Application of calcium-activated chloride channels AN01 / TMEM16A in the prevention and treatment of atherosclerosis

Abstract

This invention belongs to the field of biomedical technology and discloses the application of calcium-activated chloride channel AN01 / TMEM16A in the prevention and treatment of atherosclerosis. This invention also discloses the application of ANO1 promoters in the preparation of drugs for the treatment or prevention of atherosclerosis. Through research, this invention has found that the loss of ANO1 in vascular smooth muscle cells exacerbates the instability of atherosclerotic plaques, while overexpression of ANO1 can enhance the stability of atherosclerotic plaques and delay the progression of atherosclerosis. ANO1 in smooth muscle cells is one of the key drivers of atherosclerosis progression. This invention clarifies that targeting the non-ion channel function of ANO1 protein has important clinical translational value for screening drugs for the treatment or prevention of atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of calcium-activated chloride channel AN01 / TMEM16A in the prevention and treatment of atherosclerosis. Background Technology

[0002] Atherosclerosis is the core pathological basis of many cardiovascular and cerebrovascular diseases, such as myocardial infarction, cerebral infarction, and coronary heart disease, and is closely related to the occurrence and development of many diseases. Therefore, the prevention and treatment of atherosclerosis is considered a fundamental strategy for the prevention and treatment of cardiovascular diseases. Inhibiting the occurrence and development of atherosclerosis helps to reduce the incidence of myocardial infarction and cerebral infarction, and reduce the mortality rate of coronary heart disease.

[0003] Calcium-activated chloride channels (CaCCs) are widely present on various excitatory and non-excitatory cells and play a variety of important functions under physiological conditions, such as regulating cell excitability, contraction, cell cycle, secretion, and cell migration. They play a crucial role in maintaining tissue homeostasis.

[0004] The ANO1 gene (also known as TMEM16A) encodes a calcium-activated chloride channel protein that plays a crucial role in various physiological processes, including epithelial secretion, smooth muscle contraction, and tumorigenesis. In recent years, its abnormal expression has been closely associated with diseases such as cystic fibrosis and cancer metastasis, making it a potential therapeutic target. Studies have found that the ANO1 ion channel may be a drug target for cystic fibrosis, and activation of the ANO1 channel can compensate for the ion / fluid transport imbalance caused by mutations in the transmembrane transport regulator gene of chloride channels in epithelial tissues. ANO1 is also a cancer-related gene; inhibiting ANO1 reduces cell migration in cancer cells, including head and neck squamous cell carcinoma, gastrointestinal stromal tumors, breast cancer, and prostate cancer cells. ANO1 is the molecular basis of cerebral vascular smooth muscle cells (CaCCs), and the activity of the ANO1 ion channel is negatively correlated with blood pressure. However, the relationship between calcium-activated chloride channel ANO1 and atherosclerosis has not yet been reported. Summary of the Invention

[0005] The present invention aims to address the shortcomings of the prior art, clarify the role of ANO1 in the progression of atherosclerosis, and provide the application of ANO1 in the prevention and treatment of atherosclerosis.

[0006] The first aspect of this invention aims to provide the use of ANO1 promoters in the preparation of medicaments for treating or preventing atherosclerosis.

[0007] A second aspect of the present invention is to provide a pharmaceutical composition.

[0008] The third aspect of this invention aims to provide the application of ANO1 as a target in the screening and preparation of drugs for the treatment or prevention of atherosclerosis.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the invention provides the use of an ANO1 promoter in the preparation of a medicament for treating or preventing atherosclerosis.

[0010] In some embodiments of the present invention, the ANO1 promoter includes small molecule compounds, nucleic acids, ANO1-expressing carriers, polypeptides, proteins, or combinations thereof.

[0011] In some embodiments of the present invention, the ANO1 promoter refers to a substance that can increase the activity and / or content of a gene or its protein in vivo or in vitro.

[0012] In some embodiments of the present invention, the ANO1 promoter includes synthetic or natural compounds, proteins or nucleotides.

[0013] In some embodiments of the present invention, the ANO1 promoter includes a substance that promotes ANO1 expression.

[0014] In some embodiments of the present invention, the ANO1 promoter includes an ANO1 gene promoter and / or an ANO1 protein promoter.

[0015] In some embodiments of the present invention, the ANO1 gene or its protein is derived from mammals.

[0016] In some embodiments of the present invention, the ANO1 gene or its protein is derived from rodents, primates or humans.

[0017] In some embodiments of the present invention, the ANO1 protein comprises a full-length protein or a protein fragment.

[0018] In some embodiments of the present invention, the ANO1 protein also includes derivatives of the ANO1 protein.

