Application of Terminalia chebula tannic acid in the preparation of drugs for treating atherosclerosis

Abstract

This invention relates to the application of chebulic tannic acid in the preparation of drugs for treating atherosclerosis, belonging to the field of atherosclerosis treatment technology. This invention discovers that chebulic tannic acid can specifically bind to the catalytic pocket region of SAHH protein, significantly enhancing its efficiency in hydrolyzing SAH, in ApoE... ‑ / ‑ In a mouse model, chebulic tannic acid significantly reduced plasma SAH levels. Simultaneously, the aortic plaque area decreased in mice treated with chebulic tannic acid. This demonstrates that chebulic tannic acid has a therapeutic effect on atherosclerosis. This invention, by regulating methionine cycle homeostasis through chebulic tannic acid, not only reduces plasma SAH and tHcy levels but also effectively inhibits inflammatory responses and oxidative stress, significantly improving atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of atherosclerosis treatment technology, and in particular to the application of Terminalia chebula tannic acid in the preparation of drugs for treating atherosclerosis. Background Technology

[0002] Atherosclerosis (AS) is widely recognized as the root cause of atherosclerotic cardiovascular disease (ASCVD). Its development begins with endothelial damage and progresses through key stages including lipid infiltration, chronic inflammation, smooth muscle cell proliferation and migration, and extracellular matrix remodeling, ultimately forming atherosclerotic plaques. ASCVD directly caused by AS is the leading cause of cardiovascular and cerebrovascular disease death worldwide, accounting for up to 85% of all cardiovascular disease deaths.

[0003] Methionine, an essential sulfur-containing amino acid, is not only a raw material for protein synthesis but also a key participant in methylation reactions in organisms. Under the catalysis of methionine adenosyltransferase (MAT), methionine is converted to S-adenosylmethionine (SAM). SAM, as a core product of single-carbon metabolism and the primary methyl donor, plays a crucial role in processes such as DNA / RNA methylation. When SAM donates a methyl group through methyltransferase, it generates S-adenosyl homocysteine ​​(SAH). Subsequently, SAH is hydrolyzed by SAH hydrolase (SAHH) into adenosine and homocysteine ​​(tHcy). This reversible reaction constitutes the core regulatory site of the methionine cycle.

[0004] Existing drugs for treating atherosclerosis mostly focus on lowering lipids, reducing inflammation, or stabilizing plaques, and their therapeutic effects are effective. Therefore, it is urgent to develop new drugs with clear therapeutic targets and mechanisms. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of Terminalia chebula tannic acid in the preparation of drugs for treating atherosclerosis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the application of chebulic tannic acid in the preparation of a drug for treating atherosclerosis, wherein the chebulic tannic acid achieves its anti-atherosclerotic effect by binding to SAHH protein and increasing the enzyme activity of SAHH.

[0007] This invention discovers that Terminalia chebula tannic acid can specifically bind to the catalytic pocket region of SAHH protein, significantly enhancing its efficiency in hydrolyzing SAH, in ApoE. - / -In mouse models, a significant reduction in plasma SAH levels was observed. Simultaneously, the aortic plaque area decreased in mice treated with chebulic tannic acid. This demonstrates that chebulic tannic acid has a therapeutic effect on atherosclerosis. This invention, by regulating methionine cycle homeostasis through chebulic tannic acid, not only reduces plasma SAH and tHcy levels but also effectively reduces aortic plaque area, developing a new application for chebulic tannic acid and a novel targeted strategy for treating atherosclerosis.

[0008] Secondly, this invention provides the application of chebulic tannic acid in the preparation of SAHH agonists. Experiments have shown that the activity of SAHH in plasma was significantly increased in the chebulic tannic acid-treated group, indicating that chebulic tannic acid can directly activate SAHH enzyme activity. Its effect is independent of upstream signaling pathway regulation, but rather enhances the affinity and catalytic efficiency of SAHH for SAH through conformational optimization. Therefore, chebulic tannic acid can be used as an SAHH agonist.

[0009] Thirdly, this invention provides the application of chebulic tannic acid in the preparation of inhibitors of SAH and / or tHcy. Experiments have shown that the levels of SAH and / or tHcy in plasma were significantly downregulated in the chebulic tannic acid treatment group, indicating that chebulic tannic acid enhances the activity of SAHH and thus enhances the hydrolysis reaction of SAH, thereby reducing the levels of SAH and / or tHcy in plasma. Therefore, chebulic tannic acid can be used as an inhibitor of SAH and / or tHcy.

