Application of lobetyolin in preparation of medicine for treating atherosclerosis

Abstract

The invention discloses application of lobetyolin in preparation of a medicine for treating atherosclerosis, and belongs to the field of biological medicine. The atherosclerosis comprises atherosclerosis induced by a high methionine diet. Through verification, IL-33 in aortic tissues of ApoE <- / -> mice with high methionine diet is remarkably increased, and the expression level of IL-33 in the aortic tissues of the mice is remarkably reduced compared with that of a model group after the mice are treated by lobetyolin. It is proved that lobetyolin has a positive regulation effect on treatment of atherosclerosis. Through molecular docking analysis, the binding energy between the lobetyolin and the IL-33 is-7.3 kcal / mol, which indicates that the IL-33 and the lobetyolin have good binding stability. The invention provides a new idea for clinical treatment of atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of codonopsis pilosula in preparing medicines for treating atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is a chronic, progressive disease of the blood vessel wall mediated by lipid metabolism disorders and chronic inflammatory responses, characterized by subintimal lipid deposition, fibrous tissue proliferation, and plaque formation. AS is the primary pathological basis of cardiovascular disease. In recent years, the prevalence of unhealthy lifestyles, such as high-fat diets and lack of exercise, has further exacerbated the incidence of AS. Although AS has subtle early symptoms, progression can lead to serious complications such as coronary atherosclerotic heart disease, stroke, and peripheral arterial disease, significantly increasing disability and mortality rates. AS patients often experience chest tightness, palpitations, and intermittent claudication. Without timely intervention, it can lead to myocardial infarction, ischemic brain damage, and organ failure, while also increasing the socioeconomic burden. This disease has become a major challenge that urgently needs to be addressed in the field of public health.

[0003] Current treatment principles for atherosclerosis (AS) include lifestyle intervention, medication, revascularization, and patient education. Commonly used first-line treatments include statins (such as atorvastatin and rosuvastatin), antiplatelet agents (such as aspirin and clopidogrel), angiotensin-converting enzyme inhibitors (ACEIs), and calcium channel blockers (CCBs). While these drugs can slow AS progression and reduce the risk of cardiovascular events, they cannot completely reverse plaque formation, and long-term use may cause adverse reactions such as liver damage, myalgia, gastrointestinal bleeding, and renal dysfunction. While revascularization therapies (such as stent implantation or bypass surgery) can rapidly improve ischemic symptoms, they carry risks such as postoperative restenosis and thrombosis, are costly, and offer poor long-term prognosis for some patients. Recent studies have revealed that Traditional Chinese Medicine (TCM) offers unique advantages in the prevention and treatment of AS, with relatively few adverse reactions. It can regulate blood lipids, improve endothelial function, and reduce the progression and recurrence of AS through its effects on promoting blood circulation and removing blood stasis, resolving phlegm and reducing turbidity, and invigorating qi and unblocking meridians. Therefore, in-depth exploration of traditional Chinese medicines and their active ingredients with anti-AS potential has important research value for optimizing clinical treatment strategies and improving patients' long-term prognosis.

[0004] Homocysteine ​​(Hcy) is an essential amino acid derived from dietary metabolism. High homocysteine ​​(HHcy), a state of Hcy accumulation in the body, is closely associated with a variety of diseases. It can damage cells, tissues, and organs through various mechanisms, mediating the development and progression of cardiovascular disease and serving as an independent and significant risk factor for arteriosclerosis (AS). This suggests an urgent need for preventive and therapeutic measures against HHcy-induced atherosclerosis.

[0005] LBT, a polyacetylene compound, is one of the most important bioactive components of Codonopsis pilosula. Modern pharmacological research indicates that LBT exhibits anti-nociceptive, antibacterial, anti-inflammatory, sedative, antioxidant, antihypertensive, and immunomodulatory effects, making it a valuable ingredient for quality control of Codonopsis pilosula. It also exhibits neuroprotective, cardiovascular, and gastrointestinal effects, lipid regulation, blood sugar reduction, anti-aging, antioxidant, anti-tumor, and immunomodulatory properties. Therefore, it is a valuable ingredient for quality control of Codonopsis pilosula.