[0019] In some embodiments of the present invention, the derivatives of the ANO1 protein include modified ANO1 protein, protein molecules whose amino acid sequences are homologous to those of natural ANO1 protein and have the activity of natural ANO1 protein, dimers or polymers of ANO1 protein, and fusion proteins containing the amino acid sequence of ANO1 protein.

[0020] In some embodiments of the present invention, the term "protein molecule whose amino acid sequence is homologous to the natural ANO1 protein and has the activity of the natural ANO1 protein" refers to a protein molecule whose amino acid sequence has >85% homology to the ANO1 protein, further, >90% homology, and even further, >95% homology; and has the activity of the natural ANO1 protein.

[0021] In some embodiments of the present invention, the modified ANO1 protein is a PEGylated ANO1 protein.

[0022] In some embodiments of the present invention, the ANO1 promoter comprises a pharmaceutical formulation that enhances ANO1 gene expression.

[0023] In some embodiments of the present invention, the ANO1 promoter comprises a plasmid vector or viral vector expressing the ANO1 gene.

[0024] In some embodiments of the present invention, the ANO1 promoter is a viral vector that overexpresses the ANO1 gene.

[0025] In some embodiments of the present invention, the viral vector includes at least one of an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, or a retrovirus vector.

[0026] In some embodiments of the present invention, the serotype of the adeno-associated virus vector includes at least one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2.7M8, or AAV2-TYF mutant.

[0027] In some embodiments of the present invention, the ANO1 promoter further includes a drug delivery formulation and / or excipients.

[0028] In some embodiments of the present invention, the drug delivery formulation is selected from at least one of liposomes, chitosan, cholesterol, LNP, or nanoparticles.

[0029] In some embodiments of the present invention, the excipient is selected from at least one of phosphate buffer, mannitol or physiological saline.

[0030] A second aspect of the invention provides a pharmaceutical composition comprising an ANO1 promoter and a pharmaceutically acceptable carrier.

[0031] In some embodiments of the present invention, the ANO1 promoter comprises a pharmaceutical formulation that enhances ANO1 gene expression.

[0032] In some embodiments of the present invention, the ANO1 promoter comprises a plasmid vector or viral vector expressing the ANO1 gene.

[0033] In some embodiments of the present invention, the ANO1 promoter is a viral vector that overexpresses the ANO1 gene.

[0034] In some embodiments of the present invention, the viral vector includes at least one of an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, or a retrovirus vector.

[0035] In some embodiments of the present invention, the serotype of the adeno-associated virus vector includes at least one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2.7M8, or AAV2-TYF mutant.

[0036] In some embodiments of the present invention, the pharmaceutical composition further includes other drugs for treating or preventing atherosclerosis.

[0037] In some embodiments of the present invention, the other drugs for treating or preventing atherosclerosis include lovastatin, simvastatin, pravastatin, atorvastatin, fluvastatin, or combination preparations mainly composed of statins.

[0038] In some embodiments of the present invention, the other drugs for treating or preventing atherosclerosis include aspirin, enalapril, valsartan, metformin, or colchicine.

[0039] In some embodiments of the present invention, the pharmaceutical composition is in the form of a liquid, solid, or semi-solid.

[0040] In some embodiments of the present invention, the pharmaceutically acceptable carrier includes at least one of fillers, binders, lubricants, surfactants, coating agents, capsule shells, flavor masking agents, flavoring agents, solvents, cosolvents, suspending agents, pH adjusters, or antioxidants.

[0041] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose, etc.; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin, or polyvinylpyrrolidone, etc.; the disintegrant is selected from microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, or croscarmellose sodium, etc.; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, micronized silica gel, talc, or magnesium stearate, etc.; the flavoring agent is selected from aspartame, sucralose, flavoring, stevia, acesulfame potassium, citric acid, or sodium saccharin, etc.; the coloring agent is selected from titanium dioxide, sunset yellow, methylene blue, or pharmaceutical-grade iron oxide, etc.; and the wetting agent is selected from glycerin, Tween-80, hydrogenated castor oil, or lecithin, etc. The solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerin, propylene glycol, or vegetable oil, etc., wherein the vegetable oil is selected from soybean oil, castor oil, peanut oil, blended oil, etc.; the pH adjuster is selected from hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, or sodium tartrate, etc.; the antioxidant is selected from sodium sulfite, sodium metabisulfite, or sodium thiosulfate, etc.; the antibacterial agent is selected from phenol, benzyl alcohol, or thimerosal, etc.; the surfactant is selected from sodium dodecylbenzenesulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan, or polysorbate (Tween), etc.; the preservative is selected from methylparaben or propylparaben, etc.; the suspending agent is selected from micronized silica gel, beeswax, cellulose, solid polyethylene glycol, etc.; the absorbent is selected from at least one of calcium sulfate, dicalcium phosphate, light magnesium oxide, and calcium carbonate.

