Use of a powdery mildew compound in the preparation of a medicament for treating and / or preventing hypercholesterolemia

Abstract

The application discloses application of a piericidin compound in preparation of a medicine for treating and / or preventing hypercholesterolemia. The application proves that the piericidin compound can relieve hypercholesterolemia by constructing a 1% cholesterol (HCD) diet-induced hypercholesterolemia model in vivo, detecting serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TCHO), low-density lipoprotein (LDLC), high-density lipoprotein (HDLC) levels and liver tissue oil red staining. The piericidin compound has high affinity with PPARdelta and can act as a PPARdelta agonist to increase the transcription level of downstream genes of the PPARdelta.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of phenobarbital compounds in the preparation of drugs for the treatment and / or prevention of hypercholesterolemia. Background Technology

[0002] Hypercholesterolemia is a metabolic disorder characterized by abnormally elevated levels of total cholesterol and low-density lipoprotein cholesterol (LDL-C) in plasma, leading to abnormal lipid deposition in blood vessel walls and tissues, causing endothelial damage and inflammation. The occurrence of hypercholesterolemia is closely related to multiple factors, including genetic factors, poor dietary habits, obesity, and metabolic syndrome. To date, the molecular regulatory network of this disease is not fully elucidated. However, persistent high cholesterol load and oxidative stress often lead to impaired vascular repair, resulting in chronic inflammation of the arterial intima, smooth muscle cell proliferation, foam cell formation, and fibrous plaque deposition, ultimately developing into serious cardiovascular events such as atherosclerosis, coronary heart disease, and stroke. Currently, apart from limited lipid-lowering treatments such as statins, there is still a lack of effective means in clinical practice to completely reverse vascular plaques or eradicate lipid metabolism disorders. Therefore, precise intervention strategies targeting novel targets (such as PCSK9 and inflammatory pathways) urgently need to be explored.

[0003] Peroxisome proliferator-activated receptor delta (PPARδ) is a member of the nuclear hormone receptor superfamily and plays a central role as a transcription factor in regulating energy metabolism, fatty acid oxidation, and inflammatory responses. In the context of hypercholesterolemia, PPARδ exhibits potential therapeutic value through multiple mechanisms: Regulating lipoprotein metabolism and cholesterol reversal transport: Activation of PPARδ can upregulate the expression of key genes involved in cholesterol reversal transport (such as ABCA1 and ABCG1), promote the transport of excess cholesterol in peripheral tissues to high-density lipoprotein (HDL), and enhance the liver's uptake of cholesterol and bile acid conversion, thereby systematically reducing plasma low-density lipoprotein cholesterol (LDL-C) levels and improving the lipid profile.

[0004] Anti-inflammatory and protective effects on vascular endothelium: PPARδ can inhibit pro-inflammatory signaling pathways such as NF-κB, reduce the production of adhesion molecules (such as VCAM-1 and ICAM-1) and inflammatory factors in vascular endothelial cells, thereby alleviating vascular endothelial inflammatory damage caused by hypercholesterolemia and delaying the initial formation of atherosclerotic plaques.

[0005] Reduce oxidative stress: Activation of PPARδ can enhance the antioxidant defense capacity of cells (such as inducing superoxide dismutase, catalase, etc.), remove excess reactive oxygen species (ROS), protect vascular endothelial cells and hepatocytes from oxidative damage, and improve lipid peroxidation status.

[0006] Improving hepatic lipid deposition and insulin resistance: PPARδ is a major regulator of cellular fatty acid oxidation. Once activated, it can promote fatty acid β-oxidation in the liver and skeletal muscle, reduce the accumulation of ectopic lipids in the liver, and improve insulin sensitivity, thus intervening in the metabolic disorders that often accompany hypercholesterolemia from the metabolic source.

[0007] Inhibiting foam cell formation and plaque progression: PPARδ activation can stabilize atherosclerotic plaques and prevent their progression by regulating macrophage lipid metabolism, reducing the uptake of oxidized low-density lipoprotein (ox-LDL) and the formation of foam cells, while inhibiting abnormal proliferation of smooth muscle cells and degradation of the fibrous cap.

