CTPER polypeptide and application thereof in medicine for treating cardiovascular diseases

Abstract

The invention relates to the technical field of biological medicine, in particular to a novel polypeptide and application thereof in medicine for treating cardiovascular diseases. The polypeptide has a unique amino acid sequence and is composed of an amino acid sequence SEQ.NO: 1: Cys-Tyr-Pro-Glu-Arg (CTPER), an amino acid sequence SEQ.NO: 2: Asn-His-Met-Val-Asp (NHVD) and the like, and the cardiovascular function is improved by adjusting a nuclear factor kappa B (NF-kappa B) signal channel.

Description

1. Technical Field

[0001] The present invention belongs to the field of biomedical technology, and mainly relates to the research and development of a novel polypeptide and its application in the treatment of cardiovascular diseases. 2. Background Art

[0002] The Severe Situation of Cardiovascular Diseases Cardiovascular diseases are one of the major diseases that seriously threaten human health globally, including various types such as coronary heart disease, hypertension, heart failure, etc. These diseases bring great pain and burden to patients and also impose a heavy pressure on social medical resources. Coronary heart disease is mainly caused by atherosclerosis of the coronary arteries, leading to stenosis or obstruction of blood vessels, thereby causing myocardial ischemia, hypoxia, and even necrosis. Hypertension refers to the continuous elevation of blood pressure, and long-term hypertension can damage important organs such as the heart, kidneys, and brain. Heart failure is the gradual decline of heart function, unable to effectively pump blood to all tissues and organs of the body. II. Overview of the NF-κB Signaling Pathway Nuclear factor κB (NF-κB) is a transcription factor widely present in cells, and it plays an important role in regulating cell growth, differentiation, apoptosis, as well as immune and inflammatory responses. The NF-κB signaling pathway mainly consists of NF-κB family members, IκB protein family, and IκB kinase (IKK) complex. Under normal physiological conditions, NF-κB binds to IκB proteins to form an inactive complex, which exists in the cytoplasm. When cells are stimulated by external factors such as inflammatory factors, oxidative stress, bacterial infections, etc., the IKK complex is activated, and then phosphorylates IκB proteins, leading to the degradation of IκB proteins. After the degradation of IκB proteins, NF-κB is released and enters the nucleus, binds to specific DNA sequences, and initiates the transcription of downstream genes. III. Regulatory Role of the NF-κB Signaling Pathway in the Cardiovascular System 1. Inflammatory Response - In the cardiovascular system, the NF-κB signaling pathway is involved in the regulation of the inflammatory response. Inflammation is one of the important factors in the occurrence and development of cardiovascular diseases, which can lead to vascular endothelial cell damage, the formation of atherosclerosis, myocardial cell apoptosis, etc. - NF-κB can activate the expression of various inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), etc. These inflammatory factors can further activate the NF-κB signaling pathway, forming a positive feedback regulation and aggravating the inflammatory response. 2. Cell Proliferation and Apoptosis - The NF-κB signaling pathway can also regulate the proliferation and apoptosis of cardiovascular cells. In cardiovascular diseases, the proliferation of vascular smooth muscle cells and the apoptosis of myocardial cells are important pathological processes. - NF-κB can activate the expression of some cell cycle-related genes, promoting the proliferation of vascular smooth muscle cells. At the same time, NF-κB can also regulate the expression of some anti-apoptotic genes, inhibiting the apoptosis of myocardial cells. 3. Oxidative Stress - Oxidative stress is also one of the important factors in the occurrence and development of cardiovascular diseases. The NF-κB signaling pathway can be activated by oxidative stress and can also regulate the expression of some antioxidant genes, reducing the damage of oxidative stress to the cardiovascular system.IV. Potential of the NF-κB Signaling Pathway as a Therapeutic Target for Cardiovascular Diseases 1. NF-κB inhibitors - Due to the important role of the NF-κB signaling pathway in cardiovascular diseases, the development of NF-κB inhibitors has become a potential therapeutic strategy. Currently, some NF-κB inhibitors have been developed, such as pyrrolidine dithiocarbamate (PDTC), aspirin, etc. - These inhibitors can inhibit the activation of the NF-κB signaling pathway by inhibiting the activity of the IKK complex, preventing the degradation of IκB protein, or directly inhibiting the transcriptional activity of NF-κB. 2. Targeting upstream and downstream molecules of the NF-κB signaling pathway - In addition to directly inhibiting the NF-κB signaling pathway, its activity can also be regulated by targeting upstream and downstream molecules of the NF-κB signaling pathway. For example, the purpose of treating cardiovascular diseases can be achieved by inhibiting the production of inflammatory factors, blocking the signal transduction of oxidative stress, or regulating the expression of NF-κB downstream genes. 3. Gene therapy - Gene therapy is an emerging treatment method that can correct or compensate for gene defects or abnormal expressions in patients by introducing normal genes into the patient's cells. In the treatment of cardiovascular diseases, gene therapy can inhibit the activation of the NF-κB signaling pathway by introducing genes encoding NF-κB inhibitors into the patient's cardiovascular cells. Although the NF-κB signaling pathway has great potential as a therapeutic target for cardiovascular diseases, there are still some challenges at present. 1. Complexity of the NF-κB signaling pathway - The NF-κB signaling pathway is very complex and involves the interaction and regulation of multiple molecules. Therefore, when developing NF-κB inhibitors or therapeutic methods targeting the NF-κB signaling pathway, its complexity needs to be fully considered to avoid adverse reactions. 2. Specificity and safety of drugs - Most of the currently developed NF-κB inhibitors lack specificity and may affect other signaling pathways, resulting in adverse reactions. Therefore, more specific and safe NF-κB inhibitors need to be developed. 3. Feasibility of clinical application - Although NF-κB inhibitors and therapeutic methods targeting the NF-κB signaling pathway have shown certain potential in animal experiments and cell experiments, their safety and effectiveness need to be further verified in clinical applications. In the future, with the continuous in-depth study of the NF-κB signaling pathway, it is believed that more safe and effective treatment methods will be developed, bringing new hope for the treatment of cardiovascular diseases.