[0006] Numerous studies have demonstrated that dangshen glycosides possess a wide range of pharmacological activities, including protection against gastric mucosal damage, inhibition of breast cancer cell proliferation, and alleviation of cisplatin-induced renal injury. Furthermore, dangshen glycosides exert anti-inflammatory and immunomodulatory effects by regulating the secretion of inflammatory factors (such as TNF-α, IL-1β, IL-6, and IFN-γ) and affecting the expression of apoptosis-related proteins (Bax and Caspase). Furthermore, dangshen glycosides serve as a key pharmacological agent in the Shenqi Anti-Inflammatory Recipe for the treatment of rheumatoid arthritis. Furthermore, LBT significantly ameliorated blood-brain barrier disruption induced by cerebral ischemia-reperfusion in rats. However, to date, there have been no reports on the protective effects of dangshen glycosides against atherosclerosis. Summary of the Invention

[0007] The purpose of the present invention is to provide the use of codonopsis pilosula in the preparation of a drug for treating atherosclerosis, so as to solve the problems existing in the above-mentioned prior art. Codonopsis pilosula has a positive regulatory effect on the treatment of atherosclerosis, providing a new idea for the clinical treatment of atherosclerosis.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides application of codonopsis pilosula in preparing medicine for treating atherosclerosis.

[0010] Optionally, the atherosclerosis comprises atherosclerosis induced by a high-methionine diet.

[0011] Optionally, the dangshenoside plays a role in treating atherosclerosis by reducing the expression level of IL-33 protein.

[0012] Optionally, the dangshenoside plays a role in treating atherosclerosis by increasing the expression level of tight junction proteins.

[0013] The present invention also provides a medicine for treating atherosclerosis, which contains codonopsis pilosula and pharmaceutically acceptable excipients.

[0014] Optionally, the atherosclerosis comprises atherosclerosis induced by a high-methionine diet.

[0015] Optionally, the dangshenoside plays a role in treating atherosclerosis by reducing the expression level of IL-33 protein.

[0016] Optionally, the dangshenoside plays a role in treating atherosclerosis by increasing the expression level of tight junction proteins.

[0017] Optionally, the auxiliary materials include at least one of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant and a flavoring agent.

[0018] Optionally, the drug can be taken orally or parenterally.

[0019] The present invention discloses the following technical effects:

[0020] This study demonstrates that codonopsis pilosula can significantly treat atherosclerosis by regulating IL-33, providing a solid scientific basis for the clinical treatment of atherosclerosis. Codonopsis pilosula is a traditional and valuable Chinese medicinal material, and codonopsis pilosula is one of its main active ingredients. This study not only provides new evidence for the medicinal value of codonopsis pilosula but also has significant implications for its expanded medical applications. It is expected to promote the development of codonopsis-related industries and, in turn, boost local economic growth.

[0021] This study uses transcriptomic analysis to identify IL-33 as a key protein in inducing atherosclerosis. The successful use of combined analytical techniques has effectively expanded the scope and application of omics technologies, and has a positive impact on the updating and integration of omics analysis technologies.

[0022] The present invention used Western blot to explore the expression of IL-33, and the results showed that ApoE - / - The expression of IL-33 in the mouse aorta tissue was significantly elevated compared to the control group, while after treatment with codonopsis pilosula, the expression of IL-33 in the mouse aorta tissue was significantly reduced compared to the model group. These results demonstrate that codonopsis pilosula has a positive regulatory effect on the treatment of atherosclerosis. Further research on related pathways and targets could focus on IL-33, and the impact and mechanism of IL-33 in other cardiovascular diseases could also be explored.

[0023] The present invention used AutoDock Tools software to perform molecular docking analysis on the IL-33 protein and the ligand codonopsis glycoside. The results showed a binding energy of -7.3 kcal / mol, indicating good binding stability between IL-33 and codonopsis glycoside. Molecular docking technology is primarily used in drug development to predict the binding force between drug ingredients and targets and to identify binding sites. The results are highly accurate and reliable, and have important scientific value for in-depth exploration of the mechanism of action of drug ingredients and target pathways, effectively promoting the progress of related drug research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0025] Figure 1 2.0% methionine feeding for C57BL / 6J mice and ApoE - / - Effects of plasma Hcy levels in mice;

[0026] Figure 2 2.0% methionine feeding for C57BL / 6J mice and ApoE - / - Effects of 2.0% methionine diet on the histopathology of the aortic sinus of mice; A: H&E staining and Oil Red O staining results; B: Effects of 2.0% methionine diet on ApoE - / - Effects of 2.0% methionine diet on ApoE - / - Effects on lipid production in mice;