[0010] Fourthly, this invention provides the application of chebulic tannic acid in the preparation of drugs that reduce inflammation of atherosclerotic plaques. Experiments have shown that chebulic tannic acid can significantly reduce the inflammatory response of plaques, with a significant decrease in the CD68-positive area of ​​the aorta and the expression levels of VCAM-1 and ICAM-1 in the aortic root. This indicates that chebulic tannic acid has a significant anti-inflammatory effect and can effectively intervene in the inflammatory microenvironment during the process of atherosclerosis, thereby reducing inflammation of atherosclerotic plaques.

[0011] Fifthly, this invention provides the application of chebulic tannic acid in the preparation of drugs to alleviate oxidative stress in atherosclerotic plaques. Experiments show that chebulic tannic acid can significantly reduce the DHE-positive area and MDA content in plaque tissue, indicating that chebulic tannic acid can effectively scavenge ROS and inhibit lipid peroxidation, thereby achieving the purpose of alleviating oxidative stress.

[0012] Sixthly, this invention provides the application of Terminalia chebula tannic acid in the preparation of drugs that improve the stability of atherosclerotic plaques. Experiments show that in mice treated with Terminalia chebula tannic acid, the collagen content in the plaques is significantly increased, the SMA-positive area in the aortic root is significantly increased, and the expression levels of plaque instability factors (MMP3 and MMP9) are significantly downregulated. This indicates that Terminalia chebula tannic acid can also improve plaque stability and reduce the risk of plaque rupture.

[0013] Seventhly, this invention provides the application of chebulic tannic acid in the preparation of drugs that reduce macrophage inflammation. Experiments have shown that the levels of pro-inflammatory factors TNF-α, IL-6, IL-1β, Ccl2, and Ccl5 in macrophages of mice in the chebulic tannic acid group were reduced, as were the mRNA levels of p21, IL-1β, IL-6, ICAM-1, VCAM-1, and Ccl2. This indicates that chebulic tannic acid can inhibit macrophage inflammation, thereby achieving the purpose of reducing inflammation.

[0014] Eighthly, this invention provides the application of chebulic tannic acid in the preparation of inhibitors of macrophage lipid accumulation. Experiments show that the area of ​​Oil Red O staining in macrophages was reduced in the chebulic tannic acid group, indicating a reduction in lipid accumulation in macrophages.

[0015] Ninthly, this invention provides the application of chebulic tannic acid in the preparation of inhibitors of aortic smooth muscle cell migration or / proliferation. Experiments have shown that the migration distance of aortic smooth muscle cells and the expression levels of proliferation factors (Ki67, PCNA) mRNA were significantly reduced in mice treated with chebulic tannic acid. This indicates that chebulic tannic acid can inhibit the migration or / or proliferation of aortic smooth muscle cells.

[0016] In a tenth aspect, the present invention provides a medicament for treating atherosclerosis, the medicament comprising chebulic tannic acid.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The uniqueness of structure-activity relationship (SAR) The hydrolyzable tannin structure of Terminalia chebula tannin (containing multiple galloyl groups and HHDP groups) is the key to its ability to specifically bind to the SAHH active site and activate enzyme function, which is fundamentally different from ordinary tannins (such as gallic acid tannins).

[0018] 2. Solved the industry's technical bottlenecks Research on the epigenetic regulatory mechanisms of atherosclerosis has lagged behind. This protocol provides the first example of natural product activation of SAHH in this field, opening up a new direction for drug development. Attached Figure Description