[0003] In summary, the nuclear factor κB (NF-κB) signaling pathway plays an important regulatory role in the cardiovascular system and has become one of the research hotspots. By inhibiting the activation of the NF-κB signaling pathway, the inflammatory response, cell proliferation and apoptosis, and oxidative stress in the cardiovascular system can be regulated, providing new ideas and methods for the treatment of cardiovascular diseases. Although there are still some challenges at present, with the continuous in-depth research, the NF-κB signaling pathway is expected to become an important target for the treatment of cardiovascular diseases. III. Summary of the Invention

[0004] (I) Structure of the novel polypeptide

[0005] The novel polypeptide provided by the present invention is composed of the following amino acid sequences:

[0006] SEQ.NO 1: Cys-Tyr-Pro-Glu-Arg (CTPER);

[0007] SEQ.NO 2: Asn-His-Met-Val-Asp (NHMVD).

[0008] These sequences were designed through in-depth research on the structure and function of the target protein NF-κB and combined with binding affinity analysis.

[0009] (II) Mechanism of action of the novel polypeptide

[0010] By simulating the interaction between protein molecules, the binding mode and binding affinity between proteins are predicted. In the design of novel polypeptides, docking software can be used to predict the binding mode of polypeptides to NF-κB and screen out polypeptide sequences with high binding affinity.

[0011] Machine learning algorithms: Use machine learning algorithms to learn a large amount of known protein-protein interaction data and establish a prediction model to predict the interaction between new proteins. In the design of novel polypeptides, machine learning algorithms can be used to predict the interaction between polypeptides and NF-κB to provide guidance for the design of polypeptides.

[0012] Regulate the NF-κB signaling pathway

[0013] The novel polypeptide can specifically bind to NF-κB, thereby regulating its signaling pathway. Specifically, the amino acids in the CTPER sequence have specific chemical properties, such as the sulfhydryl group of Cys, the hydroxyl group of Tyr, the rigidity of Pro, the acidity of Glu, and the basicity of Arg. These properties help to form complementary interactions with specific binding regions of NF-κB, thereby regulating the activity of NF-κB. Similarly, the amino acids in the NHMVD sequence can also interact with NF-κB and affect its functions such as signal transduction.

[0015] Improve cardiovascular function

[0016] By regulating the NF-κB signaling pathway, the novel polypeptide can improve cardiovascular function. NF-κB is involved in processes such as inflammatory response, apoptosis, and vascular endothelial dysfunction, and is closely related to the occurrence and development of cardiovascular diseases. The novel polypeptide can inhibit the activation of NF-κB, reduce the inflammatory response, decrease apoptosis, and improve vascular endothelial function, thereby improving cardiovascular function.

[0017] Advantages of the invention:

[0018] 1. High efficiency

[0020] The novel polypeptide can significantly improve the functional state of the target protein NF-κB and effectively regulate cardiovascular-related physiological indices, such as reducing the inflammatory response and improving vascular endothelial function.

[0021] 2. Safety

[0023] Verified by in vitro and in vivo experiments, the novel polypeptide has no obvious toxicity at high doses and has no obvious adverse effects on important organs such as the liver and kidneys after long-term use.

[0024] 3. Specificity

[0026] The novel polypeptide has a highly specific binding ability to the target protein NF-κB, reducing interference with other irrelevant proteins and lowering the risk of potential side effects. IV. Specific implementation methods

[0027] Example 1

[0028] (I) Synthesis of the polypeptide

[0029] 1. Selection of amino acid monomers

[0031] Amino acid monomers with Boc protecting groups are used to ensure the activity and stability of amino acids during synthesis.

