HFD) and 4-OI treatment group. The RD group was fed with standard mouse chow for 8 weeks, and the HFD and 4-OI treatment groups were fed with a "Western diet" containing 21% (wt / wt) fat and 0.15% (wt) cholesterol (sterilized by irradiation) for 8 weeks. During the last 4 weeks of high-fat chow feeding, the 4-OI treatment group was injected intraperitoneally with 4-OI (#HY-112675, MCE, 25 mg / kg, dissolved in corn oil), and the HFD group was injected intraperitoneally with an equal amount of corn oil solution.

[0059] For surgery and tissue collection, mice were anesthetized by continuous inhalation of 2.5% isoflurane gas, and the anesthesia process was carefully monitored to avoid pain or discomfort in the mice. During this process, the body temperature of the mice was maintained at 37 ± 0.5°C using a heating pad. The aorta was collected from the base of the ascending aorta to the bifurcation of the iliac artery for measuring the atherosclerotic lesions on the aortic surface. The entire aorta was stained with oil red O (Bi Yun Tian, C0158S) to quantify AS lesions, and the lesion area on the aortic surface was quantified by Image J software.

[0060] In the embodiments of the present application, the cell culture method is as follows:

[0061] Mouse aortic vascular smooth muscle cells (VSMCs) were purchased from Wuhan Sheng Biological. The vascular smooth muscle cells were cultured in DMEM medium containing 10% horse serum (#A5256701, Thermo Fisher Scientific) at 37°C, 5% CO2. To induce the formation of foam cells, the vascular smooth muscle cells were cultured in serum-free DMEM medium containing 2% FBS for 24 hours, and then treated with human oxidized low-density lipoprotein (ox-LDL, 50 μg / mL; Yiyuan Biotechnology, China) for 24 hours.

[0062] In the embodiments of the present application, the method for measuring the lipid content is as follows:

[0063] The levels of serum total cholesterol (TC, #A110-1-1) and triglycerides (TG, #A111-1-1) were determined by using a commercial detection kit (Nanjing Jiancheng Biological Engineering Institute, China) and following the manufacturer's instructions. The levels of high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were determined using a detection kit from Yiyuan Biotechnology (China).

[0064] In the embodiments of the present application, the oil red O staining and histological analysis method is as follows:

[0065] After dissection of the mice, aortic samples were fixed with 4% paraformaldehyde, then embedded in paraffin and stained with H&E. Meanwhile, formaldehyde-fixed aortic samples were embedded with O.C.T. compound (Sakura, 4583) and stained with oil red O. For cell experiments, cells were incubated with 4% paraformaldehyde and oil red O solution for 15 minutes at room temperature in the dark, then washed with 60% isopropanol and PBS. Images were acquired using a microscope scanner (UISCAN) and oil red O positive staining was quantitatively analyzed using Image J.

[0066] In the embodiments of the present application, the sirius red and Masson staining methods are as follows:

[0067] Paraffin-embedded sections (4 microns) of aortic tissue were stained with sirius red and Masson to evaluate collagen deposition. For sirius red staining, sections were incubated in 0.1% sirius red solution in saturated salicylic acid (Sigma-Aldrich) for 1 hour, then rinsed in 0.5% acetic acid and dehydrated by gradually diluted ethanol. For Masson staining, a standard staining kit (#G1340, Solbio Biological) was used according to the manufacturer's protocol. Images were acquired using a microscope scanner (UISCAN) and collagen-positive areas were quantitatively analyzed using ImageJ.

[0068] In the embodiments of the present application, the immunofluorescence staining method is as follows:

[0069] Paraffin-embedded tissue sections (4 microns) were first deparaffinated in xylene and then rehydrated by gradually diluted ethanol solution. Sections were heated in EDTA buffer (pH 8.0) at 95°C for 20 minutes to perform antigen retrieval. After cooling to room temperature, sections were blocked with 5% BSA in PBS for 1 hour to reduce non-specific binding. Sections were incubated with primary antibodies overnight at 4°C, then incubated with fluorescently labeled secondary antibodies for 1 hour at room temperature. Cell nuclei were counterstained with DAPI for 5 minutes. Images were acquired and analyzed using LAS X software (Leica, Germany).