[0042] In some embodiments of the present invention, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0043] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0044] In some embodiments of the present invention, the non-gastrointestinal dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0045] In some embodiments of the present invention, the injectable dosage form includes at least one of the following: injection solution, injection solution, intravenous infusion injection solution, injection suspension, sterile powder for injection, intravenous injection, water injection, injection emulsion, powder injection, injection, sterile powder injection, and lyophilized powder injection.

[0046] A third aspect of the invention provides the use of ANO1 as a target in screening and preparing drugs for the treatment or prevention of atherosclerosis.

[0047] The beneficial effects of this invention are: This invention reveals that the absence of ANO1 in vascular smooth muscle cells exacerbates the instability of atherosclerotic plaques, while overexpression of ANO1 enhances plaque stability and slows the progression of atherosclerosis. The anti-atherosclerotic effect of ANO1 in smooth muscle cells does not depend on ion channel function, but rather on acting as a protein bridge, mediating a series of downstream signaling pathways to increase the number of fibroblast-like smooth muscle cells, thereby increasing plaque stability. Furthermore, the effect of ANO1 on atherosclerotic plaque stability is cell-specific; knockout of ANO1 in endothelial cells and monocytes / macrophages has no significant effect on plaque stability. ANO1 in smooth muscle cells is one of the key drivers of atherosclerotic progression and has important clinical significance.

[0048] This invention also proposes a strategy for screening drugs to treat or prevent atherosclerosis based on the non-ion channel function of the ANO1 protein. By directly binding to ANO1 and upregulating its expression, the plaque composition can be altered from within without affecting plaque size, thereby enhancing the stability of atherosclerotic plaques. Attached Figure Description

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 A schematic diagram illustrating the construction of a cell-specific ANO1 knockout mouse.

[0050] Figure 2 A schematic diagram of the construction of cell-specific ANO1 transgenic mice.

[0051] Figure 3 For ANO1 SMCKO The results of atherosclerotic plaque stability in mice are shown in the figure; where A represents ANO1 levels in mice fed a normal or high-fat diet. f / f and ANO1 SMCKO Mice, after being administered AAV-PCSK9 DYAfter processing, the following images were generated: A) Representative images of Oil Red O staining of the entire aorta and results of quantitative analysis of plaque area; B) Representative microscopic images of Oil Red O staining of the aortic root of each genotype of mouse and results of quantitative analysis, scale bar 200 μm; C) Results of plaque formation and quantitative analysis of the aortic root, scale bar 200 μm; D) Results of plaque formation and quantitative analysis of the brachiocephalic artery, scale bar 100 μm; E) Results of necrotic core area and quantitative analysis of the aortic root, scale bar 50 μm; F) Results of necrotic core area and quantitative analysis of the brachiocephalic artery, scale bar 100 μm; G) Representative images of Masson's trichrome staining of the aortic root and results of quantitative collagen analysis, scale bar 200 μm; H) Representative images of α-SMA immunofluorescence staining within the plaque of the aortic root and results of quantitative analysis, scale bar 50 μm. Data are expressed as mean ± standard deviation (n=8). A and B were analyzed using Tukey-corrected two-way ANOVA, C, D, F, G, and H were analyzed using unpaired two-tailed Student's t-test, and E was analyzed using the exact Mann-Whitney U test (normality test failed).

[0052] Figure 4 For ANO1 SMCKO The graph shows the results of metabolic parameters and inflammatory cell infiltration in mice; where A represents ANO1 levels in mice fed a normal or high-fat diet. f / f and ANO1 SMCKO The results of mouse body weight (n=12); B represents ANO1 levels in mice fed a normal or high-fat diet. f / f and ANO1 SMCKO Figure C shows the results of total cholesterol levels in mouse serum (n=8); Figure D shows the results of triglyceride levels in mice fed a normal or high-fat diet (n=8); Figure E shows the results of low-density lipoprotein levels in mice fed a normal or high-fat diet (n=8); Figure F shows representative images of CD68 cell immunofluorescence staining in aortic root plaques and the results of quantitative analysis of positive cell areas (n=8), with a scale bar of 50 μm; Figure G shows representative images of CD3 cell immunofluorescence staining in aortic root plaques and the results of quantitative analysis of positive cell areas (n=8), with a scale bar of 50 μm.

[0053] Figure 5 For ANO1 SMCKI The results of atherosclerotic plaque stability in mice are shown in the figure; where A represents ANO1 obtained from mice fed a high-fat diet for 12 weeks. f / f and ANO1 SMCKIRepresentative images and quantitative analysis results of hematoxylin-eosin staining of aortic root sections from mice (n=8), scale bar 50μm; B represents ANO1 from mice fed a high-fat diet for 12 weeks. f / f and ANO1 SMCKI Representative images and quantitative analysis results of hematoxylin-eosin staining of cephalobrachial artery sections from mice (n=8), scale bar 100 μm; C represents ANO1 obtained from mice fed a high-fat diet for 12 weeks. f / f and ANO1 SMCKI Representative images of Masson's trichrome staining of aortic root sections from mice and results of quantitative analysis (n=8), scale bar at 200 μm; D represents ANO1 obtained from mice fed a high-fat diet for 12 weeks. f / f and ANO1 SMCKI Representative images of α-SMA immunostaining and quantitative analysis results of aortic root sections from mice (n=8), scale bar 50μm.