[0019] Figure 1 Terminalia chebulic acid for ApoE - / - Schematic diagram of the effect on basal metabolism in mice (where A is a flowchart; B is a statistical analysis chart of body weight; C is a statistical analysis chart of dietary intake; D is a statistical analysis chart of blood glucose level; E is a statistical chart of cholesterol level; F is a statistical chart of triglyceride level; G is a statistical analysis chart of high-density lipoprotein cholesterol level; H is a statistical analysis chart of non-high-density lipoprotein cholesterol level, *p<0.05; ns indicates no significance). Figure 2Terminalia chebulic acid for ApoE - / - Schematic diagram of toxicity and side effects in mouse liver, kidney, and spleen (where A is a schematic diagram of HE staining of liver, kidney, and spleen (magnification 10x); B is a schematic diagram of comparison results of aspartate transaminase; C is a schematic diagram of comparison results of alanine transaminase; D is a schematic diagram of comparison results of uric acid; E is a schematic diagram of comparison results of creatinine, *p<0.05; ns indicates no significance). Figure 3 Chebulic tannins inhibit ApoE - / - Schematic diagram of mouse atherosclerosis results (where A is a schematic diagram comparing the activity of SAHH in plasma; B is a schematic diagram comparing the SAH levels in plasma; C is a schematic diagram comparing the tHcy levels; D is a representative image of HE staining, Oil Red O staining, and Maoon staining of the aortic root; E is a schematic diagram comparing the plaque area of ​​the aortic root; F is a schematic diagram comparing the relative area of ​​lesions in the aortic root; G is a schematic diagram comparing the relative collagen content; H is a schematic diagram comparing the area of ​​necrotic core). Figure 4 Schematic diagram showing the effects of Terminalia chebula tannin on reducing oxidative stress, inhibiting inflammatory response, and cell proliferation in plaques (where A is a representative image of CD68 staining, SMA staining, and DHE staining in the aortic root; B is a schematic diagram of the comparison results of CD68 positive area; C is a schematic diagram of the comparison results of SMA positive area; D is a schematic diagram of the comparison results of DHE positive area; E is a schematic diagram of the comparison results of MDA content; F is a statistical analysis of the mRNA expression levels of VCAM-1, ICAM-1, Ki67, and PCNA; G is a representative immunoblot image of the mRNA expression levels of VCAM-1, ICAM-1, Ki67, and PCNA; H is a statistical analysis of the protein expression levels of VCAM-1, ICAM-1, Ki67, and PCNA). Figure 5 This diagram illustrates the multi-target mechanism of action of Terminalia chebula tannic acid against atherosclerosis (where A is a representative diagram of aortic smooth muscle cell migration experiment; B is a statistical analysis diagram of aortic smooth muscle cell migration distance; C is a statistical analysis diagram of the expression level of PCNA mRNA in aortic smooth muscle cells; D is a representative diagram of peritoneal macrophage Oil Red O staining; E is a statistical analysis diagram of peritoneal macrophage Oil Red O staining; F is a statistical analysis diagram of the levels of pro-inflammatory factors TNF-α, IL-6, IL-1β, Ccl2, and Ccl5; G is a statistical analysis diagram of the expression levels of p21, IL-1β, IL-6, ICAM-1, VCAM-1, Ccl2, MMP3, and MMP9 mRNA). Detailed Implementation

[0020] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0021] I. Experimental Materials: Terminalia chebula tannic acid (catalog number B20496) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. ApoE knockout in 8-week-old males (ApoE...) - / - Mice and normal C57BL / 6J mice were purchased from Cyagen Biosciences Co., Ltd. Total cholesterol (TC) kit (Shanghai Bosheng), triglyceride (TG) kit (Shanghai Kanglang), low-density lipoprotein cholesterol (LDL-C) kit (Shanghai Kanglang), high-density lipoprotein cholesterol (HDL-C) kit (Shanghai Kanglang), hematoxylin-eosin (HE) staining kit (Beijing Solarbio), Oil Red O staining kit (Beijing Solarbio), frozen section reactive oxygen species detection kit - red fluorescence (Wuhan Sewell), and ELISA kit (Thermo Fisher Scientific).

[0022] II. Experimental Methods: 1. Animal experiments (ApoE) - / - Mice): The experimental procedure for the atherosclerosis model is as follows: Figure 1 As shown, ApoE - / - Atherosclerotic plaque formation was induced in mice by a high-fat diet for 8 weeks. In the animal experiment, 24 mice were randomly divided into two groups of 12 each: a control group and a chebulic tannic acid group. The specific procedures are as follows: Control group: ApoE administered - / - Mice were injected intraperitoneally with physiological saline at a volume of 100 μL per mouse, once daily.

[0023] Terminalia chebulic acid group: ApoE was administered at a dose of 10 mg / kg body weight. - / - Intraperitoneal injection in mice, 100 μL / mouse / time, once daily.

[0024] At the start of the experiment, all mice were switched to a high-fat diet (containing 21% fat and 1.5% cholesterol, catalog number WD, purchased from Guangdong Provincial Medical Laboratory Animal Center) and fed this diet for 8 weeks to establish an atherosclerosis model. During the feeding period, the mice's food intake was recorded.

[0025] After 8 weeks of high-fat feeding, mouse body weight was recorded, and blood samples were collected to measure blood glucose, blood lipid levels, aspartate aminotransferase (AST), alanine aminotransferase (ALT), uric acid, creatinine, SAHH activity, SAH and tHcy levels, and inflammatory factor levels. Mice were euthanized, and hearts and aortas were collected for protein and mRNA level detection, followed by histological evaluation and analysis to determine the formation of atherosclerosis. Liver, spleen, and kidneys were collected for HE staining to assess the toxicity and side effects of chebulic tannins. ApoE was isolated and cultured. - / - Mouse peritoneal macrophages were stained with Oil Red O to detect protein and mRNA levels. ApoE cells were then isolated and cultured. - / - Scratch assays were performed on mouse aortic smooth muscle cells to assess smooth muscle migration and evaluate the potential of the SAHH agonist chebulic tannin in inhibiting the development of atherosclerotic plaques.

