Application of nitrates in the preparation of lipid-lowering drugs

Abstract

This invention relates to the field of biomedical technology, and particularly to the application of nitrate in the preparation of lipid-lowering drugs. Nitrate can significantly inhibit the synthesis of intracellular substances including cholesterol, triglycerides, cholesterol esters and their metabolic derivatives, and clarifies the key role of the Sialin-Sialin2 axis in this process. Furthermore, nitrate regulates cholesterol metabolism by controlling the interaction between Sialin2 and key cholesterol synthesis enzymes DHCR7 and DHCR24 through the Sialin-Sialin2 axis. Simultaneously, nitrate can significantly inhibit the expression or activity of key enzymes in the triglyceride biosynthesis pathway through the Sialin2-mediated regulatory network, especially precisely regulating ACC, DGAT1 and SCD1. Nitrate blocks the de novo synthesis, desaturation processing and final esterification assembly of fatty acids from the source, thereby efficiently and targetedly reducing the generation and accumulation of lipid droplets.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a nitrate in the preparation of lipid-lowering drugs. Background Technology

[0002] Currently, statins, such as simvastatin and pravastatin, are commonly used to treat hypercholesterolemia. These are first-line drugs that inhibit HMGCR activity, but they also exhibit anti-fatty degeneration, neuroprotective, and anti-atherosclerotic effects in experimental animals. However, their use is limited due to adverse reactions, including muscle damage, liver damage, and cognitive impairment. Therefore, the search for natural products that can prevent or improve hypercholesterolemia (targeting cholesterol metabolism-related factors including SREBP2, HMGCR, CYP7A1, and LDLR) with relatively low toxicity is receiving increasing attention.

[0003] Fibrates (such as fenofibrate and gemfibrozil) are commonly used to treat hypertriglyceridemia. They accelerate lipid clearance by activating peroxisome proliferator-activated receptor α (PPAR-α) and are first-line drugs for treating hypertriglyceridemia. They have also shown effects in improving hepatic steatosis and protecting the cardiovascular system in experimental animals and clinical settings. However, their clinical use is limited by potential adverse reactions, including abnormal liver function, gastrointestinal reactions, and severe muscle toxicity (such as myopathy and rhabdomyolysis). Therefore, the search for natural substances that can prevent or improve hypertriglyceridemia (targeting triglyceride metabolism-related factors such as SREBP-1c, ACC, FAS, and DGAT) is gaining increasing attention due to their relatively low toxicity and good tolerability.

[0004] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of nitrate in the preparation of lipid-lowering drugs, aiming to solve the problems of imperfect regulatory mechanisms and lack of effective intervention targets in existing lipid-lowering drugs.

[0006] The technical solution of the present invention is as follows: The application of nitrates in the preparation of lipid-lowering drugs.

[0007] In the aforementioned application, the nitrate is an inorganic salt or a natural extract.

[0008] In the aforementioned application, the inorganic salts include one or more of sodium nitrate, potassium nitrate, and calcium nitrate; the natural extracts include beetroot extract or spinach extract.

[0009] In the aforementioned application, the lipid-lowering drug is used to inhibit one or more of cholesterol, cholesterol metabolic derivatives, triglycerides, triglyceride metabolic derivatives, cholesterol esters, and cholesterol ester metabolic derivatives.

[0010] The application described herein includes the use of the nitrate in the preparation of drugs that inhibit the expression of DHCR7 and DHCR24 enzyme proteins in the biosynthetic pathway of cholesterol and / or cholesterol metabolic derivatives.

[0011] The application described herein includes the use of the nitrate in the preparation of drugs that inhibit the expression of ACC, DGAT1, and SCD1 enzyme proteins in the biosynthetic pathway of triglycerides and / or triglyceride metabolic derivatives.

[0012] A lipid-lowering drug composition comprising nitrates and one or more of fibrates, statins, and PCSK9 inhibitors.

[0013] The lipid-lowering drug composition wherein the fibrate is selected from at least one of fenofibrate and gemfibrozil; and the statin is selected from at least one of simvastatin and pravastatin.

[0014] The lipid-lowering drug composition is formulated as one of sustained-release capsules, controlled-release tablets, transdermal patches, and oral liquids.

[0015] The lipid-lowering drug composition further includes a pharmaceutically acceptable carrier; Preferably, the pharmaceutically acceptable carrier includes at least one of a pharmaceutically acceptable excipient, a pharmaceutically acceptable additive, and a pharmaceutically acceptable adjuvant.

