Application of ilicin K in preparation of anti-atherosclerosis drugs

Abstract

The invention discloses application of ilicin K in preparation of an anti-atherosclerosis medicine, and belongs to the technical field of biological medicines. Tests find that the ilicin K can effectively reduce CD72hi macrophage infiltration in plaque lesions, inhibit inflammatory response, improve lipid metabolism, reduce plaque area and improve plaque stability, has remarkable advantages in the aspect of atherosclerosis resistance, and provides a new idea and choice for treatment and drug combination of atherosclerosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of hollyoside K in the preparation of anti-atherosclerosis drugs. Background Art

[0002] Atherosclerosis (AS) is a common pathological feature of various acute and chronic cardiovascular diseases and panvascular diseases. It is manifested as a chronic inflammatory state caused by abnormal lipid deposition in the vascular intima and is the main cause of the development of cardiovascular diseases. As a systemic inflammatory disease, atherosclerosis can still cause instability of atherosclerotic plaques and induce acute adverse cardiovascular events even if the best lipid-lowering treatment is given clinically. How to prevent the development of plaques, stabilize vulnerable plaques, and delay the progression of atherosclerotic diseases is one of the important clinical problems that need to be solved urgently. At present, there are many limitations in the commonly used treatment methods in clinical practice. For example, although statins can regulate blood lipids, they have adverse reactions; anti-inflammatory treatment has the risk of infection; and nanoparticle drug delivery systems are difficult to clinically transform. Traditional Chinese medicine has the advantage of overall regulation in the treatment of atherosclerosis, but the chemical composition of traditional Chinese medicine is complex and the material basis of its efficacy is unclear. Therefore, clarifying the effective ingredients of traditional Chinese medicine and its mechanism of action is of great significance for the development of more effective anti-atherosclerotic drugs. Hollyoside K is a natural compound extracted from plants and has been found to have multiple biological activities, but its application in anti-atherosclerosis has not been fully developed. Therefore, it is of great significance to further study the role of Hollyoside K in anti-atherosclerosis. Summary of the invention

[0003] Based on this, the object of the present invention is to provide the use of hollygoside K in the preparation of an anti-atherosclerosis drug, wherein the drug has a significant therapeutic effect on atherosclerosis.

[0004] The invention provides application of hollyoside K in preparing anti-atherosclerosis medicine.

[0005] In the present invention, the ilexoside K, also known as ilexsaponin B3, is a triterpenoid compound extracted from the root of the pure natural medicinal plant ilexoside K. It is one of the effective active ingredients of ilexoside K, has multiple biological activities and pharmacological effects, and has a more prominent advantage in lowering cholesterol.

[0006] Furthermore, the anti-atherosclerosis includes one or more of inhibiting inflammatory response, improving lipid metabolism, reducing plaque area and improving plaque stability.

[0007] Furthermore, the holly glycoside K reduces the expression of CD72 in plaque lesions. hiMacrophage infiltration and inhibition of inflammatory factor signal release can achieve the purpose of treating atherosclerosis.

[0008] Furthermore, the hollygoside K achieves the purpose of treating atherosclerosis by reducing the levels of total cholesterol, triglycerides and low-density lipoprotein in patients with atherosclerosis.

[0009] Furthermore, the holly glycoside K achieves the purpose of treating atherosclerosis by reducing the area of ​​aortic sinus plaques, increasing the content of aortic collagen fibers, improving plaque stability, and reducing lipid deposition in the aortic sinus.

[0010] The present invention also provides an anti-atherosclerosis drug, comprising hollygoside K and pharmaceutically acceptable adjuvants.

[0011] Furthermore, the drug is a solid oral preparation, a liquid oral preparation or an injection.

[0012] Furthermore, the medicine is in the form of tablets, hard capsules, soft capsules, pills or granules.