[0026] 2. In vitro cell experiments (normal C57BL / 6J mice): The specific procedures for detecting inflammatory factors in macrophages of normal C57BL / 6J mice are as follows: Thioglycolate-induced macrophages were obtained from the peritoneal cavity of normal mice 4 days after intraperitoneal injection of 1 mL of aged 4% Brewer's thioglycolate broth (BD Difco). After 6 hours of macrophage adhesion, they were cultured in RPMI medium supplemented with antibiotics and 10% fetal bovine serum (Gibco). Macrophages were treated with 10 μM chebulic tannic acid or PBS, and then treated with LPS (Invivogen) and recombinant IFNγ protein (Peprotech) for 8 hours to induce pro-inflammatory activation. Inflammatory factors (TNF-α, IL-6, IL-1β, Ccl2, Ccl5) ​​in the supernatant of cultured C57BL / 6J mouse peritoneal macrophages were detected by ELISA. After coating with capture antibody and blocking, standard or test medium, detection antibody, and enzyme-labeled secondary antibody were added sequentially for incubation and washing. Finally, TMB substrate was added for color development and termination, and OD values ​​were measured at 450 nm. The concentrations of each factor were calculated based on the standard curve, and statistical analysis was performed.

[0027] 3. Indicator Testing Instructions: (1) Effects of Terminalia chebula tannin on mouse metabolism (basal metabolism, glucose metabolism, lipid metabolism): The effects of Terminalia chebula tannin on basal metabolism, glucose metabolism, and lipid metabolism in mice were evaluated by measuring body weight, dietary intake, blood glucose, cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), and non-HDL-C. Specifically, blood glucose was used to evaluate the effect of Terminalia chebula tannin on glucose metabolism; cholesterol, triglycerides, HDL-C, and non-HDL-C were used to evaluate the effect of Terminalia chebula tannin on lipid metabolism; and body weight and dietary intake were used to evaluate the effect of Terminalia chebula tannin on basal metabolism.

[0028] (2) Effects of Terminalia chebula tannic acid on organ toxicity in mice: The toxicity of Terminalia chebula tannic acid to the liver, kidneys, and spleen of mice was evaluated by examining the morphology of the liver, kidneys, and spleen, as well as the levels of aspartate aminotransferase, alanine aminotransferase, uric acid, and creatinine.

[0029] (3) Effects of Terminalia chebula tannic acid on risk factors and plaque size of aortic atherosclerosis in mice: The effects of chebulic tannic acid on atherosclerotic risk factors and aortic plaque size were evaluated by detecting plasma SAHH activity, SAH, and tHcy levels, as well as HE, Oil Red O, and Maoon staining of the aortic root, and the aortic root plaque area, relative lesion area, and necrotic core area. Specifically, SAHH activity, SAH levels, and tHcy levels evaluated the effects of chebulic tannic acid on atherosclerotic risk factors; HE, Oil Red O, and Maoon staining of the aortic root, aortic root plaque area, relative lesion area, and necrotic core area evaluated the effects of chebulic tannic acid on aortic plaque. Increased SAHH activity and decreased SAH and tHcy levels indicated an effect of increasing SAHH activity and reducing atherosclerotic risk factors; a significant decrease in any of the aortic root plaque area, relative lesion area, or necrotic core area indicated an effect of reducing aortic plaque size.

[0030] (4) Effects of Terminalia chebula tannic acid on oxidative stress in mouse aortic plaques: The effect of Terminalia chebula tannic acid on oxidative stress in aortic plaques was evaluated by measuring the levels of DHE (dihydroethidium) and MDA (malondialdehyde) in aortic tissue. A decrease in DHE or MDA levels indicated a reduction in oxidative stress.

[0031] (5) Effects of Terminalia chebula tannic acid on aortic plaques and macrophage inflammatory responses in mice: The effects of chebulic tannins on plaque inflammation were evaluated by measuring the CD68-positive area, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule-1 (ICAM-1) in the aortic root. The effects of chebulic tannins on macrophage inflammation were evaluated by measuring macrophage cytokines (TNF-α, IL-6, IL-1β, Ccl2, Ccl5, p21, ICAM-1, and VCAM-1). A significant decrease in one or more of these indicators indicated a reduction or alleviation of aortic plaque and macrophage inflammation.