[0070] In the embodiments of the present application, the protein extraction and Western blot method is as follows:

[0071] Total proteins were extracted from VSMCs using RIPA lysis buffer, and the protein concentration was determined using a BCA protein quantification kit (brand). The protein concentration of each group was adjusted to 1 μg / μL. Subsequently, the samples were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking the membrane with 5% skim milk, the primary antibody was incubated, followed by the secondary antibody. The average gray value of the bands was statistically analyzed using ImageJ software, and the gray value of group C was homogenized. All experiments were repeated 3 times.

[0072] In the embodiments of the present application, the co-immunoprecipitation method is as follows:

[0073] VSMCs were transfected with PEI (#23966-1, Polysciences) to express FLAG-labeled p53 protein (pCMV-Trp53(mouse)-3xFLAG-Neo) and HA-labeled NQO1 protein (pCMV-3xHA-Nqo1(mouse)-Neo), respectively, for 48 hours, and co-treated with 125 μM 4-OI (#HY-112675, MedChemExpress) and Human ox-LDL (50 μg / mL, Yiyuan Biotechnologies) for 24 hours. Cells were lysed in IP buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1% Triton-100, supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor (PMSF, sigma). Finally, the total cell lysate was incubated with the specified affinity gel (MCE) antibody.

[0074] In the embodiments of the present application, the BODIPY 493 / 503 staining method is as follows:

[0075] VSMCs were fixed with 4% paraformaldehyde for 15 minutes, and then incubated with BODIPY 493 / 503 (concentration of 2 μmol / L, HY-D1614, Med Chem Express, China) for 15 minutes in the dark. The nuclei were stained with DAPI. Fluorescence images were captured using LAS X software, and analyzed using Image J software.

[0076] In the embodiments of the present application, the small interfering RNAs (siRNAs) and plasmid transfection method is as follows:

[0077] siRNA (si-p53) was provided by Shanghai Biotechnology Co., Ltd. The sequence of siRNA is shown in Table 1. These constructs were transfected into vascular smooth muscle cells by using Lipofectamine 3000 carrier of Invitrogen Co.

[0078] Table 1. si-p53 sequence

[0079]

[0080] Note: If the sequence number and sequence itself are inconsistent with this table, please refer to this table.

[0081] In the embodiments of the present application, the public database single cell data analysis method is as follows:

[0082] The keyword "atherosclerosis" was searched in the GEO database, and the expression profiles (GSE213740, GSE131778) containing AS lesions and healthy aortic tissue samples were screened. In R language 4.4.2, the uniform manifold approximation and projection (UMAP) algorithm was used for dimension reduction, the Findclusters function was used to identify clusters, the FindAllMarkers function was used to identify cluster-specific marker genes (avg_log2FC > 0.25), and finally the Nebulosa package was used for SMC subpopulation visualization.

[0083] In the embodiments of the present application, the RNA-seq and bioinformatics analysis method is as follows:

[0084] RNA-seq was performed on 4-OI treated muscle-derived foam cell samples (4-OI group) and untreated control group (Ctrl group). DESeq2 method was used to analyze differential genes (DEGs, p<0.05). Volcano plot of differential genes, gene set enrichment analysis (GSEA) were completed using the Omeicloud platform. The p value of DEG used to generate the volcano plot, clustering analysis of all significant DEGs, GESA analysis were all <0.05, |log2FC|>1.5.

[0085] In the embodiments of the present application, the statistical analysis method is as follows:

[0086] All data were expressed as mean ± standard deviation. After testing normal distribution and homogeneity of variance, single factor analysis of variance (ANOVA) and Student's t test were used for analysis. p<0.05 was considered to be significantly different. The statistical analysis used was completed using GraphPad Prism10 software (San Diego, USA).

[0087] Example 1. 4-OI improves atherosclerotic lesions and reduces plaque vulnerability

[0088] A recent study reported that IRG1 expression was up-regulated in the aortas of AS patients. Consistent with this report, our experimental results showed that IRG1 expression was significantly up-regulated in the aortas of AS mice in a high fat diet (HFD)-fed ApoE- / - mouse model of AS, as shown in detail in Figure 1 A and Figure 1 B.

