CN106063928B - Application of polypeptide or derivative thereof in treating hypertensive myocardial hypertrophy - Google Patents

Abstract

The invention discloses application of a polypeptide or a polypeptide derivative or a polypeptide chimeric peptide capable of specifically binding TRB3 in preparing a medicament or a vaccine for preventing and/or treating hypertensive myocardial hypertrophy; the amino acid sequence of the polypeptide is SEQ ID NO:1, and the amino acid sequence of the polypeptide derivative is SEQ ID NO:1 by substitution, deletion or addition of one or more amino acid residues and has the amino acid sequence similar to that shown in SEQ ID NO:1, consisting of the amino acid residue sequence of SEQ ID NO:1, and the polypeptide chimeric peptide is formed by connecting the polypeptide or the polypeptide derivative with a cell-penetrating peptide. The invention further discloses a nucleotide fragment for encoding the polypeptide or the polypeptide derivative, and application of a pharmaceutical composition containing the polypeptide or the polypeptide derivative or the polypeptide chimeric peptide and the nucleotide sequence thereof in preparing a medicament or a vaccine for preventing and/or treating hypertensive myocardial hypertrophy.

Description

Application of polypeptide or derivative thereof in treating hypertensive myocardial hypertrophy

Technical Field

The invention belongs to the technical field of medicines, and relates to application of a polypeptide or a polypeptide derivative or a polypeptide chimeric peptide in preparation of a medicine or a vaccine for preventing and/or treating hypertensive myocardial hypertrophy.

Background

Myocardial hypertrophy is the initial physiological adaptive response of blood pressure rise and afterload increase, and is also a common pathophysiological process of many cardiovascular diseases, such as hypertension, myocardial infarction, valvular heart disease, cardiomyopathy and the like. Although blood pressure can be controlled to the normal range by pharmaceutical intervention, myocardial hypertrophy will inevitably progress gradually to chronic heart failure. Myocardial hypertrophy is in fact an independent risk factor for cardiovascular disease, multiplying the mortality rate of cardiovascular disease. The treatment for myocardial hypertrophy is still limited to blood vessel expansion, myocardial contractility reduction, afterload reduction and the like, and the intervention on the myocardial hypertrophy forming process is rarely directly carried out. Myocardial hypertrophy is mainly characterized by hypertrophy of myocardial cells and change of interstitial components, heart compliance and circulation pump function are reduced, and meanwhile, occurrence and development of myocardial hypertrophy pathology are closely related to secretion of inflammatory factors interleukin 6 and interleukin 17A.

Autophagy is an important defense mechanism against body lesions, and classical autophagy is defined as clearance of intracellular redundant, misfolded proteins and damaged organelles via the lysosomal pathway, preventing oxygen radical accumulation and inflammation P62 is a "wagon protein" important in autophagy, P62 protein domain contains a Ubiquitin-related domain (UBA) that binds to ubiquitinated proteins ((Ubiquitin, Ub), as a "wagon" recruits damaged or protein to the membrane of the autophagy tt 3, which is mainly achieved by L of the P62 protein (IR L C28-interactino), and is also inhibited by intracellular autophagy t 829 2, which promotes the formation of cytoplasmic t β + t.

TRB3(Tribbles Homologue3) was one of the members of the Tribbles homologous protein family, was first identified in Drosophila melanogaster, and was found to inhibit mitosis, regulate cell proliferation, migration and morphogenesis during development. In mammals, there are three Tribbles homologous proteins: TRB1, TRB2 and TRB3, which are pseudo kinase protein family members. All three proteins contain a Ser/Thr protein kinase-like domain (Kinaselikedomain, KD), but lack the binding site for ATP and catalytic residues, and thus have no kinase activity. Nevertheless, the Tribbeles proteins have adaptor-like functions and are involved in the assembly of multiple protein complexes. Among the mammalian Tribbeles family members, TRB3 has been studied most extensively, and its interacting proteins include transcription factors, ubiquitin ligases, type II BMP receptors on cell membranes, and MAPK, PI3K signaling pathway members. Through interaction with these proteins, TRB3 is involved in regulation of glycolipid metabolism, adipocyte differentiation, apoptosis, stress, collagen expression, and the like. Recent evidence indicates that TRB3 has an important regulation effect in the occurrence and development processes of diseases related to hypertensive myocardial hypertrophy, and the expression of TRB3 in heart tissues of hypertensive mice induced by abdominal aorta ligation is obviously increased, which suggests that TRB3 may be a potential target for treating hypertensive myocardial hypertrophy and diseases related to hypertensive myocardial hypertrophy.