[0016] Beneficial Effects: This invention provides the application of nitrate in the preparation of lipid-lowering drugs. The nitrate can significantly inhibit the synthesis of intracellular cholesterol, triglycerides, cholesterol esters and their metabolic derivatives, and clarifies the key role of the Sialin-Sialin2 axis in this process. Furthermore, nitrate regulates cholesterol metabolism by controlling the interaction between Sialin2 and the key cholesterol synthesis enzymes DHCR7 and DHCR24 through the Sialin-Sialin2 axis. At the same time, nitrate can significantly inhibit the expression or activity of key enzymes in the triglyceride biosynthesis pathway through the Sialin2-mediated regulatory network, especially precisely regulating acetyl-CoA carboxyltransferase (ACC), diacylglycerol acyltransferase 1 (DGAT1) and stearoyl-CoA desaturase 1 (SCD1). Nitrate blocks the de novo synthesis, desaturation processing and final esterification assembly of fatty acids from the source, thereby efficiently and targetedly reducing the generation and accumulation of lipid droplets. In addition, this invention utilizes nitrates to prepare lipid-lowering drugs, which have a complete regulatory mechanism and effective intervention targets. Furthermore, while nitrates play a role in degrading molecules such as cholesterol and triglycerides, they have no toxic side effects on liver and kidney function, demonstrating extremely high biosafety. This invention effectively proposes new indications for nitrates in "safe lipid-lowering" and improving fatty liver. Attached Figure Description

[0017] Figure 1 Example 1 shows a schematic diagram and characterization diagram of the in vivo experiment demonstrating the inhibition of cholesterol and derivative synthesis in mice by nitrate; Figure 2 This is a schematic diagram and characterization diagram of the in vitro experimental analysis of the effect of nitrate on the synthesis of intracellular cholesterol and its derivatives in Example 1; Figure 3 This is a characterization diagram of the nitrate-stimulated Sialin1 / 2 response in Example 2; Figure 4 This is a characterization diagram of nitrate-stimulated endocytosis enriching Sialin2 on the endoplasmic reticulum in Example 2. Figure 5 This is a characterization diagram of the interaction between Sialin2 and VAPA and their localization in the endoplasmic reticulum in Example 3; Figure 6 This is a characterization diagram showing that Sialin2 can inhibit cholesterol synthesis in response to nitrate stimulation in Example 3; Figure 7 Example 4 presents a flowchart and result characterization diagram of the multi-omics study revealing the inhibition of triglyceride synthesis by nitrate; Figure 8 This is a characterization diagram of the key enzyme in triglyceride synthesis regulated by nitrate through the Sialin-Sialin2 axis in Example 5. Figure 9This is a characterization diagram used in Example 6 to verify the excellent hepatic and renal safety of nitrates. Detailed Implementation

[0018] This invention provides an application of nitrates in the preparation of lipid-lowering drugs. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0019] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0020] Cholesterol and its derivatives are essential components of eukaryotic cell membranes, playing a crucial role in maintaining membrane fluidity and cell integrity, and serving as precursors for the synthesis of steroid hormones, bile acids, and vitamin D. However, abnormally elevated levels of cholesterol and its derivatives are closely associated with a variety of serious diseases, including atherosclerosis, cardiovascular disease, and metabolic syndrome. Intracellular cholesterol and its derivative levels are precisely regulated by multiple mechanisms, including biosynthesis, uptake, esterification, and efflux. The biosynthetic pathway of cholesterol and its derivatives is primarily located in the endoplasmic reticulum and is completed through a series of enzymatic reactions. Key enzymes such as HMG-CoA reductase (HMGR) are the rate-limiting enzymes in this pathway and are also targets for statin drugs. Furthermore, key enzymes involved in terminal synthesis are potential targets for inhibiting tumor development.

[0021] Despite their widespread clinical use, statins have several limitations, including hepatotoxicity, muscle side effects, and heterogeneity in response. More importantly, some patients are insensitive to statin treatment or develop resistance. Therefore, exploring novel regulatory mechanisms of cholesterol and its derivatives biosynthesis and identifying potential intervention targets is of significant scientific and clinical value for developing new strategies to lower cholesterol and its derivatives.

[0022] In addition, triglycerides (TG) are the main form of energy storage in the body, playing a central role in maintaining energy metabolism balance, and also participating in the formation of intracellular lipid droplets, providing thermal insulation, and maintaining normal organ function. However, under conditions of high fat intake such as long-term high-fat diets or metabolic disorders, abnormally elevated triglyceride levels (i.e., hypertriglyceridemia) are closely related to the development of various serious diseases, including non-alcoholic fatty liver disease (NAFLD), atherosclerosis, cardiovascular disease, acute pancreatitis, and metabolic syndrome.

[0023] The homeostasis of intracellular and extracellular triglycerides is precisely regulated by a variety of complex mechanisms, mainly including the uptake of exogenous free fatty acids, de novo lipogenesis of endogenous fatty acids, esterification and assembly of triglycerides, formation and accumulation of lipid droplets, as well as lipolysis and fatty acid β-oxidation. Among these, the biosynthetic pathway of triglycerides is mainly located in the endoplasmic reticulum of the liver and adipose tissue, and is completed by a series of key enzymatic reactions. Key enzymes such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and diacylglycerol acyltransferase (DGAT) are the core regulatory sites of this synthetic pathway.

