A polypeptide and its use

Abstract

The present invention provides a polypeptide, whose amino acid sequence is shown in SEQ ID NO.1. The present invention also provides a DNA molecule encoding the above polypeptide. The present invention also provides a recombinant vector containing the above DNA molecule. The present invention also provides the use of the above polypeptide in the preparation of a drug for treating myocardial fibrosis. Compared with the existing polypeptides for treating myocardial fibrosis, the polypeptide of the present invention is secreted in large quantities under physiological conditions after exercise, so the polypeptide has no obvious adverse reactions to the organism. In normal organisms and organisms in the early stages of the disease, myocardial fibrosis can be alleviated by increasing the amount of exercise. In the middle and late stages, myocardial fibrosis can be alleviated by administering the polypeptide in vitro, especially under conditions of restricted exercise. Therefore, the present invention can inhibit the transformation of cardiac fibroblasts into myofibroblasts and alleviate myocardial fibrosis.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a polypeptide and its use. Background Art

[0002] Heart failure (HF) is the terminal stage of various heart diseases. It not only seriously affects the quality of life of patients, but also causes malignant arrhythmias and even sudden death. With the aging of the global population, the total number of HF patients continues to rise, and it has become a serious public health problem worldwide. At present, the clinical treatment of HF mainly includes drug therapy, interventional therapy and heart transplantation, but these methods have limitations to varying degrees, making the 5-year mortality rate of patients with chronic HF still as high as about 50%. Therefore, it is of great significance to find safer and more effective strategies for the prevention and treatment of HF.

[0003] Myocardial fibrosis leads to systolic and diastolic dysfunction under many cardiac pathological and physiological conditions. Existing studies have found that ① ghrelin inhibits myocardial fibrosis by regulating Act A / FS imbalance, reduces left ventricular remodeling, and ultimately improves cardiac function; ② matrix protein peptide p1159 activates the RhoA pathway through the membrane receptor integrin α4, thereby increasing the migration of cardiac fibroblasts and reducing adverse left ventricular remodeling after myocardial infarction by regulating the deposition, arrangement and perfusion of fibrotic scars; ③ apelin-13 inhibits oxidative stress by inhibiting the PI3K / Akt signaling pathway, improves angⅡ-induced cardiac dysfunction, impaired cardiac hemodynamics, and reduces cardiac fibroblast fibrosis.

[0004] The beneficial effects of long-term regular exercise on the cardiovascular system have become a consensus in the industry. In recent years, with the development of relevant clinical research, the good preventive and therapeutic effects of exercise on heart failure have received more and more attention. Studies have shown that regular and moderate exercise can reduce myocardial fibrosis, reduce cardiac stiffness, and improve myocardial diastolic and systolic function. It can not only effectively reduce the risk of heart failure, but also reduce the all-cause mortality and hospitalization rate of patients with heart failure, and significantly improve patient prognosis. At present, more and more studies have found that exercise, as a safe physiological stimulus, has a good preventive and therapeutic effect on heart failure.

[0005] It has been found that exercise can stimulate muscles to release a variety of substances, including proteins and peptides, which are important mediators of their effects. Studies have shown that peptides play an important role in human physiology and clinical diagnosis and treatment, and have now become one of the hot directions in biopharmaceutical development and clinical research. Peptides are ideal for drug development due to their low toxicity, low immunogenicity, high safety, strong specificity, and low energy consumption in production. At present, peptide drugs have been widely used in many disease areas and have achieved good therapeutic effects. Therefore, we interpreted and explored exercise from the peptide level, and found peptides that have an inhibitory effect on myocardial fibrosis. So far, there have been no cases where exercise-related peptide drugs have been reported to have excellent therapeutic effects on myocardial fibrosis. Summary of the invention

[0006] In view of the above technical problems in the prior art, the present invention provides a polypeptide and its use, which is intended to solve the technical problem that the drugs in the prior art are not effective in treating myocardial fibrosis.

[0007] The present invention provides a polypeptide, whose amino acid sequence is shown as SEQ ID NO.1.

[0008] Specifically:

[0009] MPFSRTEDGKIYQRAFGGHSLKFGKGRQAHRCCCVADRTGHSILHTLYGRVRLPPVHLRQDT.

[0010] The present invention also provides a DNA molecule encoding the above polypeptide.

[0011] The invention also provides a recombinant vector containing the above DNA molecule.

[0012] The present invention also provides a host cell comprising the above-mentioned recombinant vector.

[0013] The present invention also provides an expression system, which contains the above-mentioned recombinant vector, or a genome in which the above-mentioned exogenous DNA molecule is integrated.

[0014] The present invention also provides the use of the above polypeptide in preparing a medicine for treating myocardial fibrosis.

[0015] The present invention also provides a pharmaceutical composition, the active ingredient of which is the above polypeptide.

[0016] Furthermore, the composition also contains pharmaceutical excipients.

[0017] Furthermore, the composition is in the form of tablets, capsules or injections.

[0018] Compared with the existing polypeptides for treating myocardial fibrosis, the polypeptide of the present invention is secreted in large quantities under physiological conditions after exercise, so the polypeptide has no obvious adverse reaction to the organism. In normal organisms and organisms in the early stage of the disease, myocardial fibrosis can be reduced by increasing the amount of exercise. In the middle and late stages, myocardial fibrosis can be reduced by in vitro administration of the polypeptide, especially under conditions of restricted exercise. Therefore, the present invention can inhibit the transformation of cardiac fibroblasts into myofibroblasts and reduce myocardial fibrosis.