It was shown that TRB3 binds to the autophagy substrate p62 and blocks autophagy. Therefore, research and development of TRB3 protein inhibitors or substances for blocking combination of TRB3 protein and P62 protein have good prospects in drug development for restoring autophagy flow and treating hypertensive myocardial hypertrophy and related diseases.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a polypeptide or a polypeptide derivative or a polypeptide chimeric peptide capable of specifically binding TRB3, a nucleotide fragment for coding the polypeptide or the derivative or the polypeptide chimeric peptide thereof, and application of a pharmaceutical composition containing the polypeptide or the polypeptide derivative or the polypeptide chimeric peptide or the nucleotide fragment thereof in preparing a medicament or a vaccine for treating and/or preventing hypertensive myocardial hypertrophy.

The first aspect of the technical scheme provided by the invention is as follows: the application of a polypeptide or polypeptide derivative or polypeptide chimeric peptide capable of specifically binding TRB3 in the preparation of a medicament or vaccine for preventing and/or treating hypertensive myocardial hypertrophy; the method is characterized in that:

the amino acid sequence of the polypeptide is SEQIDNO: 1, and the amino acid sequence of the polypeptide derivative is SEQ ID NO:1 is substituted, deleted or added by one or more amino acid residues and has the amino acid sequence similar to that shown in SEQ ID NO:1, the amino acid residue sequence of SEQ ID NO:1, and the polypeptide chimeric peptide is formed by connecting the polypeptide or the polypeptide derivative with a cell-penetrating peptide.

In the present invention, the polypeptide derivative refers to a polypeptide (sequence shown in SEQ ID NO: 1) in which amino acid substitutions, deletions or additions are appropriately introduced, as long as the altered amino acid sequence can still form a polypeptide capable of specifically binding to TRB3 and the polypeptide still maintains the activity before alteration.

In the invention, the amino acid sequence of the cell-penetrating peptide is shown in any one of SEQ ID NO. 2-SEQ ID NO. 7 in a sequence table.

In the present invention, the cell-penetrating peptide is a cell-penetrating peptide as conventionally described in the art, as long as it can assist the polypeptide to be delivered into a cell to function, and in general, the cell-penetrating peptide is a short peptide molecule consisting of 10 to 30 amino acids, the cell-penetrating peptide is preferably a Pep2 polypeptide having an amino acid sequence shown in SEQ ID NO:2, that is, in the present invention, the polypeptide chimeric peptide is preferably a chimeric polypeptide in which a Pep2 polypeptide is linked to a sequence shown in SEQ ID NO:1, the cell-penetrating peptide may also be a TAT peptide (YGRKKRRQRRR having an amino acid sequence shown in SEQ ID NO: 3) of an HIV-1 virus reverse transcription activator (Trascript-activator of Tar) protein, a transcription factor Antp peptide (IWFQNRKZWKK having an amino acid sequence shown in SEQ ID: 4) of a Drosophila homeoprotein, a transcription factor Antp peptide (RQRKFQNRKWKK) having an amino acid sequence shown in SEQ ID NO: 4), a PekXK-1 peptide (EWKK), a TW peptide having an amino acid sequence shown in SEQ ID NO:7, preferably an amino acid sequence shown in SEQ ID NO:7, or more preferably a TWXkXArg 2, or a TWXArg 6 amino acid sequence shown in TW-RG-7.

The second aspect of the technical scheme provided by the invention is as follows: the application of a nucleotide fragment in preparing a medicament or vaccine for preventing and/or treating hypertensive myocardial hypertrophy is characterized in that the nucleotide fragment is a nucleotide fragment which can encode a polypeptide or a polypeptide derivative or a polypeptide chimeric peptide capable of specifically binding TRB 3;

wherein said nucleotide fragments comprise all bioinformatically acceptable nucleotide fragments encoding a polypeptide or polypeptide derivative or polypeptide chimeric peptide that specifically binds TRB 3;

wherein, the amino acid sequence of the polypeptide is SEQIDNO: 1, and the amino acid sequence of the polypeptide derivative is SEQ ID NO:1 is substituted, deleted or added by one or more amino acid residues and has the amino acid sequence similar to that shown in SEQ ID NO:1, the amino acid residue sequence of SEQ ID NO:1, and the polypeptide chimeric peptide is formed by connecting the polypeptide or the polypeptide derivative with a cell-penetrating peptide.

