CN110563810B - Polypeptide and composition for resisting myocardial ischemia and myocardial anoxia, application thereof and polypeptide medicament - Google Patents
Polypeptide and composition for resisting myocardial ischemia and myocardial anoxia, application thereof and polypeptide medicament Download PDFInfo
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- CN110563810B CN110563810B CN201910792484.0A CN201910792484A CN110563810B CN 110563810 B CN110563810 B CN 110563810B CN 201910792484 A CN201910792484 A CN 201910792484A CN 110563810 B CN110563810 B CN 110563810B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to an anti-myocardial ischemia and anti-hypoxia polypeptide, a composition, application thereof and a polypeptide medicament. Wherein the sequence of the anti-myocardial ischemia and anoxia polypeptide has the following structure: the amino acid sequence shown as SEQ ID NO.1 or the amino acid sequence with at least 70 percent of homology with the amino acid sequence shown as SEQ ID NO. 1. The polypeptide for resisting myocardial ischemia and hypoxia can obviously reduce the activity level and release of lactic dehydrogenase in cells and can protect the cells; meanwhile, the polypeptide for resisting myocardial ischemia and hypoxia can obviously improve the survival rate of cells under the condition of hypoxia. Animal experiments show that the polypeptide can obviously reduce Lactate Dehydrogenase (LDH) and Creatine Kinase (CK) in rat serum, has obvious protective effect on myocardial cell ischemia injury, prevents and/or treats coronary atherosclerotic heart disease, and reduces the mortality rate of myocardial infarction.
Description
Technical Field
The invention relates to the field of biomedicine, and in particular relates to an anti-myocardial ischemia and anti-hypoxia polypeptide, a composition, application thereof and a polypeptide medicament.
Background
Coronary atherosclerotic heart disease is a heart disease caused by myocardial ischemia, hypoxia or necrosis due to stenosis or obstruction of a blood vessel cavity caused by atherosclerotic lesions generated in coronary vessels, and is generally called as "coronary heart disease". "coronary heart disease" ranks the first cause of death in the United states and many developed countries. In China, the morbidity and mortality of coronary heart disease are both in a high level, and great burden is caused to social economy and family life.
At present, the treatment method of the coronary heart disease is mainly divided into revascularization treatment and drug treatment, and the drugs for treating the coronary heart disease mainly comprise: (1) nitrate ester drugs: nitroglycerin, isosorbide dinitrate, etc. are commonly used; (2) antithrombotic drugs: the anti-platelet drug mainly comprises aspirin, clopidogrel, tirofiban and the like, can inhibit platelet aggregation and avoid blood vessel blockage caused by thrombosis; the anticoagulant drug comprises heparin, sodium sulfonate, bivalirudin, etc.; (3) beta-blockers: common drugs include metoprolol, atenolol, bisoprolol, carvedilol, and the like; (4) calcium channel blockers: common medicines include verapamil, nifedipine, amlodipine, diltiazem and the like; (5) renin angiotensin system inhibitors: including Angiotensin Converting Enzyme Inhibitors (ACEI), angiotensin 2 receptor Antagonists (ARB) and aldosterone antagonists; (6) lipid regulating drugs: simvastatin, atorvastatin and the like are commonly used; (7) fibrinolytic drugs: mainly includes streptokinase, urokinase, tissue plasminogen activator, etc. However, most of the above drugs are chemical drugs, and have disadvantages of great side effects, high drug accumulation in organs, and easy induction of severe immune reactions.
With the rapid development of the proteomics in recent years, more and more endogenous polypeptides with diagnostic and therapeutic potential are identified. The polypeptide is not only a protein degradation product, but also an important bioactive molecule, plays an indispensable role in vivo, and is applied to the treatment in the fields of tumors, neurodegenerative diseases, osteoporosis, metabolism, immune diseases and the like. Compared to other chemical drugs, for example: compared with trimetazidine hydrochloride, the polypeptide has simple structure, chemical and biological diversity, high specificity and affinity, low organ accumulation and less serious immune reaction, thereby becoming one of hot spots of new drug research and having good market prospect. At present, no small molecule polypeptide drug is reported to be used for treating coronary atherosclerotic heart disease caused by myocardial ischemia and hypoxia.