[0008] In summary, PPARδ, as a multifunctional regulatory node, plays a crucial role in the treatment of hypercholesterolemia and its complications (atherosclerosis) by coordinating multiple pathways, including lipid metabolism, anti-inflammation, anti-oxidation, and vascular protection. Targeted activation of PPARδ is considered a promising strategy for lipid-lowering and anti-atherosclerotic treatment. Currently, specific PPARδ agonists are still in preclinical and early clinical research stages, but their unique advantages in lipid metabolism regulation are attracting increasing attention. Summary of the Invention

[0009] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide the use of phenobarbital compounds in the preparation of medicaments for the treatment and / or prevention of hypercholesterolemia.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides the use of phenobarbital compounds and their pharmaceutically acceptable salts in the preparation of medicaments for the treatment and / or prevention of hypercholesterolemia, wherein the chemical structure of the phenobarbital compounds is shown in formula (I).

[0011] Formula (I).

[0012] Furthermore, the hypercholesterolemia is hypercholesterolemia induced by a diet high in cholesterol and sodium cholate.

[0013] In another aspect, the present invention provides the use of pyromycin compounds and their pharmaceutically acceptable salts in the preparation of PPARδ agonists, the chemical structures of said pyromycin compounds being shown in formula (I).

[0014] Formula (I).

[0015] In the technical solution of the present invention, the pteridoxycin-like compounds can upregulate downstream genes of PPARδ; the downstream genes include ANGPTL4, PDK4 and PLIN2.

[0016] In another aspect, the present invention provides the use of phenobarbital compounds and their pharmaceutically acceptable salts in the preparation of drugs that upregulate downstream genes of PPARδ, the chemical structures of the phenobarbital compounds being shown in formula (I).

[0017] Formula (I).

[0018] Furthermore, the downstream genes include ANGPTL4, PDK4, and PLIN2.

[0019] In the technical solution of the present invention, the pharmaceutically acceptable salt is selected from any one of hydrochloride, sulfate, phosphate, acetate, and citrate.

[0020] The pyromycin compounds in this invention are derived from marine fungi. Streptomyces sp. SCSIO NS104 (JMC. 2019, 62 (15), 7058-7069). This invention discloses for the first time that this pyrophoric acid compound can specifically target PPARδ and regulate its downstream transcriptional levels to achieve an anti-hypercholesterol effect. This invention demonstrates, through optical surface plasmon resonance (SPR) and cell thermal transfer ablation (CETSA)-Western immunoblotting (WB) experiments, that this pyrophoric acid compound can interact with the PPARδ protein. Furthermore, it can increase the transcriptional levels of downstream PPARδ genes and improve metabolic disorders in hypercholesterolemia, thus serving as a lead compound for the development of anti-hypercholesterol drugs.

[0021] Compared with the prior art, the advantages of the present invention are as follows: 1. This invention constructs a hypercholesterolemia model induced by a 1% cholesterol (HCD) diet in vivo, and detects serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TCHO), low-density lipoprotein (LDLC), high-density lipoprotein (HDLC) levels and liver tissue oil red staining to confirm that the pyrophoric acid compound can alleviate hypercholesterolemia.

[0022] 2. This invention is the first to demonstrate the PPARδ agonist effect of this pyromycin-like compound, providing a new candidate compound for the preparation of drugs to prevent and treat hypercholesterolemia.

[0023] 3. The present invention finds that piericidin compounds have high affinity for PPARδ and increase the transcription level of its downstream genes, which provides a new direction for the field of hypercholesterolemia treatment and has broad development and application prospects. Brief Description of the Drawings

[0024] Figure 1 is the overall liver morphology diagram and the Oil Red O staining diagram of liver tissue of each group of mice in Example 1 of the present invention.

[0025] Figure 2 shows the serum levels of ALT, TCHO, LDLC and HDLC of each group of mice in Example 1 of the present invention.

[0026] Figure 3 is the relative expression level of PPARδ downstream genes in the liver of each group of mice in Example 1 of the present invention.