The third aspect of the technical scheme provided by the invention is as follows: use of a pharmaceutical composition for the manufacture of a medicament or vaccine for the prevention and/or treatment of hypertensive myocardial hypertrophy, wherein the pharmaceutical composition may comprise a polypeptide or polypeptide derivative or polypeptide chimeric peptide capable of specifically binding to TRB3 and/or a nucleotide fragment thereof, alone or in combination, and a pharmaceutically acceptable carrier or excipient;

wherein, the amino acid sequence of the polypeptide is SEQIDNO: 1, and the amino acid sequence of the polypeptide derivative is SEQ ID NO:1 is substituted, deleted or added by one or more amino acid residues and has the amino acid sequence similar to that shown in SEQ ID NO:1, the amino acid residue sequence of SEQ ID NO:1, and the polypeptide chimeric peptide is formed by connecting the polypeptide or the polypeptide derivative with a cell-penetrating peptide.

In the invention, the polypeptide derivative or the polypeptide chimeric peptide and the nucleotide fragment can be independently or jointly used with other antihypertensive myocardial hypertrophy drugs.

In the present invention, the pharmaceutical composition may comprise a physiologically or pharmaceutically acceptable carrier, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient, preferably one or more selected from chitosan and its derivatives, carbomer and liposome. Therefore, in the present invention, the polypeptide or the derivative of the polypeptide or the polypeptide chimeric peptide is preferably combined with the pharmaceutical excipient to form a pharmaceutical composition. The pharmaceutical composition may be in various forms conventionally described in the art, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, non-aqueous solution or suspension, more preferably a tablet, capsule, granule, injection or infusion, etc. The route of administration of the pharmaceutical composition is preferably injection or oral administration, which preferably comprises: intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

The dosage of the pharmaceutical composition of the present invention in treatment depends on the age and condition of the patient, and is preferably 0.1-15 mg/kg, more preferably 5-10 mg/kg, and most preferably 5mg/kg, and the administration is preferably performed once or several times a day.

In the invention, the hypertensive myocardial hypertrophy refers to the conventional pathological states in the field, including abnormal ventricular remodeling, myocardial cell volume increase, cardiac mass increase, fibrous tissue hyperplasia and high expression of inflammatory factors interleukin 6 and interleukin 17A.

On the basis of the common knowledge in the field, the above-mentioned preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available, unless otherwise specified.

Advantageous technical effects

The polypeptide or the polypeptide derivative can be specifically combined with TRB3, so that the interaction between TRB3 and P62 protein is blocked, autophagy flow is recovered, the chimeric peptide Pep2-A2 of the peptide segment can treat hypertensive myocardial hypertrophy of mice, and the polypeptide or the polypeptide derivative has a very remarkable curative effect.

Drawings

FIG. 1 is a graph showing the dynamic binding of protein TRB3 to polypeptide A2 by surface plasmon resonance. The right panel is the binding curve for A2 and TRB 3.

FIG. 2 shows the effect of Pep2-A2, a chimeric peptide of polypeptide A2, on the interaction between proteins TRB3 and P62, at the cellular level, as demonstrated by co-immunoprecipitation.

FIG. 3 shows the increase of cardiac weight and myocardial cell size in hypertensive myocardial hypertrophy mice over the control group. The upper graph is typical heart size, the lower graph is typical myocardial cell size, and the right graph is the statistical result of weight and area.

Fig. 4 shows the wall thickness and the change of cardiac function of the ventricle of hypertensive myocardial hypertrophic mice, the left graph is a typical echocardiogram, and the right graph is the statistical results of the ventricular posterior wall (L VPWd), the diastolic function of tissue Doppler (E/E'), and the Ejection Fraction (EF) of the mice.

FIG. 5 is a pathological typical graph showing the proliferation of fibrous tissues and the infiltration of inflammatory cells in the heart of hypertensive myocardial hypertrophy mice.

FIG. 6 shows the result of the competitive enzyme-linked immunosorbent assay (E L ISA) to verify the increase of the expression level of interleukin 6 and 17A in the hypertensive myocardial hypertrophy lesion site.

FIG. 7 shows that the level of TRB3 accumulation in the hypertensive myocardial hypertrophy lesion is increased by immunofluorescence.

FIG. 8 shows the increase of TRB3 expression in hypertensive myocardial hypertrophy lesion by immunoblotting.

FIG. 9 is a graph showing that administration of Pep2-A2 reduced mortality in hypertensive myocardial hypertrophy mice.