Disclosure of Invention
Based on the polypeptide, the polypeptide can effectively prevent and/or treat coronary atherosclerotic heart disease.
An anti-myocardial ischemia-hypoxia polypeptide, the sequence of which has:
an amino acid sequence as shown in SEQ ID NO.1, or
Has an amino acid sequence with at least 70 percent of homology with the amino acid sequence shown in SEQ ID NO. 1.
Cell experiments show that the polypeptide for resisting myocardial ischemia and hypoxia can obviously reduce the activity level and release of lactic dehydrogenase in cells and can protect the cells; meanwhile, the polypeptide for resisting myocardial ischemia and hypoxia can obviously improve the survival rate of cells under the condition of hypoxia. Animal experiments of myocardial ischemia caused by rat coronary artery ligation show that the anti-myocardial ischemia and anoxia polypeptide can obviously reduce Lactate Dehydrogenase (LDH) and Creatine Kinase (CK) in rat serum and has obvious protective effect on myocardial cell ischemia injury, and the anti-myocardial ischemia and anoxia polypeptide can prevent and/or treat coronary atherosclerotic heart disease and reduce the death rate of myocardial infarction by integrating results of cell experiments and animal experiments.
In one embodiment, the sequence of the anti-myocardial ischemia and anoxia polypeptide has an amino acid sequence represented by the formula X-P, wherein X is Val-Arg or Arg, and P is the amino acid sequence shown in SEQ ID NO. 1.
In one embodiment, the sequence of the anti-myocardial ischemia and anoxia polypeptide has an amino acid sequence represented by the formula P-Y, wherein P is an amino acid sequence shown in SEQ ID NO.1, and Y is Ile or Ile-Ile.
In one embodiment, the sequence of the anti-myocardial ischemia-hypoxia polypeptide has an amino acid sequence represented by the formula X-P-Y, wherein X is Val-Arg or Arg, P is the amino acid sequence shown in SEQ ID NO.1, and Y is Ile or Ile-Ile.
In one embodiment, the anti-myocardial ischemia and anoxia polypeptide sequence is an amino acid sequence shown as SEQ ID No.1 or an amino acid sequence with at least 70% homology with the amino acid sequence shown as SEQ ID No. 1.
In one embodiment, the anti-myocardial ischemia-hypoxia polypeptide sequence is an amino acid sequence represented by the formula X-P, an amino acid sequence represented by the formula P-Y or an amino acid sequence represented by the formula X-P-Y, wherein X is Val-Arg or Arg, P is an amino acid sequence represented by SEQ ID NO.1, and Y is Ile or Ile-Ile.
The invention also provides a polypeptide composition for resisting myocardial ischemia and anoxia, which comprises at least two of polypeptide for resisting myocardial ischemia and anoxia, which has an amino acid sequence shown as SEQ ID No.1, polypeptide for resisting myocardial ischemia and anoxia, which has an amino acid sequence shown as SEQ ID No.2, polypeptide for resisting myocardial ischemia and anoxia, which has an amino acid sequence shown as SEQ ID No.3, polypeptide for resisting myocardial ischemia and anoxia, which has an amino acid sequence shown as SEQ ID No.4, and polypeptide for resisting myocardial ischemia and anoxia, which has an amino acid sequence shown as SEQ ID No. 5.
The invention also provides a medicament for preventing and/or treating coronary atherosclerotic heart disease, wherein the active ingredient of the polypeptide medicament comprises the anti-myocardial ischemia and anoxia polypeptide or the anti-myocardial ischemia and anoxia polypeptide composition.
The invention also provides application of the polypeptide resisting myocardial ischemia and anoxia or the polypeptide composition resisting myocardial ischemia and anoxia in preparation of medicines for preventing and/or treating coronary atherosclerotic heart disease.
The invention also provides application of the anti-myocardial ischemia and anoxia polypeptide or the anti-myocardial ischemia and anoxia polypeptide composition in preparation of a medicine for preventing and/or treating myocardial ischemia and anoxia.