[0032] (6) Effect of Terminalia chebula tannic acid on the stability of aortic plaques in mice: The effects of Terminalia chebula tannic acid on aortic plaque stability were evaluated by detecting the relative collagen content in the aortic root, the SMA-positive area of ​​smooth muscle in the aortic root, and the expression levels of plaque instability factors (MMP3 and MMP9 in macrophages). A significant increase in relative collagen content, a significant increase in SMA-positive area, or a significant decrease in plaque instability factors indicated an effect on improving aortic plaque stability.

[0033] (7) Effects of Terminalia chebula tannin on proliferation and migration of mouse aortic smooth muscle cells: The effects of Terminalia chebula tannin on smooth muscle cell proliferation and migration were evaluated by detecting the levels of aortic root smooth muscle cell proliferation markers (Ki67, PCNA) and by performing a smooth muscle cell migration assay. Decreased expression levels of cell proliferation markers or shortened cell migration distance indicated an inhibitory effect on smooth muscle cell proliferation or migration.

[0034] (8) Effect of Terminalia chebula tannin on lipid accumulation in macrophages: The effect of Terminalia chebula tannin on lipid accumulation in macrophages was evaluated by detecting the Oil Red O staining area in macrophages. A decrease in the Oil Red O staining area in macrophages indicated a reduction in lipid accumulation.

[0035] 4. The specific testing methods are as follows: (1) Blood lipid test: Mice were fasted for 12 hours before their serum was collected. The serum was then analyzed using an automated biochemical analyzer (Hitachi, MODEL 7100) with enzymatic methods to determine various parameters in the plasma, including cholesterol, triglycerides, high-density lipoprotein cholesterol, and non-high-density lipoprotein cholesterol.

[0036] (2) Detection of SAH and tHcy in plasma: The levels of SAH and tHcy in plasma were determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS, UltiMate 3000 UHPLC / TSQ Vantage, Thermo Fisher Scientific). The plasma SAHH activity was quantitatively detected by HPLC MS / MS and calculated by dividing the amount of product by the reaction time and the amount of protein (nmol·h⁻¹·mg⁻¹).

[0037] (3) Pathological examination of the aortic sinus: Mouse hearts were exposed, perfused with PBS via the left ventricle, and the heart and aorta were carefully separated and fixed in paraformaldehyde. 8 μm thick paraffin sections were prepared using an automated semi-thin paraffin sectioner (Shanghai Leica). The aortic sinus paraffin sections were stained with hematoxylin and eosin (HE) and Masoon stain, respectively. After cryopreservation, the heart tissue was prepared into 8 μm sections using a tissue sectioner. The aortic sinus was located based on the tricuspid valve anatomical landmark at the junction of the heart and aorta.

[0038] Hematoxylin-eosin (H&E) staining: After baking paraffin sections at 70℃ for 30 minutes, dewax them twice with an environmentally friendly dewaxing solution, 5 minutes each time. Then, sequentially hydrate the sections in 100%, 90%, 80%, and 70% ethanol for 5 minutes each. Stain the sections with hematoxylin solution for 1 minute, rinse with tap water, differentiate in 1% hydrochloric acid alcohol for 1–3 seconds, and then soak in tap water for 5–10 minutes to regain blue color. Stain the sections with eosin solution for 15 seconds, dehydrate them in a gradient of 80%, 90%, 95%, and 100% ethanol for 5 minutes each, clear them twice with an environmentally friendly dewaxing solution, 5 minutes each time, mount with neutral resin, and photograph under a microscope.

[0039] Oil Red O staining: Frozen sections of mouse aortic sinus were incubated at room temperature for 2-3 minutes, fixed with 4% PFA for 30 minutes, rinsed twice with distilled water (3 minutes each time), infiltrated with 70% ethanol for 20-30 seconds, and then stained with Oil Red O staining solution in a sealed container for 15 minutes. After staining, the sections were allowed to differentiate in 70% ethanol for 15-20 seconds, and washed with distilled water for 1 minute. The cell nuclei were counterstained with Mayer hematoxylin for 1 minute, washed rapidly three times with tap water, and then blued again with running water for 10 minutes. After wiping away the moisture around the sample, the sections were mounted, then mounted with glycerol gelatin, and photographed under a microscope.