[0089] To explore the potential function of IRG1 in HFD-induced AS, the inventors supplemented its metabolite itaconic acid (which is encoded by IRG1 to exert its biological effects) exogenously and further evaluated its effect on the progression of AS. Serological tests showed that the contents of total cholesterol, triglycerides and LDL in the serum of 4-OI group mice were significantly lower than those of the AS model group, and the content of HDL was significantly higher than that of the AS model group, suggesting that 4-OI can effectively reduce serum lipid levels, as shown in detail in Figure 1 C. HE and oil red O staining results showed that the plaque area and distribution range of the aortas of 4-OI group mice were significantly reduced compared with the AS model group. Sirius red staining and Masson staining results showed that collagen deposition in the plaque was significantly reduced after 4-OI treatment, as shown in detail in Figure 1 D Figure 2 I, in addition, the protein expression level of matrix metalloproteinase 9 (a marker of plaque vulnerability in AS) was significantly down-regulated, as shown in detail in Figure 3 J and Figure 4 K, indicating that 4-OI can enhance plaque stability. The above results show that 4-OI treatment improves lipid metabolism disorder in AS mice to some extent, reduces local lipid deposition, and may reduce plaque vulnerability and inhibit lesion progression.

[0090] Example 2. 4-OI may reduce foam cell formation by regulating ABCA1

[0091] Foam cells are one of the main sources of lipid accumulation in AS plaques. This embodiment further detects the expression changes of foam cell-related markers to explore whether 4-OI plays a role by affecting foam cell formation. Western blot and immunofluorescence results showed that compared with the control group (RD), the expression of foam / macrophage marker CD68 was significantly up-regulated and the expression of vascular smooth muscle cell marker actin alpha 2 (ACTA2) was significantly down-regulated in the AS model group of mice. 4-OI intervention significantly reduced CD68 expression and up-regulated ACTA2 expression, as shown in detail in Figure 5 , suggesting that 4-OI may be involved in the improvement of AS lesions by reducing foam cell formation.

[0092] During the process of AS plaque formation, foam cells derived from VSMCs can account for more than 80% of the total foam cells in advanced lesions. Therefore, the changes in CD68 and ACTA2 expression can reflect the changes in the proportion of myogenic foam cells in the plaque. To further clarify this possible mechanism, the present application searches the GEO database for the keywords "atherosclerosis" and "vascular smooth muscle cells", screens and analyzes single-cell transcriptome sequencing data containing AS and healthy aortic samples. The analysis results show that there are 5 VSMC subgroups in AS plaques, as shown in Figure 5 , wherein the expression levels of CD68 and ATP-binding cassette transporter A1 (ABCA1) in the SMC2 subgroup are significantly up-regulated, as shown in Figure 5 . Further confirmed by immunofluorescence staining, there is a subgroup of ABCA1⁺CD68⁺ VSMCs in the AS plaque area, as shown in Figure 6 -A. In addition, the Western blot results show that compared with the control group, the expression of ABCA1 in the aorta of the AS model group mice is significantly down-regulated, and after 4-OI treatment, the expression level can be significantly up-regulated, as shown in Figure 6 -B and Figure 6 -C. ABCA1 is a key cholesterol transporter protein that has been shown to play an important role in regulating intracellular cholesterol efflux and preventing lipid accumulation. The above results show that 4-OI treatment can reduce the formation of myogenic foam cells in AS plaques, which may be related to the enhancement of ABCA1-mediated cholesterol efflux and the reduction of lipid accumulation in VSMCs.

[0093] Example 3. 4-OI up-regulates p53 to enhance ABCA1-mediated cholesterol efflux