Drawings
FIG. 1 is a graph showing the comparison of the effect of lactate dehydrogenase on various test groups in the lactate dehydrogenase activity test;
FIG. 2 is a graph comparing the effect of various test groups on cell viability in a trypan blue stained cell viability assay;
FIG. 3 is a graph comparing the effect of serum lactate dehydrogenase on various test groups in rat animal model experiments;
fig. 4 is a graph comparing the effect of creatine kinase on various test groups in rat animal model experiments.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides a polypeptide for resisting myocardial ischemia and anoxia, wherein an amino acid sequence shown as SEQ ID No.1 is in a polypeptide sequence for resisting myocardial ischemia and anoxia.
Wherein the amino acid sequence shown in SEQ ID NO.1 is as follows:
Leu-Ala-Pro-Asp-Tyr-Asp-Ala-Leu-Asp
-Val-Ala-Asn-Lys-lle-Gly。
in one embodiment, the anti-myocardial ischemia-hypoxia polypeptide sequence of the present invention is SEQ ID No. 1: Leu-Ala-Pro-Asp-Tyr-Asp-Ala-Leu-Asp-Val-Ala-Asn-Lys-lle-Gly, and the polypeptide E which is the polypeptide for resisting myocardial ischemia and anoxia and consists of the amino acid sequence shown in SEQ ID NO. 1.
It is understood that the sequence of the anti-myocardial ischemia and anoxia polypeptide of the present invention has the amino acid sequence shown in SEQ ID No.1, which includes but is not limited to the amino acid sequence shown in SEQ ID No.1, for example, one or more amino acid fragments can be inserted, substituted, or deleted in the amino acid sequence shown in SEQ ID No. 1.
In another embodiment of the invention, the sequence of the polypeptide for resisting myocardial ischemia and anoxia has an amino acid sequence with at least 70% homology with the amino acid sequence shown in SEQ ID NO. 1.
The term "homology" in the context of the present invention refers to the percentage identity between two polypeptide parts. Homology refers to the degree of identity with a given amino acid sequence and can be expressed as a percentage. In the present disclosure, homologous sequences having the same or similar activity as a given amino acid sequence may be represented by "% homology". Homology between sequences from one portion to another can be determined by techniques known in the art. For example, standard software for calculating parameters such as score, identity and similarity, in particular BLAST 2.0, may be used. For example, 70% homology as used herein refers to a sequence that is identical to 70% sequence identity as determined by a well-defined algorithm, and thus a homologue of a given sequence has greater than or equal to 70% sequence identity over the length of the given sequence. The homology referred to in the present invention includes, but is not limited to, insertion, substitution, deletion of one or more amino acid fragments in a polypeptide sequence.
In one embodiment, the anti-myocardial ischemia and anoxia polypeptide sequence has an amino acid sequence represented by the formula X-P, wherein X is Val-Arg or Arg, and P is the amino acid sequence shown in SEQ ID NO. 1.
In one embodiment, the anti-myocardial ischemia and anoxia polypeptide sequence has an amino acid sequence represented by the formula P-Y, wherein P is an amino acid sequence shown in SEQ ID NO.1, and Y is Ile or Ile-Ile.
In one embodiment, the anti-myocardial ischemia and anoxia polypeptide sequence has an amino acid sequence represented by the formula X-P-Y, wherein X is Val-Arg or Arg, P is the amino acid sequence shown in SEQ ID NO.1, and Y is Ile or Ile-Ile.
In one embodiment, the anti-myocardial ischemia and anoxia polypeptide sequence is an amino acid sequence shown as SEQ ID No.1 or an amino acid sequence with at least 70% homology with the amino acid sequence shown as SEQ ID No. 1.
In one embodiment, the anti-myocardial ischemia and anoxia polypeptide sequence is an amino acid sequence with at least 80% homology with the amino acid sequence shown in SEQ ID No. 1. Further, the polypeptide sequence for resisting myocardial ischemia and anoxia is an amino acid sequence which has at least 85% homology with the amino acid sequence shown in SEQ ID NO. 1. Further, the polypeptide sequence for resisting myocardial ischemia and anoxia is an amino acid sequence which has at least 90% homology with the amino acid sequence shown in SEQ ID NO.1, and further, the polypeptide sequence for resisting myocardial ischemia and anoxia is an amino acid sequence which has at least 95% homology with the amino acid sequence shown in SEQ ID NO. 1.