[0013] Optionally, the drug is a tablet, a dispersible tablet, an enteric-coated tablet, a chewable tablet, an orodisintegrating tablet, a capsule, a sugar-coated tablet, a granule, a dry powder, an oral solution, a small water injection for injection, a lyophilized powder injection for injection, a large infusion or a small infusion.

[0014] The beneficial effects of the present invention are:

[0015] The present invention provides an application of hollyoside K in the preparation of an anti-atherosclerotic drug. The present invention uses hollyoside K to study and find that hollyoside K can effectively reduce CD72 in plaque lesions. hi Macrophage infiltration, reduction of inflammatory factor release, improvement of lipid metabolism, reduction of plaque area and lipid deposition, and enhancement of plaque stability showed significant advantages in anti-atherosclerosis, confirming that hollyoglycan K has potential therapeutic value for atherosclerosis, providing a strong experimental basis and theoretical basis for the use of hollyoglycan K in the anti-inflammatory treatment of atherosclerosis, and providing a new direction for the development of anti-atherosclerosis drugs.

[0016] The anti-atherosclerosis drug of the present invention has the advantages of definite efficacy, clear composition, no obvious toxic side effects, convenient administration, low price, etc. Compared with the existing traditional Chinese medicine for treating atherosclerosis, the anti-atherosclerosis drug of the present invention has simple and clear chemical composition and clear action target, which makes it easier to clarify its action mechanism in pharmacological research and easier to control the quality of the drug in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 This is a comparison chart of the effects of hollyglucoside K on body weight and blood lipids in mice with atherosclerosis, where ( Figure 1 A) shows the weight change trend of mice in different groups. Figure 1 BD) correspond to the inter-group comparison of serum TC, TG, and LDL-C levels;

[0019] Figure 2 Figure 2 is the effect of hollyglucoside K on aortic plaque morphology and lipid deposition in atherosclerotic mice, where ( Figure 2 AB) HE staining results show the changes in aortic plaque area. Figure 2 CD) is the result of Oil Red O staining, showing the changes in lipid red staining area;

[0020] Figure 3 The effect of hollyglucoside K on the stability and inflammatory response of atherosclerotic plaques in mice, where ( Figure 3 A) Masson staining results show the content of aortic collagen fibers and plaque stability. ( Figure 3 BC) are ELISA test results showing the changes in serum IL-1β and TNF-α levels;

[0021] Figure 4 CD72 in atherosclerotic mice and patient tissues hi Comparative display of macrophages regulated by holly glycoside K, where ( Figure 4 A) CD72 in human carotid artery plaque lesions hi Macrophage signal analysis diagram, ( Figure 4 B) CD72 in aorta tissue of AS mice hi Macrophage signal analysis diagram, ( Figure 4 C) Effect of hollyoside K on CD72 in aorta tissue of AS mice hi Effect diagram of macrophage infiltration. DETAILED DESCRIPTION

[0022] The invention provides application of hollyoside K in preparing anti-atherosclerosis medicine.

[0023] The present invention does not specifically limit the source and preparation method of hollyoside K. Common commercial products in the art can be used, or it can be prepared according to known extraction methods or preparation methods. The hollyoside K used in the embodiments of the present invention is preferably purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0024] The present invention also provides an anti-atherosclerosis drug, comprising hollygoside K and pharmaceutically acceptable adjuvants.

[0025] The drug of the present invention can use hollygoside K as an active ingredient, and can also be combined with other active ingredients for treating atherosclerosis as active ingredients, and supplemented with pharmaceutically acceptable excipients to prepare multiple dosage forms of the drug. The present invention does not specifically limit the dosage form of the drug and the type and amount of the excipients.