[0094] To further clarify the upstream regulatory mechanism of 4-OI promoting ABCA1 expression and enhancing cholesterol efflux, this embodiment uses oxidized low density lipoprotein (ox-LDL) to stimulate VSMCs to construct an in vitro myogenic foam cell model. The lipid droplet accumulation is observed by BODIPY 493 / 503 staining, as shown in Figure 6 -A, and the protein expression levels of related markers CD68 and ACTA2 are detected, as shown in Figure 6 -B Figure 6-D, confirming the successful establishment of the model. Studies have shown that p53 plays a key regulatory role in various diseases, and its loss of function or mutation is closely related to a significant increase in AS susceptibility. Notably, p53 can promote cholesterol efflux and inhibit intracellular cholesterol accumulation by regulating the ABCA1 / SREBP-2 pathway, thereby regulating cell lipid metabolism. This effect is initially seen in tumor-related research, but considering the importance of cholesterol homeostasis in AS progression, we speculate that p53 may also be involved in the AS pathological process by regulating ABCA1 expression, thereby mediating the effects of 4-OI on VSMC cholesterol efflux and foam formation. Therefore, the present application further detects the expression of p53 in myogenic foam cells and contractile VSMCs. Western blot and immunofluorescence show that p53 expression is significantly down-regulated after foam induction, and 4-OI treatment can effectively restore its expression, as shown in Figure 6 -E Figure 6 -G. Cell oil red O staining shows that 4-OI can reduce ox-LDL-induced VSMC foam formation, as shown in Figure 6 -H and Figure 6 -I. To further clarify the key role of p53 in the regulatory effect of 4-OI, we used siRNA to knock down the expression of p53 in VSMCs. Western blot verified the effectiveness of the knockdown, as shown in Figure 7 -J and Figure 7 -K, accompanied by down-regulation of ACTA2 and up-regulation of CD68, as shown in Figure 7 -L Figure 7 -N, suggesting that down-regulation of p53 can promote VSMC foam formation. Oil red O staining results further show that the reversing effect of 4-OI on foam formation is basically eliminated after p53 knockdown, as shown in Figure 8 -A and Figure 9 -B, indicating that the absence of p53 weakens the protective effect of 4-OI. Western blot results show that p53 knockdown significantly inhibits the up-regulation of ABCA1 induced by 4-OI, as shown in Figure 10 -C and Figure 11 -D. The above results show that the regulatory effect of 4-OI on ABCA1-mediated cholesterol efflux is achieved by up-regulating p53, and the p53 / ABCA1 pathway may play an important role in the process of VSMC foam formation.

[0095] Example 4. 4-OI stabilizes p53 / ABCA1 to inhibit VSMC foam formation by activating the antioxidant pathway

[0096] To further explore the potential mechanism of 4-OI regulating p53 / ABCA1 axis to affect the formation of muscle-derived foam cells, the inventors performed RNA-seq on the ox-LDL-induced muscle-derived foam cell model (Ctrl group) and the 4-OI treatment group (4-OI group). The results showed that there were 290 differentially expressed genes between the two groups (p<0.05, |log2FC|>2), and clustering analysis showed that the gene expression profiles were significantly separated, as shown in Figure 12 . Through comparison with the TRRUST database, it was found that p53 upstream molecules Hmox1 (encoding HO-1), Cdkn1c and Pdgfrb were significantly up-regulated in the 4-OI group, as shown in Figure 12 . Among them, Cdkn1c and Pdgfrb are cell cycle and proliferation factors regulated by p53, and their increased expression helps to maintain the contractile phenotype of VSMCs, thereby inhibiting their foam. GSEA showed that the porphyrin metabolism and ROS response pathways were significantly enriched in the 4-OI group, as shown in Figure 12 , suggesting that 4-OI not only activates the p53 downstream network, but also synchronously mobilizes the antioxidant defense mechanism of the cell. Hemeoxygenase-1 (HO-1) is the core enzyme of porphyrin metabolism, and its degradation product can scavenge ROS and exert antioxidant protection. Studies have shown that the transcriptional activation of HO-1 depends on the regulation of nuclear factor erythroid 2-related factor 2 (NRF2), and the IRG1-itaconate axis is an important activation pathway of NRF2. NRF2 is a core transcription factor for cellular antioxidant stress response, and has been confirmed to play an antioxidant role through HO-1, NAD(P)H Quinone Dehydrogenase 1 (NQO1) and Glutathione S-transferase alpha 2 (Gsta2). RNA-Seq analysis results showed that Hmox1, NQO1 and Gsta2 were significantly up-regulated in the 4-OI group, as shown in Figure 13 and Figure 13 -A. Western blot further verified that 4-OI significantly enhanced the nuclear localization of NRF2 and up-regulated the protein expression of HO-1 and NQO1, as shown in Figure 13 -B Figure 13 -E, indicating that the NRF2-HO-1 / NQO1 pathway was activated.