In one embodiment, the anti-myocardial ischemia-hypoxia polypeptide sequence is an amino acid sequence represented by the formula X-P, an amino acid sequence represented by the formula P-Y or an amino acid sequence represented by the formula X-P-Y, wherein X is Val-Arg or Arg, P is an amino acid sequence represented by SEQ ID NO.1, and Y is Ile or Ile-Ile.
In this embodiment, the sequence of the anti-hypoxic myocardial ischemia polypeptide is an amino acid sequence represented by the formula X-P-Y, and when X is Val-Arg and Y is Ile-Ile, the sequence of the anti-hypoxic myocardial ischemia polypeptide is SEQ ID NO. 2:
Val-Arg-Leu-Ala-Pro-Asp-Tyr-Asp-Ala-Leu-Asp-Val-Ala-Asn-Lys-lle-Gly-lle-lle, wherein the polypeptide A is the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.2, and comprises 19 amino acids, the molecular weight is 2056g/mol, the isoelectric point is 4.17, and the average hydrophobicity is 0.48.
In this embodiment, the anti-hypoxic myocardial ischemia is an amino acid sequence represented by the formula X-P-Y, wherein when X is Arg and Y is Ile-Ile, the anti-hypoxic myocardial ischemia is a polypeptide of SEQ ID NO. 3: Arg-Leu-Ala-Pro-Asp-Tyr-Asp-Ala-Leu-Asp-Val-Ala-Asn-Lys-lle-Gly-lle-lle, wherein the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.3 is named as polypeptide B, and comprises 18 amino acids, the molecular weight is 1957g/mol, the isoelectric point is 4.17, and the average hydrophobicity is 0.28.
In this embodiment, the anti-hypoxic myocardial ischemia is an amino acid sequence represented by the formula P-Y, and when Y is Ile-Ile, the anti-hypoxic myocardial ischemia is an amino acid sequence represented by SEQ ID NO. 4:
Leu-Ala-Pro-Asp
-Tyr-Asp-Ala-Leu-Asp-Val-Ala-Asn-Lys-lle-Gly-lle-lle, an anti-hypoxic myocardial ischemia polypeptide consisting of the amino acid sequence shown in SEQ ID No.4 comprising 17 amino acids, having a molecular weight of 1801g/mol, an isoelectric point of 3.6 and an average hydrophobicity of 0.56 is designated polypeptide C.
In this example, the anti-hypoxic myocardial ischemia is represented by the amino acid sequence represented by the formula X-P, and when X is Val-Arg, the anti-hypoxic myocardial ischemia is represented by the amino acid sequence of SEQ ID NO. 5: Val-Arg-Leu-Ala-Pro-Asp-Tyr-Asp-Ala-Leu-Asp-Val-Ala-Asn-Lys-lle-Gly, wherein the polypeptide for resisting myocardial ischemia and anoxia, which consists of the amino acid sequence shown in SEQ ID NO.5, is named as polypeptide D, and comprises 17 amino acids, the molecular weight is 1830g/mol, the isoelectric point is 4.17, and the average hydrophobicity is 0.01.
It is understood that the amino acid sequences corresponding to the polypeptide A, the polypeptide B, the polypeptide C, the polypeptide D and the polypeptide E in the invention all have at least 70% of sequence homology, i.e., the polypeptide A, the polypeptide B, the polypeptide C, the polypeptide D and the polypeptide E are homologous polypeptides. It is understood that the polypeptides having at least 70% homology with the polypeptide E include, but are not limited to, the polypeptide A, the polypeptide B, the polypeptide C and the polypeptide D, and may be other polypeptides having similar physiological activities not listed in the present invention, that is, the polypeptides having at least 70% homology with the polypeptide E also have the effects of reducing Lactate Dehydrogenase (LDH) and Creatine Kinase (CK) in rat serum, having a significant protective effect on myocardial ischemia injury, preventing and/or treating coronary atherosclerotic heart disease, and reducing the mortality of myocardial infarction.
