Application of Spexin active polypeptide in preparation of medicine for preventing and treating atherosclerosis

Abstract

The invention relates to application of a Spexin active polypeptide in preparation of a medicine for preventing and treating atherosclerosis, and belongs to the technical field of biological medicine manufacturing. The invention provides a new drug application of a Spexin active polypeptide, namely an application of the Spexin active polypeptide in preparation of a drug for preventing and treating atherosclerosis, and the amino acid sequence of the Spexin active polypeptide is NWTPQAMLYLKGAQ. The invention proves that the Spexin active polypeptide can reduce the aortic plaque area of a high-fat diet ApoE- / -mouse, reduce lipid deposition at the aortic root, reduce the plaque area at the aortic root and increase the plaque stability at the aortic root; the blood fat level in blood plasma of the high-fat diet ApoE- / -mice and the level of related inflammatory factors in the blood plasma and aorta can be reduced, and the progress of atherosclerotic plaques can be slowed down. The new clinical application of the Spexin active polypeptide is developed, and the Spexin active polypeptide has important transformation significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical manufacturing, and particularly relates to the application of Spexin active polypeptide in the preparation of drugs for preventing and treating atherosclerosis. Background Art

[0002] Atherosclerosis refers to the deposition of lipid components and inflammatory substances on the inner wall of arterial blood vessels, forming plaque that looks like porridge, narrowing and hardening the arteries, and obstructing blood flow. Atherosclerosis is a slowly progressing multifocal chronic immune-inflammatory disease, the most common disease in the cardiovascular and cerebrovascular systems, mainly affecting large and medium-sized arteries in the body, such as coronary arteries, carotid arteries, cerebral arteries, renal arteries, etc. Epidemiological studies have revealed environmental and genetic risk factors related to the formation of atherosclerotic lesions, such as dyslipidemia, hypertension, diabetes, obesity, and smoking. Atherosclerosis may begin in childhood and remain latent or asymptomatic for many years before middle or old age, while clinical symptoms often manifest through acute thromboembolic events.

[0003] Although standardized medical treatments such as lipid-lowering therapy and antiplatelet therapy, as well as non-medical treatments such as stent implantation and coronary artery bypass grafting, have made great progress and significantly improved the prognosis of patients, however, many patients still suffer from impaired cardiac function due to acute or chronic complications of atherosclerosis, which in turn leads to the prevalence of ischemic cardiomyopathy and heart failure.

[0004] Spexin active polypeptide is a novel bioactive peptide hormone widely distributed in the central nervous system and peripheral tissues. It can be secreted into the blood and play a role by changing the physiological functions of organs and tissues. Studies have shown that Spexin active polypeptide not only participates in various physiological functions such as energy metabolism and mood regulation, but also can effectively protect cardiomyocytes from metabolic disorders and mitochondrial dysfunction caused by hypoxia, and shows its protective effect on the heart in animal models of myocardial infarction. In addition, Spexin active polypeptide also shows potential in the regulation of arrhythmia. However, the application of Spexin active polypeptide in the prevention and treatment of atherosclerosis has not been reported. Summary of the Invention

[0005] The present invention provides a new pharmaceutical use of Spexin active polypeptide, that is, the application of Spexin active polypeptide in the preparation of drugs for preventing and treating atherosclerosis.

[0006] The technical solution of the present invention:

[0007] The application of Spexin active polypeptide in the preparation of drugs for preventing and treating atherosclerosis, wherein the amino acid sequence of the Spexin active polypeptide is NWTPQAMLYLKGAQ.

[0008] Furthermore, the atherosclerosis is coronary atherosclerosis, carotid atherosclerosis, lower extremity artery atherosclerosis, renal artery atherosclerosis, cerebral artery atherosclerosis or mesenteric artery atherosclerosis.

[0009] Furthermore, the drug uses Spexin active polypeptide as the sole active ingredient or one of the active ingredients.