[0024] Currently, commonly used drugs for hypertriglyceridemia (such as fibrates, which mainly target PPAR-α receptors) can accelerate lipid clearance, but they are often accompanied by side effects such as liver and kidney damage, gastrointestinal reactions, or muscle toxicity. Therefore, in-depth research into the regulatory mechanisms of triglyceride metabolism under hyperlipidemia and the search for safe, effective, and low-side-effect novel lipid-lowering active substances or potential intervention targets are important issues that urgently need to be addressed in the field of cardiovascular and metabolic diseases.

[0025] Although fibrates and niacin, among other triglyceride-lowering drugs, are widely used clinically, they still have many limitations, including the aforementioned liver toxicity, muscle side effects, and heterogeneity in patient response. More importantly, some patients with severe hypertriglyceridemia are insensitive to existing drug treatments or cannot tolerate long-term use due to side effects. Therefore, exploring novel regulatory mechanisms of triglyceride metabolism under hyperlipidemia and identifying safe and effective potential intervention targets or natural active substances are of significant scientific and clinical value for developing new strategies for lowering triglycerides and treating lipid metabolism disorders.

[0026] Increasing evidence suggests that fruit extracts rich in dietary fiber and flavonoids have positive effects on lipid metabolism and cardiovascular disease in laboratory animals, and that daily diets rich in specific bioactive components (such as leafy green vegetables and root vegetables rich in inorganic nitrates) have a positive regulatory effect on lipid metabolism and cardiovascular health in laboratory animals.

[0027] Based on this, the present invention provides an application of nitrate in the preparation of lipid-lowering drugs.

[0028] In this embodiment, nitrate can significantly inhibit the synthesis of intracellular substances including cholesterol, triglycerides, cholesterol esters, and their metabolic derivatives, and the key role of the Sialin-Sialin2 axis in this process has been clarified. Furthermore, nitrate regulates cholesterol metabolism by controlling the interaction between Sialin2 and the key cholesterol synthesis enzymes DHCR7 and DHCR24 through the Sialin-Sialin2 axis. At the same time, nitrate can significantly inhibit the expression or activity of key enzymes in the triglyceride biosynthesis pathway through the Sialin2-mediated regulatory network, especially by precisely controlling acetyl-CoA carboxyltransferase (ACC), diacylglycerol acyltransferase 1 (DGAT1), and stearoyl-CoA desaturase 1 (SCD1). Nitrate blocks the de novo synthesis, desaturation processing, and final esterification assembly of fatty acids at the source, thereby efficiently and specifically reducing the generation and accumulation of lipid droplets. In addition, this invention utilizes nitrates to prepare lipid-lowering drugs, which have a complete regulatory mechanism and effective intervention targets. Furthermore, while nitrates play a role in degrading molecules such as cholesterol and triglycerides, they have no toxic side effects on liver and kidney function, demonstrating extremely high biosafety. This invention effectively proposes new indications for nitrates in "safe lipid-lowering" and improving fatty liver.

[0029] It should be noted that Sialin refers to... SLC17A5 The gene-encoded nitrate transport protein is a nitrate transport channel located on the cell membrane, responsible for transporting nitrate into saliva in the salivary glands. Sialin2 is produced by the hydrolysis of Sialin protein and is involved in the transport of sialic acid on the lysosomal membrane, participating in the regulation of cell function.

[0030] Specifically, the present invention uses nitrates to prepare lipid-lowering drugs, which has the following advantages: 1) Breaking away from the traditional understanding that nitrate is only a precursor or signaling molecule for NO, this invention proposes that nitrate directly regulates the synthesis of cholesterol, triglycerides, cholesterol esters and their metabolic derivatives through the endosome membrane transport protein Sialin / Sialin2. It is found that nitrate competitively inhibits the sialic acid transport function of Sialin, rather than simply acting as an ion channel, thereby reshaping the endosome-lysosome cholesterol and derivative efflux pathway and constructing a ternary regulatory model of "inorganic ions-membrane transport proteins-metabolic homeostasis". Furthermore, the specific function of Sialin2 in cholesterol and derivative metabolism can be further explored, namely, promoting the degradation of related synthetic enzymes through Rab7-dependent endosome sorting (non-classical autophagy pathway).

[0031] 2) Redefining the metabolic regulatory role of the endosome system, demonstrating that the endosome is not only a material sorting hub but also a core regulatory platform for cholesterol and derivative synthesis: integrating endosome-endoplasmic reticulum membrane contact and lipid droplet formation through Sialin-mediated nitrate signaling. The "metabolic buffer pool" hypothesis is proposed: the dynamic balance of nitrate concentration in the endosome lumen can buffer fluctuations in dietary cholesterol and derivatives, providing a new target for intervention in metabolic syndrome.