[0027] Figure 3 Oil red O staining shows the effect of LBT on plaque formation in the aortic sinus of AS mice; A: Control group; B: Control + LBT 20 mg / kg group; C: HCY group; D: LBT 5 mg / kg group; E: LBT 10 mg / kg group; F: LBT 20 mg / kg group; G: folic acid group; H: ratio of aortic sinus plaque area in AS mice;

[0028] Figure 4 H&E staining shows the effect of LBT on lipid formation in aortic sinus plaques of AS mice; A: Control group; B: Control + LBT 20 mg / kg group; C: HCY group; D: LBT 5 mg / kg group; E: LBT 10 mg / kg group; F: LBT 20 mg / kg group; G: folic acid group; H: lipid area in aortic sinus plaques of AS mice;

[0029] Figure 5 The macroscopic Oil Red O staining shows the effect of LBT on lipid formation in aortic sinus plaques of AS mice; A: Control group; B: Control + LBT 20 mg / kg group; C: HCY group; D: LBT 5 mg / kg group; E: LBT 10 mg / kg group; F: LBT 20 mg / kg group; G: Folic acid group; H: Aortic sinus plaque area of ​​AS mice;

[0030] Figure 6 ApoE - / - Heat map of differential gene expression among the control, HCY, and LBT groups of mice;

[0031] Figure 7 is the expression of IL-33 in the aorta tissue of mice in each group;

[0032] Figure 8 This is a molecular docking visualization diagram; the figure below shows the interaction between IL-33 and codonopsis pilosula in the upper frame;

[0033] Figure 9 The effect of LBT on HUVECs cell viability induced by Hcy;

[0034] Figure 10 The effect of LBT on the TEER value of HUVECs induced by Hcy;

[0035] Figure 11 Effects of LBT on tight junction expression in HUVECs induced by Hcy; A: representative protein bands of ZO-1 and Occludin; B: quantitative analysis of ZO-1 expression; C: quantitative analysis of Occludin expression. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Codonopsis pilosula molecular formula: C 20 H 28 O8, molecular weight: 396.433, appearance: light yellow crystals, chemical structure is as follows:

[0042]

[0043] Example 1

[0044] 1. Experimental Materials

[0045] 1.1 Main experimental drugs and instruments

[0046] Codonopsis pilosula (purity > 98%, purchased from Shanghai Yuanye Biotechnology Co., Ltd., J30HB186713), folic acid (purchased from Shanghai Yuanye Biotechnology Co., Ltd., J30HB186713); maintenance feed containing 2.0% high methionine was purchased from Beijing Xiaoshu Youtai Biotechnology Co., Ltd.; BCA protein quantification kit (KGPBCA) was purchased from Jiangsu Keygen Biotechnology Co., Ltd.; Western Blot primary and secondary antibody diluent (15127C17) was purchased from Boster Biotechnology; rabbit anti-IL-33 monoclonal antibody and rabbit anti-GAPDH monoclonal antibody were purchased from Wuhan Sanying Company; cryo-grinder, JXFSTPRP-CL, was purchased from Shanghai Jingxin Co., Ltd.; microplate reader (Multiskan G0) was purchased from Thermo Fisher Scientific, USA; high-speed low-temperature centrifuge (5430R) was purchased from Eppendorf GmbH, Germany.

[0047] 1.2 Animal Handling

[0048] Male C57BL / 6J mice and SPF-grade ApoE of the same background - / - Mice (18-22 g, 6 weeks old) were purchased from Beijing Weitonglihua Co., Ltd. and housed at the Animal Experimental Center of Ningxia Medical University. The feeding conditions included standard feed, tap water, room temperature maintained at (24 ± 2) °C, humidity of 50-60%, and daily light and dark time of 12 h each.

[0049] 1.3 Experimental Animal Grouping and Dosing

[0050] Six-week-old male C57BL / 6J mice were divided into a normal group and a high methionine group; six-week-old male ApoE - / - Mice were randomly divided into seven groups: normal group (Control), codonopsis glycoside control group (Control + LBT 20 mg / kg), model group (HCY), model group + codonopsis glycoside (5 mg / kg / d, 10 mg / kg / d, 20 mg / kg / d) groups (LBT 5 mg / kg, LBT 10 mg / kg, LBT 20 mg / kg), and model group + folic acid drinking water (75 μg / kg) positive drug group (folic acid). A 12-h light / dark cycle was maintained at constant temperature and humidity.