[0032] 2. Solid-phase synthesis

[0034] The amino acid monomers are sequentially linked to the solid-phase carrier. After each coupling reaction, the Boc group is removed through a deprotection step. A step-by-step synthesis method is adopted to ensure the accuracy and purity of the polypeptide.

[0035] 3. Cleavage and purification

[0037] After all amino acids are linked, it is cleaved from the solid-phase carrier to obtain the crude polypeptide. Its purity and molecular weight are confirmed by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis. Multiple purification methods, such as reverse-phase high-performance liquid chromatography and ion-exchange chromatography, are used to improve the purity of the polypeptide.

[0038] (II) Structural Verification Experiments

[0039] Mass Spectrometry Analysis

[0041] 1. Instrument: Use a high-resolution mass spectrometer (such as MALDI-TOF MS) for analysis.

[0042] 2. Sample Preparation: Dissolve each synthesized peptide segment in a mixed solvent of methanol and water to prepare a solution with an appropriate concentration.

[0043] 3. Experimental Results: The results of mass spectrometry analysis show that the molecular weights of the peptide segments are consistent with the theoretical values, further confirming the correctness of their structures. Evaluate the synthesis accuracy of the polypeptide by comparing the difference between the measured molecular weight and the theoretical molecular weight.

[0044] Circular Dichroism (CD) Analysis

[0045] 1. Instrument: Use a circular dichroism spectrometer for analysis.

[0046] 2. Sample Preparation: Dissolve each synthesized peptide segment in phosphate buffer to prepare a solution with an appropriate concentration.

[0047] 3. Experimental Results: The results of circular dichroism analysis show that each peptide segment has specific secondary structure characteristics, which are consistent with the expected structures. Analyze the secondary structure of the polypeptide, such as α-helix, β-sheet, etc., to understand its structural stability.

[0048] (III) Identification and Related Studies of the Target Protein NF-κB

[0049] 1. Identification of NF-κB

[0051] Identify NF-κB in cardiovascular tissues through proteomics and bioinformatics methods. Use techniques such as immunohistochemistry and Western blot to verify the expression of NF-κB in the cardiovascular system,

[0052] especially its high expression in vascular endothelial cells and cardiomyocytes.

[0053] 2. Determination of Binding Sites

[0055] Determine the binding sites of the novel polypeptide and NF-κB through molecular docking technology. Use computer simulation software to predict the binding mode and affinity of the polypeptide and NF-κB, providing a basis for further optimizing the polypeptide structure.

[0056] (IV) In Vitro Activity Tests

[0058] Cell Culture

[0059] 1. Cell lines: Human umbilical vein endothelial cell line (HUVEC cells), human cardiomyocyte cell line (HCM cells).

[0060] 2. Culture conditions:

[0061] 1. HUVEC cells were cultured in ECM medium containing 10% fetal bovine serum, 1% penicillin - streptomycin, at 37°C, 5% CO2.

[0062] 2. HCM cells were cultured in RPMI - 1640 medium containing 15% fetal bovine serum, 1% penicillin - streptomycin, at 37°C, 5% CO2.

[0063] Peptide treatment

[0064] 1. Each synthesized polypeptide was dissolved in DMSO to prepare solutions with different concentrations.

[0065] 2. Each polypeptide with different concentrations was added to the media of HUVEC cells and HCM cells and incubated for 24 hours.

[0066] Experimental results

[0067] 1. NF - κB activity: The nuclear translocation of NF - κB was detected to reflect its activity state.

[0068] The results showed that each polypeptide significantly inhibited the activity of NF - κB. The specific data are as follows:

[0069]

[0070] · Vascular endothelial cell function:

[0071] · Cell proliferation ability: The proliferation ability of HUVEC cells was detected by the MTT method. The results showed that each polypeptide significantly promoted the proliferation of HUVEC cells. The specific data are as follows:

[0072]

[0073] · Endothelial cell permeability: It was detected by the Transwell permeability assay. The results showed that each polypeptide regulated the endothelial cell permeability to a certain extent. The specific data are as follows:

[0074]

[0075] · Cardiomyocyte function:

[0076] · Cell viability: The viability of HCM cells was detected by the MTT method. The results showed that each polypeptide significantly increased the viability of HCM cells. The specific data are as follows:

[0077]

[0078] · Apoptosis rate: The apoptosis rate of HCM cells was detected by flow cytometry. The results showed that each polypeptide significantly reduced the apoptosis rate of HCM cells. The specific data are as follows:

[0079]

[0080] (V) Verification in animal models

[0082] Animal model

[0083] · Model: Atherosclerosis mouse model (prepared by induction with a high-fat diet).

[0084] · Experimental group: Each polypeptide treatment group.

[0085] · Control group: Normal saline treatment group.

[0086] · Positive control group: Atorvastatin calcium tablets, a commonly used lipid-lowering drug, treatment group.

[0087] Drug administration method

[0088] · Each polypeptide or normal saline was administered daily by intraperitoneal injection for eight consecutive weeks.