[0054] Figure 6 For ANO1 ECKO The results of atherosclerotic plaque stability in mice are shown in the figure; where A represents ANO1 levels in mice fed a normal or high-fat diet. f / f and ANO1 ECKO A) Representative images of Oil Red O staining of the entire aorta in mice and results of quantitative analysis of plaque area (n=8); B) Representative microscopic images of Oil Red O staining of the aortic root in mice of various genotypes and results of quantitative analysis (n=8), scale bar 200μm; C) Results of plaque formation and quantitative analysis in the aortic root (n=8), scale bar 200μm; D) Results of necrotic core area and quantitative analysis in the aortic root (n=8), scale bar 50μm; E) Representative images of Masson's trichrome staining in the aortic root and results of quantitative collagen analysis (n=8), scale bar 200μm; F) Representative images of CD68 cell immunofluorescence staining within the plaque in the aortic root and positive cells. The results of quantitative analysis of cell regions (n=8), scale bar is 50μm; G is a representative image of CD3 cell immunofluorescence staining in the aortic root plaque and the results of quantitative analysis of positive cell regions (n=8), scale bar is 50μm; H is the result of body weight of mice after being fed a normal or high-fat diet (n=12); I is the result of total cholesterol level of mice after being fed a normal or high-fat diet (n=8); J is the result of triglyceride level of mice after being fed a normal or high-fat diet (n=8); K is the result of low-density lipoprotein level of mice after being fed a normal or high-fat diet (n=8); L is the result of high-density lipoprotein level of mice after being fed a normal or high-fat diet (n=8).

[0055] Figure 7 For Ano1 MKOThe results of atherosclerotic plaque stability in mice are shown in the figure; where A represents ANO1 levels in mice fed a normal or high-fat diet. f / f and ANO1 MKO A) Representative images of Oil Red O staining of the entire aorta in mice and results of quantitative analysis of plaque area (n=8); B) Representative microscopic images of Oil Red O staining of the aortic root in mice of various genotypes and results of quantitative analysis (n=8), scale bar 200μm; C) Results of plaque formation and quantitative analysis in the aortic root (n=8), scale bar 200μm; D) Results of necrotic core area and quantitative analysis in the aortic root (n=8), scale bar 50μm; E) Representative images of Masson's trichrome staining in the aortic root and results of quantitative collagen analysis (n=8), scale bar 200μm; F) Representative images of CD68 cell immunofluorescence staining within the plaque in the aortic root and positive cells. The results of quantitative analysis of cell regions (n=8), scale bar is 50μm; G is a representative image of CD3 cell immunofluorescence staining in the aortic root plaque and the results of quantitative analysis of positive cell regions (n=8), scale bar is 50μm; H is the result of body weight of mice after being fed a normal or high-fat diet (n=12); I is the result of total cholesterol level of mice after being fed a normal or high-fat diet (n=8); J is the result of triglyceride level of mice after being fed a normal or high-fat diet (n=8); K is the result of low-density lipoprotein level of mice after being fed a normal or high-fat diet (n=8); L is the result of high-density lipoprotein level of mice after being fed a normal or high-fat diet (n=8). Detailed Implementation

[0056] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0057] the term ANO1, also known as TMEM16A or DOG1, is a calcium-activated chloride channel protein and belongs to the Anoctamin (a class of membrane proteins) family.