[0097] Example 5. 4-OI inhibits p53 / ABCA1 pathway by promoting NQO1-p53 interaction to stabilize p53 ubiquitination

[0098] Based on the experimental results of Examples 1-4, the present application focuses on the key effector molecule NQO1 downstream of NRF2 for research, aiming to further explore the specific mechanism of 4-OI activating the NRF2 / NQO1 pathway and then regulating the p53 / ABCA1 pathway. First, we found that in the ox-LDL-induced myogenic foam cell model, after adding the NQO1 inhibitor dicumarol, the up-regulation of p53 and ABCA1 by 4-OI was significantly weakened: Western blot results showed that compared with the untreated myogenic foam cell group, the p53 protein expression level in the dicumarol group was significantly decreased, and the expression of ABCA1 was also not significantly increased. Oil red O staining results showed that the intracellular lipid droplet accumulation in the dicumarol group was aggravated, suggesting that inhibiting NQO1 weakened the protective effect of 4-OI on foam cell formation, see Figure 13 -A Figure 13 -F. In addition, immunofluorescence staining showed that NQO1 and p53 in myogenic foam cells treated with 4-OI were significantly co-localized, suggesting that they might form a functional complex, see Figure 14 -G. Co-IP experiments further confirmed this speculation: the specific band of NQO1 was detected in the protein complex pulled down using anti-p53 antibody, suggesting that 4-OI promoted the formation of NQO1-p53 complex, see ​ -H and ​ -I.

[0099] Previous studies have shown that the formation of NQO1-p53 complex can inhibit the ubiquitination modification-proteasome degradation pathway of p53, and increase the expression level of p53 protein in cells. To verify whether this mechanism is involved in the regulation of p53 by 4-OI, anti-ubiquitin antibody was used for IP, and the protein expression level was detected by Western blot. The results showed that: compared with the contractile VSMCs, the ubiquitination level of p53 in ox-LDL-induced myogenic foam cells was significantly increased, and 4-OI treatment could reduce the ubiquitination level of p53; when NQO1 was inhibited, the above inhibitory effect was significantly weakened, and the ubiquitination level of p53 was close to that of untreated myogenic foam cells, see ​ -J. The above results show that 4-OI mainly inhibits the ubiquitination degradation of p53 by enhancing NQO1-p53 binding, thereby increasing the stability of p53.

[0100] In summary, the present application found that 4-OI activates the NRF2-HO-1 / NQO1 antioxidant network, promotes the formation of NQO1-p53 complex and blocks the ubiquitination of p53, thereby increasing the stability of p53, further promoting the expression of ABCA1, and ultimately inhibiting the foam of VSMCs, and the related mechanism diagram is as follows ​The mechanism provides clear upstream regulatory basis for the anti-AS effect of 4-OI, and further emphasizes the core position of p53 / ABCA1 pathway in regulating VSMCs foam.

Claims

1. Use of 4-octyl itaconic acid in the preparation of a medicament for preventing and / or treating atherosclerosis.

2. Use according to claim 1, characterized in that, The atherosclerosis includes atherosclerosis induced by hyperlipidemia.

3. Use according to claim 1, characterized in that, The medicament prevents and / or treats atherosclerosis by reducing the formation of VSMCs-derived foam cells.

4. Use according to claim 1, characterized in that, The 4-octyl itaconic acid is used in the preparation of a medicament having any one of the following 1)~5) effects: 1) inhibiting the progression of atherosclerotic lesions; 2) inhibiting lipid accumulation in atherosclerotic plaques; 3) reducing the size and distribution range of aortic plaque; 4) reducing plaque vulnerability; 5) reducing serum lipid levels; 6) promoting cholesterol efflux.

5. Use of 4-octyl itaconic acid in the preparation of a foam cell formation inhibitor.

6. Use according to claim 5, characterized in that, The foam cells are derived from vascular smooth muscle cells.

7. Use of 4-octyl itaconic acid in the preparation of a medicament for preventing and / or treating atherosclerosis by inhibiting the formation of VSMCs-derived foam cells.

8. Use according to claim 7, characterized in that, The 4-octyl itaconic acid improves atherosclerotic lesions by activating the NRF2-HO-1 / NQO1 antioxidant network, promoting the formation of NQO1-p53 complexes, inhibiting the ubiquitination and degradation of p53, increasing the stability of p53, further promoting the expression of ABCA1, and inhibiting the foam of vascular smooth muscle cells.

9. A pharmaceutical composition for preventing and / or treating atherosclerosis, characterized by, The pharmaceutical composition comprises 4-octyl itaconic acid and a pharmaceutically acceptable carrier or excipient.

10. The use according to claim 1, characterized in that, The dosage form of the medicament includes any one or more of tablets, capsules, granules, powders, oral liquids, pills, and injections.

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