[0089] Experimental results

[0090] · Blood lipid levels: The blood lipid levels of the mice were detected, including total cholesterol (TC), triglyceride

[0091] (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol

[0092] (HDL-C). The results showed that the blood lipid levels in each polypeptide treatment group were significantly lower than those in the control group and the positive control group. The specific data are as follows:

[0093]

[0094]

[0095] · Degree of atherosclerotic lesions: The degree of atherosclerotic lesions in the mice was observed by pathological section. The results showed that the degree of atherosclerotic lesions in each polypeptide treatment group was significantly lower than those in the control group and the positive control group. The specific data are as follows:

[0096]

[0097] (VI) Safety assessment

[0098] 1. Acute toxicity test

[0099] · By administering a single large dose, the survival rate and behavioral performance of mice were observed. The results showed that there was no obvious toxicity for each polypeptide at high doses. The specific data are as follows:

[0100]

[0101]

[0102] 1. Long-term toxicity test

[0103] · By continuously administering the drug for eight weeks, indicators such as the body weight, blood routine, liver and kidney functions of rats were observed.

[0104] The results showed that there were no obvious adverse reactions for each polypeptide under long-term use. The specific data are as follows:

[0105] The novel polypeptide provided by the present invention has a unique amino acid sequence, can specifically regulate the NF-κB signaling pathway, and provides new ideas and methods for the treatment of cardiovascular diseases; the novel polypeptide can significantly improve cardiovascular function, effectively regulate cardiovascular-related physiological indexes, such as reducing inflammatory response, improving vascular endothelial function, etc.; verified by in vitro and in vivo experiments, the novel polypeptide has no obvious toxicity at high doses, and has no obvious adverse effects on important organs such as the liver and kidney under long-term use, and has high safety; the novel polypeptide has a high specific binding ability to the target protein NF-κB, reduces the interference with other irrelevant proteins, and reduces the risk of potential side effects.

[0106] Data table of comparative synergistic test

[0108]

[0109] In order to verify the effect of the novel polypeptide of the present invention in the treatment of cardiovascular diseases, a series of experiments were carried out. The experimental results showed that the novel polypeptide can effectively regulate the NF-κB signaling pathway, improve cardiovascular function, and reduce the risk factors of cardiovascular diseases. At the same time, the polypeptide has no obvious toxicity at high doses, and has no obvious adverse effects on important organs such as the liver and kidney under long-term use, and has high safety and reliability.

[0110] Example 2: Novel polypeptide based on soybean extract

[0111] 1. Structure of the novel polypeptide

[0112] The novel polypeptide provided in this example is composed of the following amino acid sequence: SEQ.NO 3: Ala-Gly-

[0113] Ser-Thr-Val-Leu-Ile-Phe-Lys-His(AGSTVLIFKH).

[0114] 2. Extraction method

[0115] 1. Pretreatment of raw materials: Select high-quality soybeans, crush them, and reflux and defat them with n-hexane to remove the oil components in the soybeans. Add defatted soybean powder to phosphate buffer solution with pH 8.0 at a ratio of 1:10 (w / v), stir and extract at 40 °C for 4 hours, and perform ultrasonic assistance (power 200W, ultrasonic time 10 minutes) every 1 hour during this period to fully dissolve the soy protein.

[0116] 2. Enzymatic hydrolysis reaction: Centrifuge the extract (8000 rpm, 15 minutes), take the supernatant, adjust the pH to 7.5, add alkaline protease (enzyme to substrate mass ratio 1:100), and perform enzymatic hydrolysis at 50 °C

[0117] for 6 hours. After the enzymatic hydrolysis is completed, place the reaction solution in a 95 °C water bath to inactivate the enzyme for 10 minutes.

[0118] 3. Separation and purification: After the enzymatic hydrolysate is concentrated by rotary evaporation, ultrafiltration is carried out through an ultrafiltration membrane with a molecular weight cut-off of 3000 Da, and the retentate is collected. The retentate is further separated by gel filtration chromatography (Sephadex G-25), using 0.1 mol / L sodium chloride solution as the eluent, controlling the flow rate at 0.5 mL / min, and collecting the active elution peak. The active peak is purified by reverse-phase high performance liquid chromatography (RP-HPLC), using a C18 chromatographic column, and gradient elution is carried out with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, and the target polypeptide peak is collected.

[0119] 3. Screening process

[0120] 1. Preliminary activity screening: Act on the in vitro cultured human vascular endothelial cell line (HUVEC cells) with each purified polypeptide component, and use the MTT method to detect the cell proliferation situation, and preliminarily screen out the polypeptide components that have a promoting effect on cell proliferation.

[0121] 2. NF-κB activity detection: For the polypeptide components preliminarily screened out, further detect their effects on NF

[0122] -κB activity. Detect the phosphorylation level of NF-κB by Western blot, and screen out the polypeptides that can significantly regulate NF-κB activity.