[0040] Masson staining: Frozen sections were thawed, fixed with paraformaldehyde for 15 minutes, and washed with deionized water. They were stained with Weigert's serotonin solution for 10 minutes, washed with 1% hydrochloric acid alcohol for approximately 5 minutes, and stained with Biebrich Scarlet-Acid Fuchsin for 10 minutes. Differentiation was performed with 1% phosphomolybdic acid solution, followed by aniline blue staining for 5 minutes. Sections were then rinsed with 0.2% glacial acetic acid aqueous solution, gently washed with PBS, mounted with glycerol gelatin, and photographed under a microscope. ImageJ image analysis software was used for quantitative analysis, measuring the absolute area and corresponding lumen area of ​​the aortic sinus AS plaque. To eliminate the influence of individual differences, the ratio of plaque area to lumen area was calculated to assess the size of the AS plaque.

[0041] (4) Immunofluorescence staining was used to detect the expression of differentiation cluster 68 (CD68) and smooth muscle actin (SMA) in aortic sinus plaque tissue: Paraffin sections were dewaxed, rehydrated, and antigen retrieval was performed. 5% BSA was added to block non-specific sites, and then the sections were incubated overnight at 4°C with specific primary antibodies CD68 and SMA, respectively. After rinsing with PBS buffer, secondary antibodies of the corresponding species (proteintech, RGAR003, RGAM003) were added, and the sections were incubated at room temperature in the dark for 60 min. Subsequently, the cell nuclei were counterstained with DAPI staining solution, and the samples were fixed with anti-fluorescence quenching mounting medium before image acquisition under a fluorescence microscope.

[0042] The level of peroxides (superoxide anions) in the aortic sinus was detected by chemiimmunofluorescence. Frozen sections of the aortic sinus were incubated with dihydroethidium (DHE) solution in the dark for 60 min, rinsed with PBS, mounted, and the superoxide fluorescence signal was observed under a fluorescence microscope. The average fluorescence intensity was detected using ImageJ software.

[0043] (5) Detection of VCAM-1, ICAM-1, Ki67 and PCNA proteins in the aortic root: The expression levels of VCAM-1, ICAM-1, Ki67 and PCNA proteins in the aortic root of mice were detected by immunoblotting.

[0044] (6) mRNA detection: The mRNA expression levels of mouse aortic root (VCAM-1, ICAM-1, Ki67 and PCNA) and macrophages (p21, IL-1β, IL-6, ICAM-1, VCAM-1, Ccl2, MMP3, MMP9) were detected by real-time quantitative PCR (qRT-PCR).

[0045] (7) Oxidative stress detection: The thiobarbituric acid (TBA) method is used to detect malondialdehyde (MDA). Under acidic and high-temperature conditions, MDA reacts with TBA to produce a red product that absorbs or fluoresces at a specific wavelength. The intensity of the signal is used to quantify MDA.

[0046] (8) Oil Red O staining of macrophages: Isolate and culture ApoE - / - Mouse peritoneal macrophages were treated with ox-LDL (25 μg / ml) and then with 10 μM chebulic tannic acid or PBS for 24 hours. After fixation with paraformaldehyde for 20 minutes, the cells were washed three times with PBS (1 minute each time). Oil Red O working solution was added and stained at room temperature in the dark for 30 minutes. The cells were washed three times with deionized water (1 minute each time). The nuclei were counterstained with hematoxylin for 1 minute, washed with running tap water for 5 minutes to regain blue color, mounted with glycerol and gelatin, and photographed under a microscope.

[0047] (9) Detection of aortic smooth muscle cell migration ability: Isolate and culture ApoE - / - Mouse aortic smooth muscle cells were plated into a high-density monolayer, synchronized with low serum, and then vertically scratched with a pipette tip. After washing with PBS, the culture medium was replaced with a medium containing the treatment factor. The cells were then treated with 10 μM chebulic tannic acid or PBS for 48 hours. The cells were photographed at fixed locations, and the scratch area was measured and the migration rate was calculated using ImageJ software.

[0048] III. Experimental Results 1. Effects of Terminalia chebula tannin on mouse metabolism (basal metabolism, glucose metabolism, lipid metabolism): like Figure 1 As shown, compared with the saline group, the body weight of the Terminalia chebula tannic acid group ( Figure 1 B) Dietary intake ( Figure 1 C) Blood glucose level ( Figure 1 D) Cholesterol levels ( Figure 1 E), triglyceride levels ( Figure 1 F), High-density lipoprotein cholesterol levels ( Figure 1 G), Non-high-density lipoprotein cholesterol levels ( Figure 1 No significant changes were observed in H). This indicates that chebulic tannic acid at this dose did not significantly interfere with the basal metabolic and glucose-lipid metabolism parameters of mice.