[0051] Mice were fed a maintenance diet containing 2.0% methionine to establish a high-methionine diet-induced atherosclerosis model. They were divided into four groups: NC: C57 mice + standard diet; NHM: C57 mice + methionine diet; AC: ApoE - / - Mice + standard diet; AHM: ApoE - / - Mice were fed with methionine feed (model group) for 16 weeks from the beginning of the experiment.

[0052] After successful modeling, the model groups were given corresponding dangshen glycosides (5 mg / kg / d, 10 mg / kg / d, 20 mg / kg / d) and folic acid drinking water (75 μg / kg) for 12 weeks, the normal group was given an equal amount of normal saline and maintenance feed, and the dangshen glycoside control group was gavaged with dangshen glycoside 20 mg / kg / d and fed with maintenance feed.

[0053] 1.4 Preparation of high-methionine diet-induced atherosclerosis model

[0054] Mice were fed a maintenance diet containing 2.0% methionine to establish a high-methionine diet-induced atherosclerosis model.

[0055] 2. Detection of mouse blood lipid levels and mouse plasma Hcy levels

[0056] Experimental methods

[0057] Blood was collected from mouse eyes and directly injected into EP tubes pre-filled with 2015 mg / mL EDTA anticoagulant. The cells were centrifuged at 3000 rpm for 15 minutes at room temperature. The supernatant, representing plasma, was collected and stored at -80°C until further use. Levels of total Hcy, total cholesterol (CHO), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were measured using an automated biochemical analyzer.

[0058] Experimental results

[0059] After 16 weeks of feeding with 2.0% methionine diet, C57BL / 6J mice and ApoE - / - There was no significant difference in the blood lipid levels of mice of the same species, and ApoE - / - The plasma cholesterol and low-density lipoprotein levels of C57BL / 6J mice were significantly higher than those of C57BL / 6J mice ( Table 1 ), which was caused by the disorder of cholesterol metabolism caused by ApoE gene knockout.

[0060] Table 1 Levels of four blood lipids in mice

[0061] Group TG (mmol / L) CHO (mmol / L) HDL (mmol / L) LDL (mmol / L) NC 0.78±0.24 3.25±1.07 1.81±0.20 0.26±0.04 NHM 0.84±0.29 3.01±1.52 1.82±0.22 0.27±0.03 AC 0.79±0.23 11.84±2.96 1.79±0.18 5.87±0.72 AHM 0.83±0.21 12.52±2.87 1.89±0.23 5.90±1.84

[0062] After feeding with 2.0% methionine, C57BL / 6J mice and ApoE - / - The plasma Hcy levels of mice were significantly higher than those of the corresponding normal control group ( Figure 1 ), indicating that high homocysteine ​​was successfully induced in mice.

[0063] 3. H&E staining, Oil Red O staining, and gross Oil Red O staining to observe changes in tissue pathology results

[0064] Preparation of aortic sinus frozen sections:

[0065] The aortic sinuses removed from each group of mice were dehydrated by soaking in a 30% sucrose solution for one week. Filter paper was used to absorb the surface moisture of the tissue. An appropriate amount of OCT embedding medium was added to the embedding cassette. The tissue was placed in the embedding medium with the section facing up, and the section was adjusted to a horizontal level. The embedding cassette was placed on the quick-freezing stage of a cryostat and the OCT embedding medium was frozen until it turned white and hardened. The tissue block was mounted on the cryostat and serial cross-sections were made along the aortic root, marking the three aortic valves. 500 mm of 6 mm thick frozen sections were collected and mounted on clean, anti-slip slides, with two sections collected per slide. After fixation with 4% paraformaldehyde for 30 minutes, the sections were stored at -20°C until further use.

[0066] H&E staining:

[0067] (1) Place the frozen sections at room temperature for 10 minutes, then soak them in distilled water for 10 minutes to wash away the embedding agent.

[0068] (2) Soak the sections in Harris hematoxylin solution for 3 minutes, rinse the elution box containing the sections gently with tap water, and observe under a microscope at any time. After the cell nuclei turn blue, differentiate them with 1% hydrochloric acid alcohol for a few seconds, and rinse again with running water.

[0069] (3) Place the slices in a dye vat containing eosin solution and soak for 3 minutes.

