CN110801510B

CN110801510B - Use of polypeptides

Info

Publication number: CN110801510B
Application number: CN201810863713.9A
Authority: CN
Inventors: 郝丽英; 胡慧媛; 雷帅; 周诗
Original assignee: China Medical University
Current assignee: China Medical University
Priority date: 2018-08-01
Filing date: 2018-08-01
Publication date: 2022-06-24
Anticipated expiration: 2038-08-01
Also published as: CN110801510A

Abstract

The invention belongs to the technical field of polypeptide medicines in biochemistry, and particularly relates to application of a polypeptide Ahf-caltide, namely application of a polypeptide Ahf-caltide in preparation of a medicine for treating heart failure. The polypeptide Ahf-caltide can obviously improve the survival rate of heart failure rats, improve the electrophysiological properties of the heart failure rats, reduce the level of troponin in myocardial tissues, improve the cardiac quality index, relieve the hypertrophy and fibrosis of the myocardial tissues, and down-regulate the expression of heart failure marker proteins BNP and alpha-SMA, thereby having obvious therapeutic effect on heart failure.

Description

Use of polypeptides

Technical Field

The invention belongs to the technical field of polypeptide medicines in biochemistry, and particularly relates to application of a polypeptide Ahf-caltide, namely application of a polypeptide Ahf-caltide in preparation of a medicine for treating heart failure.

Technical Field

Heart failure (HF, abbreviated as Heart failure) is a complex clinical syndrome caused by the reduction of the blood pumping function of the Heart due to the systolic and/or diastolic dysfunction caused by various reasons such as hypertension, coronary Heart disease, valvular disease, etc., and is the terminal stage of various cardiovascular diseases. With the aging population and the change of life style of people, the incidence of heart failure is rising year by year and becomes a main public health problem harming human health worldwide, so that it is urgent to understand the etiology and pathogenesis of heart failure and find out potential treatment targets.

The understanding of heart failure in medicine goes through the development process from organs to cells to genes, the pathophysiological changes of the heart failure are very complex, the understanding is continuous and deep, the mechanism of retention of body fluid is mainly used in the 40 th century, the mechanism of dysfunction of a pump is used in the 60 th century, the over-activation of neuroendocrine cytokine systems is emphasized in the 80 th century, and the heart muscle reconstruction is recognized as an important mechanism for the development of the heart failure until now. The understanding of the pathophysiological mechanism of heart failure is greatly deepened, and the pathological change of the heart failure is realized to be a very complex dynamic process, including the metabolic change of systemic neuro-humoral regulatory factors, myofibril loss, myocardial cell apoptosis, collagen synthesis increase, cytokine activation, cell signal transduction disorder and the like, and the myocardial remodeling and the reduction of ventricular relaxation performance caused by a series of changes, so that the heart function is compensated to generate the clinical symptoms of the heart failure.

Voltage-gated L-type calcium channels (LTCC) are the major ion channels in the myocardial cell membrane, and most of the calcium ions enter the myocardial cells through the LTCC during the plateau phase of action potential. The influx of calcium ions induces the sarcoplasmic reticulum to release more calcium ions, a process that plays an important role in the excitation-contraction coupling of the myocardium. Calmodulin (CaM) as intracellular calcium ion sensor mediated Ca_V1.2 calcium dependent modulation of channels, CaM being capable of interacting with Ca_V1.2 multiple site binding of the alpha 1 subunit of calcium channels, including Ca_V1.2 the C-terminal IQ motif, pre-IQ motif and Ca_V1.2 sequences I-II loop, and the like, thereby regulating the activity of LTCC. Previous patch-clamp experimental studies found that calcium currents were recorded using the intimal-outward mode, i.e. they gradually decreased over time when intracellular factors were absent, a phenomenon known as the 'run-down' phenomenon. Domain L (CS) of intracellular factor Calpain (CS)_L) Can recover calcium current in a dose-dependent manner, and further research shows that CS_LThe action of activating calcium channels is mediated primarily by eleven amino acids. Subsequent validation of CS by Pull-down technique_LCapable of binding to IQ and pre-IQ motifs at the carboxy terminus of calcium channels, which serve as calmodulin (CaM) binding sites. CS_LCan compete with CaM in a dose-dependent manner for the IQ motif on the carboxy terminus of the voltage-dependent calcium channel and act as a partial agonist.