[0123] 3. Sequence identification: Perform mass spectrometry analysis (MALDI-TOF MS) on the active polypeptides, and combine with the Edman degradation method to determine their amino acid sequences.

[0124] 4. Physical chromatographic characteristics of small molecule peptides

[0125] 1. Gel filtration chromatography: On a Sephadex G-25 gel column, the elution volume of the polypeptide was between 4 and 6 mL, and the relative molecular mass was estimated to be between 1000 and 2000 Da, which was consistent with the theoretical value.

[0126] 2. Reverse-phase high performance liquid chromatography: On a C18 column, with acetonitrile-water (containing 0.1% trifluoroacetic acid) gradient elution, the polypeptide peaks at acetonitrile concentrations of 30%-35%, with a retention time of about 12-14 minutes. The peak is symmetrical and sharp, indicating a high purity.

[0127] 5. Mechanism of action of novel peptides

[0128] From the perspective of plant extraction, soybeans contain a variety of bioactive ingredients. During the extraction process, the polypeptide may synergize with other ingredients to affect the intracellular signal transduction pathway. For the NF-κB signaling pathway, the methyl group of Ala, the small side chain of Gly, the hydroxyl group of Ser and Thr, the basic group of Lys, etc. in its amino acid composition may regulate the activity of NF-κB by forming hydrogen bonds, ionic bonds or hydrophobic interactions with specific areas on the surface of NF-κB molecules.

[0129] 6. Beneficial effects of the invention

[0130] 1. High efficiency: significantly regulates NF-κB activity, improves the proliferation and migration ability of vascular endothelial cells, and enhances the repair ability of blood vessels.

[0131] 2. Safety: It is derived from plant extracts and has undergone multiple purification processes. It contains no chemical synthetic impurities. Animal experiments have proven that high-dose use has no obvious toxic side effects on the body.

[0132] 3. Specificity: It has a high affinity for NF-κB and can specifically bind to and regulate its signaling pathway, reducing interference with other unrelated proteins.

[0133] 7. Synergistic test

[0134] 1. Experimental design: A mouse model of atherosclerosis was selected and divided into three groups. The control group was given normal saline, the experimental group was given the novel polypeptide of the present invention (AGSTVLIFKH), and the comparative group was given traditional drugs (such as atorvastatin calcium tablets).

[0135] 2. Experimental results: After four weeks of administration, the blood lipid levels (total cholesterol, triglycerides, and low-density lipoprotein cholesterol) of the experimental group mice were significantly reduced, and the increase in high-density lipoprotein cholesterol was more obvious compared with the control group. The area of ​​atherosclerotic lesions was significantly smaller than that of the control group and the comparison group, and the levels of inflammatory factors (such as tumor necrosis factor-α and interleukin-6) were significantly lower than those of the control group and the comparison group.

[0136] It is also significantly lower than the control group, indicating that this novel polypeptide has a synergistic effect in improving cardiovascular function.

[0137] Example 3: Novel polypeptide extracted from ginseng

[0138] 1. Structure of the novel polypeptide

[0139] The amino acid sequence of the novel polypeptide is: SEQ.NO 4: Pro-Gln-Asn-Asp-Glu-Cys-

[0140] Met-Trp-Tyr-Arg (PQNDECMWYR).

[0141] 2. Extraction method

[0142] 1. Raw material treatment: Wash and dry ginseng, then crush it into fine powder. Reflux extract with 70% ethanol three times, 2 hours each time. Combine the extracts and concentrate under reduced pressure until the alcohol smell disappears. Dilute the concentrated solution with water, then extract with petroleum ether three times to remove lipophilic impurities. The aqueous phase is then extracted with n-butanol three times, collect the n-butanol phase, and concentrate under reduced pressure to obtain the crude ginsenoside.

[0143] 2. Protein extraction and enzymatic hydrolysis: Dissolve the crude ginsenoside in phosphate buffer solution with pH 7.4, add an appropriate amount of trypsin (the mass ratio of enzyme to substrate is 1:80), and carry out enzymatic hydrolysis at 37°C for 8 hours. Stir continuously during the enzymatic hydrolysis process. After the enzymatic hydrolysis is completed, heat at 90°C for 15 minutes to inactivate the enzyme.

[0144] 3. Separation and purification: Centrifuge the enzymatic hydrolysate (10000 rpm, 20 minutes), take the supernatant, ultrafilter through an ultrafiltration membrane with a molecular weight cut-off of 5000 Da, and collect the filtrate. The filtrate is separated by strong cation exchange chromatography (SP-Sepharose FF), and gradient elution is carried out with 0-1 mol / L sodium chloride solution to collect the active elution peak. The active peak is further purified by reverse-phase high-performance liquid chromatography (RP-HPLC). Using a C18 column, gradient elution is carried out with methanol-water (containing 0.1% formic acid) as the mobile phase to collect the target polypeptide peak.