[0058] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0059] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0060] Example Experimental methods 1. Constructing cell-specific knock-in / knockout mouse models (1) Construction of cell-specific ANO1 knockout mice like Figure 1 As shown, using bacterial artificial chromosome clones from the C57BL / 6J library as templates, mouse genome fragments containing homologous arms and conditional knockout regions were synthesized via polymerase chain reaction. The targeting vector was designed by inserting two loxP sites flanking exon 12 of ANO1 and introducing a neomycin selection frame flanked by the Frt site. This operation deleted 53 amino acids from the second transmembrane domain and the extracellular loop between the first and second transmembrane domains, resulting in a linearized vector. After the linearized vector was transformed into 129Sv / J embryonic stem cells, positive clones were selected using neomycin. Embryonic stem cell clones were validated by Southern blotting and then microinjected into C57BL / 6J mouse blastocysts. The injected blastocysts were transferred to the uterus of pseudopregnant female mice, and chimeric male mice were identified by coat color. After removing the resistance selection frame, the progeny were successfully backcrossed into ANO1. f / f Homozygous state. Myh11-CreER T2 (Item No.: 019079) Cdh5-Cre (Item No.: 017968) Lyz2-Cre (Catalog No.: 004781) Transgenic mice were purchased from the Jackson Laboratory and backcrossed into the C57BL / 6 genetic background for at least nine generations. ANO1 smooth muscle cells (ANO1...) SMCKO ), endothelial cells (ANO1) ECKO ), mononuclear cells (Ano1) MKO Knockout mice via ANO f / f Mice and Myh11-CreER T2 , Cdh5-Cre , Lyz2-Cre Mouse hybridization was used to obtain smooth muscle-specific ANO1 knockout mice. Further induction was achieved through daily intraperitoneal injections of tamoxifen (TAM, 75 mg / kg / d; Sigma-Aldrich, St. Louis, MO) for 5 consecutive days. The control group consisted of ANO1 endothelial cell or monocyte knockout mice. f / f Mice. The control group of ANO1 smooth muscle cell knockout mice was ANO1. f / fMice were injected with peanut oil. Genotyping was performed using PCR amplification with two pairs of flux primers and one pair of Cre primers. The primers were as follows: Flox region 1: forward 5'-GGTATCACCCAAGGTAACCATCCA-3' (SEQ ID NO:1), reverse 5'-CAACCCTCTCTATCCCTGTCACATG-3' (SEQ ID NO:2); flux region 2: forward 5'-TGATTCTGATAGCAAATGAGGCAGAT-3' (SEQ ID NO:3), reverse 5'-AGGTTATCATAGCTCAGTTCACAAGCTTT-3' (SEQ ID NO:4). Myh11-CreER T2 : Forward 5'-TGACCCCATCTCTTCACTCC-3' (SEQ ID NO:5), Reverse 5'-AGTCCCTCACATCCTCAGGTT-3' (SEQ ID NO:6). Cdh5-Cre : Forward 5'-CCAGGCTGACCAAGCTGAG-3' (SEQ ID NO:7), Reverse 5'-CCTGGCGATCCCTGAACA-3' (SEQ ID NO:8). Lyz2-Cre : Forward 5'-CCCAGAAATGCCAGATTACG-3' (SEQ ID NO:9), Reverse 5'-CTTGGGCTGCCAGAATTTCTC-3' (SEQ ID NO:10).

[0061] (2) Construction of cell-specific ANO1 knock-in mice like Figure 2 As shown, a transgenic construct containing ANO1 cDNA was inserted into the pRP.ExBi-CMV-LoxP-Stop-LoxP vector and microinjected into fertilized mouse embryos. This strain did not overexpress ANO1 due to the presence of a stop codon system, thus obtaining ANO1 knock-in-floxed (ANO1 knock-in-floxed) mice. f / + -KI) mice. ANO1 f / + -KI mice and Myh11-CreER T2 Mice were mated to obtain smooth muscle cell-specific ANO1 knock-in mice (ANO1 knock-in mice). SMCKI Gene knock-in was induced in smooth muscle cells by daily intraperitoneal injection of tamoxifen (TAM, 75 mg / kg / day; Sigma-Aldrich, St. Louis, MO) for 5 consecutive days. The control group (ANO1) f / f-KI mice were injected with peanut oil only. Genotyping was performed using a pair of flox primers and a pair of Cre primers for PCR amplification. The primers were as follows: Flox: forward 5'-GAGTACTTCTCGGGGACCCTCA-3' (SEQ ID NO:11), reverse 5'-TCATGTCTGGATCCCCATCAAGC-3' (SEQ ID NO:12). Myh11-CreER T2 The primers are the same as above.

[0062] 2. Induced atherosclerosis model ANO1 f / f ANO1 SMCKO ANO1 ECKO ANO1 MKO ANO1 f / + -KI and ANO1 SMCKI Mice were injected via tail vein with AAV8-D374Y-PCSK9 (AAV-PCSK9) at 6-8 weeks of age. DY 1×10 11 Atherosclerosis was induced by feeding animals with a dose of vg / animal, followed by feeding with a normal diet (11.4% calories, D10001; Research Diets) or a high-fat diet (HFD; 40% calories from fat, 11.4% calories from sucrose, 1.25% cholesterol; D12492; Research Diets) for 12 weeks.

[0063] The amino acid sequence of AAV8-D374Y-PCSK9 is as follows: MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGASSYCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ (SEQ ID NO:13).