The polypeptide Ahf-caltide is an undecapeptide consisting of 3Glu, 1Gly, 3Lys, 2Pro, 1His and 1Thr, has the sequence of EGKPKEHTEPK (SEQ ID1) and the molecular weight of 1279.43, and is found to have the effect of restoring the calcium channel activity of run-down in previous researches. I have confirmed by studies the therapeutic effect of the polypeptide Ahf-caltide on isoproterenol (Isoprenaline, ISO) induced heart failure in Sprague Dawley (SD) rats. At present, no report is found about the application of the polypeptide Ahf-caltide in treating heart failure.

Disclosure of Invention

The invention aims to provide a new application of polypeptide Ahf-caltide, namely an application in preparing a medicament for treating heart failure. The polypeptide Ahf-caltide can obviously improve the survival rate of heart failure rats, improve the electrophysiological properties of the heart failure rats, reduce the level of troponin in myocardial tissues, improve the cardiac quality index, relieve the hypertrophy and fibrosis of the myocardial tissues, and down-regulate the expression of heart failure marker proteins BNP and alpha-SMA, thereby having obvious therapeutic effect on heart failure.

The invention adopts the following technical scheme: the polypeptide Ahf-caltide is used for preparing the medicine for treating the heart failure.

The drug comprises the polypeptide Ahf-caltide and various pharmaceutical excipients, and is any pharmaceutically acceptable dosage form, preferably powder injection.

The medicament is in any pharmaceutically therapeutically acceptable dose.

The heart failure includes various heart failures caused by excessive activation of the sympathetic nervous system and renin-angiotensin system, hypertension or myocardial ischemia and anoxia, etc.

The medicine can improve the symptoms and pathological changes of heart failure, including improving myocardial remodeling, electrophysiological property change, myocardial enzymology change, heart index, heart failure marker protein expression, and the like, and improve the survival rate of heart failure.

Use of the polypeptide Ahf-caltide as a medicament for the treatment of any disease based on the principle of binding to a CaV channel. The medicament comprises an amino acid mutant based on a polypeptide Ahf-caltide sequence.

Compared with the prior art, the invention has the following technical advantages:

the polypeptide of the invention has small molecular weight, easy artificial synthesis, easy tissue absorption and strong specificity, and is a safe, high-efficiency and ideal anti-heart-failure drug. The polypeptide drug restores the steady state of calcium ions in cells mainly by regulating the expression of an L-shaped calcium channel, thereby improving the reconstruction of myocardial tissues, restoring the functions of systole and diastole and playing a role in treating heart failure.

For ease of understanding, the Ahf-caltide invention will be described in detail below with reference to specific figures and examples. It is specifically intended that the specification and figures be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Drawings

FIG. 1 shows the effect of Ahf-caltide on the survival curve of heart failure rats.

FIG. 2 shows the effect of Ahf-caltide on II-lead ECG of rat.

FIG. 3 the results of the effect of Ahf-caltide on HE staining of ventricular muscle tissue in rats.

FIG. 4 results of Masson staining of rat ventricular muscle tissue with different doses of Ahf-caltide polypeptide.

FIG. 5 the results of Masson staining of rat ventricular muscle tissue with prophylactic and therapeutic polypeptides.

FIG. 6 shows the result of detecting BNP protein expression levels of different groups of myocardial tissues by a Western blot method.

FIG. 7 shows the result of Western blot to detect the expression level of SMA protein in different myocardial tissues.

Detailed Description

The present invention will be further explained by the following detailed description in conjunction with the drawings, without thereby limiting the invention to the scope of the embodiments described. The following experimental methods without specifying specific conditions were selected according to conventional methods and conditions or according to the commercial instructions.