[0010] Furthermore, the content of Spexin active polypeptide in the drug is 0.1 wt% - 99 wt%.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients and / or carriers.

[0012] Furthermore, the drug can reduce the area of arterial plaques.

[0013] Furthermore, the drug can reduce lipid deposition in the arterial root.

[0014] Furthermore, the drug can increase the stability of arterial plaques.

[0015] Furthermore, the drug can reduce the levels of blood lipids and inflammatory factors in plasma.

[0016] Furthermore, the drug can reduce the level of arterial inflammatory factors.

[0017] Advantages of the present invention:

[0018] Through animal experiments, the present invention confirms that Spexin active polypeptide can reduce the aortic plaque area, reduce lipid deposition in the aortic root, reduce the plaque area in the aortic root, and increase the stability of the plaque in the aortic root of ApoE mice fed a high-fat diet; and can reduce the blood lipid level and the levels of related inflammatory factors in plasma and aorta of ApoE mice fed a high-fat diet, thereby slowing down the progression of atherosclerotic plaques. The present invention applies Spexin active polypeptide to the preparation of drugs for preventing and treating atherosclerosis, develops its new clinical uses, has the characteristics of biological pleiotropy, high safety, and low toxicity and side effects, and is expected to become a new and effective means for the treatment of atherosclerosis, having important translational significance. - / - - / - Description of the Drawings

[0019] Figure 1 Photographs of gross oil red O staining of the heart aorta of four groups of mice in Example 1;

[0020] Figure 2 Comparison chart of the proportion of the gross plaque area of the heart aorta of four groups of mice in Example 1;

[0021] Figure 3 ​Photographs of Oil Red O staining of the aortic root of the hearts of four groups of mice in Example 1;

[0022] Figure 4 Comparison chart of the lipid content ratio in the aortic root of the hearts of four groups of mice in Example 1;

[0023] Figure 5 Photographs of HE staining of the aortic root of the hearts of four groups of mice in Example 2;

[0024] Figure 6 Comparison chart of the plaque area ratio in the aortic root of the hearts of four groups of mice in Example 2;

[0025] Figure 7 Photographs of Masson staining of the aortic root of the hearts of four groups of mice in Example 2;

[0026] Figure 8 Comparison chart of the collagen content ratio in the aortic root of the hearts of four groups of mice in Example 2;

[0027] Figure 9 Comparison chart of the Spexin content in the plasma of four groups of mice in Example 3;

[0028] Figure 10 Comparison chart of the contents of TC, TG and LDL-C in the plasma of four groups of mice in Example 3;

[0029] Figure 11 Comparison chart of the contents of inflammatory factors IL-1β, IL-6 and TNF-α in the plasma of four groups of mice in Example 3;

[0030] Figure 12 Comparison chart of the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the aortic samples of the hearts of four groups of mice in Example 4;

[0031] Figure 13 Schematic diagram of the grouping and administration methods of four groups of mice. Detailed implementation manners

[0032] The technical solutions of the present invention will be further described below in conjunction with the embodiments, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the protection scope of the present invention. For the process equipment or devices not specifically noted in the following embodiments, conventional equipment or devices in the art are used. If not specifically indicated, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically indicated, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0033] Statistical analysis of the experimental data in Examples 1 - 4:

[0034] Statistical analysis was performed using GraphPad Prism 10.0 software (GraphPad Software, Inc, La Jolla, CA). Continuous variables were expressed as mean ± standard error of the mean (SEM). Categorical variables were expressed as numbers and percentages. Comparisons between two groups were evaluated by Student's non-paired t-test or Wilcoxon (Mann-Whitney U) test, and normality test was performed using Shapiro-Wilk. Variables over two groups were analyzed by one-way analysis of variance, followed by Tukey test. Chi-square test was used for categorical variables. Univariate and multivariate logistic regression were used for clinical correlation analysis. P < 0.05 indicated a statistically significant difference between the two groups.