[0032] Furthermore, it was revealed that nitrate can regulate the triglyceride biosynthesis pathway. Through the Sialin2-mediated regulatory network, nitrate can significantly inhibit the expression or activity of key enzymes in the triglyceride biosynthesis pathway, especially precisely regulating acetyl-CoA carboxyltransferase (ACC), diacylglycerol acyltransferase 1 (DGAT1), and stearoyl-CoA desaturase 1 (SCD1). Nitrate blocks the de novo synthesis, desaturation processing, and final esterification assembly of fatty acids at the source, thereby efficiently and specifically reducing the formation and accumulation of lipid droplets.

[0033] 3) Propose indications for cholesterol-lowering drugs and their derivatives, and improve liver targeting through sustained-release formulation modifications. This could also promote the development of Sialin2 allosteric agonists, achieving low-dose, long-acting regulation by enhancing nitrate binding stability.

[0034] Furthermore, this invention provides a novel intervention target and strategy for the treatment of hypertriglyceridemia and related metabolic syndromes (such as non-alcoholic fatty liver disease). Most importantly, unlike existing clinically used lipid-lowering drugs (such as fibrates or statins), which are often accompanied by abnormal liver function, kidney damage, or muscle toxicity, this invention demonstrates that nitrates, while significantly degrading triglycerides, have no toxic side effects on liver and kidney function, exhibiting extremely high biocompatibility. This invention is the first to propose a new indication for nitrates in "safe lipid-lowering" and improving fatty liver; simultaneously, the development of novel allosteric agonists or modulators targeting Sialin2 will open up an important new pathway for the development of highly effective, low-toxicity innovative triglyceride-lowering drugs.

[0035] In some embodiments, the nitrate is an inorganic salt or a natural extract. The inorganic salt is a pharmaceutically acceptable inorganic nitrate; using a natural extract expands the application from prescription drugs to health foods, foods for special medical purposes (FSMP), or daily dietary supplements.

[0036] In some embodiments, the inorganic salts include, but are not limited to, one or more of sodium nitrate, potassium nitrate, and calcium nitrate; the natural extracts include, but are not limited to, beetroot extract or extracts from leafy green vegetables such as spinach.

[0037] In some embodiments, the lipid-lowering drug is used to inhibit one or more of, but not limited to, cholesterol, cholesterol metabolic derivatives, triglycerides, triglyceride metabolic derivatives, cholesterol esters, and cholesterol ester metabolic derivatives.

[0038] In some embodiments, the nitrate is used in the preparation of medicaments that inhibit the expression of DHCR7 and DHCR24 enzyme proteins in the biosynthetic pathways of cholesterol and / or cholesterol metabolic derivatives.

[0039] In some embodiments, the nitrate is used in the preparation of medicaments that inhibit the expression of ACC (acetyl-CoA carboxylase), DGAT1 (diacylglycerol acyltransferase 1), and SCD1 (stearoyl-CoA desaturase 1) proteins in the biosynthetic pathway of triglycerides and / or their metabolic derivatives.

[0040] In addition, the present invention also provides a lipid-lowering drug composition, comprising nitrates, and one or more of fibrates, statins, and PCSK9 inhibitors.

[0041] In this embodiment, nitrates have the advantage of being non-hepatic and non-nephrotoxic, and can be used in combination with existing lipid-lowering drugs to develop compound formulations, namely lipid-lowering drug compositions. This not only produces a synergistic lipid-lowering effect, but also significantly reduces the dosage of traditional lipid-lowering drugs while ensuring efficacy, thereby reducing or eliminating liver and kidney damage and muscle toxicity caused by traditional lipid-lowering drugs.

[0042] In some embodiments, the fibrate drug is selected from, but is not limited to, at least one of fenofibrate and gemfibrozil; the statin drug is selected from, but is not limited to, at least one of simvastatin and pravastatin.

[0043] In some embodiments, the lipid-lowering drug composition is formulated as, but not limited to, sustained-release capsules, controlled-release tablets, transdermal patches, or oral liquids. Using these dosage forms can maintain stable nitrate concentrations in the body and avoid fluctuations in blood drug concentrations.

[0044] In some implementations, taking advantage of the fact that triglycerides are mainly synthesized in the liver, nanotechnology (such as liposomes, polymer nanoparticles, or liver-targeting peptide-modified carriers) can be used to encapsulate nitrates or Sialin2 agonists to achieve liver-specific targeted delivery, thereby further reducing systemic drug dosage and improving local efficacy.

[0045] In some embodiments, the lipid-lowering drug composition further includes a pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable carrier includes at least one of a pharmaceutically acceptable excipient, a pharmaceutically acceptable additive, and a pharmaceutically acceptable adjuvant.