[0064] 3. Histological analysis After euthanasia, mice were perfused with physiological saline via the left ventricle to remove circulating blood. The aortic root and brachiocephalic artery were carefully dissected and fixed in 4% paraformaldehyde (PFA) at 4°C for 24 hours. The fixed tissues were embedded in paraffin or Tissue-Tek OCT embedding medium (Sakura, Tokyo, Japan) and sectioned (6-8 μm thick) for histological analysis. Standard staining procedures were used, including hematoxylin-eosin staining, Masson's trichrome staining, and Oil Red O staining (standard reagents purchased from Wuhan Sewell Biotechnology Co., Ltd.), to assess tissue morphology, fibrosis, and lipid accumulation. For hematoxylin-eosin staining, tissue sections were first dewaxed to water (paraffin sections were dewaxed with xylene and hydrated with graded ethanol), then the cell nuclei were stained with hematoxylin for 5-10 minutes, rinsed with running water, differentiated with 1% hydrochloric acid ethanol for a few seconds, and then blued with a weakly alkaline solution; then the cytoplasm was stained with eosin for 1-3 minutes, and finally dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. Masson's trichrome staining: After dewaxing the sections to water, stain the nuclei with iron hematoxylin for 5-10 minutes, differentiate with hydrochloric acid and ethanol, and then return to blue. Next, stain with Ponceau S and acidic fuchsin for 5-10 minutes to make the muscle fibers and cytoplasm appear red. Then differentiate with 1% phosphomolybdic acid for 1-3 minutes, and finally counterstain collagen fibers with aniline blue for 1-2 minutes. After dehydration with ethanol and clearing with xylene, mount the sections. Oil Red O staining: After fixing the sections with 4% paraformaldehyde, infiltrate with 60% isopropanol to enhance lipid binding. Stain with Oil Red O working solution (0.3%-0.5% Oil Red O isopropanol solution filtered before use) in the dark for 10-15 minutes. Differentiate with 60% isopropanol for a few seconds to remove non-specific staining, counterstain the nuclei with hematoxylin, return to blue with running water, and mount with glycerol and gelatin. For whole-vessel smear analysis of atherosclerotic lesions, the aorta from the heart to the bifurcation of the iliac artery was dissected under a stereomicroscope, fixed in 4% PFA at 4°C for 24 hours, then longitudinally cut and stained with Oil Red O. Images were acquired using a digital camera and quantitatively analyzed using ImageJ software.

[0065] 4. Immunofluorescence staining The immunofluorescence staining procedure on paraffin-embedded tissue sections begins with a standard dewaxing and hydration process. Specifically, the sections are immersed sequentially in xylene I and xylene II for 10-15 minutes each to completely remove paraffin. They are then hydrated sequentially with a gradient of anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol, each step lasting approximately 5 minutes. Finally, the sections are washed with distilled water. After hydration, the crucial antigen retrieval step follows: the sections are immersed in citrate buffer (pH 6.0) and heated at 95°C for 20 minutes to fully expose the antigenic epitopes. To increase permeability, the sections are incubated in PBS containing 0.5% Triton X-100 for 10 minutes, followed by blocking with 10% goat serum (C0265, Beyotime) at room temperature for 30 minutes. Sections were incubated overnight at 4°C with the following primary antibodies: anti-CD3 (ab16669, 1:100; Abcam), anti-CD68 (ab955, 1:100; Abcam), and anti-α-SMA (67735-1-Ig, 1:100; Proteintech, Rosemont, IL). After primary antibody incubation, sections were washed and incubated for 1 hour at room temperature in the dark with secondary antibodies (1:200, Beyotime) labeled with Alexa Fluor 488 (catalog number: A0428) or 594 (catalog number: A1143). Cell nuclei were counterstained with DAPI (C1002, Beyotime) for 10 minutes. Stained sections were mounted with anti-fluorescence quenching mounting medium (AC28L532, Beyotime) and imaged using a laser scanning confocal microscope (Zeiss LSM800, Munich, Germany).

[0066] 5. Serum biochemical analysis Blood samples were collected from mice after they were fasted overnight under isoflurane anesthesia. The concentrations of total cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein, alanine aminotransferase, and aspartate aminotransferase were quantitatively determined using a fully automated biochemical analyzer (Chemray 800; Raydu Life Sciences, Shenzhen, China).

[0067] 6. Statistical Analysis All non-sequencing data are expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism8 software (GraphPad Software, La Jolla, CA). Normality of the data was assessed using the Shapiro-Wilk or Kolmogorov-Smirnov tests. For normally distributed comparisons among multiple groups, one-way ANOVA was used, followed by Dunnett's or Tukey's tests, or two-way ANOVA was used followed by Tukey's multiple comparisons. Nonparametric data were analyzed using the Mann-Whitney U exact test. Correlation analysis was performed using the Pearson correlation test. Statistical significance was set at [value missing]. P <0.05. The graph plots the data points and provides precise values ​​for the main comparisons. P value.