[0035] Example 1

[0036] In this example, the preventive and therapeutic effects of Spexin active polypeptide on atherosclerosis were investigated through animal experiments.

[0037] The Spexin active polypeptide used in this example was purchased from Shanghai Qiangyao Biotechnology Co., Ltd. In the experiment, the Spexin active polypeptide was prepared into a concentration of 1 mg / ml with normal saline and intraperitoneally injected at 50 mg / kg / d.

[0038] I. Experimental animals

[0039] 8-week-old ApoE - / - mice (mouse model with complete knockout of APOE gene) were purchased from Liaoning Changsheng Technology Co., Ltd. (Liaoning). All animals were housed in a specific pathogen-free facility, with the temperature controlled at (20 - 25°C) and humidity at (40 - 70%), a 12-hour light cycle, and free access to standard laboratory food or a high-fat diet (21% fat, 1.25% cholesterol) and tap water.

[0040] II. Grouping and administration method

[0041] After 1 week of adaptive feeding, as Figure 13 shown, all mice were randomly divided into four groups:

[0042] 1. CD (Chow diet: standard diet) + Saline group: normal diet, intraperitoneal injection of normal saline to mice;

[0043] 2. CD + Spexin group: normal diet, intraperitoneal injection of Spexin (50 μg / kg / day) to mice;

[0044] 3. HFD (High-fat diet) + Saline group: While feeding the mice with a high-fat diet to establish an atherosclerosis model, the mice were intraperitoneally injected with saline.

[0045] 4. HFD + Spexin group: While feeding the mice with a high-fat diet to establish an atherosclerosis model, the mice were intraperitoneally injected with Spexin (50 μg / kg / day).

[0046] The administration was continued for twelve weeks.

[0047] III. Gross Oil Red O staining of the heart aorta of the mice in the third and fourth groups

[0048] 1. Specimen collection and fixation:

[0049] The heart was removed from the four groups of experimental animals, and the entire blood vessel was removed along the root of the aorta, and then fixed with 4% paraformaldehyde for more than 24 hours.

[0050] 2. Dissection of blood vessels:

[0051] It is best to remove the fat outside the blood vessel and dissect it as soon as it is removed from the animal body. Such blood vessels are more elastic and easy to operate; if it has been fixed, take out the blood vessel, wash it slightly with PBS, and gently remove the peripheral fat from one end with a special dissection forceps until there is no other fat except small blood vessels. Along the opposite side of the three arteries of the aortic arch, gently dissect the blood vessel, and also dissect the bifurcation at the tail. Then cut a small section along the lower side of the three arteries, and dissect the three arteries along this opening respectively.

[0052] 3. Staining:

[0053] (1) Prepare the Oil Red working solution: Mix the saturated Oil Red staining solution: pure water = 3:2, filter it and then use.

[0054] (2) Immerse the blood vessel in 60% isopropanol for 3 - 5 s first, and then immerse it in the working solution in step (1) and stain it at room temperature for 10 - 60 min.

[0055] (3) Take out the blood vessel and differentiate the background color outside the blood vessel with 60% isopropanol, wash it with water while differentiating until the fat plaque inside the blood vessel wall is clearly visible in bright red and the background is white or almost colorless.

[0056] 4. Photographing:

[0057] Spread the stained blood vessel on a glass slide, absorb the moisture with filter paper, place the scale, and then place the glass slide on a black background board and take a photo with a single-lens reflex camera in a photography studio. The results are as Figure 1 shown.

[0058] 5. The positive staining area of Oil Red O and the total area of the aorta were statistically analyzed using Image J, and analyzed and graphed using Graphpad Prism 10. The results are as Figure 2 shown.

[0059] IV. Oil Red O staining of the aortic root of the heart in four groups of mice

[0060] 1. Specimen collection:

[0061] Fresh aortic root tissues of mice were fixed in 4% paraformaldehyde for more than 24 hours. The tissues were taken out of the fixative and the target tissues were slightly trimmed with a scalpel in a fume hood.