Example 1

The polypeptide Ahf-caltide with the sequence EGKPKEHTEPK is synthesized by a solid phase polypeptide synthesis method. The purity, amino acid residue composition and molecular weight of the synthesized polypeptide are determined by High Performance Liquid Chromatography (HPLC) purification, mass spectrum and electrospray mass spectrum, and the synthesized product is proved to be the target peptide, and the purification purity of 97.27 percent under 220mn by adopting a C18 nonpolar column.

1. Preparation and grouping of animal models

190-220 g of 32 healthy male SD rats (purchased from the department of laboratory animals of Chinese university of medical science) are randomly divided into four groups:

(1) control group (n = 6): injecting normal saline;

(2) ISO model group (n = 10): injection ISO;

(3) lesion-treatment group (Post-treatment) (n = 8): 2 weeks + 4 days after ISO injection, with Ahf-caltide polypeptide;

(4) prophylactic treatment group (Pre-treatment) (n = 8): ahf-caltide polypeptide was administered for 1 week followed by simultaneous administration of ISO.

ISO used subcutaneous injections at a concentration of 1 mg/mL and a dose of 3 mg/kg once daily for 4 weeks. The polypeptide Ahf-caltide is injected into abdominal cavity, the concentration is 4 mg/mL, the dosage is 3 mg/kg in low dose group, 15 mg/kg in medium dose group and 75 mg/kg in high dose group. And finishing molding after 4 weeks.

Comparing the survival of rats in each group, it was found that the survival rate of rats to which Ahf-caltide polypeptide was administered for the prevention or treatment was significantly increased as compared to the ISO heart failure model group (as shown in fig. 1), confirming that the polypeptide can improve the survival rate of heart failure rats.

2. Electrocardiogram observation

Each group of rats was anesthetized with descending isoflurane in a small animal anesthesia machine, with the limbs and head supinely fixed on an operating table, with Electrocardiogram (ECG) electrodes subcutaneously connected to the limbs, and recording standard II-lead ECG. As shown in FIG. 2, the amplitude and time limit of P wave in the ISO heart failure model group are significantly increased, while the amplitude and time limit of P wave in ECG are significantly lower than those in the ISO heart failure model group after Ahf-cadtide polypeptide is administered for prevention or treatment, i.e., the polypeptide can restore the electrophysiological properties of heart of rat with ISO-induced heart failure and improve heart failure.

3. Animal sacrifice and material selection

(1) Anesthesia and sacrifice: the rats were sacrificed by decapitating after deep anesthesia with 1% sodium pentobarbital at 50 mg/kg.

(2) Material taking: exposing the chest and heart, cutting the right auricle, injecting proper amount of normal saline at the apex of the heart to flush out the blood in the heart, cutting the heart, and flushing in normal saline for later use.

4. Measurement of CK

Collecting blood from abdominal aorta of rats of different groups before sacrifice, anticoagulating with heparin sodium at 3000 r.min^-1Centrifuging for 10 min, collecting supernatant, and storing at-80 deg.C. The CK content in each group of serum was determined by performing the procedures according to the CK kit instructions.

As shown in Table 1, compared with the Control group, the content of CK in the serum of the rat in the ISO model group is obviously increased (P is less than 0.05); compared with the ISO model group, the content of CK in the serum of the rats in the polypeptide prevention group or the treatment group is obviously reduced and is close to the normal level (P < 0.05), and the polypeptide is proved to be capable of improving the myocardial enzymological change of the heart failure rats.

5. Rat cardiac quality index determination

The residual blood of the heart is washed away by precooled normal saline, the surrounding connective tissues and blood vessels are cut off, and the wet mass of the heart is weighed after the water is absorbed by filter paper. Cardiac mass index = cardiac wet mass/body mass.

As shown in Table 2, the heart mass index of rats in the ISO model group was significantly increased (P < 0.001) compared to that in the Control group, while the heart mass index of rats in the polypeptide prevention or treatment group was significantly decreased compared to that in the ISO model group, confirming that the polypeptide had the effect of improving heart mass.