[0070] (4) Soak the sections in 95% ethanol, 95% ethanol II for 15 minutes, anhydrous ethanol for 5 minutes, anhydrous ethanol II for 15 minutes, xylene I for 5 minutes, xylene II for 5 minutes, and finally remove the sections from the last xylene staining tank and dry them in a fume hood. Add neutral gum to the tissue sections and cover them with slides of appropriate size.

[0071] (5) Scan the slides using an Aperio digital pathology scanner.

[0072] Oil Red O staining:

[0073] (1) Take the frozen sections out of the refrigerator, place them at room temperature for 10 minutes to warm up, and soak them in distilled water for 10 minutes.

[0074] (2) Soak in freshly prepared 60% isopropyl alcohol for 10 minutes.

[0075] (3) Soak the sections in freshly prepared Oil Red 0 working solution for 15 minutes.

[0076] (4) Rinse with 60% isopropyl alcohol for a few seconds to remove floating color.

[0077] (5) Soak in Harris hematoxylin solution for 2 minutes and observe under a microscope until the cell nucleus turns obviously blue. Place the elution box containing the slices under the tap and rinse gently with running water. Differentiate with 1% hydrochloric acid alcohol for a few seconds and rinse again with running water.

[0078] (6) Rinse with distilled water.

[0079] (7) Quickly seal the slides with warm liquid glycerol gelatin sealing medium.

[0080] (8) Leave at room temperature to allow the glycerin gelatin to cool completely and solidify.

[0081] (9) Scan the slides using an Aperio digital pathology scanner.

[0082] Calculation of aortic sinus plaque area and lipid index:

[0083] H&E staining was performed, and the plaque area and lumen cross-sectional area were measured using Image Pro Plus software. The ratio of the two was calculated to represent the relative size of the AS lesion area, i.e., the AS lesion area ratio. The area positive for Oil Red O staining was used to represent the lipid content within the plaque. These data were measured using Image Pro Plus 6.0 software.

[0084] result:

[0085] After 16 weeks of feeding with 2.0% methionine, C57BL / 6J mice showed no obvious plaques and lipid formation in the aortic sinus, while ApoE - / - The formation of plaque and lipid in the aortic sinus of mice was significantly higher than that of the control group, indicating that ApoE - / - The atherosclerosis model was successfully induced in mice by feeding them 2.0% methionine for 16 weeks ( Figure 2 ).

[0086] H&E staining showed ApoE - / - After mice were fed a 2.0% methionine diet, the plaque area in the aortic sinus increased significantly. Subsequently, different doses of dangshen glycosides were given for intervention. The results showed that 20 mg / kg of dangshen glycosides could significantly reduce the plaque area (P<0.001) ( Figure 3 ).

[0087] Oil red O staining showed ApoE - / - After mice were fed a 2.0% methionine diet, lipid deposition in the aortic sinus increased significantly. Subsequently, different doses of codonopsis pilosula were given to intervene. The results showed that 20 mg / kg of codonopsis pilosula could significantly reduce lipid deposition (P<0.01) ( Figure 4 ).

[0088] Macroscopic oil red O shows ApoE - / - After mice were fed a 2.0% methionine diet, aortic lipid deposition increased significantly. Subsequently, different doses of codonopsis pilosula were given for intervention. The results showed that 20 mg / kg of codonopsis pilosula could significantly reduce the formation of aortic lipids (P<0.001) ( Figure 5 ).

[0089] 4. Transcriptomics detection of IL-33 protein expression in mouse aorta tissue

[0090] Experimental methods

[0091] The transcriptomic analysis involves the sample processing and analysis process of the normal group, model group and administration group. First, total RNA is extracted from each group of mice, and the Trizol reagent method is used for RNA extraction. The concentration and integrity of the RNA are detected by NanoDrop spectrophotometer and agarose gel electrophoresis to ensure that the RNA quality meets the requirements of subsequent experiments. Subsequently, the Illumina platform is used to construct the RNA-Seq library, including RNA fragmentation, cDNA synthesis, end repair, adapter ligation and other steps. After the construction is completed, the quality of the library is detected, and after passing the quality detection, high-throughput sequencing is performed. After sequencing, the raw data obtained is subjected to quality assessment by FastQC software, and low-quality sequences and adapter sequences are removed by Trimmomatic software. Then, the clean data is aligned to the mouse reference genome by HISAT2 software, the expression of each gene is counted by HTSeq software, and finally the DESeq2 software is used for differential expression analysis to screen out genes with significant differences between the normal group, model group and administration group. Further functional annotation and enrichment analysis are performed, including GO functional annotation and KEGG pathway enrichment analysis, to reveal the differences in gene expression between groups and their potential biological functions.

