阅读说明：本技术 抗心肌缺血缺氧多肽、组合物及其应用和多肽药物 (Polypeptide and composition for resisting myocardial ischemia and myocardial anoxia, application thereof and polypeptide medicament ) 是由 钱玲梅 李沄 冯梦文 尹安雯 于 2019-08-26 设计创作，主要内容包括：本发明涉及一种抗心肌缺血缺氧多肽、组合物及其应用和多肽药物。其中,所述抗心肌缺血缺氧多肽的序列具有：如SEQ ID NO.1所示的氨基酸序列,或具有与所述SEQ ID NO.1所示的氨基酸序列具有至少70％同源性的氨基酸序列。上述抗心肌缺血缺氧多肽可显著降低细胞内乳酸脱氢酶的活性水平和释放,可对细胞起到保护作用；同时该抗心肌缺血缺氧多肽可显著提高缺氧情况下的细胞存活率。动物实验表明,该多肽可显著降低大鼠血清中的乳酸脱氢酶(LDH)和肌酸激酶(CK),对心肌细胞缺血损伤有显著的保护作用,预防和/或治疗冠状动脉粥样硬化性心脏病,降低心肌梗死的死亡率。(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.)

1. An anti-myocardial ischemia-hypoxia polypeptide, wherein the sequence of the anti-myocardial ischemia-hypoxia polypeptide comprises:

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.

2. The anti-myocardial ischemia-hypoxia polypeptide of claim 1, wherein the sequence of the anti-myocardial ischemia-hypoxia polypeptide has an amino acid sequence represented by the formula M-Q, wherein M is Arg-Ala, and Q is the amino acid sequence represented by SEQ ID No. 1.

3. The polypeptide of claim 1, wherein the sequence of the polypeptide has an amino acid sequence represented by the formula Q-N, wherein Q is an amino acid sequence represented by SEQ ID NO.1, and N is Gln or Gln-Lys.

4. The polypeptide of claim 1, wherein the sequence of the polypeptide has an amino acid sequence represented by formula M-Q-N, wherein M is Arg-Ala, Q is the amino acid sequence represented by SEQ ID No.1, and N is Gln or Gln-Lys.

5. The polypeptide of claim 1, wherein the sequence of the polypeptide is 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.

6. The polypeptide of claim 1, wherein the sequence of the polypeptide is represented by formula M-Q, formula Q-N or formula M-Q-N, wherein M is Arg-Ala, Q is the amino acid sequence of SEQ ID No.1, and N is Gln or Gln-Lys.

7. The polypeptide composition for resisting myocardial ischemia and anoxia is characterized by comprising 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.

8. A polypeptide drug for preventing and/or treating coronary atherosclerotic heart disease or preventing and/or treating myocardial ischemia, wherein the active ingredient of the polypeptide drug comprises the anti-myocardial ischemia and anoxia polypeptide of any one of claims 1-6 or the anti-myocardial ischemia and anoxia polypeptide composition of claim 7.

9. use of the polypeptide of any one of claims 1-6 or the polypeptide composition of claim 7 for preventing and/or treating coronary atherosclerotic heart disease.

10. Use of the polypeptide of any one of claims 1-6 or the polypeptide composition of claim 7 for preventing and/or treating myocardial ischemia/anoxia.

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.

At present, with the rapid development of the proteomics in recent years, the role of the polypeptide substance in the treatment of diseases is receiving more and more attention due to its diverse biological characteristics. With the continuous development of peptide screening, synthesis, stabilization and modification technologies, a large number of endogenous polypeptides with diagnostic and therapeutic potential are identified and transformed into clinical disease diagnosis and treatment. Compared to other chemical drugs, for example: compared with trimetazidine hydrochloride, the polypeptide has the characteristics of strong tissue penetration, good solubility, stability, easy absorption, ideal oral administration or injection, poor immunogenicity, high specificity and affinity, low organ accumulation, less serious immunoreaction and the like, can be prepared in large quantities, becomes one of hot spots for research of new medicaments, and has good market prospect. At present, polypeptide medicines with twenty amino acids or less are not reported to be used for treating coronary atherosclerotic heart disease caused by myocardial ischemia and anoxia.

Disclosure of Invention

Based on the polypeptide, the small-molecule anti-myocardial ischemia-hypoxia 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 patients with myocardial infarction by combining results of cell experiments and animal experiments.

In one embodiment, the sequence of the polypeptide for resisting myocardial ischemia and anoxia has an amino acid sequence represented by the formula M-Q, wherein M is Arg-Ala, and Q is the amino acid sequence shown in SEQ ID NO. 1.