FIG. 10 treatment with Pep2-A2 reduced cardiac weight and cardiomyocyte size in hypertensive myocardial hypertrophy mice. The upper graph is typical of heart size, the lower graph is typical of cardiomyocyte size, and the right graph is the statistical result of weight and area.

FIG. 11 shows the statistical results of the ventricular wall thickness reduction and the cardiac function improvement in hypertensive myocardial hypertrophy mice after administration of Pep2-A2 treatment, the left is a typical echocardiogram, and the right is the mouse ventricular posterior wall (L VPWd), the diastolic function of tissue Doppler (E/E'), and the Ejection Fraction (EF).

FIG. 12 is a graph showing that the aggregation of the hypertensive myocardial hypertrophy lesion P62 is reduced after administration of Pep2-A2 treatment by immunofluorescence.

FIG. 13 shows that the expression of P62 at the hypertensive myocardial hypertrophy lesion site was reduced after the administration of Pep2-A2 as demonstrated by the immunoblotting method.

FIG. 14 improvement of cardiac fibroplasia and inflammatory cell infiltration following treatment with Pep 2-A2. The left graph is a typical pathological graph, and the right graph is a statistical result of the infiltration area of fibrous tissues and inflammatory cells.

FIG. 15 reduction of IL 6, 17A levels in myocardial hypertrophy lesion following Pep2-A2 administration.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

In the following examples, the full names or corresponding Chinese names of the partial substances are as follows:

AAC: ligation of abdominal aorta

I L Interleukin

BSA: bovine serum albumin

DMEM: culture medium containing various amino acids and glucose

SDS (sodium dodecyl sulfate): sodium dodecyl sulfate

PVDF: polyvinylidene fluoride

PBST: PhosphateBuffered Salinewith Tween-20, pH7.5,10 ×, decontamination buffer

EC L electrochemiluminescence

TRB 3: tribbeles homologous protein 3

DAPI: polyvinylidene fluoride

A2: the sequence is shown as SEQIDNO: 1 is a polypeptide

Pep 2: cell-penetrating peptide shown as SEQ ID NO. 2 in sequence table

In the following examples, the chimeric polypeptide formed by connecting the polypeptide A2 with the Pep2 cell-penetrating peptide is called Pep2-A2, and the polypeptide or the chimeric peptide is artificially synthesized by Beijing Saibutsu Gene technology, Inc. A2 is connected to the C-terminus of Pep2 by two glycine chains. The sequence structure of the chimeric peptide is as follows:

the sequence of Pep2-A2 is:

"N" -His-L eu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-Gly-Gly-Trp-L eu-Thr-Arg-L eu-L eu-Gln-Thr-L ys- "C" (the sequence is shown as SEQ ID NO:8 in the sequence table).

The room temperature described in the examples below is as conventional in the art and is typically 15-25 ℃.

Example 1: screening the peptide fragment combined with the TRB3 protein and identifying the biological function thereof.

The specific screening method and identification of the peptide fragment combined with TRB3 protein refer to the following patents: polypeptide specifically binding to TRB3 protein, screening method, identification and use thereof (title of the invention), application No.: 201310206907.9. the method comprises the following specific steps:

(1) and screening the peptide fragment bound with the TRB3 protein by using a surface plasmon resonance method.

Firstly, P62 protein is segmented and cut into different polypeptide segments, and peptide segment synthesis is carried out by a polypeptide solid phase synthesizer, and the process is carried out by Beijing Saibaosheng Gene Co. Examples the whole screening process was carried out in a surface plasmon resonance apparatus BiacoreT 200.

The screening method comprises the following steps:

(1) the purified protein TRB3 (available from R & D) was coupled to a CM5 chip (available from GE) via an amino group, unbound protein was washed off at a flow rate of 10. mu. L/min, and the chip surface was equilibrated for 2 hours.

(2) Different concentrations of 250. mu. L polypeptide fragment (200,50,12.5,6.25nM) were injected automatically, the entire procedure was carried out at 25 ℃ and the buffer used was HBS-EP buffer (0.01MHEPES,0.15MNaCl,3mM EDTA, 0.005% surfactant).

(3) Binding curves of the polypeptides with different concentrations and TRB3 are simulated by Biacore T200 self-contained analysis software, and a peptide fragment A2 with strong binding capacity with TRB3 protein in figure 1 is obtained. The sequence of A2 is as follows:

A2：Gly-Gly-Trp-Leu-Thr-Arg-Leu-Leu-Gln-Thr-Lys

the abscissa in FIG. 1 represents the reaction time in seconds. The ordinate represents the reaction intensity of the reaction chip surface with the polypeptide, and the unit is RU. The result shows that the A2 peptide fragment has higher affinity with TRB3 protein in the peptide fragment intercepted from the P62 protein domain.