It is understood that the anti-myocardial ischemia-hypoxia polypeptide of the present invention can be prepared by a solid phase synthesis method commonly used by those skilled in the art, and will not be described herein in detail.
Cell experiments show that the polypeptide for resisting myocardial ischemia and hypoxia can obviously reduce the activity level and release of lactic dehydrogenase in cells and can protect the cells; meanwhile, the polypeptide for resisting myocardial ischemia and hypoxia can obviously improve the survival rate of cells under the condition of hypoxia. Animal experiments of myocardial ischemia caused by rat coronary artery ligation show that the anti-myocardial ischemia and anoxia polypeptide can obviously reduce Lactate Dehydrogenase (LDH) and Creatine Kinase (CK) in rat serum and has obvious protective effect on myocardial cell ischemia injury, and the anti-myocardial ischemia and anoxia polypeptide can prevent and/or treat coronary atherosclerotic heart disease and reduce the death rate of myocardial infarction by integrating results of cell experiments and animal experiments.
The invention also provides a polypeptide composition for resisting myocardial ischemia and anoxia, which comprises at least two of polypeptide for resisting myocardial ischemia and anoxia, which has an amino acid sequence shown as SEQ ID No.1, polypeptide for resisting myocardial ischemia and anoxia, which has an amino acid sequence shown as SEQ ID No.2, polypeptide for resisting myocardial ischemia and anoxia, which has an amino acid sequence shown as SEQ ID No.3, polypeptide for resisting myocardial ischemia and anoxia, which has an amino acid sequence shown as SEQ ID No.4, and polypeptide for resisting myocardial ischemia and anoxia, which has an amino acid sequence shown as SEQ ID No. 5. The polypeptide composition for resisting myocardial ischemia and anoxia can prevent and/or treat coronary atherosclerotic heart disease and reduce mortality of myocardial infarction.
The invention also provides a medicament for preventing and/or treating coronary atherosclerotic heart disease, wherein the active ingredient of the polypeptide medicament comprises the anti-myocardial ischemia and anoxia polypeptide or the anti-myocardial ischemia and anoxia polypeptide composition. The medicine containing the anti-myocardial ischemia and anoxia polypeptide or polypeptide composition has the effect of preventing and/or treating coronary atherosclerotic heart disease.
The invention also provides application of the anti-myocardial ischemia and anoxia polypeptide or the anti-myocardial ischemia and anoxia polypeptide composition in preparation of medicines for preventing and/or treating coronary atherosclerotic heart disease.
The invention also provides application of the anti-myocardial ischemia and anoxia polypeptide or the anti-myocardial ischemia and anoxia polypeptide composition in preparation of a medicine for preventing and/or treating myocardial ischemia and anoxia.
In order to make the objects and advantages of the present invention more apparent, the present invention will be described in further detail below in conjunction with cellular experiments and animal experiments. It should be understood that the specific experiments described herein are merely illustrative and are not intended to limit the present invention.
Unless otherwise specified, the reagents used in the following experiments were commercially available, and the procedures were conventional.
Design of experiments
Cell experiments for protection of hypoxic cardiomyocytes
1. Test cell
Rat cardiomyocytes H9c2 were selected for the experiments and purchased from American Type Culture Collection (ATCC).
2. Method of cellular hypoxia
Grouping experiments: divided into a hypoxia group, an experimental group, a control group and a trimetazidine group.
Control group: inoculating cells with a six-hole plate, changing a sugar-free and serum-free DMEM complete culture medium after the density reaches 80%, and culturing in an ordinary oxygen incubator.
Hypoxia group: inoculating cells with a six-hole plate, changing a sugar-free and serum-free DMEM medium after the density reaches 80%, and placing the DMEM medium in an anoxic box to perform anoxic treatment for 10 hours under the condition.
Experimental groups: the method comprises the following steps of dividing the test cells into a polypeptide group A (corresponding to the polypeptide A of the invention), a polypeptide group B (corresponding to the polypeptide B of the invention), a polypeptide group C (corresponding to the polypeptide C of the invention), a polypeptide group D (corresponding to the polypeptide D of the invention) and a polypeptide group E (corresponding to the polypeptide E of the invention), specifically, inoculating the cells by using a six-well plate, changing a sugar-free and serum-free DMEM culture medium after the density reaches 80%, respectively selecting the polypeptides in each experimental group to pre-treat H9C2 cells at the concentration of 50 mu mol/L, and after 2H, placing the cells in an anoxic box to perform anoxic treatment for 10H according to the conditions.