[0046] In some implementations, in addition to direct nitrate supplementation, small molecule compounds, peptides, or allosteric modulators that can specifically upregulate Silin2 expression or enhance the binding affinity of Silin2 to nitrate can be screened and developed. Alternatively, gene editing technology, mRNA technology, or adeno-associated virus (AAV) vector technology can be used to specifically overexpress the Silin2 gene in hepatocytes or adipocytes, thereby achieving long-term inhibition of triglyceride synthases such as ACC, DGAT1, and SCD1 without additional high-dose nitrate supplementation.

[0047] In some embodiments, the nitrate can also be used to prepare drugs for treating non-alcoholic fatty liver disease (NAFLD / NASH), obesity, atherosclerosis, or insulin resistance syndrome.

[0048] In some embodiments, the present invention may also design a short peptide that mimics a specific domain of Sialin2 or DHCR7 / DHCR24 to competitively bind to or stabilize the interaction between Sialin2 and DHCR7 / 24, thereby specifically regulating cholesterol and derivative synthesis and avoiding other systemic effects that nitrates may cause.

[0049] In some embodiments, this invention targets congenital cholesterol synthesis disorders caused by mutations in the DHCR7 or DHCR24 genes (such as Smith-Lemli-Opitz syndrome). While these diseases require cholesterol supplementation, the nitrates proposed in this invention can be used to regulate the activity or stability of mutant proteins, improving the pathological state. Non-alcoholic fatty liver disease (NAFLD): Hepatic cholesterol and derivative deposition is a key driver of NAFLD. Nitrates inhibit cholesterol and derivative synthesis through the hepatocyte Sialin-Sialin2 axis, potentially serving as a novel strategy for treating NAFLD / NASH. Preferably, nitrates are used in combination with statins: statins work by inhibiting HMG-CoA reductase, while this invention acts on downstream DHCR7 / 24. The combination may produce a synergistic effect and may reduce the dosage of statins, lowering their muscle toxicity and other side effects. Nitrates are also used in combination with PCSK9 inhibitors: This can simultaneously regulate the synthesis and clearance of cholesterol and its derivatives, achieving a more potent lipid-lowering effect.

[0050] In some implementations, the delivery method or form of nitrates is modified to improve efficacy, targeting, or reduce side effects. Liver-targeted: Nitrates are encapsulated in GalNAc (N-acetylgalactosamine)-modified liposomes or nanoparticles. Since the liver is the primary site of cholesterol and derivative synthesis, and hepatocytes highly express desialyl glycoprotein receptors, GalNAc modification enables active targeting to hepatocytes, increasing the concentration of nitrates in the liver. Enteric-coated or sustained-release formulations: Orally administered nitrates may be absorbed or degraded in the stomach. Enteric-coated formulations or sustained-release microparticles are designed to ensure that nitrates effectively reach the small intestine and are absorbed into the bloodstream, or directly act on local cholesterol and derivative metabolism in the intestine. Nitrate ions are chemically modified to create prodrugs. These prodrugs release active nitrates only after specific enzymatic cleavage in vivo (especially in the liver), achieving "on-demand release" or reducing systemic blood pressure effects (because nitrates also have vasodilatory effects).

[0051] In addition, this invention also provides a diagnostic kit for detecting cholesterol synthesis pathway activity. By detecting the binding strength of Sialin2 and DHCR7 / 24 in biological samples, the patient's cholesterol synthesis status or sensitivity to nitrate drugs can be determined.

[0052] In this embodiment, based on the mechanism of action of nitrates, they are transformed into a detection tool to determine a patient's cholesterol synthesis status or sensitivity to nitrate drugs. Companion diagnostics: Before administering nitrate treatment for hypercholesterolemia, the expression levels of Sialin and Sialin2 in the patient's liver tissue or peripheral blood are measured. High expression levels may indicate greater sensitivity to nitrate treatment, thereby identifying a favorable patient population and enabling personalized medicine.

[0053] In some embodiments, the application of nitrates in lowering blood lipids can be indirectly achieved, including: 1) Application in animal husbandry: Adding an appropriate amount of nitrates to animal feed can inhibit the synthesis of cholesterol and its derivatives in animals (such as pigs and chickens), thereby producing low-cholesterol meat or eggs, which meets the market demand for healthy food.

[0054] 2) Disease prevention or health food (medical food or health food): Nitrate (or its precursor, such as nitrate-rich beetroot extract) is used as a functional food ingredient to regulate the blood lipid levels of specific populations (such as those with mild hypercholesterolemia).

[0055] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention.

[0056] In the following examples, all nitrates are sodium nitrate.

[0057] Example 1 This embodiment reveals, through multi-omics research, that nitrates inhibit the synthesis of cholesterol and its derivatives, as detailed below: 1) In vivo experiments revealed that nitrates inhibit the synthesis of cholesterol and its derivatives in mice. A schematic diagram of the model and characterization are shown below. Figure 1 As shown, where, Figure 1 In the diagram, a represents the animal model, b represents the enrichment analysis of liver tissue proteomics, c represents the differential lipid analysis of liver tissue lipidomics, d represents the enrichment analysis of liver tissue metabolomics, and e represents the expression map of cholesterol and its derivatives.