[0062] 2. Dehydration:

[0063] The tissues were transferred to 15% sucrose solution for dehydration for 1 - 2 days, and then transferred to 30% sucrose solution for dehydration for 1 - 2 days. The dehydrated tissues could sink to the bottom of the tube and not float in the solution.

[0064] 3. OCT embedding:

[0065] The target position of the tissue was taken out of 30% sucrose, and the surface moisture was wiped dry. The temperature of the cryostat was set to -18°C to -22°C. An appropriate amount of OCT embedding medium was extruded onto the circular base in the cryostat. Before the embedding medium solidified, the tissue was immersed in the embedding medium.

[0066] 4. Sectioning:

[0067] After the embedding medium solidified, the circular base with the OCT block was placed on the sample holder. The section thickness was adjusted to 30μm for trimming, and when the tissue was exposed, the section thickness was adjusted to 8μm to start sectioning.

[0068] 5. Baking the sections:

[0069] The cut sections were inserted into the staining rack and placed in an oven at 37°C for baking for 2 hours.

[0070] 6. Staining:

[0071] (1) The sections were placed in Oil Red O staining solution 1 (Oil Red, stored at room temperature) prepared 12 hours in advance and filtered before use, and stained for 6 minutes;

[0072] (2) Without washing with water, the sections were directly transferred to 60% isopropanol for differentiation for 5 - 8 seconds;

[0073] (3) Without washing with water, the sections were directly transferred to 60% isopropanol for differentiation for 5 - 8 seconds;

[0074] (4) The sections were placed in a staining cylinder and washed with water and the water was changed;

[0075] (5) Take out the sections and stain them with Oil Red O staining solution 2 (hematoxylin, stored at room temperature) in a staining cylinder for 3 - 5 min;

[0076] (6) Wash the staining cup with water until the sections are colorless;

[0077] (7) Immerse the sections in Oil Red O staining solution 3 (differentiating solution, stored at room temperature) in a staining cylinder for 2 - 3 s, and wash quickly with water;

[0078] (8) Immerse the sections in Oil Red O staining solution 4 (blue - returning solution, stored at room temperature) in a staining cylinder for 2 - 3 s, and wash quickly with water;

[0079] 7. Mounting:

[0080] Dry the excess water on the sections with filter paper and then mount the sections with glycerol gelatin.

[0081] 8. Take pictures and save with a high - definition camera, and the results are as Figure 3 shown; Use Image J software to count the positive area of Oil Red O staining, plot and analyze with Graphpad Prism 10, and the results are as Figure 4 shown.

[0082] From Figures 1 to 4 shown, through gross observation of the mouse aorta and Oil Red O staining of the aortic root, it was found that compared with the ApoE - / - mice in the CD + Saline group, the aortic plaque area of ApoE - / - mice in the HFD + Saline group increased significantly; compared with the ApoE - / - mice in the HFD + Saline group, the aortic plaque area of ApoE - / - mice in the HFD + Spexin group decreased, and lipid deposition in the aortic root decreased. This indicates that Spexin active polypeptide can significantly reduce the aortic plaque area (P < 0.0001) and lipid deposition in the aortic root (P < 0.0001) of ApoE - / - mice in the high - fat diet group.

[0083] Example 2

[0084] This example investigated the preventive and therapeutic effects of Spexin active polypeptide on atherosclerosis through animal experiments.