Trimetazidine group: inoculating cells with a six-hole plate, changing a sugar-free serum-free DMEM medium after the density reaches 80%, pretreating H9c2 cells with 10 mu mol/L trimetazidine, and after 2 hours, placing the cells in an anoxic box to perform anoxic treatment for 10 hours under the condition.
3. Experimental methods
3.1 Activity detection of lactate dehydrogenase
And (3) sucking the cell culture medium, centrifuging at 8000rpm for 5min, adding 120 mu l of sample and 60 mu l of working solution into each hole of a 96-hole plate, incubating at room temperature for 30min, and detecting the absorbance at 490nm by using an enzyme-labeling instrument.
3.2 Trypan blue staining
All H9c2 cells were collected from each group, stained according to the procedure of trypan blue stained cell viability assay kit, and counted on a blood cell counting plate after staining, and the cell death rate was blue cell count/total cell count × 100%.
4. Data and statistical processing:
data in this experiment were processed using SPSS13.0 software and expressed as mean. + -. standard deviation (mean. + -. SD), and significant differences were expressed as p <0.05 between groups compared by One-way ANOVA and t-test analysis.
Animal experiments
1. Experimental animals: SD rats were 28 in total, male, clean grade, body weight: 200- "220 g", provided by Shanghai Sphere-Bikeka laboratory animals Co.
2. Grouping experiments: experimental rats were randomly grouped (n ═ 7): a sham group, a model group, a polypeptide a group (corresponding to the polypeptide a of the present invention), a polypeptide B group (corresponding to the polypeptide B of the present invention), a polypeptide C group (corresponding to the polypeptide C of the present invention), a polypeptide D group (corresponding to the polypeptide D of the present invention), a polypeptide E group (corresponding to the polypeptide E of the present invention), and a Trimetazidine (TMZ) group. The administration dose is calculated by body weight, each polypeptide group is administered according to 6mg/kg, Trimetazidine (TMZ) group is administered according to 5mg/kg, the medicine is prepared into injection by using normal saline as a solvent, and the tail vein injection is administered.
3. Rat coronary artery ligation
The rats are anesthetized by 10% chloral hydrate intraperitoneal injection, the rats are fixed on an animal table in a supine position after full anesthesia, the fourth rib and the fifth rib are cut along the left side of the sternum at the position of 2mm, the fourth rib and the fifth rib are cut off after blunt separation until the intercostal space, the thoracic cavity is exposed, the pericardium is cut off, the chest wall is pulled by a small draw hook, and the heart is fully exposed. A No. 0/3 suture is passed through the anterior descending branch of the left coronary artery by an atraumatic needle, the blood vessel of the left anterior descending branch is ligated, the heart is put back into the thoracic cavity, the air in the thoracic cavity is discharged, and the thoracic cavity is closed rapidly. The whole process should be completed within 30 seconds. Myocardial ischemia is caused after ligation of anterior descending coronary artery of a rat, and only threading is performed in a sham operation group without ligation. 30 minutes before coronary artery ligation, the polypeptide A group, the polypeptide B group, the polypeptide C group, the polypeptide D group, the polypeptide E group and the Trimetazidine (TMZ) group are administrated by tail vein injection, the administration dose of each polypeptide group is 6mg/Kg, the administration dose of the Trimetazidine (TMZ) group is 0.0115mmol/Kg, and the sham operation group and the model group are injected with physiological saline with the same volume. The ligature is cut off after the coronary artery is ligated for 40min, and the blood supply is recovered. After 5 hours, 3ml of blood is taken from each group of rats through femoral artery, supernatant is obtained by centrifugation, serum creatine kinase and lactate dehydrogenase activity are measured, and the rats are continuously raised to observe the survival conditions.
4. Data and statistical processing:
data in this experiment were processed using SPSS13.0 software and expressed as mean ± standard deviation (mean ± SD), and significant differences were expressed as p <0.05 between groups compared by t-test analysis.