[0026] In order to further illustrate the present invention, the application of hollyoside K provided by the present invention in the preparation of anti-atherosclerotic drugs is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0027] In the embodiments of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0028] Example 1

[0029] 1. Drug source

[0030] Hollyoside K standard was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0031] 2. Establishment and grouping of animal models

[0032] 8-week-old SPF grade healthy male apolipoprotein E gene knockout (ApoE - / -) mice were randomly divided into atherosclerosis group (AS group) and ilexoside K gavage treatment group (Ilexoside K group), and the same number of C57BL / 6J mice were taken as normal group (Normal group), with a body weight of 21-25g; the mice in AS group and Ilexoside K group were fed with high-fat diet (composed of 22.5% protein, 45% carbohydrate, 20% fat, 1.25% cholesterol, 0.5% sodium cholate) for 8 weeks to establish atherosclerosis mouse model, and the mice in Normal group were fed with normal control diet (composed of 19.2% protein, 67.3% carbohydrate, 4.3% fat) as control group. From the 9th week, the mice were gavaged once a day, with a volume of 0.2ml each time. Among them, the mice in Ilexoside K group were gavaged with Ilexoside K solution (12mg / kg), and the mice in AS group and Normal group were gavaged with equal volume of normal saline, and the gavage time lasted for 8 weeks. During the entire experiment, the overall condition of each group of mice was observed every morning and evening, including general condition, mental and hair condition, weight changes, and changes in food and water intake.

[0033] 3. General indicators

[0034] During the administration process, the changes in body weight of mice in each group were recorded, and general conditions such as eating habits, hair growth, reaction speed, etc. were observed to evaluate the effects of hollygoside K on the overall state of mice.

[0035] After 8 weeks of drug intervention, the mice in the Normal group had a good mental state, smooth and shiny hair, no hair loss, and good activity. Compared with the Normal group, the mice in the AS group had a significant increase in body weight (P<0.05), dull hair, partial hair loss, fatigue, and slow reaction. Compared with the AS group, the hair loss and activity of the mice in the lexoside K group were improved, the reaction was acceptable, and the body weight was significantly reduced (P<0.05). This shows that lexoside K can slow down the weight gain of mice with atherosclerosis, improve mental state, activity, etc. (results are shown in Figure 1 A).

[0036] 4. Blood lipid level detection

[0037] After the administration, the blood of the mice was obtained by removing the eyeballs, and the serum was taken according to the instructions of the kit. The colorimetric method was used to detect the levels of total cholesterol (TC) and triglyceride (TG) in the mouse serum, and the direct method was used to determine the level of low-density lipoprotein (LDL-C) to evaluate the regulatory effect of hollygoside K on lipid metabolism in mice with atherosclerosis.

[0038] Compared with the Normal group, the serum TC, TG, and LDL-C levels of mice in the AS group were significantly increased (P<0.05); compared with the AS group, the serum TC, TG, and LDL-C levels of the Ilexoside K group were significantly decreased (P<0.05). This indicates that Ilexoside K can reduce the serum TC, TG, and LDL-C levels of AS mice (the results are shown in Figure 1 BD).

[0039] 5. Aortic plaque lesions

[0040] The aortic root tissue of mice was carefully collected and fixed for more than 24 hours, then dehydrated with gradient ethanol, embedded in paraffin and sliced. After the slices were dewaxed to water, they were stained with HE, dehydrated with gradient ethanol, soaked in xylene to make them transparent, and finally sealed with neutral gum. Pictures were collected under a microscope to observe the aortic wall structure, plaque morphology and other pathological conditions.

[0041] HE staining results showed that the media structure of the aorta wall of mice in the Normal group was continuous, with uniform thickness, smooth and flat intima, orderly arrangement of endothelial cells, and no atherosclerotic plaque formation; compared with the Normal group, the media structure of the aorta wall of mice in the AS group was disordered, endothelial cells partially fell off, the intima was significantly thickened, obvious atherosclerotic plaque protrusions appeared, the lumen became smaller, necrotic cores formed in the plaques, and the calculated plaque area and lumen ratio increased significantly (P<0.05); compared with the AS group, the pathological morphological changes in the Ilexoside K group were alleviated, the media structure was more neatly arranged, and medium-sized atherosclerotic plaques were observed (P<0.05). This shows that Ilexoside K can significantly reduce the area of ​​aortic sinus plaques (results are shown in Figure 2 AB).