(2) The co-immunoprecipitation method demonstrated that the chimeric peptide Pep2-a2 could compete for binding of protein p62 to protein TRB3 at the cellular level.

The peptide fragment A2 is connected with cell-penetrating peptide Pep2 (sequence is H L YVSPW) to form a new chimeric peptide Pep2-A2, and the peptide fragment is synthesized by Sebai Gene technology GmbH, and the purity is more than 98%.

The co-immunoprecipitation reagents were as follows:

lysate A solution comprising 0.6057g of Tris base, 1.7532g of NaCl, 0.1017g of MgCl 2.6H 2O, 0.0742g of EDTA, 10m of L glycerol and 10m of L10% NP40 is added with deionized water to 150m of L, the pH value is adjusted to 7.6 by hydrochloric acid, the volume is determined to be 191m of L, the mixture is fully mixed, filtered by a 0.45 mu m filter membrane and stored at 4 ℃.

Lysate B liquid, 200 mu L2M β -phosphoglycerol, 4M L2.5.5 MNaF, 2M L100 mMNaVO3, 2M L100 mMPMSF, 200 mu L1 MDTT, 1mg/M L L eu, Pep and Apr which are respectively 200 mu L in total volume, 9M L in total volume, mother liquid is stored at-20 ℃, before use, the mother liquid of each component in the B liquid is thawed, and is respectively added into the A liquid according to the composition proportion and mixed evenly.

ProteinA/GPlus-Agarose is available from Santacruz, USA.

The specific operation steps are as follows:

(1) the human epithelial kidney 293T cells are spread on a 90mm large dish, 1mg/ml of polypeptide Pep2-A2 is added after the cells are attached to the wall, and the cells are collected after incubation for 12 hours.

(2) The cells were lysed with a co-immunoprecipitation lysate, and about 4-10mg of total cell protein was harvested and each histone was adjusted to the same concentration. Each group of proteins was sampled at 200. mu.g, and left as cell lysate Input as a control.

(3) Adding 2 mu g of P62 antibody or NormalIgG of the same species as the P62 antibody into the residual protein, adding 10 mu L protein A/GPlus-Agarose, fully resuspending, slowly rotating and shaking at 4 ℃, overnight, centrifuging at 4 ℃, 3000rpm for 5min, carefully sucking the supernatant, if a small amount of supernatant can not be sucked into the Agarose, adding 0.5m L immune coprecipitation washing solution, mixing, standing for 1min in an ice bath, centrifuging at 4 ℃, 3000rpm for 30sec, carefully sucking the supernatant, repeatedly washing for 5 times, standing for 5min before the last centrifugation, carefully sucking the supernatant, adding 20-30 mu L2 × SDS gel loading buffer, mixing, denaturing at 95 ℃ for 3min, quickly transferring to the ice bath, cooling, centrifuging at 12000rpm for 2min at room temperature, obtaining a supernatant which is a precipitated protein sample, and taking part or all of the supernatant to perform polyacrylamide gel electrophoresis.

Example 2: and (3) establishing and evaluating a hypertensive myocardial hypertrophy mouse model.

(1) Establishment of hypertensive myocardial hypertrophy mouse model

SPF grade ICR mice (male, 7-8 weeks old) were purchased from Beijing Witonglihua laboratory animal technology, Inc., and were housed in the pharmaceutical research institute laboratory animal center of Chinese academy of medicine, with constant temperature, humidity and free diet. After the mice are bred adaptively for one week, the mice are anesthetized by intraperitoneal injection with 10ml/kg of 4% chloral hydrate, the abdominal cavity is opened under the aseptic condition, the abdominal aorta is separated, the mice are fastened with an injection needle with the outer diameter of 0.6mm by a No. 4 silk thread along the abdominal aorta above the right renal artery, the needle is removed rapidly, and the abdominal cavity is closed by suturing layer by layer. Sham (sham) groups did not undergo silk ligation, and other treatments were identical. And (5) normally feeding for 30 days after operation, and establishing a model.

(2) Evaluation of hypertensive myocardial hypertrophy mouse model.

1) And (3) measuring the heart weight and the size of the myocardial cells of the hypertensive myocardial hypertrophy mouse.