[0068] Experimental results (1) Smooth muscle cell loss and ANO1 exacerbate the instability of atherosclerotic plaques. To investigate the effect of ANO1 in smooth muscle cells on the stability of atherosclerotic plaques, ANO1 was constructed... f / f Mice (with the same phenotype as wild-type under normal conditions) and smooth muscle cell-specific ANO1 knockout mice (ANO1 SMCKO ), and give AAV-PCSK9 DY The ANO1 was used to treat the induced atherosclerosis model. f / f and ANO1 SMCKO The entire aorta of mice was stained with Oil Red O, and the aortic root and brachiocephalic artery were stained with Masson's trichrome, Oil Red O, and α-SMA immunofluorescence. The experimental results are as follows: Figure 3 As shown, ANO1 SMCKO The mouse's overall aorta ( Figure 3 (middle A) and aortic root ( Figure 3 The area of ​​plaque in the middle B) and the aortic root ( Figure 3 C) and brachiocephalic artery ( Figure 3 The lesion area of ​​D) and ANO1 f / f The mice were similar, but their aortic root ( Figure 3 (Central E) and brachiocephalic artery ( Figure 3 The necrotic core area in the F-plaque expanded, accompanied by collagen deposition. Figure 3 (G) and α-smooth muscle actin positive (α-SMA) + The number of smooth muscle cells is reduced. Figure 3 (H). This indicates that the loss of ANO1 in smooth muscle cells exacerbates the instability of atherosclerotic plaques.

[0069] (2) The absence of ANO1 in smooth muscle cells had no significant effect on body weight, metabolic parameters, or inflammatory cell infiltration in atherosclerotic mice. To further investigate the effects of ANO1 deficiency in smooth muscle cells on body weight, metabolic parameters, and inflammatory cell infiltration in atherosclerotic mice, body weight, blood lipid levels, and inflammatory cell infiltration levels were measured. The experimental results are as follows: Figure 4 As shown, ANO1 SMCKO With ANO1 f / f Mouse weight ( Figure 4 (A) Blood lipid profile including total cholesterol ( Figure 4 B), triglycerides ( Figure 4 Medium C), low density lipoprotein (LDL) Figure 4 (D) and high-density lipoprotein (HDL) Figure 4 (E), plaque inflammatory cells CD68 mononuclear cells ( Figure 4 neutrophil CD3 (F) and neutrophil CD3 ( Figure 4 Infiltration of G and ANO1 f / f The results were comparable in mice. This indicates that the absence of ANO1 in smooth muscle cells had no significant effect on mouse body weight, blood lipids, or inflammatory cell infiltration levels.

[0070] (3) Smooth muscle cell-specific overexpression of ANO1 improves the stability of atherosclerotic plaques. To investigate the effect of smooth muscle cell-specific ANO1 overexpression on improving the stability of atherosclerotic plaques, a smooth muscle cell-specific ANO1 overexpression mouse (ANO1) was constructed. SMCKI ), and give AAV-PCSK9 DY The atherosclerosis model was induced using ANO1 mice fed a high-fat diet for 12 weeks. f / f and ANO1 SMCKI Sections of the aortic root and brachiocephalic artery from mice were stained with hematoxylin-eosin, Masson's trichrome stain, and α-SMA immunostaining. The experimental results are as follows: Figure 5 As shown, with ANO1 f / f Compared to mice, ANO1 SMCKI aortic root of mouse ( Figure 5 (middle A) and brachiocephalic artery ( Figure 5 In plaque B, the necrotic core area is reduced, accompanied by collagen deposition. Figure 5 Positive for C) and α-smooth muscle actin (α-SMA) + Increased number of smooth muscle cells Figure 5 (D). This indicates that smooth muscle cell-specific overexpression of Ano1 improves the stability of atherosclerotic plaques.

[0071] (4) ANO1 in endothelial cells has no significant effect on the stability of atherosclerotic plaques. To investigate the effect of endothelial cell ANO1 on the stability of atherosclerotic plaques, an endothelial cell-specific ANO1 knockout mouse (ANO1 knockout mouse) was constructed. ECKO ), and give AAV-PCSK9 DY The ANO1 was used to treat the induced atherosclerosis model. f / f and ANO1 ECKO The entire aorta of mice was stained with Oil Red O. The aortic root and brachiocephalic artery were stained with Masson's trichrome, Oil Red O, and α-SMA immunofluorescence. Mouse body weight, blood lipid levels, and the level of inflammatory cell infiltration were measured. Experimental results are as follows: Figure 6 As shown, ANO1 ECKO The mouse's overall aorta ( Figure 6 (middle A) and aortic root ( Figure 6 The area of ​​plaque in the middle B) and the aortic root ( Figure 6 (C) lesion area, aortic root ( Figure 6 In the necrotic core area of ​​the middle D region, collagen deposition was observed at the aortic root. Figure 6 (E), plaque inflammatory cells CD68 mononuclear cells ( Figure 6 neutrophil CD3 (F) and neutrophil CD3 ( Figure 6 Infiltration status of G in the middle, body weight ( Figure 6 (H), total cholesterol ( Figure 6 I), triglycerides ( Figure 6 (J), low-density lipoprotein (LDL) Figure 6 (K) and high-density lipoprotein (HDL) Figure 6 (L) and Ano1 f / f Mice were comparable. This indicates conditional knockout of ANO1 in endothelial cells (ANO1). ECKO It has no significant effect on atherosclerosis.