[0085] On the basis of the modeling and treatment in Example 1, this example further performed HE staining on the aortic roots of the hearts of four groups of mice. The specific staining steps are as follows:

[0086] 1. Specimen collection:

[0087] Fresh mouse heart aortic root tissue was fixed in 4% paraformaldehyde for more than 24 hours. The tissue was taken out of the fixative and the target tissue was slightly trimmed with a scalpel in a fume hood;

[0088] 2. Dehydration:

[0089] The tissue was transferred to a 15% sucrose solution for dehydration for 1 - 2 days, and then transferred to a 30% sucrose solution for dehydration for 1 - 2 days. The dehydrated tissue could sink to the bottom of the tube and not float in the solution;

[0090] 3. OCT embedding:

[0091] The target position of the tissue was taken out of the 30% sucrose, and the surface moisture was wiped dry. The temperature of the cryostat was set to -18°C to -22°C. An appropriate amount of OCT embedding medium was extruded onto the circular base in the cryostat. Before the embedding medium solidified, the tissue was immersed in the embedding medium;

[0092] 4. Sectioning:

[0093] After the embedding medium solidified, the circular base with the OCT block was placed on the sample holder. The section thickness was adjusted to 30μm for trimming, and after the tissue was exposed, the section thickness was adjusted to 8μm to start sectioning;

[0094] 5. Staining and dehydration:

[0095] (1) The sections were returned to room temperature, fixed in methanol for 15 minutes, and stained in HE staining solution 1 (hematoxylin, stored at room temperature) in the staining jar for 3 - 5 minutes;

[0096] (2) The sections were taken out and washed with water in a staining cup until the sections were colorless;

[0097] (3) The sections were placed in HE staining solution 2 (differentiating solution, stored at room temperature) in the staining jar for 3 - 5 seconds, and quickly washed with water;

[0098] (4) The sections were placed in HE staining solution 3 (blueing solution, stored at room temperature) in the staining jar for 3 - 5 seconds, and quickly washed with water;

[0099] (5) The sections were successively placed in 85% ethanol, 95% ethanol, HE staining solution 4 (eosin, stored at room temperature), absolute ethanol I, absolute ethanol II, absolute ethanol III, n-butanol, xylene I, and xylene II in the staining jars, and soaked in each jar for 3 - 5 minutes.

[0100] 6. Sealing the slides:

[0101] The sections were taken out, quickly dried in a draft, and sealed with neutral balsam.

[0102] 7. Observation and photography under the microscope and saving. The results are as Figure 5 shown; The images were analyzed with Image J. The results are as Figure 6As shown

[0103] On the basis of the modeling and treatment in Example 1, in this example, Masson staining was further performed on the aortic roots of the hearts of four groups of mice. The specific staining steps are as follows:

[0104] 1. Specimen collection:

[0105] The fresh aortic root tissue of the mouse heart was fixed in 4% paraformaldehyde for more than 24 hours. The tissue was taken out of the fixative and the target tissue was slightly trimmed with a scalpel in the fume hood;

[0106] 2. Dehydration:

[0107] The tissue was transferred to a 15% sucrose solution for dehydration for 1 - 2 days, and then transferred to a 30% sucrose solution for dehydration for 1 - 2 days. The dehydrated tissue could sink to the bottom of the tube and not float in the solution;

[0108] 3. OCT embedding:

[0109] The target position of the tissue was taken out of the 30% sucrose, and the surface moisture was wiped dry. The temperature of the cryostat was set to -18°C to -22°C. An appropriate amount of OCT embedding medium was extruded onto the circular base in the cryostat. Before the embedding medium solidified, the tissue was immersed in the embedding medium;

[0110] 4. Sectioning:

[0111] After the embedding medium solidified, the circular base with the OCT block was placed on the sample holder. The section thickness was adjusted to 30μm for trimming, and after the tissue was exposed, the section thickness was adjusted to 8μm to start sectioning.