Hypertensive myocardial hypertrophy control and diseased mouse hearts were obtained and weighed, and tissues and paraffin sections of the hypertensive myocardial hypertrophy control and diseased sites were obtained and stained with hematoxylin & eosin (H & E) (performed by tokyo snowbant technologies ltd.). The film was observed and photographed using an optical microscope, and the scale was 20 μm.

As a result, as shown in fig. 3, the heart of the hypertensive myocardial hypertrophy mouse was enlarged as compared with the control group, and the cardiomyocytes were hypertrophied.

2) The echocardiography test evaluates the heart function and morphology of the mice.

On the day before the experiment is finished, a mouse is subjected to anesthesia by intraperitoneal injection of pentobarbital sodium at a dose of 50mg/kg, the chest and abdomen of the mouse are subjected to unhairing treatment, the mouse is fixed on an ultrasonic detection table in a supine position, a VisualSonicsVevo770(VisualSonics, Canada) ultrasonic system is used for detection, a 10MHz ultrasonic probe is used for carrying out B-type ultrasonic monitoring on the long axis direction of the heart of the mouse, the position of papillary muscles of the left ventricle is determined, the heart is converted into the short axis direction for monitoring, an M-type ultrasonic change diagram and a Doppler ultrasonic diagram of 10-20 cardiac cycles of the mouse are recorded, and Vevo770 software is used for respectively measuring various cardiac structure and cardiac function indexes such as the ventricular posterior wall (L VPWd) of the end diastole mouse, the diastolic function (E/E') of tissue Doppler, the Ejection Fraction (EF) and the like.

As a result, as shown in fig. 4, the wall thickness of the posterior ventricle of the end-diastolic phase of hypertensive myocardial hypertrophy mice was increased, the diastolic function of tissue doppler was enhanced, and the ejection fraction was decreased, compared to the control group.

3) The heart of the hypertensive myocardial hypertrophic mouse has fibrous tissue hyperplasia and inflammatory cell infiltration.

Hypertensive myocardial hypertrophy control and diseased mouse hearts were obtained, fixed with 4% paraformaldehyde, paraffin-embedded and transected, stained with hematoxylin-eosin (HE), and overall cardiac inflammatory cell infiltration changes were evaluated. After the tissue section is stained by the masson, the tissue section is observed under a polarized light microscope, the collagen deposition condition of the heart fiber is determined, the fibrosis degree is judged, and a high-definition pathological picture (magnified by 200 times) specially stained by the masson is obtained by applying a high-definition color pathological image-text analysis system SpotAdvanced3.0. The area of collagen staining after sirius red staining was measured for each field using Image-proplus 5.1. 6-8 samples were analyzed in each group, 10 fields were randomly selected for each sample, and the mean value represents the content and expression intensity of collagen tissue of an animal in heart tissue. Groups were compared for "absolute area of collagen" by nonparametric analysis of variance.

The results are shown in fig. 5, in which the hypertensive myocardial hypertrophy mice exhibited more severe tissue fibrosis and immune cell infiltration than the control group.

4) The expression level of interleukin 6 and 17A at the lesion part of hypertensive myocardial hypertrophy is verified by a competitive enzyme-linked immunosorbent assay (E L ISA).

The specific operation steps are as follows:

(1) mice I L-2, I L-4, I L-6, TNF-gamma, I L-17, I L-10 protein and BSA were diluted to 10. mu.l/ml with PBS, 100. mu.l was added to each well, and 96-well E L ISA plates were coated overnight at 4 ℃.

(2) Washed three times with PBS containing 0.1% Tween-20. Plates were coated with 200. mu.l blocking solution (10% BSA-PBS) and coated for 2h at 37 ℃.

(3) Pouring out the coating solution, adding 200 μ l of protein solution into the diseased part of hypertensive myocardial hypertrophy and the normal part of the control group, and incubating at 37 deg.C for 1 h.

(4) Five washes with PBS containing 0.1% Tween-20 were performed. 0.1ml of a freshly diluted enzyme-labeled antibody was added to each reaction well. Incubating at 37 ℃ for 0.5-1 hour.

(5) The reaction was stopped by washing six times with PBS containing 0.1% Tween-20. A substrate developing solution (100 mmol/L sodium acetate, pH6.0, 10. mu.l of 30% hydrogen peroxide per 50ml of buffer, 100. mu.g/ml of TMB) was prepared, 100. mu.l was added to each well, and incubation was carried out at room temperature for 5 min. 50. mu.l of 0.1M dilute sulfuric acid was added to each well.