Results of the experiment
1. Polypeptide reduces the activity of lactate dehydrogenase
Referring to FIG. 1, the damage of cells was detected by using a lactate dehydrogenase cytotoxicity assay kit. The results show that:
compared with a corresponding control group (1.196 +/-0.245), the absorbance value (OD-490nm,2.485 +/-0.392) of the anoxic LDH group is obviously increased, and the difference has statistical significance (P is less than 0.001); compared with the pure hypoxia group, the release of the lactate dehydrogenase of the experimental group added with the polypeptide [ A (1.323 +/-0.318), B (1.420 +/-0.202), C (1.660 +/-0.298), D (1.335 +/-0.408) and E (1.573 +/-0.315) ] is obviously reduced, and the difference has statistical significance (P is less than 0.01); compared with Trimetazidine (TMZ) (1.314 + -0.281), the release amount of lactate dehydrogenase is not obviously changed when the polypeptide group is added. In conclusion, the polypeptide A, the polypeptide B, the polypeptide C, the polypeptide D and the polypeptide E can obviously reduce the level of lactate dehydrogenase, play a role in protecting hypoxic cell damage, and have slightly better effect than trimetazidine.
2. Polypeptide increases cell survival rate
Referring to fig. 2, the cell viability was measured by using a trypan blue staining cell viability measurement kit. The results show that:
compared with the mortality of a control group (8.11 +/-1.75%), the mortality of the hypoxia group (75.6 +/-3.56%) is obviously increased, and the difference has statistical significance (P is less than 0.001), which indicates that the hypoxia modeling is successful; compared with the hypoxia group, the cell death rate caused by hypoxia is reduced by 40-50% in each polypeptide group [ A (21.31 +/-1.54%), B (28.68 +/-4.10%), C (26.15 +/-4.23%), D (23.31 +/-4.92%), E (25.37 +/-2.58%) ], which indicates that the polypeptide A, the polypeptide B, the polypeptide C, the polypeptide D and the polypeptide E can reduce the cell death caused by hypoxia, and the effect is equivalent to that of the trimetazidine group (19.67 +/-1.98%).
3. Polypeptide group for reducing content of creatine kinase and lactate dehydrogenase in blood serum of myocardial ischemia rat
With reference to fig. 3 and 4, the content of creatine kinase and lactate dehydrogenase in serum of rats in each group is detected, and the result shows that:
compared with the serum creatine kinase [ (2732 +/-73.71) IU/L ] and lactate dehydrogenase [ (1518 +/-42.00) IU/L ] of rats in the sham operation group, the model group (the serum creatine kinase [ (6817 +/-350.6) IU/L) and the lactate dehydrogenase [ (5610 +/-236.6) IU/L ]) is obviously increased, and the difference has statistical significance (P is less than 0.001), which indicates that myocardial ischemia of rats is caused after coronary artery ligation, myocardial cells are damaged, and serum creatine kinase and lactate dehydrogenase are released into blood.
Compared with the model group, the contents of serum creatine kinase [ A (3894 +/-232.7) IU/L, B (3678 +/-245.6) IU/L, C (3489 +/-80.6) IU/L, D (3552 +/-260.7) IU/L, E (3715 +/-200.4) IU/L ] and lactate dehydrogenase [ A (3226 +/-194.9) IU/L, B (3510 +/-358.3) IU/L, C (2830 +/-190.6) IU/L, D (2597 +/-179.5) IU/L, E (3254 +/-200.6) IU/L ] in the polypeptide group A, the polypeptide group B, the polypeptide group C, the polypeptide group D and the polypeptide group E are obviously reduced, and the difference has statistical significance (P is less than 0.01). In conclusion, the polypeptide A, the polypeptide B, the polypeptide C, the polypeptide D and the polypeptide E can reduce serum creatine kinase and lactate dehydrogenase of myocardial ischemia rats, have protective effect on myocardial cell ischemia injury, and have the effect similar to the intervention results of trimetazidine (serum creatine kinase [ (3257 +/-190.8) IU/L ], lactate dehydrogenase [ (2694 +/-186.3) IU/L ]).