[0145] 3. Screening process

[0146] 1. Primary screening of cell model: Act on the in vitro cultured cardiomyocyte cell line (HCM cells) with the purified polypeptide, detect cell viability and apoptosis, and screen out the polypeptides that have a protective effect on cardiomyocytes.

[0147] 2. Verification of NF-κB pathway: Use the luciferase reporter gene experiment to detect the effect of the screened polypeptides on the activity of the NF-κB signaling pathway, and determine the polypeptides that can regulate NF-κB activity.

[0148] 3. Sequence analysis: The amino acid sequence of the polypeptide was determined by tandem mass spectrometry (MS / MS) and amino acid composition analysis.

[0149] 4. Physical chromatographic characteristics of small molecule peptides

[0150] 1. Cation exchange chromatography: On an SP-Sepharose FF column, the polypeptide was eluted at a sodium chloride concentration of 0.3 - 0.4

[0151] mol / L, indicating that it has certain cation exchange characteristics, which are related to the positively charged amino acid residues (such as Arg) in the polypeptide.

[0152] 2. Reverse phase high performance liquid chromatography: On a C18 column, with a gradient elution of methanol - water (containing 0.1% formic acid)

[0153] The polypeptide eluted when the methanol concentration was 40% - 45%, with a retention time of about 15 - 17 minutes and high peak purity, indicating that it has good separation characteristics under reverse phase conditions.

[0154] 5. Mechanism of action of the novel polypeptide

[0155] Ginseng is rich in various active ingredients such as saponins. In the complex component system of ginseng, this polypeptide may jointly regulate the physiological functions of cells with other components. For the NF-κB signaling pathway, the cyclic structure of Pro, the sulfhydryl group of Cys, the phenolic hydroxyl group of Tyr, and the guanidine group of Arg in the polypeptide can interact with the corresponding sites on the NF

[0156] -κB molecule, affecting the formation or nuclear translocation process of its dimer, thereby regulating the NF-κB signaling pathway.

[0157] 6. Advantages of the invention

[0158] 1. High efficiency: It can effectively inhibit the overactivation of NF-κB, reduce the release of inflammatory mediators, improve the energy metabolism of cardiomyocytes, and enhance myocardial contractility.

[0159] 2. Safety: Ginseng is a traditional plant with both medicinal and edible uses. The extraction process of this polypeptide is safe and reliable. Animal experiments show that long-term use has no obvious effect on liver and kidney functions and no obvious adverse reactions.

[0160] 3. Specificity: It acts precisely on the NF-κB signaling pathway, specifically binds to the NF-κB molecule, and has minimal interference with other intracellular signaling pathways.

[0161] 7. Synergistic effect test

[0162] 1. Experimental design: A mouse model of myocardial ischemia-reperfusion injury was used. A control group, an experimental group of the novel polypeptide (PQNDECMWYR), and a positive control (a commonly used myocardial protection drug) were set up

[0163] Control group

[0164] 2. Experimental results: After myocardial ischemia-reperfusion in the experimental group of mice, the myocardial infarction area was significantly smaller than that in the control group and the comparison group. The levels of myocardial injury markers (such as creatine kinase isoenzyme, cardiac troponin I) in the serum were significantly lower than those in the comparison group, indicating that this novel polypeptide has a synergistic effect in myocardial protection and can more effectively reduce myocardial ischemia-reperfusion injury.

[0165] Example 4: Novel polypeptide based on tea extract

[0166] 1. Structure of the novel polypeptide

[0167] The amino acid sequence of the novel polypeptide is: SEQ.NO 5: Leu-Ala-Asp-Glu-Gly-His-Lys-Pro-Ser-Val (LADEGHKPSV).

[0168] 2. Extraction method

[0169] 1. Tea pretreatment: Soak the tea leaves in hot water (mass ratio of tea leaves to water is 1:100), soak at 80 °C for 30 minutes, and stir constantly during this period to dissolve components such as proteins in the tea leaves. Filter and collect the soaking solution, and concentrate it under reduced pressure to 1 / 5 of the original volume.

[0170] 2. Enzymolysis and separation: Adjust the pH of the concentrated solution to 6.0, add papain (mass ratio of enzyme to substrate is 1:120), and enzymolyze at 45 °C for 5 hours. After the enzymolysis is completed, heat to 85 °C to inactivate the enzyme for 10 minutes. Centrifuge the enzymolysis solution (9000 rpm, 18 minutes) to take the supernatant, ultrafilter through an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, and collect the retentate. The retentate is separated by hydrophobic interaction chromatography (Phenyl-Sepharose 6FF), eluted with an ammonium sulfate concentration gradient (2 - 0 mol / L), and the active components are collected. The active components are further purified by reverse-phase high-performance liquid chromatography (RP-HPLC), using a C18 column, and gradient eluted with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, and the target polypeptide peak is collected.