(6) Results are plotted as a histogram of the OD450 values of the sample wells.

The results are shown in FIG. 6, compared with the control group, the content of I L-6 and I L-17 in the pathological tissues of the hypertensive myocardial hypertrophy mouse is higher than that of the control group, and the changes of the rest inflammatory indexes are not obvious.

5) The immunofluorescence method proves that the TRB3 level of the hypertensive myocardial hypertrophy lesion part is increased.

The specific operation is as follows:

(1) frozen sections of hypertensive myocardial hypertrophy control and lesion site were obtained (performed by nivale technologies ltd., beijing).

(2) PBS was washed 5 times.

(3) Incubate with TRB3 primary antibody overnight at 4 ℃.

(4) The next day, PBS was washed 5 times.

(5) Incubation was carried out with specific secondary antibody (purchased from gold bridge, Kyoto, China) at 37 ℃ for half an hour and washing 5 times with PBS.

(6) The slides were mounted with DAPI (available from beijing shoji gold bridge, ltd) containing an anti-quencher.

(7) After the slides were air dried, TRB3 protein was scanned by confocal laser microscopy, as shown in fig. 6, with a scale of 50 microns.

The results are shown in FIG. 7, in which the level of TRB3 in the hypertensive myocardial hypertrophy lesion was increased as compared to the control group.

6) The immunoblotting method proves that the expression of TRB3 in the hypertensive myocardial hypertrophy lesion part is increased.

The specific operation steps are as follows:

(1) cutting with small scissors, cracking hypertensive myocardial hypertrophy (contrast and lesion tissue), centrifuging at 4 deg.C and 12000rpm for 30min, and carefully collecting supernatant;

(2) adjusting the protein concentration to be the same according to a volume ratio of 4: 1 Add 5xloadingbuffer (from Beijing prilley Gene Co., Ltd., 5xSDS-PAGE non-reducing protein Loading buffer B1030)

(3) Loading to SDS electrophoresis gel, taking gel after electrophoresis, and transferring the protein to a PVDF membrane by using an electrotransfer instrument.

(4) Blocking with 10% BSA, adding diluted specific anti-TRB 3 primary antibody (from CST, USA) at the ratio of the antibody specification, and incubating at 4 deg.C for 12 h.

(5) PBST was washed in a shaker at room temperature for 6X8min, horseradish-labelled secondary antibody was diluted according to the instructions and incubated at room temperature for 2 h.

(6) Wash with PBST in shaker at room temperature for 6x8 min.

(7) The total cell protein number was consistent as indicated by consistent gray scale of GAPDH plaques using EC L luminescence liquid exposure and EC L4000 (available from Beijing prilley Gene Co., Ltd., SuperEC L Plus hypersensitivity luminescence liquid P1010) for imaging.

As a result, the level of TRB3 in the hypertensive myocardial hypertrophy lesion shown in FIG. 8 was increased as compared with that in the control group.

Example 3: pep2-A2 was administered to treat hypertensive myocardial hypertrophy in mice.

The establishment and evaluation of a hypertensive myocardial hypertrophy model in mice are shown in example 2, which is to test the treatment effect after Pep2-A2 treatment is given on the basis of the mouse model established in example 2.

(1) The mortality rate of hypertensive myocardial hypertrophy mice is reduced after administration of Pep2-A2 treatment.

The specific operation steps are as follows:

SPF grade ICR mice (male, 7-8 weeks old) were purchased from Beijing Witonglihua laboratory animal technology, Inc., and were housed in the pharmaceutical research institute laboratory animal center of Chinese academy of medicine, with constant temperature, humidity and free diet. After one week of adaptive feeding, mice were randomly divided into 3 groups by weight: control group 20, abdominal aorta ligation (AAC) and control polypeptide (dose: 5mg/kg, i.p.) group 20 were administered, abdominal aorta ligation (AAC) and Pep2-A2 treatment (dose: 5mg/kg, i.p.) group 20 were administered. Mice deaths were recorded daily.

The results are shown in fig. 9, and the mortality of mice was reduced after administration of Pep2-a2 treatment.

(2) After being treated by Pep2-A2, the weight of the heart and the size of myocardial cells of a hypertensive myocardial hypertrophy mouse can be reduced.

The administration was carried out in the same manner as in example 3(1), and the measurement of the heart weight and the size of cardiomyocytes were carried out in the same manner as in (1) of example 2).