[0027] Figure 4 shows the results of SPR experiment and CETSA-WB assay for detecting the binding of S14 to PPARδ protein in Example 2 of the present invention. Detailed Description of the Embodiments

[0028] The following embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the present invention as claimed, but merely to represent selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts shall fall within the protection scope of the present invention.

[0029] In the present invention, unless otherwise specified, all equipment, raw materials and the like are commercially available or commonly used in the industry. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0030] Example 1: In this example, the effect of piericidin compound S14 on liver function of HCD mice was investigated. The structure of piericidin compound S14 is shown in formula (I), and the specific process is as follows: Experimental animals: The present invention uses male C57 BL / 6 mice, SPF grade, with a body weight of 22-25 g, provided by the Laboratory Animal Center of Southern Medical University (License No.: SCXK (Guangdong) 2016-0041). All mice are housed in an environment with a temperature of 25±1°C and a humidity of 55±5%. The day and night are alternately light and dark (12 h light, 12 h dark), with free access to drinking water and food. The mice were housed in an SPF-grade animal room for one week before the experiment to adapt to the environment.

[0031] Formula (I).

[0032] 1. Compound S14 alleviates hypercholesterolemia caused by HCD. 1.1 Establishment of HCD-induced hypercholesterolemia model and grouping of patients for drug administration HCD-induced hypercholesterolemia mouse models were fed 1% cholesterol + 0.5% sodium cholate (Dez Biotech) for 14 days to establish a bile duct injury model.

[0033] Thirty male C57 BL / 6 mice were randomly divided into five groups of six each: normal diet (ND), HCD diet, HCD diet + S14-L (0.5 mg / kg), HCD diet + S14-H (1 mg / kg), and HCD diet + atorvastatin. The HCD + S14-L (0.5 mg / kg) and HCD + S14-H (1 mg / kg) groups were intraperitoneally injected with the corresponding S14 solution every other day for 14 consecutive days. During this period, the normal diet (ND) group and the HCD model group were intraperitoneally injected with an equal dose of blank control solvent (1% DMSO / PBS). Fourteen days after modeling, blood was collected via the orbital venous plexus into centrifuge tubes containing heparin sodium. The tubes were centrifuged at 8000 rpm for 5 min (4℃), and the supernatant plasma was transferred to new centrifuge tubes for plasma biochemical analysis. The mice were then euthanized, and their liver tissue was removed, a portion of which was used for assessing fatty degeneration of the liver tissue.

[0034] 1.2 Assessment of steatosis in liver tissue (Oil Red O staining) Collected organ tissues were fixed in 4.5% formalin, dehydrated using a sucrose gradient, and then embedded in OCT embedding medium to prepare frozen sections. The sections were stained with Oil Red O, and lipid deposition within the tissues was observed using an optical microscope.

[0035] 1.3 Detection of serum ALT, TCHO, LDLC and HDLC levels The tests were performed using ALT, TCHO, LDLC, and HDLC kits produced by Nanjing Jiancheng Biotechnology Research Institute, and the specific assay methods were performed entirely in accordance with the instructions.

[0036] 1.4 Experimental Results like Figure 1 As shown, S14 significantly alleviated high-cholesterol diet (HCD)-induced lipid deposition and steatosis in the liver of mice, and reduced hepatocyte ballooning degeneration and intralobular inflammatory infiltration. Plasma lipid parameters were detected using serum TCHO, LDL-C, and HDL-C assay kits. Figure 2As shown, compared with the ND group, the HCD model group had significantly higher serum ALT, TCHO, and LDL-C levels and lower HDL-C levels, while S14 could dose-dependently reverse these lipid abnormalities. The results indicate that S14 can effectively alleviate HCD-induced hypercholesterolemia and its associated hepatic lipid accumulation.

[0037] 2. Effects of compound S14 on downstream genes of PPARδ and its role in a hypercholesterolemia model. 2.1 Detection of liver PPARδ and its downstream mRNA levels Total RNA was extracted from mouse liver tissue using an animal tissue total RNA extraction kit (Chengdu Fuji Biotechnology Co., Ltd.), and the extraction method was strictly followed according to the manufacturer's instructions. After extraction, the total RNA was reverse transcribed into cDNA, and changes in its transcriptional expression level were detected using qPCR.