6. Tissue sections and HE staining

Material taking and fixing, trimming and dehydrating, dipping in wax and embedding, slicing and baking, dewaxing and dyeing, and observing the change of the morphology of the myocardial cells under a light microscope. The specific method is as follows

(1) Paraffin section dewaxing to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5min-75% alcohol 5min, and washing with tap water.

(2) Hematoxylin staining: staining the slices with hematoxylin staining solution for 3-5min, washing with tap water, differentiating with differentiation solution, washing with tap water, returning blue to blue with blue returning solution, and washing with running water.

(3) Eosin staining: the slices are dehydrated for 5min respectively by 85 percent and 95 percent gradient alcohol, and are dyed for 5min in eosin dye solution.

(4) Dewatering and sealing: placing the slices in anhydrous ethanol I5 min-anhydrous ethanol II 5 min-anhydrous ethanol III 5 min-dimethyl I5 min-xylene II 5min, sealing with neutral gum.

(5) Microscopic examination and image acquisition and analysis.

Cardiomyocyte size was counted using Image J Image processing software and comparative analysis between groups using ANOVA in GraphPad Prism 6 software indicated that the difference was statistically significant when P < 0.05.

The HE staining result is shown in FIG. 3, the myocardial cells of Control group are arranged neatly and compactly, the structure is clear and complete, and the cell nucleus is compact and clear; the cardiac muscle cells of the ISO heart failure model group are obviously hypertrophied (P <0.001, compared with a control group), the arrangement is irregular, the nucleus is enlarged, the arrangement of muscle fibers is disordered, and part of cardiac muscle is changed in a wavy manner; compared with the ISO heart failure model group, the myocardial cells of the rats in the polypeptide prevention and treatment group are regularly arranged, the shape is regular, the cell volume is reduced (P is less than 0.001), and the swelling is not obvious. The polypeptide is proved to have the effects of reducing myocardial hypertrophy and improving myocardial tissue structure.

7. Masson staining

Masson staining, also known as Masson staining, is one of the most classical methods of staining connective tissue, using a mixture of two or three anionic dyes to reveal fibers and inflammatory factors in the tissue. The Masson dyed myofibers are red, the collagen fibers are blue, and the method is mainly used for distinguishing collagen fibers from myofibers and comprises the following specific method.

(2) Dyeing with potassium dichromate: the slices were soaked in potassium dichromate overnight and washed with tap water.

(3) And (3) hematoxylin staining: mixing the solution A and the solution B in equal ratio to obtain a hematoxylin staining solution, slicing the stained solution into hematoxylin for 3min, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue to the blue solution, and washing with running water.

(4) Ponceau acid fuchsin dyeing: the slices are dip-dyed in ponceau acid fuchsin for 5-10 min and rinsed with tap water.

(5) Phosphomolybdic acid staining: and dip-dyeing with phosphomolybdic acid aqueous solution for 1-3 min.

(6) And (3) aniline blue dyeing: after phosphomolybdic acid is washed, the mixture is directly dyed in aniline blue dye solution for 3-6 min.

(7) Differentiation: the slices were differentiated with 1% glacial acetic acid and dehydrated in two jars of absolute ethanol.

(8) Transparent sealing sheet: placing the slices in a third jar with anhydrous ethanol for 5min, transparent xylene for 5min, and sealing with neutral gum.

(9) Microscopic examination and image acquisition and analysis.

Area of myocardial tissue collagen fibrosis was counted using Image J Image processing software, multiple group comparative analysis was performed using ANOVA in GraphPad Prism 6 software, indicating that the difference was statistically significant when P < 0.05.