In one embodiment, the sequence of the anti-myocardial ischemia-hypoxia polypeptide has an amino acid sequence represented by the formula Q-N, wherein Q is an amino acid sequence shown in SEQ ID NO.1, and N is Gln or Gln-Lys.

In one embodiment, the sequence of the anti-myocardial ischemia-hypoxia polypeptide has an amino acid sequence represented by the formula M-Q-N, wherein M is Arg-Ala, Q is the amino acid sequence shown in SEQ ID NO.1, and N is Gln or Gln-Lys.

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 M-Q, an amino acid sequence represented by the formula Q-N or an amino acid sequence represented by the formula M-Q-N, wherein M is Arg-Ala, Q is the amino acid sequence represented by SEQ ID NO.1, and N is Gln or Gln-Lys.

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 polypeptide medicament for preventing and/or treating coronary atherosclerotic heart disease or preventing and/or treating myocardial ischemia, 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 embodiment of the invention provides a polypeptide for resisting myocardial ischemia and anoxia, wherein the polypeptide sequence for resisting myocardial ischemia and anoxia has an amino acid sequence shown as SEQ ID NO. 1.

Wherein the amino acid sequence shown in SEQ ID NO.1 is as follows: Ser-Thr-Ser-Lys-Ser-Glu-Ser-Ser.

in one embodiment, the anti-myocardial ischemia-hypoxia polypeptide sequence of the present invention is SEQ ID No. 1: an amino acid sequence shown by Ser-Thr-Ser-Lys-Ser-Glu-Ser-Ser, and an anti-myocardial ischemia and hypoxia polypeptide consisting of the amino acid sequence shown by SEQ ID NO.1 is named as polypeptide E.

It is understood that the polypeptide sequence of the invention having the amino acid sequence shown in SEQ ID No.1 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 polypeptide 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 M-Q, wherein M is Arg-Ala, and Q is the amino acid sequence shown in SEQ ID NO. 1.

In one embodiment, the anti-myocardial ischemia-hypoxia polypeptide sequence has an amino acid sequence represented by the formula Q-N, wherein Q is an amino acid sequence shown in SEQ ID NO.1, and N is Gln or Gln-Lys.

In one embodiment, the anti-myocardial ischemia-hypoxia polypeptide sequence has an amino acid sequence represented by the formula M-Q-N, wherein M is Arg-Ala, Q is the amino acid sequence shown in SEQ ID NO.1, and N is Gln or Gln-Lys.

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 M-Q, an amino acid sequence represented by the formula Q-N or an amino acid sequence represented by the formula M-Q-N, wherein M is Arg-Ala, Q is the amino acid sequence represented by SEQ ID NO.1, and N is Gln or Gln-Lys.

In this embodiment, the anti-hypoxic polypeptide sequence is an amino acid sequence represented by the formula M-Q-N, and when M is Arg-Ala and N is Gln-Lys, the anti-hypoxic polypeptide sequence is SEQ ID NO. 2: Arg-Ala-Ser-Thr-Ser-Lys-Ser-Glu-Ser-Ser-Gln-Lys, wherein the polypeptide A is an anti-myocardial ischemia and hypoxia polypeptide consisting of an amino acid sequence shown in SEQ ID NO.2, the polypeptide comprises 12 amino acids, the molecular weight is 1295g/mol, the isoelectric point is 10.58, and the average hydrophobicity is-1.85.

In this embodiment, the anti-hypoxic myocardial ischemia is represented by the amino acid sequence represented by the formula M-Q, and when M is Arg-Ala, the anti-hypoxic myocardial ischemia is represented by the amino acid sequence of SEQ ID NO. 3: Arg-Ala-Ser-Thr-Ser-Lys-Ser-Glu-Ser-Ser, wherein the polypeptide B is an anti-myocardial ischemia and hypoxia polypeptide consisting of an amino acid sequence shown in SEQ ID NO.3, and comprises 10 amino acids, the molecular weight is 1039g/mol, the isoelectric point is 10.09, and the average hydrophobicity is-1.48.