[0171] 3. Screening process

[0172] 1. In vitro activity screening: Act on the vascular smooth muscle cells cultured in vitro with the purified polypeptide, detect the cell proliferation and migration abilities, and screen out the polypeptides that have a regulatory effect on the functions of vascular smooth muscle cells.

[0173] 2. Determination of NF-κB activity: Use the ELISA method to detect the effect of the screened polypeptides on the secretion of NF-κB-related inflammatory factors (such as tumor necrosis factor-α), and determine the polypeptides that can regulate NF-κB activity.

[0174] 3. Sequence determination: The amino acid sequence of the polypeptide was determined using an automatic amino acid analyzer and mass spectrometry.

[0175] 4. Physical chromatographic characteristics of small molecule peptides

[0176] 1. Hydrophobic interaction chromatography: On a Phenyl-Sepharose 6FF column, the polypeptide was eluted at an ammonium sulfate concentration of 0.8 - 1.0 mol / L, indicating its certain hydrophobicity, which is related to hydrophobic amino acid residues such as Leu and Ala in the polypeptide.

[0177] 2. Reverse-phase high performance liquid chromatography: On a C18 column, with a gradient elution of acetonitrile - water (containing 0.1% trifluoroacetic acid), the polypeptide eluted when the acetonitrile concentration was 35% - 40%, with a retention time of about 13 - 15 minutes, a good peak shape, and a high purity.

[0178] 5. Mechanism of action of the novel polypeptide

[0179] Tea contains various antioxidant components such as tea polyphenols. The polypeptide produced during the fermentation process synergistically acts with other components. For the NF-κB signaling pathway, the amino acid residues in the polypeptide can interact with the key sites on the NF-κB molecule, inhibit its activation, and thereby reduce the expression of inflammation-related genes. For example, the basic groups of Lys and His can bind to the acidic region on the NF-κB molecule, change its conformation, and affect its activity.

[0180] 6. Advantages of the invention

[0181] 1. High efficiency: Significantly reduce the inflammatory response, improve vascular endothelial function, increase the release of vasodilator factors (such as nitric oxide), and reduce vascular resistance.

[0182] 2. Safety: The tea fermentation process is natural and the product is safe. Verified by toxicity experiments, there is no obvious toxicity to animals at high doses, and there are no adverse effects on the body with long-term use.

[0183] 3. Specificity: Specifically act on NF-κB, reduce the non-specific effect on other intracellular proteins, and reduce the risk of potential side effects.

[0184] 7. Synergistic effect test

[0185] 1. Experimental design: Select a hypertensive rat model and divide it into a control group, a novel polypeptide

[0186] (LADEGHKPSV) experimental group, and a traditional antihypertensive drug control group.

[0187] 2. Experimental results: After eight weeks of drug administration, the blood pressure of the rats in the experimental group decreased significantly, and the amplitude of blood pressure fluctuation was smaller than that of the control group. The NF-κB activity in the vascular tissue was significantly lower than that of the control group and the comparison group. At the same time, the proliferation of vascular smooth muscle cells was significantly inhibited, indicating that this novel polypeptide has a synergistic effect in reducing blood pressure and improving vascular remodeling.

[0188] Example 5: Novel polypeptide extracted from corn

[0189] 1. Structure of the novel polypeptide

[0190] The amino acid sequence of the novel polypeptide is: SEQ.NO 6: Ile-Thr-Glu-Asn-Pro-Gly-Ala

[0191] -Ser-Tyr-Cys (ITENPGASTC).

[0192] 2. Extraction method

[0193] 1. Pretreatment of corn protein powder: Corn protein powder was dissolved in sodium hydroxide solution with pH 9.0, stirred and extracted at 55 °C for 3 hours, during which intermittent ultrasound (power 150 W, ultrasound time 8 minutes) was carried out to fully dissolve the protein. Centrifuge (7000 rpm, 12 minutes) to take the supernatant, and adjust the pH to 7.0 with hydrochloric acid.

[0194] 2. Enzymolysis and separation: Add neutral protease (enzyme to substrate mass ratio 1:150), and carry out enzymolysis at 40 °C

[0195] for 7 hours. After the enzymolysis is completed, heat to 95 °C to inactivate the enzyme for 10 minutes. After the enzymolysis solution is concentrated by rotary evaporation, ultrafiltration is carried out through an ultrafiltration membrane with a molecular weight cut-off of 4000 Da, and the retentate is collected. The retentate is separated by ion exchange chromatography (DEAE-Sepharose FF)

[0196] and gradient elution is carried out with 0 - 0.5 mol / L sodium chloride solution to collect the active peak. The active peak is further purified by reverse-phase high performance liquid chromatography (RP-HPLC), using a C18 column, and gradient elution is carried out with methanol-water (containing 0.1% formic acid) as the mobile phase to collect the target polypeptide peak.

[0197] 3. Screening process

[0198] 1. Screening at the cellular level: The purified polypeptide was applied to human umbilical vein endothelial cells (HUVEC) cultured in vitro, and the cell viability, apoptosis and nitric oxide release were detected to screen out the polypeptide that has a positive effect on the function of vascular endothelial cells.