[0058] By constructing high-fat diet mouse models and nitrate feeding models (such as...) Figure 1 As shown in a), proteins were extracted from the liver, and mass spectrometry analysis revealed significant changes in cholesterol and derivative metabolism-related proteins in the liver (e.g., [reference needed]). Figure 1 As shown in b), further lipid extraction from liver tissue and analysis revealed consistent results with proteomic enrichment analysis. Cholesterol and its derivative metabolic pathways were significantly downregulated, and among the differentially expressed lipids, cholesterol and its derivatives were significantly downregulated in liver tissue (e.g., ...). Figure 1 (as shown by c, d, and e in the diagram).

[0059] 2) In vitro experiments analyzed the effects of nitrate on intracellular cholesterol and derivative synthesis. Schematic diagrams of the model and characterization are shown below. Figure 2 As shown, where, Figure 2 In the diagram, a represents the cell modeling image, b represents the differential lipid heatmap, c represents the expression of cholesterol and its derivatives in cells, d represents the cell metabolomics enrichment analysis, and e represents the distribution of differential lipids in cells.

[0060] Lipomics were analyzed after HEK293T cells were treated with 4 mM nitrate for 4 h. Figure 2 As shown in a), multiple lipids are downregulated, and lipid synthesis-related pathways are significantly downregulated, with cholesterol and its derivatives being significantly downregulated (e.g., ...). Figure 2 (as shown in b, c, d, and e).

[0061] Example 2 This embodiment explores the localization of the nitrate-induced Sialin-Sialin2 signaling axis in the endoplasmic reticulum, as detailed below: 1) The nitrate-stimulated Sialin 1 / 2 response is shown in the figure below. Figure 3 As shown, where, Figure 3In the diagram, a is the WB plot of the nitrate intervention gradient experiment, b and c are the quantitative analysis plots of Sialin1 / 2 protein, d is the bar chart of differentially expressed genes in the WT, WTN, KO, and KON intervention groups, e is the Venn plot of differentially expressed genes, and f is the volcano plot of differentially expressed genes.

[0062] Intervention with 0 mM, 1 mM, and 4 mM of Sialin2 for 4 h significantly upregulated Sialin2 at 4 mM (e.g., Figure 3 (As shown in a, b, and c in the figure), HEK293T cells were treated with 4 mM nitrate for 4 h, and then the transcriptome was analyzed. Significant knockout was observed. SLC17A5 In HEK293T cells, differentially expressed genes were significantly downregulated under nitrate stimulation. In wild-type HEK293T cells, downregulation of genes related to cholesterol and its derivatives metabolism was observed, such as… INSIG1 , HMGCS1 etc. (e.g.) Figure 3 (as shown by d, e, and f in the figure).

[0063] 2) Nitrate-stimulated endocytosis enriches Sialin2 on the endoplasmic reticulum, as shown in the characterization diagram. Figure 4 As shown, where Figure 4 In the diagram, a is the volcano plot of Sialin2-TurboID differential protein, b is the GO-biological function enrichment analysis of Sialin2-TurboID differential protein, c is the Wiki enrichment analysis of Sialin2-TurboID differential protein, d and e are the immunofluorescence staining and quantitative analysis of Sialin2 and the late endosome marker RAB7, and f and g are the adjacent linkage diagram and quantitative analysis of RAB7 and Sialin2 after nitrate intervention with MDA-MB-231.

[0064] After constructing the Sialin2-TurboID plasmid and transfecting cells, biotinylation of pull-down proteins significantly upregulated endoplasmic reticulum-related proteins (e.g., ...). Figure 4 As shown in a), differentially expressed genes are enriched in endocytic transport and lipid metabolism-related pathways (such as...). Figure 4 (As shown in b and c). Further, by constructing the EGFP-Sialin2 plasmid, immunofluorescence staining revealed that the late endosome marker RAB7 and Sialin2 showed significant co-localization and enhancement under nitrate stimulation (e.g., ...). Figure 4 As shown in d and e), the adjacent linkage technique further verified that the interaction between intracellular Sialin2 and RAB7 was enhanced after nitrate intervention (as shown in d and e). Figure 4 (as shown by f and g in the figure).

[0065] Example 3 This embodiment explores the mechanism by which Sialin2 interacts with DHCR7 and DHCR24 to regulate cellular cholesterol metabolism, as detailed below: 1) Sialin2 interacts with VAPA and is localized in the endoplasmic reticulum, as shown in the characterization diagram below. Figure 5 As shown, where, Figure 5 In the diagram, a represents the differential protein expression of the top 50 proteins in Sialin2-TurboID; b, c, and d represent the immunoblotting and quantitative analysis of VAPA protein on the endoplasmic reticulum under nitrate stimulation; and e and f represent confocal microscopy images of co-transfected EGFP-VAPA and mCherry-Sialin2 plasmid.