[0049] 2. Effects of Terminalia chebula tannic acid on organ toxicity in mice: like Figure 2 As shown in the HE staining results, there were no significant morphological changes in the liver, kidneys, and spleen of mice in the Terminalia chebula tannic acid treatment group compared to the saline group. Figure 2A). The tissue structure was clear, the cells were neatly arranged, and no pathological changes such as inflammatory infiltration, necrosis, or fibrosis were observed. Furthermore, compared with the control group, the aspartate aminotransferase (AST) level in the chebulic tannic acid group mice was significantly lower. Figure 2 B), alanine transaminase ( Figure 2 C), uric acid ( Figure 2 D) and creatinine ( Figure 2 The levels of E) were not significantly different, indicating that administration of Terminalia chebula tannins had no obvious toxicity or side effects on the liver, kidneys, and spleen.

[0050] 3. Effects of Terminalia chebula tannic acid on risk factors and plaque size in the aorta of mice: like Figure 3 As shown, compared with the control group, the activity of SAHH in the plasma of mice treated with Terminalia chebula tannic acid (SAHH) was significantly reduced. Figure 3 A) Increased levels of SAH in plasma ( Figure 3 B) and tHcy levels ( Figure 3 C) All decreased, suggesting that Terminalia chebula tannic acid can increase the activity of SAHH, thereby reducing the risk factors for atherosclerosis.

[0051] Representative images of mouse aortic root stained with HE, Oil Red O, and Maoon staining ( Figure 3 D) and the area of ​​plaque at the aortic root ( Figure 3 E), relative area of ​​lesion ( Figure 3 F) and the area of ​​the necrotic core ( Figure 3 Statistical analysis (H) showed that, compared with the control group, the aortic plaque area, relative lesion area, and necrotic core area were significantly reduced in the chebulic tannic acid group. This indicates that chebulic tannic acid can reduce the aortic plaque area.

[0052] 4. Effects of Terminalia chebula tannic acid on oxidative stress in mouse aortic plaques: like Figure 4 As shown, by ApoE - / - Representative image of DHE staining in the root of the mouse aorta ( Figure 4 A), and statistical analysis of DHE-positive area and MDA content show that: compared with the control group, the DHE-positive area in the Terminalia chebula tannic acid group ( Figure 4 D) and MDA content ( Figure 4 E) was significantly reduced, indicating that plaque oxidative stress was inhibited and lipid peroxidation was alleviated.

[0053] 5. Effects of Terminalia chebula tannic acid on arterial plaques and macrophage inflammatory responses in mice: like Figure 4 As shown, by ApoE - / - Representative image of CD68 staining in the root of the mouse aorta ( Figure 4A), and the statistical analysis of CD68 positive area shows that: compared with the control group, the CD68 positive area in the chebulic tannic acid group was significantly reduced ( Figure 4 B) indicates a significant reduction in macrophage infiltration at the aortic root and a decrease in plaque inflammation.

[0054] Statistical analysis of the mRNA expression levels of VCAM-1 and ICAM-1 in the aortic root (Figure) Figure 4 F), representative immunoblot images ( Figure 4 G) and statistical analysis of protein expression levels (G) Figure 4 (H) It was found that, compared with the control group, the mRNA and protein expression levels of VCAM-1 and ICAM-1 in the aortic root of the chebulic tannic acid group were significantly reduced. This indicates that chebulic tannic acid can alleviate or relieve the inflammatory response of macrophages.

[0055] Statistical analysis of the levels of pro-inflammatory factors TNF-α, IL-6, IL-1β, Ccl2, and Ccl5 (Figure) Figure 5 F) indicates that, compared with the control group, the level of pro-inflammatory factors in the macrophage culture medium of the chebulic tannic acid group was significantly reduced.

[0056] Statistical analysis of mRNA expression levels of p21, IL-1β, IL-6, ICAM-1, VCAM-1, and Ccl2 in macrophages (Figure) Figure 5 As shown in G), compared with the control group, the expression levels of inflammatory genes (p21, IL-1β, IL-6, ICAM-1, VCAM-1, Ccl2) in the chebulic tannic acid group were significantly reduced. This indicates that chebulic tannic acid can alleviate or relieve the inflammatory response of macrophages.

[0057] 6. Effects of Terminalia chebula tannic acid on the stability of aortic plaques in mice: Based on the relative collagen content ( Figure 3 Statistical analysis of G showed that, compared with the control group, the collagen content in the Terminalia chebula tannin group was significantly increased. (Based on ApoE...) - / - Representative image of SMA staining in the root of the mouse aorta ( Figure 4 A) and the statistical chart of positive area of ​​SMA ( Figure 4 C) It can be seen that, compared with the control group, the SMA positive area in the Terminalia chebula tannic acid group was significantly increased.