The results are shown in table 1 and fig. 10, and the heart weight and cardiomyocyte size of the hypertensive myocardial hypertrophy mouse were reduced after administration of Pep2-a 2.

TABLE 1 mouse Heart weights

	Control group	Model set	Model group + Pep2-A2
				Heart weight (g)	0.18±0.01	0.22±0.02	0.19±0.01

(3) After being treated by Pep2-A2, the ventricular wall thickness of hypertensive myocardial hypertrophy mouse is reduced and the cardiac function is improved.

The administration was performed in the same manner as in example 3(1), and the ventricular wall thickness and cardiac function were measured in the same manner as in example 2 (2).

The results are shown in table 2, fig. 10, and decrease ventricular wall thickness and improve cardiac function in hypertensive myocardial hypertrophy mice after administration of Pep2-a2 treatment.

TABLE 2 mouse ventricular wall thickness and ejection fraction

	Control group	Model set	Model group + Pep-A2
				Myocardial cell area (mm)2)	2.2±0.5	4.3±0.8	2.3±0.6
LVPW，d(mm)	0.8±0.05	1.15±0.07	0.90±0.05
				E/E’	35±5	60±5	38±7
EF(％)	60±5	38±6	50±7

(4) The immunofluorescence method proves that the expression of the hypertensive myocardial hypertrophy lesion part P62 is reduced after the Pep2-A2 is given for treatment.

The administration method was the same as in example 3(1), and the immunofluorescence method was the same as in example 2 (2) (5).

The results are shown in fig. 12, which reduces the accumulation of hypertensive myocardial hypertrophy lesion P62 following treatment with Pep2-a 2.

(5) The immunoblotting method proves that the expression of TRB3 in the hypertensive myocardial hypertrophy lesion part is increased.

The administration method was the same as in example 3(1), and the immunoblotting method was the same as in example 2 (2) 6).

The results are shown in fig. 13, which reduces the expression of P62 at the hypertensive myocardial hypertrophy lesion after administration of Pep2-a2 treatment.

(6) Treatment with Pep2-A2 improved cardiac fibroplasia and inflammatory cell infiltration.

The administration method was the same as in example 3(1), and the measurement of cardiac fibrous tissue proliferation and inflammatory cell infiltration were the same as in example 2 (2) 3).

The results are shown in fig. 14, reducing cardiac fibroplasia and inflammatory cell infiltration area following treatment with Pep2-a 2.

(7) After being treated by Pep2-A2, the content level of interleukin 6 and 17A at the pathological part of myocardial hypertrophy is reduced.

The administration method was the same as in example 3(1), and the competitive ELISA assay was the same as in example 2 (2) 4).

The results are shown in table 3 and fig. 15, and the IL-6 and IL-17A levels in the myocardial hypertrophy lesion are reduced after the Pep2-A2 treatment, which indicates that the inflammatory microenvironment of the lesion tissues is improved.

TABLE 3 Heart cytokine content (unit: pg/ml)

Claims (4)

1. The application of a polypeptide or polypeptide chimeric peptide capable of specifically binding TRB3 in preparing a medicament for treating hypertensive myocardial hypertrophy; the method is characterized in that:

the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1, and the polypeptide chimeric peptide is formed by connecting the polypeptide with cell-penetrating peptide; the amino acid sequence of the cell-penetrating peptide is shown as any one of SEQ ID NO 2-SEQ ID NO 7 in a sequence table.

2. The application of a pharmaceutical composition in preparing a medicament for treating hypertensive myocardial hypertrophy, wherein the pharmaceutical composition comprises a polypeptide or a polypeptide chimeric peptide capable of specifically binding TRB3 and a pharmaceutically acceptable carrier or excipient in a single or combined mode; wherein the amino acid sequence of the polypeptide is SEQ ID NO:1, the polypeptide chimeric peptide is a chimeric peptide formed by connecting the polypeptide with a cell-penetrating peptide; the amino acid sequence of the cell-penetrating peptide is shown as any one of SEQ ID NO 2-SEQ ID NO 7 in a sequence table.

3. The use of claim 2, wherein said polypeptide, said polypeptide or polypeptide chimeric peptide is used in combination with other antihypertensive myocardial hypertrophy drugs.

4. The use according to claim 2, wherein said hypertensive myocardial hypertrophy is abnormal remodeling of the ventricles and/or increased heart mass and/or fibrosis of the heart tissue and/or increased expression of the inflammatory factor interleukin 6 and/or increased expression of the inflammatory factor interleukin 17A caused by hypertensive myocardial hypertrophy.

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