Masson staining results are shown in figures 4 and 5, and show that the myocardial cells in the Control group have no fibrosis, the myocardial fascicles in the ISO heart failure model group are separated and surrounded by blue-stained fibrous connective tissues, and the myocardial fibrosis is obviously enhanced, the myocardial fibrosis range is reduced after the treatment of the polypeptide Ahf-caltide, and particularly the effect of the medium-dose polypeptide (15 mg/kg) is most obvious (P < 0.001). The polypeptide was confirmed to be capable of significantly reducing the degree of fibrosis in heart failure rats.

8、Western blot

Extracting myocardial tissue protein, quantifying protein, preparing glue, adding sample, performing electrophoresis, transferring a membrane, sealing, performing primary antibody incubation (BNP 1:500 dilution; SMA 1:200 dilution; GAPDH 1:1500 dilution) at 4 ℃ overnight, taking out secondary antibody incubation from 4 ℃, adding a proper amount of TBST, placing on a horizontal shaking bed, and washing the membrane for 3 times, wherein each time lasts for 10 min. Adding secondary antibody (goat anti-mouse 1:10000 diluted, goat anti-rabbit 1:10000 diluted) and incubating for 2h at room temperature. And (3) carrying out quantitative analysis on the Western Blot result by protein detection luminescence identification.

Measuring the gray value of the protein electrophoresis band by using Image J Image processing software, wherein the ratio of the gray value of the protein electrophoresis band to the gray value of the GAPDH band is the relative value of the target protein expression quantity. Comparative analysis between groups was performed using ANOVA in GraphPad Prism 6 software, indicating that the difference was statistically significant when P < 0.05.

The difference of BNP protein expression levels of different groups of myocardial tissues is detected by a Western blot method, and the result is shown in figure 6, wherein the BNP protein expression level of the ISO model group is higher than that of the Control group (P < 0.01); the BNP expression quantity of the injury treatment group is obviously lower than that of the ISO model group (P < 0.05); the BNP expression quantity of the prevention and treatment group is obviously lower than that of the ISO model group (P < 0.001). The polypeptide is proved to be capable of changing the expression of the heart failure marker protein BNP.

Western blot detection of the differences of the SMA protein expression levels of different groups of myocardial tissues shows that the results are shown in FIG. 7, and the SMA protein expression of the ISO heart failure model group is obviously higher than that of the Control group (P < 0.001); the SMA expression quantity of the injury treatment group is lower than that of an ISO model group (P < 0.01); the SMA expression quantity of the prevention treatment group is obviously lower than that of an ISO model group (P < 0.001). The polypeptide is proved to be capable of changing the expression of heart failure index protein SMA.

SEQUENCE LISTING

<110> university of Chinese medical science

<120> use of polypeptide

<130> 1

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 11

<212> PRT

<213> unknown

<400> 1

Glu Gly Lys Pro Lys Glu His Thr Glu Pro Lys

1 5 10

Claims

1. Use of polypeptide Ahf-caltide as a medicament for the treatment of heart failure, wherein the medicament comprises an active fragment of polypeptide Ahf-caltide, and the sequence of polypeptide Ahf-caltide is EGKPKEHTEPK.

2. The use of the polypeptide Ahf-caltide of claim 1 for the preparation of a medicament for the treatment of heart failure, wherein the medicament further comprises various pharmaceutical excipients.

3. The use of the polypeptide Ahf-caltide as defined in claim 1, in the preparation of a medicament for the treatment of heart failure, wherein the medicament is in any pharmaceutically-therapeutically acceptable dosage form.

4. The use of the polypeptide Ahf-caltide of claim 1 for preparing a medicament for the treatment of heart failure, wherein the medicament is a powder for injection.

5. The use of the polypeptide Ahf-caltide of claim 1 for the preparation of a medicament for the treatment of heart failure, wherein the heart failure comprises heart failure due to myocardial injury.

6. Use of the polypeptide Ahf-caltide as defined in claim 1 for the preparation of a medicament for the treatment of heart failure, characterized in that the medicament can improve heart failure conditions, i.e. myocardial remodeling, alteration of electrophysiological properties, alteration of cardiomyopathy, cardiac index and expression of marker proteins for heart failure, while improving the survival rate of heart failure.