In this embodiment, the anti-hypoxic polypeptide sequence is an amino acid sequence represented by the formula Q-N, and when N is Gln-Lys, the anti-hypoxic polypeptide sequence is the amino acid sequence shown in SEQ ID NO. 4: Ser-Thr-Ser-Lys-Ser-Glu-Ser-Ser-Gln-Lys, wherein the polypeptide C is an anti-myocardial ischemia and hypoxia polypeptide consisting of an amino acid sequence shown in SEQ ID NO.4, and comprises 10 amino acids, the molecular weight is 1068g/mol, the isoelectric point is 9.88, and the average hydrophobicity is-1.95.

in this embodiment, the anti-myocardial ischemia-hypoxia polypeptide sequence is an amino acid sequence represented by the formula Q-N, and when N is Gln, the anti-myocardial ischemia-hypoxia polypeptide sequence is represented by SEQ ID No. 5: Ser-Thr-Ser-Lys-Ser-Glu-Ser-Ser-Gln, wherein the polypeptide for resisting myocardial ischemia and anoxia, which consists of an amino acid sequence shown in SEQ ID NO.5, is named as polypeptide D, the polypeptide contains 9 amino acids, the molecular weight is 939g/mol, the isoelectric point is 7, and the average hydrophobicity is-1.73.

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 polypeptide 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 a polypeptide medicament for preventing and/or treating myocardial ischemia, and the active component 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 polypeptide or the polypeptide composition for resisting myocardial ischemia and anoxia has the effect of preventing and/or treating myocardial ischemia.

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: 200-220g, supplied by Shanghai Spiral-BiKai 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 administrated according to 6mg/kg, Trimetazidine (TMZ) group is administrated according to 5mg/kg, the medicine is prepared into injection by using normal saline as a solvent, and the medicine is administrated by tail vein injection.

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. The No. 0/3 suture is threaded through the anterior descending branch of the left coronary artery by an atraumatic suture 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:

The absorbance value (3.185 +/-0.292) of the LDH in the hypoxia group is obviously increased compared with that in the control group [ (1.340 +/-0.165), and the difference has statistical significance (P is less than 0.001); compared with the pure hypoxia group, the release of the lactate dehydrogenase after the polypeptide is added [ A (1.823 +/-0.258), B (2.320 +/-0.145), C (2.053 +/-0.198), D (2.471 +/-0.337) and E (2.315 +/-0.253) ] is obviously reduced, and the difference has statistical significance (P is less than 0.05); while the levels of lactate dehydrogenase were comparable in the group of polypeptide A to those in the group of Trimetazidine (TMZ) (1.657. + -. 0.153), with slightly higher levels in the groups of polypeptide B, C, D, E relative to those in the group of trimetazidine. In conclusion, the polypeptide A, the polypeptide B, the polypeptide C, the polypeptide D and the polypeptide E can obviously reduce the activity of lactate dehydrogenase and protect hypoxic cell damage.

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 corresponding control group (18.29 +/-3.351%), the mortality of the hypoxia group (82.01 +/-5.446%) 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 pure hypoxia group, the cell death rate caused by hypoxia is reduced by 30-40% in each polypeptide group [ A (32.14 +/-4.005%), B (52.73 +/-4.960%), C (44.68 +/-5.025%), D (48.38 +/-5.870%), E (40.14 +/-3.767%) ] or trimetazidine group (TMZ) (28.16 +/-2.774%), which shows that the anti-myocardial ischemia hypoxia polypeptide can reduce the cell death caused by hypoxia, and the polypeptide A has the effect equivalent to that of trimetazidine.

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 a sham operation group (serum creatine kinase [ (3142 +/-123.71) IU/L ] and lactate dehydrogenase [ (1685 +/-98.5) IU/L ]), the model group (serum creatine kinase [ (7518 +/-359.4) IU/L ] and lactate dehydrogenase [ (6309 +/-395.8) IU/L ]) is obviously increased, and the difference has statistical significance (P is less than 0.001), so 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 (4100 +/-411.9) IU/L, B (4448 +/-397.2) IU/L, C (4574 +/-156.8) IU/L, D (4225 +/-387.9) IU/L, E (4133 +/-325.8) IU/L ] and lactate dehydrogenase [ A (3663 +/-241.6) IU/L, B (4400 +/-497.4) IU/L, C (4552 +/-200.6) IU/L, D (4124 +/-186.5) IU/L, E (3986 +/-258.7) 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 for resisting myocardial ischemia and anoxia can reduce the content of serum creatine kinase and lactate dehydrogenase of myocardial ischemia rats, has protective effect on myocardial cell ischemia injury, and has similar effect to the intervention results of trimetazidine (serum creatine kinase [ (3802 +/-300.4) IU/L) and lactate dehydrogenase [ (3302 +/-222.6) 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> 2019-08-26

<160> 5

<170> SIPOSequenceListing 1.0

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