[0042] 6. Determination of aortic lipid red staining area

[0043] The aortic root tissue of mice was obtained for OCT embedding, and serial frozen sections (5 μm) were stained with Oil Red O. Images were collected using a digital pathology section scanner, and Image J software was used to calculate and analyze the percentage of plaque red staining area, that is, the percentage of aortic lipid red staining area (%) = Oil Red staining area / aortic lumen area × 100%, to evaluate the effect of hollygoside K on aortic lipid deposition.

[0044] Oil red O staining results showed that no red-stained lipid accumulation was observed under the intima of the aortic root of mice in the Normal group; compared with the Normal group, significant lipid deposition was observed in the aortic root of mice in the AS group, and the red-stained area was significantly increased (P<0.01); compared with the AS group, the lipid red-stained area in the Ilexoside K group was significantly reduced (P<0.05). This indicates that Ilexoside K can significantly reduce lipid deposition in the aortic sinus of AS mice (results shown in Figure 2 shown on CD).

[0045] 7. Assessment of aortic plaque lesion stability

[0046] The aortic root tissue of mice was collected and fixed for more than 24 hours, dehydrated with gradient ethanol, embedded in paraffin, and sliced. After dewaxing to water, Masson staining was performed. After staining, it was dehydrated with gradient ethanol, soaked in xylene to make it transparent, and sealed with neutral gum. Pictures were collected under a microscope to observe the structural changes of elastic fibers and collagen fibers in the aortic wall and evaluate the stability of plaques.

[0047] The results of Masson staining showed that the elastic fibers of the aorta wall in the Normal group were intact and continuous, and the blue collagen was neatly arranged and evenly dense; the elastic fibers of the aorta wall in the AS group were interrupted, the collagen content was reduced, the arrangement was disordered, and the collagen in the plaque was loosely distributed in a reticular manner; compared with the AS group, the aorta elastic fibers in the Ilexoside K group were arranged more regularly, and the content of aortic collagen fibers increased significantly. This shows that Ilexoside K can significantly increase the content of aortic collagen fibers and improve the stability of plaques (results are shown in Figure 3 A).

[0048] 8. Inflammatory factor detection

[0049] After the administration, the eyeballs were removed and blood was collected. The serum was taken according to the instructions of the ELISA kit to measure the levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the mouse serum to evaluate the regulatory effect of hollygoside K on the inflammatory response.

[0050] ELISA test results showed that after 8 weeks of intervention, the levels of serum IL-1β and TNF-α in the AS group were significantly increased compared with those in the Normal group (P<0.05); compared with the AS group, the levels of serum IL-1β and TNF-α in the Ilexoside K group were significantly decreased (P<0.05), indicating that Ilexoside K can alleviate the inflammatory response of atherosclerosis model mice (results are shown in Figure 3 BC).

[0051] 9. Single-cell sequencing data processing and CD72 hi Macrophage signaling analysis

[0052] Single-cell sequencing of mouse aorta was performed, and single-cell sequencing of human carotid atherosclerosis was obtained from the GEO database. The sequencing data was reduced in dimension, clustered, and cell types were divided for cell annotation, and the expression of CD72 gene in each sample was extracted. Data analysis was based on the Seurat analysis package of R language, and the presto difference test method was used for gene difference analysis, and violin plots were drawn to visualize the differences.

[0053] The single-cell sequencing data of the carotid artery lesions and adjacent normal arterial tissues in AS patients were analyzed and compared. The results showed that compared with the normal control arterial tissues, CD72 hi Macrophages are highly expressed in the carotid artery plaque lesions of AS patients (results as shown in Figure 4 A).