[0092] Experimental results

[0093] Transcriptomic analysis of gene expression differences in each group, the heatmap result Figure 6 ) shows that the expression of IL-33 protein in the model group is significantly higher than that in the model group, and the expression of IL-33 protein in the administration group is significantly lower than that in the model group. The results show that the therapeutic effect of gypenoside on atherosclerosis after administration may be through the regulation of IL-33 signaling pathway.

[0094] 5, Western blotting detection of IL-33 protein expression in mouse aortic tissue

[0095] Experimental method

[0096] The required group of aortic tissue was taken out from the refrigerator, and 100 mg of mouse aortic tissue was weighed from each group, and 1 mL of protein lysis buffer was added to the pre-cooled centrifuge tube. The homogenizer was opened to grind and lyse the aortic tissue, and the tissue homogenate was obtained. The tissue homogenate was placed in a centrifuge, and the supernatant was transferred to a centrifuge tube. Then, the protein was quantified by BCA kit. SDS-PAGE gel electrophoresis: (1) preparation of separation gel and concentration gel (2) electrophoresis, and after electrophoresis, wet transfer was performed. After transfer, the milk powder blocking method was used, and the incubator was incubated at room temperature for 2 hours. After blocking, primary antibody and secondary antibody were incubated, and after each antibody incubation, the membrane was washed with PBST three times, each for 10 minutes. Finally, the gel image analysis imaging system was used to scan and image analyze the PVDF membrane, and the gray value of the target band was recorded and analyzed.

[0097] Experimental results

[0098] In order to examine the relationship between IL-33 and atherosclerosis, the expression of IL-33 in aortic tissue of AS mice was further detected by Western blot. Figure 7 As shown in the results, the expression level of IL-33 in the aorta tissue of the model group mice was significantly higher than that of the normal control group (P<0.001), and the expression level of IL-33 was significantly decreased after administration of codonopsis pilosula (P<0.05).

[0099] 6. Molecular Docking: IL-33 and Codonopsis pilosula

[0100] Experimental methods

[0101] The IL-33 protein and the ligand codonopsis glycoside were docked using AutoDockTools. The IL-33 protein structure was downloaded from the PDB database, and the codonopsis glycoside structure was downloaded from the TCMSP database. These structures were then imported into the software to calculate binding energies.

[0102] Experimental results

[0103] If the binding energy is less than 0, it means that the ligand and receptor can bind. The smaller the binding energy value, the higher the binding activity. The binding energy of molecular docking is -7.3kcal / mol, indicating that the binding activity of IL-33 and codonopsis pilosula is stable ( Figure 8 ).

[0104] Example 2

[0105] 1. Effect of Codonopsis pilosula on homocysteine-induced cell viability of human umbilical vein endothelial cells

[0106] Experimental methods

[0107] Cell Culture and Model Preparation: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro. HUVECs were obtained from human umbilical veins after collagenase type I digestion and cultured in 0.1% gelatin-coated flasks at 37°C in a 5% CO2 atmosphere in Medium 199. Medium 199 contained 20% fetal bovine serum, penicillin (100 μ / mL), streptomycin (100 μ / mL), and heparin (50 μ / mL), supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 5 ng / mL endothelial growth factor.

[0108] HUVECs were randomly divided into 7 groups: control group, control group + leucoside (LBT, 100 μg / mL), Hcy group (100 μmol), LBT group (25 μg / mL, 50 μg / mL, 100 μg / mL), and folic acid group (50 μg / mL). Cells in the control group were incubated under normal growth conditions. HUVECs in the Hcy group were incubated with culture medium containing 100 μmol Hcy for 24 hours. The LBT group was pre-incubated with different concentrations of LBT for 24 hours and then incubated with 100 μmol Hcy for 24 hours. Homocysteine-induced human umbilical vein endothelial cells were obtained.