[0066] Using the Sialin2-TurboID technique, it was revealed that Sialin2 participates in intracellular transport processes and interacts with key proteins in the MCSs complex on the endoplasmic reticulum, namely VAPA and the endoplasmic reticulum marker protein CNX (e.g., Figure 5 As shown in a, b, c, and d). Confocal microscopy images and quantitative analysis diagrams also confirm the local co-localization of Sialin2 and VAPA, with VAPA mediating the localization of Sialin2 on the endoplasmic reticulum (e.g., ...). Figure 5 (as shown in e and f).

[0067] 2) Sialin2 can inhibit cholesterol synthesis in response to nitrate stimulation, as shown in the figure below. Figure 6 As shown, where Figure 6 In the diagram, a is the Venn plot of differentially expressed TurboID protein in the differential proteome of nitrate-treated cells; b is a heatmap of differentially expressed proteins related to cholesterol metabolism in the intersection; c and d are Filipin staining and quantitative analysis after nitrate intervention and Sialin2 transfection; and e is a statistical graph of whole-cell cholesterol expression quantification.

[0068] By intersecting differentially expressed proteins from nitrate-treated cells with Sialin2-TurboID protein, eight of the differentially expressed proteins were found to be closely related to cholesterol metabolism. Furthermore, knocking out the SLC17A5 gene did not significantly alter these proteins, indicating that Sialin2 interacts with these proteins to influence cholesterol metabolism (e.g., Figure 6 (As shown in a and b). Furthermore, by reintroducing Sialin2 into SLC17A5 gene knockout cells, the function of inhibiting cholesterol synthesis was restored, while the function of Sialin (KRI / AAA) reintroduction was not significantly restored (e.g., ...). Figure 6 (as shown in c, d, and e).

[0069] Example 4 This embodiment reveals, through multi-omics research, that nitrates inhibit triglyceride synthesis, as detailed below: To investigate the regulatory role of nitrate on lipid metabolism, this study performed combined lipidomics and proteomics analysis on liver tissue from mice on a high-fat diet (HFD) for 8 weeks with nitrate intervention. The flowchart and results characterization diagram are shown below. Figure 7 As shown, where, Figure 7 In the diagram, 'a' represents the experimental design flowchart, 'b' represents the liver lipid profile of high-fat mice fed with nitrate, 'c' and 'd' represent the liver proteome of high-fat mice fed with nitrate, and 'e' represents the lipid profile of HEK293T mice treated with nitrate.

[0070] The lipidomics heatmap results visually demonstrate that nitrate intervention significantly improved lipid accumulation in the liver, with the abundance of the vast majority of lipid molecules showing a significant downregulation (e.g., Figure 7 As shown in a and b). Further proteomics GO biological process (BP) enrichment analysis revealed the underlying lipid metabolism network remodeling mechanism: under the pathological state of HFD, biological processes related to fatty acid metabolism (including monocarboxylic acid metabolism, long-chain and ultra-long-chain fatty acid metabolism, acyl-CoA metabolism, etc.) were significantly activated (e.g. Figure 7 c); Conversely, processes related to the biosynthesis of cholesterol and sterols were significantly downregulated (e.g., Figure 7 (As shown in d and e). Furthermore, in vitro experiments further confirmed this phenotype; HEK293T cells treated with nitrate for 4 hours also exhibited a significant decrease in the abundance of a large number of lipid molecules in their lipidomics profile (e.g., ...). Figure 2 (As shown). In summary, multi-omics studies based on in vivo and in vitro models consistently demonstrate that nitrates can effectively intervene in abnormal fatty acid metabolism networks, thereby significantly inhibiting the synthesis and accumulation of lipids such as triglycerides.

[0071] Example 5 This embodiment verifies that nitrate regulates the key enzyme in triglyceride synthesis via the Sialin-Sialin2 axis, and its characterization diagram is shown below. Figure 8 As shown, where, Figure 8 In the figures, a and b represent the effects of nitrate treatment under high-fat conditions on the protein expression of key enzymes in triglyceride synthesis (GPAT4, DGAT1, SCD1) in Huh7 cells and their quantitative analysis; c and d represent the effects of knockdown of Sialin (si-sialin) on the expression of lipid synthesis-related proteins in Huh7 cells and their quantitative analysis; and e represents the effects of knockdown of Sialin (si-sialin) on the mRNA expression of lipid synthesis-related genes in Huh7 cells.

[0072] Given that nitrates can effectively reduce triglyceride levels in hyperlipidemic mice, it is hypothesized that they may exert their effects by regulating key lipid synthesis pathways. To verify this hypothesis, nitrate treatment was applied to Huh7 cells induced by hyperlipidemic conditions for 12 hours in vitro. Western blot (WB) results showed that nitrate treatment significantly inhibited the protein expression of key triglyceride synthesis enzymes DGAT1 and SCD1. Furthermore, cellular omics analysis further confirmed that nitrate treatment significantly reduced intracellular triglyceride accumulation (e.g., ...). Figure 8 (as shown in a and b in the diagram).