[0054] Analysis of single-cell transcriptional profiles of AS mice, selection of macrophages and monocytes for dimensionality reduction clustering, and analysis of CD72 hi Macrophage signaling; CD72 in aortic tissue of AS model mice compared with normal mice hi Macrophage signal was highly expressed (results such as Figure 4 B).

[0055] 10. CD72 hi Macrophage infiltration observation

[0056] The paraffin sections of the aorta root were dehydrated, antigen repaired, and blocked, and then CD72 and F4 / 80 antibodies were added. The sections were incubated overnight at 4°C in a wet box. After washing, the secondary antibody was added, and the sections were incubated at room temperature in the dark for 50 minutes. DAP1 was added to counterstain the nuclei. After sealing, the sections were observed with a digital pathology section scanner, and the CD72 hi Macrophage infiltration.

[0057] The results of immunofluorescence staining showed that compared with the Normal group, obvious CD72 hi Compared with the AS group, the CD72 in the aorta tissue of the mice in the Ilexoside K group was significantly higher than that in the AS group (P<0.05). hi The level of macrophage infiltration was significantly reduced (P<0.05), indicating that Ilexoside K can reduce the expression of CD72 in the aorta tissue of atherosclerosis model mice. hi Macrophage infiltration (results such as Figure 4 C).

[0058] In summary, hollyglucoside K can significantly improve multiple indicators of atherosclerotic mice. In general, it slows down the weight gain of mice, improves their mental state and activity; in lipid metabolism, it reduces serum TC, TG, and LDL-C levels; in plaque lesions, it reduces the aortic sinus plaque area and lipid deposition, increases collagen fiber content and improves plaque stability; in inflammatory response, it reduces serum IL-1β and TNF-α levels; and it can effectively reduce CD72 in the aorta tissue of atherosclerotic mice. hi Macrophage infiltration and CD72 hi Macrophages are highly expressed in carotid plaque lesions of patients with atherosclerosis and in aortic tissues of AS mice, which fully demonstrates the therapeutic potential of hollyoside K for atherosclerosis. The present invention provides a strong experimental basis and theoretical basis for the anti-inflammatory treatment of atherosclerosis with hollyoside K, and provides a new direction for the development of anti-atherosclerosis drugs.

[0059] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0060] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of hollyoside K in the preparation of anti-atherosclerotic drugs.

2. The use of hollyoside K according to claim 1 in the preparation of anti-atherosclerotic drugs, characterized in that: The anti-atherosclerosis includes one or more of inhibiting inflammatory response, improving lipid metabolism, reducing plaque area and improving plaque stability.

3. The use of hollyoside K according to claim 1 in the preparation of anti-atherosclerotic drugs, characterized in that: Hollyoside K reduces CD72 in plaque lesions hi Macrophage infiltration and inhibition of inflammatory factor signal release can achieve the purpose of treating atherosclerosis.

4. The use of hollyoside K according to claim 1 in the preparation of an anti-atherosclerotic drug, characterized in that: The hollyglucoside K achieves the purpose of treating atherosclerosis by reducing the levels of total cholesterol, triglycerides and low-density lipoprotein in patients with atherosclerosis.

5. The use of hollyoside K according to claim 1 in the preparation of anti-atherosclerotic drugs, characterized in that: The holly glycoside K achieves the purpose of treating atherosclerosis by reducing the area of ​​aortic sinus plaques, increasing the content of aortic collagen fibers, improving plaque stability, and reducing aortic sinus lipid deposition.

6. An anti-atherosclerotic drug, characterized in that: The invention comprises hollygoside K and pharmaceutically acceptable auxiliary materials.

7. The drug according to claim 6, characterized in that The medicine is a solid oral preparation, a liquid oral preparation or an injection.

8. The drug according to claim 6, characterized in that The medicine is in the form of tablets, hard capsules, soft capsules, dropping pills or granules.

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