[0058] Statistical analysis of the mRNA expression levels of MMP3 and MMP9 in macrophages (Figure) Figure 5 G) It was found that, compared with the control group, the expression levels of plaque instability factors (MMP3 and MMP9) in the chebulic tannic acid group were significantly reduced. These results indicate that the aortic plaque stability of mice in the chebulic tannic acid group was increased.

[0059] 7. Effects of Terminalia chebula tannic acid on the proliferation and migration of mouse aortic smooth muscle cells: Statistical analysis of Ki67 and PCNA mRNA expression levels (Figure) Figure 4 F), representative immunoblot images ( Figure 4 G) and statistical analysis of protein expression levels (G) Figure 4 H) It can be seen that compared with the control group, the mRNA and protein expression levels of Ki67 and PCNA in the aorta of the chebulic tannic acid group were restricted, indicating that chebulic tannic acid has the effect of inhibiting smooth muscle cell proliferation.

[0060] Representative diagram from the aortic smooth muscle cell migration experiment ( Figure 5 A) Statistical analysis chart of migration distance ( Figure 5 B) and a statistical analysis of the expression level of PCNA mRNA in aortic smooth muscle cells (Phase B) Figure 5 C) It can be seen that these indicators were significantly reduced in the chebulic tannic acid group compared with the control group, indicating that the migration and proliferation capacity of smooth muscle cells were significantly reduced.

[0061] In summary, these results indicate that chebulic tannic acid has the effect of inhibiting the proliferation or migration of smooth muscle cells.

[0062] 8. Effects of Terminalia chebula tannin on lipid accumulation in macrophages: Representative image of peritoneal macrophages stained with Oil Red O ( Figure 5 D) and its statistical analysis chart ( Figure 5 E) It was found that, compared with the control group, the Oil Red O staining area in macrophages of the chebulic tannic acid group was reduced, indicating a decrease in lipid accumulation in macrophages. These results suggest that chebulic tannic acid has the effect of reducing lipid accumulation in macrophages.

[0063] In summary, these results indicate that chebulic tannins promote the stability and health of atherosclerosis by reducing oxidative stress, inhibiting inflammatory responses, and suppressing pathological cell proliferation. Therefore, intervention with chebulic tannins helps stabilize the blood vessel wall and protect plaque stability, thus exerting a multifaceted anti-atherosclerotic effect.

[0064] IV. Conclusion Terminalia chebulic acid does not affect ApoE ⁻ / ⁻ Under safe conditions affecting basal metabolism and liver and kidney function in mice, it can effectively inhibit the progression of atherosclerosis and enhance plaque stability. The core molecular starting point for its therapeutic effect is the activation of SAHH activity, thereby reducing SAH and tHcy levels.

[0065] Mechanistically, Terminalia chebula tannins exert multi-target effects: locally, they can alleviate oxidative stress and inhibit macrophage infiltration and pathological migration and proliferation of smooth muscle cells; at the systemic level, they can reduce serum pro-inflammatory factor levels; and crucially, they can simultaneously downregulate the expression of macrophage matrix degrading enzymes MMP3 and MMP9. These effects collectively lead to a reduction in plaque area, an increase in collagen content, and a decrease in the necrotic core.

[0066] In summary, this invention reveals that Terminalia chebula tannic acid synergistically exerts anti-inflammatory, antioxidant, and plaque-stabilizing effects by activating multiple SAHH-mediated pathways, making it a highly promising candidate drug for anti-atherosclerosis.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of Terminalia chebula tannic acid in the preparation of drugs for treating atherosclerosis, characterized in that, The chebulic tannic acid exerts its anti-atherosclerotic effect by binding to SAHH protein and increasing the enzyme activity of SAHH.

2. Application of Terminalia chebula tannic acid in the preparation of SAHH agonists.

3. Application of Terminalia chebula tannic acid in the preparation of inhibitors of SAH and / or tHcy.

4. The application of Terminalia chebula tannic acid in the preparation of drugs to reduce inflammation of atherosclerotic plaques.

5. Application of Terminalia chebula tannic acid in the preparation of drugs to reduce oxidative stress in atherosclerotic plaques.

6. Application of Terminalia chebula tannic acid in the preparation of drugs that improve the stability of atherosclerotic plaques.

7. Application of Terminalia chebula tannic acid in the preparation of drugs to reduce macrophage inflammation.

8. Application of Terminalia chebula tannic acid in the preparation of inhibitors of macrophage lipid accumulation.

9. Application of Terminalia chebula tannic acid in the preparation of inhibitors of aortic smooth muscle cell migration or / proliferation.

10. A drug for treating atherosclerosis, characterized in that, The drug includes chebulic tannic acid.

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