[0072] (5) ANO1 in monocytes / macrophages had no significant effect on the stability of atherosclerotic plaques. To investigate the effect of ANO1 from monocytes / macrophages on the stability of atherosclerotic plaques, ANO1 knockout mice (ANO1 knockout mice) specifically derived from monocytes / macrophages were constructed. MKO ), and give AAV-PCSK9 DY The ANO1 was used to treat the induced atherosclerosis model. f / f and ANO1 MKOThe entire aorta of mice was stained with Oil Red O. The aortic root and brachiocephalic artery were stained with Masson's trichrome, Oil Red O, and α-SMA immunofluorescence. Mouse body weight, blood lipid levels, and the level of inflammatory cell infiltration were measured. Experimental results are as follows: Figure 7 As shown, ANO1 MKO The mouse's overall aorta ( Figure 7 (middle A) and aortic root ( Figure 7 The area of ​​plaque in the middle B) and the aortic root ( Figure 7 (C) Lesion area, aortic root ( Figure 7 In the necrotic core area of ​​the middle D region, collagen deposition was observed at the aortic root. Figure 7 (E), plaque inflammatory cells CD68 mononuclear cells ( Figure 7 neutrophil CD3 (F) and neutrophil CD3 ( Figure 7 Infiltration status of G in the middle, body weight ( Figure 7 (H), total cholesterol ( Figure 7 I), triglycerides ( Figure 7 (J), low-density lipoprotein (LDL) Figure 7 (K) and high-density lipoprotein (HDL) Figure 7 (L) and ANO1 f / f Mice were comparable. This indicates conditional knockout of ANO1 in monocytes / macrophages (ANO1). MKO It has no significant effect on atherosclerosis.

[0073] In summary, this invention reveals that ANO1 in vascular smooth muscle cells can enhance the stability of atherosclerotic plaques, thereby delaying the progression of atherosclerosis, but has no significant effect on plaque size or blood lipid levels. ANO1 exhibits cell specificity; ANO1 in endothelial cells and monocytes / macrophages has no significant effect on plaque stability. Smooth muscle cell homeostasis is crucial for plaque stability. Mechanistic studies have found that the anti-atherosclerotic effect of ANO1 in smooth muscle cells does not depend on ion channel function, but rather on acting as a protein bridge, mediating a series of downstream signaling pathways to increase the number of fibroblast-like smooth muscle cells, thereby increasing plaque stability.

[0074] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of ANO1 promoters in the preparation of drugs for the treatment or prevention of atherosclerosis.

2. The application according to claim 1, characterized in that, The ANO1 promoters include small molecule compounds, nucleic acids, ANO1-expressing vectors, peptides, proteins, or combinations thereof.

3. The application according to claim 2, characterized in that, The ANO1 promoter includes pharmaceutical formulations that enhance ANO1 gene expression.

4. The application according to claim 3, characterized in that, The ANO1 promoter includes a plasmid vector or viral vector expressing the ANO1 gene; Preferably, the viral vector includes at least one of an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, or a retrovirus vector.

5. The application according to any one of claims 1-4, characterized in that, The ANO1 promoter also includes drug delivery formulations and / or excipients.

6. The application according to claim 5, characterized in that, The drug delivery formulation is selected from at least one of liposomes, chitosan, cholesterol, LNP, or nanoparticles; Preferably, the excipient is selected from at least one of phosphate buffer, mannitol, or physiological saline.

7. A pharmaceutical composition, characterized in that, This includes ANO1 promoters and pharmaceutically acceptable carriers.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes other drugs for the treatment or prevention of atherosclerosis; Preferably, the other drugs used to treat or prevent atherosclerosis include lovastatin, simvastatin, pravastatin, atorvastatin, fluvastatin, or compound preparations mainly composed of statins. Preferably, the other drugs used to treat or prevent atherosclerosis include aspirin, enalapril, valsartan, metformin, or colchicine.

9. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutically acceptable carriers include at least one of the following: fillers, binders, lubricants, surfactants, coating agents, capsule shells, flavor masking agents, flavoring agents, solvents, solubilizers, suspending agents, pH adjusters, or antioxidants; Preferably, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

10. The application of ANO1 as a target in screening and preparing drugs for the treatment or prevention of atherosclerosis.

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