[0199] 2. Verification at the molecular level: The selected polypeptide was detected by real-time fluorescence quantitative PCR for NF

[0200] Effect on -κB related downstream gene expression to determine polypeptides capable of regulating the NF-κB signaling pathway.

[0201] 3. Sequence identification: Use tandem mass spectrometry (MS / MS) technology combined with amino acid composition analysis to determine the amino acid sequence of the polypeptide.

[0202] 4. Physical chromatographic characteristics of small molecule peptides

[0203] 1. Ion exchange chromatography: On a DEAE-Sepharose FF column, the polypeptide elutes at a sodium chloride concentration of 0.2 - 0.3

[0204] mol / L, indicating that it carries a certain negative charge, which is related to acidic amino acid residues such as Glu in the polypeptide.

[0205] 2. Reverse phase high performance liquid chromatography: On a C18 column, with methanol-water (containing 0.1% formic acid)

[0206] gradient elution, the polypeptide elutes when the methanol concentration is 32% - 37%, with a retention time of approximately 14 - 16 minutes, a sharp peak shape, and a purity meeting the requirements.

[0207] 5. Mechanism of action of the novel polypeptide

[0208] Corn contains a variety of nutrients, and this polypeptide plays a role in the complex component system of corn. For the NF-κB signaling pathway, the branched chain structure of Ile, the acidic group of Glu, the phenolic hydroxyl group of Tyr, and the sulfhydryl group of Cys in the polypeptide can interact with the amino acid residues on the surface of the NF-κB molecule, affecting the activity of NF-κB. For example, the sulfhydryl group of Cys may form a disulfide bond with certain cysteine residues in the NF-κB molecule, thereby regulating its activity.

[0209] 6. Beneficial effects of the invention

[0210] 1. High efficiency: Effectively regulate the NF-κB signaling pathway, reduce the production of inflammatory factors, improve the survival and function of cardiomyocytes, and enhance the pumping ability of the heart.

[0211] 2. Safety: The corn source is safe and reliable, the polypeptide extraction process is green and environmentally friendly, and animal experiments have confirmed that there is no obvious toxicity at high doses, and long-term use has no adverse effects on important organs.

[0212] 3. Specificity: Has a highly specific binding ability to NF-κB, can accurately regulate its signaling pathway, and reduce interference with other intracellular signaling pathways.

[0213] 7. Synergistic test

[0214] 1. Experimental design: A diabetic cardiomyopathy mouse model was used, and a control group, a new polypeptide

[0215] (ITENPGASTC) experimental group, and a comparison group of commonly used drugs for improving myocardial metabolism were set up.

[0216] Experimental results: The NF-κB activity in the myocardial tissue of the mice in the experimental group was significantly lower than that in the control group and the comparison group, and the degree of myocardial fibrosis was significantly reduced. Cardiac ultrasound examination showed that the cardiac function indexes (such as left ventricular ejection fraction, left ventricular shortening fraction) of the mice in the experimental group were significantly better than those in the comparison group, indicating that the new polypeptide has a synergistic effect in improving diabetic cardiomyopathy.

Claims

1. A CTPER polypeptide, characterized in that It is composed of the amino acid sequence SEQ.NO 1: Cys - Tyr - Pro - Glu -Arg (CTPER) , SEQ.NO 2: Asn - His - Met - Val - Asp (NHMVD) .

2. The novel polypeptide according to claim 1, characterized in that The polypeptide also includes SEQ.NO 3: Ala-Gly-Ser-Thr-Val-Leu-Ile-Phe-Lys-His (AGSTVLIFKH), SEQ.NO 4: Pro-Gln-Asn-Asp-Glu-Cys-Met-Trp-Tyr-Arg (PQNDECMWYR), SEQ.NO 5: Leu-Ala-Asp-Glu-Gly-His-Lys-Pro-Ser-Val (LADEGHKPSV), SEQ.NO6: Ile-Thr-Glu-Asn-Pro-Gly-Ala-Ser-Tyr-Cys (ITENPGASTC).

3. The novel polypeptide according to claim 1, characterized in that The polypeptide is used to regulate the nuclear factor κB (NF-κB) signaling pathway.

4. The novel polypeptide according to claim 1, characterized in that The polypeptide improves cardiovascular function by binding to NF-κB and is used for treating cardiovascular diseases.

5. The novel polypeptide according to claim 1, characterized in that The polypeptide is prepared by solid phase synthesis, including sequentially connecting amino acid monomers with Boc protective groups to a solid phase carrier, followed by cleavage and purification.

6. A pharmaceutical composition, characterized in that It comprises the novel polypeptide according to claim 1 and a pharmaceutically acceptable carrier.

7. Use of the novel polypeptide according to claim 1 in the preparation of drugs for treating cardiovascular diseases.

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