[0112] 5. Staining:

[0113] (1) The sections were incubated overnight in Masson stain 1 (CGSJ, stored at room temperature) in a microwave repair box;

[0114] (2) The sections were taken out and quickly washed with tap water in a staining cup until colorless;

[0115] (3) The sections were placed in Masson stain 2 (ponceau, stored at room temperature, preheated at 65°C for 30 minutes before staining) in a microwave repair box, put back into the oven for staining for 3 - 5 minutes, and then washed 2 - 3 times with tap water in a staining cup;

[0116] (4) The sections were stained in Masson stain 3 (phosphomolybdic acid, stored at room temperature) for 30 seconds to 1 minute;

[0117] (5) The sections were taken out, slightly drained, and stained in Masson stain 4 (aniline blue, stored at room temperature, preheated at 65°C for 30 minutes before staining and put back into the oven for preheating after use) for 5 - 20 seconds;

[0118] 6. Dehydration:

[0119] Transfer the sections into three cylinders of absolute ethanol in the microwave repair box, 5 - 10 s for each cylinder; transfer the sections into n-butanol in the microwave repair box for 10 - 20 s;

[0120] 7. Sealing the sections:

[0121] Transfer the sections into xylene (special) Ⅰ in the staining cylinder for 5 min; transfer them into xylene (special) Ⅱ in the staining cylinder for 5 min; after quickly drying in the air outlet, seal the sections with neutral balsam;

[0122] 8. Place the sections under the microscope for observation, photographing and saving. The results are as Figure 7 shown; analyze the pictures with Image J. The results are as Figure 8 shown.

[0123] As Figures 5 to 8 shown, through HE staining and Masson staining of the aortic root of mouse hearts, it was found that compared with ApoE - / - mice in the CD + Saline group, the plaque area at the aortic root of ApoE - / - mice in the HFD + Saline group increased significantly, and the plaque stability decreased significantly; compared with ApoE - / - mice in the HFD + Saline group, the plaque area at the aortic root of ApoE - / - mice in the HFD + Spexin group decreased, and the plaque stability increased significantly. This indicates that the Spexin active polypeptide can significantly reduce the plaque area at the aortic root of ApoE - / - mice in the high-fat diet group (P < 0.0001), and improve the plaque stability at the aortic root of ApoE - / - mice in the high-fat diet group (P < 0.001).

[0124] Example 3

[0125] In this example, the preventive and therapeutic effects of the Spexin active polypeptide on atherosclerosis were investigated through animal experiments.

[0126] On the basis of the modeling and treatment in Example 1, this example further measured the levels of Spexin, blood lipids and inflammatory factors in the plasma of four groups of mice by ELISA method. The specific detection methods are as follows:

[0127] 1. Collection of mouse blood:

[0128] Prepare a 2-ml EDTA anticoagulant tube before blood collection. First, anesthetize the mouse, then use ophthalmic forceps to remove the mouse's eyeball, collect the blood in a 2-ml EDTA anticoagulant tube, gently shake it up and down several times, centrifuge it in an ultracentrifuge at 3500 rpm for 15 minutes, extract the supernatant into a 1.5-ml enzyme-free EP tube, and store it in a -80°C refrigerator.

[0129] 2. In this experiment, an enzyme immunoassay kit was used to detect the plasma Spexin level; a practical enzyme immunoassay kit was used to detect the plasma blood lipid and inflammatory factor levels. The detection steps of the above kits are the same. Before detection, first perform sample preparation: Take out the mouse plasma sample from the refrigerator and thaw it.

[0130] (1) Take out the required strip from the aluminum foil bag that has been equilibrated at room temperature for 20 minutes, and seal the remaining strips with a self-sealing bag and return them to 4°C;

[0131] (2) Set up the standard wells and sample wells, and add 50 μl of standards with different concentrations to each standard well;

[0132] (3) First add 10 μl of the sample to be tested to the sample well, and then add 40 μl of sample diluent; do not add to the blank well;

[0133] (4) Except for the blank well, add 100 μl of the detection antibody labeled with horseradish peroxidase (HRP) to each well in the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate them in a 37°C water bath or incubator for 60 minutes;

[0134] (5) Discard the liquid, pat it dry on absorbent paper, fill each well with washing solution, let it stand for 1 minute, discard the washing solution, pat it dry on absorbent paper, and repeat the washing process 5 times;

[0135] (6) Add 50 μl of substrate A and B to each well, and incubate it in the dark at 37°C for 15 minutes;

[0136] (7) Add 50 μl of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0137] (8) Draw a standard curve and calculate the concentration values of each sample according to the curve equation.