[0109] Cell viability was determined using the (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After different treatments, the cell culture medium was replaced with MTT at a final concentration of 0.5 mg / mL. After incubation at 37°C for 4 h, the reaction solution was discarded, and 150 μL of DMSO was added to each well to dissolve the formamide crystals. After shaking for 10 min, the absorbance at 570 nm was recorded using a microplate reader (Epoch, BioTek, Winooski, VT, USA) with a reference wavelength of 650 nm. The ratio of the absorbance to the control value represents cell viability.

[0110] Experimental results

[0111] After Hcy treatment, the cell viability of HUVECs decreased. LBT at concentrations of 50 μg / mL and 100 μg / mL had a protective effect on HUVECs under Hcy conditions ( Figure 9 ).

[0112] 2. Effects of Codonopsis pilosula on the barrier function of human umbilical vein endothelial cells induced by homocysteine

[0113] Experimental methods

[0114] Transendothelial electrical resistance (TEER) measurement: HUVECs were cultured on gelatin-coated transwell inserts in 24-well plates for 6 days. The transendothelial electoral resistance (TEER) of the HUVEC monolayer was monitored daily using a Millicell-ERS voltammeter (Millipore, USA). Results for the experimental groups were measured after subtracting the values ​​from a blank, cell-free filter. After removing the culture medium, 200 μL of EB solution (0.67 mg of EB powder dissolved in 4% bovine serum albumin (BSA) solution) was added to the Millicell cell culture insert, and 600 μL of 4% BSA solution was added to the outer chamber. Cells were incubated in EB solution for 1 hour. The outer chamber was then collected and the absorbance at 620 nm was measured spectrophotometrically using an Infinite M200 Pro plate reader (Tecan, NC, USA). EB leakage in each group was calculated using a standard curve and expressed as a percentage of the control value.

[0115] Immunoblotting: Cells were quickly collected after Hcy induction. The prepared cells (n=6, each group) were homogenized in a glass homogenizer in a 1:10 (w / v) frozen protein extraction buffer. Soluble proteins were collected, centrifuged at 12000×g for 10 min at 4°C, and the supernatant was taken. Protein quantification was then performed using a BCA kit. SDS-PAGE gel electrophoresis: separation gel and stacking gel were prepared for electrophoresis, and wet transfer was performed after electrophoresis. After transfer, milk powder blocking was used and incubated on a shaker at room temperature for 2 hours. After blocking, primary and secondary antibodies were incubated. After each antibody incubation, the membrane was washed three times with PBST for 10 minutes each. Finally, the PVDF membrane was scanned and image analyzed using a gel image analysis imaging system, and the grayscale value of the target band was recorded and analyzed.

[0116] Experimental results

[0117] LBT increased the transendothelial resistance value of HUVECs induced by Hcy

[0118] HUVECs were cultured in tranwell inserts under normoxic conditions for 6 days until TEER reached its peak. After Hcy treatment, TEER was analyzed. Figure 10 As shown in the figure, TEER decreased in the Hcy group, and LBT (50 μg / mL, 100 μg / mL) reversed TEER.

[0119] LBT reversed Hcy-induced loss of tight junctions in HUVECs

[0120] like Figure 11 As shown in the results, LBT could reverse the loss of tight junction proteins (ZO-1 and Occludin) in HUVECs induced by Hcy.

[0121] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of codonopsis pilosula in the preparation of drugs for treating atherosclerosis.

2. The use according to claim 1, characterized in that The atherosclerosis includes atherosclerosis induced by a high-methionine diet.

3. The use according to claim 1, characterized in that The dangshen glycoside plays a role in treating atherosclerosis by reducing the expression level of IL-33 protein.

4. The use according to claim 1, wherein The dangshen glycoside plays a role in treating atherosclerosis by increasing the expression level of tight junction proteins.

5. A drug for treating atherosclerosis, characterized in that: The medicine comprises codonopsis pilosula and pharmaceutically acceptable excipients.

6. The drug according to claim 5, wherein The atherosclerosis includes atherosclerosis induced by a high-methionine diet.

7. The drug according to claim 5, characterized in that The dangshen glycoside plays a role in treating atherosclerosis by reducing the expression level of IL-33 protein.

8. The drug according to claim 5, wherein The dangshen glycoside plays a role in treating atherosclerosis by increasing the expression level of tight junction proteins.

9. The drug according to claim 5, wherein The auxiliary materials include at least one of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

10. The drug according to claim 5, wherein The administration of the drug includes oral administration or parenteral administration.

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