[0073] To further investigate its mechanism of action, the nitrate transporter siline was knocked down in Huh7 cells. Subsequent Western blotting and qPCR results showed that the absence of siline led to a significant decrease in ACC phosphorylation levels (p-ACC), thereby relieving the inhibition of lipid synthesis and significantly enhancing triglyceride synthesis (e.g., Figure 8 (as shown by c, d, and e in the figure).

[0074] Example 6 This embodiment verifies that nitrate has excellent hepatic and renal safety, as shown in the characterization diagram below. Figure 9 As shown, where, Figure 9 In the figure, af represents the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin (ALB), and uric acid (UA) levels in wild-type mice and high-fat mice after nitrate feeding, respectively.

[0075] The presence of nitrates on the liver and kidneys was verified by analyzing liver and kidney function in serum from wild-type mice and high-fat mice fed with nitrates. Figure 9 As shown in Figure af, after feeding nitrates, there were no significant differences in the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin (ALB), and uric acid (UA) between wild-type mice and high-fat mice. This indicates that nitrates have no significant toxic effects on the liver and kidneys of mice and that nitrates have excellent liver and kidney safety.

[0076] In summary, this invention provides an application of nitrate in the preparation of lipid-lowering drugs. The nitrate significantly inhibits the synthesis of intracellular cholesterol, triglycerides, cholesterol esters, and their metabolic derivatives, and clarifies the crucial role of the Sialin-Sialin2 axis in this process. Furthermore, nitrate regulates cholesterol metabolism by modulating the interaction between Sialin2 and the key cholesterol synthesis enzymes DHCR7 and DHCR24 through the Sialin-Sialin2 axis. Simultaneously, nitrate, through a Sialin2-mediated regulatory network, significantly inhibits the expression or activity of key enzymes in the triglyceride biosynthesis pathway, particularly precisely regulating acetyl-CoA carboxyltransferase (ACC), diacylglycerol acyltransferase 1 (DGAT1), and stearoyl-CoA desaturase 1 (SCD1). Nitrate blocks the de novo synthesis, desaturation processing, and final esterification assembly of fatty acids at the source, thereby efficiently and specifically reducing the formation and accumulation of lipid droplets. In addition, this invention utilizes nitrates to prepare lipid-lowering drugs, which have a complete regulatory mechanism and effective intervention targets. Furthermore, while nitrates play a role in degrading molecules such as cholesterol and triglycerides, they have no toxic side effects on liver and kidney function, demonstrating extremely high biosafety. This invention effectively proposes new indications for nitrates in "safe lipid-lowering" and improving fatty liver.

[0077] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. Application of nitrates in the preparation of lipid-lowering drugs.

2. The application according to claim 1, characterized in that, The nitrate is an inorganic salt or a natural extract.

3. The application according to claim 2, characterized in that, The inorganic salts include one or more of sodium nitrate, potassium nitrate, and calcium nitrate; the natural extracts include beetroot extract or spinach extract.

4. The application according to claim 1, characterized in that, The lipid-lowering drug is used to inhibit one or more of the following: cholesterol, cholesterol metabolic derivatives, triglycerides, triglyceride metabolic derivatives, cholesterol esters, and cholesterol ester metabolic derivatives.

5. The application according to claim 1, characterized in that, The use of the nitrate in the preparation of drugs that inhibit the expression of DHCR7 and DHCR24 enzyme proteins in the biosynthetic pathways of cholesterol and / or cholesterol metabolic derivatives.

6. The application according to claim 1, characterized in that, The use of the nitrate in the preparation of drugs that inhibit the expression of ACC, DGAT1 and SCD1 enzyme proteins in the biosynthetic pathway of triglycerides and / or triglyceride metabolic derivatives.

7. A lipid-lowering drug composition, characterized in that, This includes nitrates, as well as one or more of fibrates, statins, and PCSK9 inhibitors.

8. The lipid-lowering drug composition according to claim 7, characterized in that, The fibrates are selected from at least one of fenofibrate and gemfibrozil; the statins are selected from at least one of simvastatin and pravastatin.

9. The lipid-lowering drug composition according to claim 7, characterized in that, The lipid-lowering drug composition is formulated as one of sustained-release capsules, controlled-release tablets, transdermal patches, or oral liquids.

10. The lipid-lowering drug composition according to claim 7, characterized in that, The lipid-lowering drug composition also includes a pharmaceutically acceptable carrier; Preferably, the pharmaceutically acceptable carrier includes at least one of a pharmaceutically acceptable excipient, a pharmaceutically acceptable additive, and a pharmaceutically acceptable adjuvant.

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