[0138] Figure 9 This is the comparison chart of the Spexin content in the plasma of the four groups of mice in this example; Figure 10 This is the comparison chart of the contents of TC, TG, and LDL-C in the plasma of the four groups of mice in this example; Figure 11 This is the comparison chart of the contents of inflammatory factors IL-1β, IL-6, and TNF-α in the plasma of the four groups of mice in this example; As Figures 9 to 11 shown, compared with the ApoE - / - mice in the CD+Saline group, the ApoE in the HFD+Saline group- / - The Spexin level in the plasma of mice decreased, while the levels of plasma TC, TG, and LDL-C increased, and the levels of plasma IL-1β, IL-6, and TNF-α also increased; compared with the ApoE - / - mice in the HFD+Saline group, the levels of plasma TC, TG, and LDL-C in the ApoE - / - mice in the HFD+Spexin group decreased, and the levels of plasma IL-1β, IL-6, and TNF-α also decreased.

[0139] This indicates that a high-fat diet significantly decreased the Spexin level in the plasma of ApoE - / - mice in the HFD+Saline group (P < 0.0001), while the Spexin active polypeptide was able to significantly decrease the levels of blood lipids and inflammatory factors in the plasma of ApoE - / - mice in the high-fat diet group (P < 0.05).

[0140] Example 4

[0141] In this example, the preventive and therapeutic effects of the Spexin active polypeptide on atherosclerosis were investigated through animal experiments.

[0142] On the basis of the modeling and treatment in Example 1, the mRNA levels of related inflammatory factors in the aortic samples of four groups of mice were further detected by qRT-PCR. The specific detection method is as follows:

[0143] 1. Aortic tissue collection from mice:

[0144] After opening the chest of the mice, remove the redundant organs and connective tissues. Insert a pre-cooled 1 ml syringe into the left ventricle for perfusion to remove the residual blood in the blood vessels, and perform perfusion 2 - 3 times. Use micro forceps to clamp the left and right common iliac arteries respectively, and use micro scissors to sever the left and right common iliac arteries. Slightly lift the left and right common iliac arteries with micro forceps, and slowly cut off the aorta of the mice together with the heart from bottom to top along the spine of the mice with micro scissors. Place it in pre-cooled PBS, and remove the connective tissues around the aorta under a microscope. Store the dissected aortic tissue in paraformaldehyde or in a -80°C refrigerator;

[0145] 2. RNA extraction from mouse aortic tissue:

[0146] Weigh 20 mg of mouse aortic tissue and grind it into powder in a mortar with liquid nitrogen. All the following centrifugation steps are carried out at 4°C. Transfer it to a 1.5-ml enzyme-free EP tube, add 400 μl of Buffer R-I, mix well, then add 150 μl of Buffer R-II, and centrifuge at 12,000 g for 5 min. Take the supernatant, mix 250 μl of isopropanol, transfer the mixture to a preparation tube and place it in a 2-ml centrifuge tube, centrifuge at 6,000 g for 1 min, and discard the liquid. Add 500 μl of Buffer W1 to the preparation tube, centrifuge at 12,000 g for 1 min, and discard the liquid. Add 700 μl of Buffer W2 solution to the preparation tube, centrifuge at 12,000 g for 1 min, and discard the liquid. Repeat once, and centrifuge the empty tube for 2 min. Drop 70 μl of Buffer TE in the center of the preparation tube, let it stand, and then centrifuge at 12,000 g for 1 min to obtain RNA and detect its concentration;