4. Polypeptide A reduces mortality of myocardial ischemia rats
As shown in table 1, on day 6 after the coronary artery ligation of the rats, 6 rats died in 7 model groups, 3 rats died in the polypeptide a group and 4 rats died in the trimetazidine group, respectively, and the number of deaths in the rats in the polypeptide a group was significantly reduced compared to the rats in the model group. In conclusion, the polypeptide A can reduce the mortality rate of the myocardial infarction rats and has a protective effect on myocardial ischemia and hypoxia injury.
TABLE 1 Effect of myocardial ischemia reperfusion mortality in groups of rats
The experimental results show that at the cellular level, the polypeptide of the invention can reduce the death rate of hypoxic myocardial cells and the activity level and release of lactate dehydrogenase, and has a protective effect on cells. On the animal level, the polypeptide can reduce serum creatine kinase and lactate dehydrogenase of myocardial ischemia rats of coronary artery ligation rats, can reduce the death rate of the myocardial ischemia rats, and has a protective effect on myocardial ischemia and hypoxia injury. The effect of the polypeptide of the invention in cell and animal experiments is equal to or even better than that of trimetazidine.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Hospital of Shanghai city
<120> polypeptide for resisting myocardial ischemia and myocardial anoxia, composition and application thereof, and polypeptide medicament
<141> 2019-08-19
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<213> Artificial Sequence
<400> 5
Val Arg Leu Ala Pro Asp Tyr Asp Ala Leu Asp Val Ala Asn Lys Ile
1 5 10 15
Gly
Claims (6)
1. The polypeptide for resisting myocardial ischemia and anoxia is characterized in that the polypeptide sequence for resisting myocardial ischemia and anoxia is an amino acid sequence shown as SEQ ID NO. 1.
2. The polypeptide of claim 1, wherein the polypeptide sequence is represented by formula X-P, P-Y or X-P-Y, wherein X is Val-Arg or Arg, P is the amino acid sequence of SEQ ID No.1, and Y is Ile or Ile-Ile.
3. The polypeptide composition for resisting myocardial ischemia and anoxia is characterized by comprising at least two of polypeptide with an amino acid sequence shown as SEQ ID No.1, polypeptide with an amino acid sequence shown as SEQ ID No.2, polypeptide with an amino acid sequence shown as SEQ ID No.3, polypeptide with an amino acid sequence shown as SEQ ID No.4 and polypeptide with an amino acid sequence shown as SEQ ID No. 5.
4. A medicament for preventing and/or treating coronary atherosclerotic heart disease, wherein the active ingredient of the polypeptide medicament comprises the anti-ischemic-hypoxic polypeptide of any one of claims 1-2 or the anti-ischemic-hypoxic polypeptide composition of claim 3.
5. Use of the polypeptide of any one of claims 1-2 or the polypeptide composition of claim 3 for preventing and/or treating coronary atherosclerotic heart disease.
6. Use of the polypeptide of any one of claims 1-2 or the polypeptide composition of claim 3 for preventing and/or treating myocardial ischemia/anoxia.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107602691A (en) * | 2017-08-22 | 2018-01-19 | 徐州医科大学 | Purposes of the derivative polypeptide series of pigment epidermal derived factors for protection ischemic myocardium |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107602691A (en) * | 2017-08-22 | 2018-01-19 | 徐州医科大学 | Purposes of the derivative polypeptide series of pigment epidermal derived factors for protection ischemic myocardium |
Non-Patent Citations (4)
| Title |
|---|
| EAW51131.1;Venter,J.C. et al.;《NCBI》;20150323;第1-2页 * |
| ProBNP‐derived peptides in cardiac disease;J. P. Goetze;《Scandinavian Journal of Clinical and Laboratory Investigation》;20090708;第64卷(第5期);第497-510页 * |
| 多肽 pATMtide 在先天性心脏病中的作用;冯梦文等;《临床与病理杂志》;20181231;第38卷(第2期);第227-231页 * |
| 多肽在心血管疾病中的研究进展及展望;程子杰等;《转化医学电子杂志》;20171231;第4卷(第2期);第77-80页 * |
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