[0038] 2.2 Experimental Results like Figure 3 As shown, S14 significantly increased the expression levels of downstream genes of PPARδ induced by HCD. The results indicate that S14 can alleviate HCD-induced hypercholesterolemia by targeting PPARδ.

[0039] Example 2: This embodiment investigated the ability of the pteropterin S14 compound to bind to the PPARδ protein, as detailed below: 1. SPR method for analyzing the affinity of S14 for PPARδ protein 1.1 SPR Method Human PPARδ protein was purchased from Cloud Clone (Catalog No. RPC041Hu02, China). The human PPARδ protein was immobilized on a nanocapture sensor chip with a bare gold coating. S14 was diluted in PBS buffer at concentrations ranging from 15.625 to 4000 nM and injected in a multi-cycle analysis mode to analyze the affinity of S14 for PPARδ protein.

[0040] 1.2 Cell thermal transfer assay (CETSA) To assess the binding affinity of S14 to PPARδ, cell thermal shift assays were performed as previously described. HepG2 cells were seeded in large dishes (1×10⁶ cells / year). 6When HepG2 cells reached ~70% confluence, 1‰ DMSO or S14 (10 μM) was added, and the cells were incubated for 4 h. The cells were washed three times with PBS, then PBS containing protease inhibitors was added, and the cells were scraped off with a scraper and transferred to cryovials. The cell suspension was freeze-thawed five times using liquid nitrogen. The cell lysates were centrifuged at 13,000 × g for 20 min at 4°C. The supernatants were divided into equal aliquots and heated at different temperatures for 3 min each. The heated supernatants were centrifuged at 13,000 × g for 15 min at 4°C to remove denatured proteins. The supernatants were collected, and the expression level of PPARδ protein was analyzed using Western blotting to verify its thermostability.

[0041] 1.3 Experimental Results The results are as follows Figure 4 As shown in the figure, the SPR results indicate that S14 binds to PPARδ protein in a concentration-dependent manner, with a dissociation equilibrium constant ( K D The concentration was 605 nM. CETSA-WB results showed that, compared with the control group, S14 significantly improved the thermal stability of PPARδ, further supporting the direct binding of S14 to PPARδ as its target.

[0042] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of pterocaryonid compounds and their pharmaceutically acceptable salts in the preparation of medicaments for the treatment and / or prevention of hypercholesterolemia, characterized in that, The chemical structure of the pteridoxycin-like compounds is shown in formula (I); Equation (I).

2. The application according to claim 1, characterized in that, The hypercholesterolemia mentioned refers to hypercholesterolemia induced by a diet high in cholesterol and sodium cholate.

3. The application according to claim 1, characterized in that, The pharmaceutically acceptable salt is selected from any one of hydrochloride, sulfate, phosphate, acetate, and citrate.

4. The use of pterocaryonin compounds and their pharmaceutically acceptable salts in the preparation of PPARδ agonists, characterized in that, The chemical structure of the pteridoxycin-like compounds is shown in formula (I); Equation (I).

5. The application according to claim 3, characterized in that, The pteridoxycin compounds can upregulate downstream genes of PPARδ; the downstream genes include ANGPTL4, PDK4 and PLIN2.

6. The application according to claim 1, characterized in that, The pharmaceutically acceptable salt is selected from any one of hydrochloride, sulfate, phosphate, acetate, and citrate.

7. The use of pteropterinoid compounds and their pharmaceutically acceptable salts in the preparation of drugs that upregulate downstream genes of PPARδ, characterized in that, The chemical structure of the pteridoxycin-like compounds is shown in formula (I); Equation (I).

8. The application according to claim 7, characterized in that, The downstream genes include ANGPTL4, PDK4, and PLIN2.

9. The application according to claim 7, characterized in that, The pharmaceutically acceptable salt is selected from any one of hydrochloride, sulfate, phosphate, acetate, and citrate.

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