[0147] 3. Reverse transcription and qRT-PCR:

[0148] (1) Reverse transcription: Measure the concentration with NanoDrop 2000, and dilute each sample to the same concentration; Prepare a 20-μl reverse transcription system: Take 1 μg of mRNA, add 5 μl of 5× reverse transcription reagent, and make up to 20 μl with RNase-free water; Carry out reverse transcription according to the following program: 30°C for 5 min, 55°C for 15 min, 85°C for 5 min;

[0149] (2) qRT-PCR: The reaction system is 20 μl: 4 pmol of upstream primer, 4 pmol of downstream primer and 2 μl of cDNA, make up to 10 μl with RNase-free water, and 10 μl of qPCR SYBR GREEN reagent. The reaction conditions are pre-denaturation at 95°C for 2 min, denaturation at 95°C for 10 s, annealing / extension at 60°C for 30 s, and set 40 cycles for denaturation and annealing / extension. Record the data and calculate the expression levels of each index.

[0150] Figure 12 This is the comparison chart of the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the heart aortic samples of four groups of mice in this example; As Figure 12 shown, compared with ApoE - / - mice in the CD+Saline group, the mRNA levels of IL-1β, IL-6 and TNF-α in the aorta of ApoE - / - mice in the HFD+Saline group increased; compared with ApoE - / - mice in the HFD+Saline group, ApoE - / -The mRNA levels of IL-1β, IL-6, and TNF-α in the aorta of mice decreased. This indicates that the Spexin active polypeptide can significantly reduce the mRNA levels of inflammatory factors in the aorta of ApoE- / - mice in the high-fat diet group (P < 0.01), and can be used to prevent and treat the progression of atherosclerotic plaques.

Claims

1. Use of Spexin active polypeptide in the preparation of a drug for preventing and treating atherosclerosis, characterized in that, The amino acid sequence of the Spexin active polypeptide is NWTPQAMLYLKGAQ.

2. Use of the Spexin active polypeptide according to claim 1 in the preparation of a medicament for preventing and treating atherosclerosis, characterized in that, The atherosclerosis is coronary atherosclerosis, carotid atherosclerosis, lower extremity atherosclerosis, renal artery atherosclerosis, cerebral atherosclerosis or mesenteric artery atherosclerosis.

3. Use of the Spexin active polypeptide according to claim 1 or 2 in the preparation of a drug for preventing and treating atherosclerosis, characterized in that, The drug takes the Spexin active polypeptide as the sole active ingredient or one of the active ingredients.

4. Use of the Spexin active polypeptide according to claim 3 in the preparation of a drug for preventing and treating atherosclerosis, characterized in that, The content of the Spexin active polypeptide in the drug is 0.1wt% - 99wt%.

5. Use of the Spexin active polypeptide according to claim 4 in the preparation of a drug for preventing and treating atherosclerosis, characterized in that, The drug also includes pharmaceutically acceptable excipients and / or carriers.

6. Use of the Spexin active polypeptide according to claim 5 in the preparation of a medicament for preventing and treating atherosclerosis, characterized in that, The drug can reduce the area of arterial plaques.

7. Use of the Spexin active polypeptide according to claim 6 in the preparation of a drug for preventing and treating atherosclerosis, characterized in that, The drug can reduce lipid deposition at the arterial root.

8. Use of the Spexin active polypeptide according to claim 7 in the preparation of a medicament for preventing and treating atherosclerosis, characterized in that, The drug can increase the stability of arterial plaques.

9. Use of the Spexin active polypeptide according to claim 8 in the preparation of a drug for preventing and treating atherosclerosis, characterized in that, The drug can reduce the levels of blood lipids and inflammatory factors in plasma.

10. Use of the Spexin active polypeptide according to claim 9 in the preparation of a medicament for preventing and treating atherosclerosis, characterized in that, The drug can reduce the level of arterial inflammatory factors.

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