[0019] Compared with the existing technology, the technical effect of the present invention is positive and obvious. Compared with the existing drugs for treating myocardial fibrosis, the polypeptide of the present invention has fewer side effects, low immunogenicity, low production energy consumption, and the polypeptide can cover patients with multiple risk factors, and can better manage and treat patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The homology of EICMP1 in vertebrates is shown.

[0021] Figure 2 It shows that EICMP1 inhibits the transformation of cardiac fibroblasts into myofibroblasts; A. Cell proliferation level; B. mRNA expression level of myofibroblast-related markers; C. Protein expression level of myofibroblast-related markers; D. Protein expression level of SRF and Calcineurin.

[0022] Figure 3 Shown that EICMP1 antagonizes myocardial fibrosis induced by hypertension in mice; A. heart-to-weight ratio of mice; B. H&E staining of mouse heart; C. Masson staining of mouse heart. DETAILED DESCRIPTION

[0023] Example 1 Screening of polypeptides

[0024] We constructed an endurance exercise mouse model (endurance exercise characterized by low-intensity, long-term training, such as jogging, swimming, etc., which is considered to be the main form of exercise to improve heart function). Eight-week-old C57BL / 6 male mice were used as research subjects to construct an endurance exercise model. The mice first underwent adaptive exercise training for 2 days by running at a speed of 10 meters / minute for 5 minutes every day. Then the mice started running exercise training from an initial speed of 10 meters / minute every day, and the speed increased by 1 meter / minute every 20 minutes until exhaustion. The exercise training lasted for 14 days.

[0025] In the present invention, normally raised non-exercised mice were used as a control group.

[0026] After modeling, the heart tissues of mice in each group were collected for peptide group detection. We performed bioinformatics analysis and screening on the sequencing results according to Log2 (fold change)>1 and P<0.05, and found a peptide that was significantly increased in the heart tissue of mice after exercise. The amino acid peptide encoded by it has high homology in many vertebrates such as humans, mice, and rats ( Figure 1 ), showing evolutionary conservation, suggesting that it may have important biological functions. No function or mechanism has been reported for this peptide so far, so we named it EICMP1 (Exercise-induced cardiac micropeptide 1).

[0027] The sequence of the polypeptide is:

[0028] MPFSRTEDGKIYQRAFGGHSLKFGKGRQAHRCCCVADRTGHSILHTLYGRVRLPPVHLRQDT (SEQ IDNO.1)

[0029] The preparation method of the above polypeptide is prepared by a conventional method, which is as follows:

[0030] Peptides are synthesized from the C-terminus to the N-terminus.

[0031] 1. Weigh 3 g of RINK resin (substitution degree 0.3 mmol / g) into a 150 ml reactor and soak it with 50 ml of dichloromethane (DCM).

[0032] 2. After 2 hours, wash the resin with 3 times the volume of the resin in nitrogen-dimethylformamide (DMF), then drain it. Repeat this process four times and drain the resin for later use.

[0033] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF 3 times the volume of the resin and then drain it.

[0034] 4. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin has color, it means that the deprotection is successful.

[0035] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50 ml centrifuge tube, add 20 ml of DMF to dissolve them, then add 3 ml of N, N-diisopropylcarbodiimide (DIC) and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for reaction.

[0036] 6. After 2 hours, cap the resin with a certain amount of acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash it four times with DMF 3 times the volume of the resin, and drain it for later use.

[0037] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it with DMF four times and then drain it.

[0038] 8. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin has color, it means that the deprotection is successful.

[0039] 9. Weigh an appropriate amount of the second amino acid and HOBT into a 50 ml centrifuge tube, add 25 ml of DMF to dissolve them, then add 2.5 ml of DIC and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for reaction.

[0040] 10. After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops of test A and test B, react at 100°C for 1 minute). If the resin is colorless, it means the reaction is complete; if the resin has color, it means the condensation is incomplete and the reaction should continue.

[0041] 11. After the reaction is complete, wash the resin four times with DMF, then drain it, add a certain amount of 20% piperidine (piperidine / DMF=1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF, then drain it to check whether the protection is removed.

[0042] 12. Follow steps 9-11 to connect the following amino acids.

[0043] 13. After the last amino acid is attached, remove the protection, wash four times with DMF, and then drain the resin with methanol. Then use 95 cutting solution (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) to cut the peptide from the resin (add 10ml cutting solution per gram of resin), and centrifuge and precipitate four times with ice ether (cutting solution: ether = 1:9). Finally, use HPLC to separate and purify, and then freeze-dry to obtain a certain purity of the peptide.

[0044] 14. The purification conditions are as follows:

[0045] Stationary phase: C18 chromatographic column (commercially available product);

[0046] Mobile phase configuration:

[0047] Pump A: V(tfa) / V(water)=1 / 1000;

[0048] Pump B: V(TFA) / v(acetonitrile)=1 / 1000;

[0049] Flow rate: 10ml / min;

[0050] Retention time: between 20-30 minutes.

[0051] Example 2 Application of polypeptides

[0052] 1. Peptide inhibits the transformation of cardiac fibroblasts into myofibroblasts

[0053] In order to study the effect of peptides on myocardial fibrosis, we isolated primary cardiac fibroblasts, marked the cells by CD11b and MEFSK4 staining, removed CD11b-stained cells by flow cytometry, and purified the fibroblasts. We used 1µM Ang Ⅱ to stimulate for 24h to construct an in vitro model, and divided the cells into a control group, an EICMP1 group, an Ang Ⅱ+control group, and an Ang Ⅱ+EICMP1 group. We detected the proliferation levels of cardiac fibroblasts at 0, 24, 48, and 72h by CCK-8. The results showed that Ang Ⅱ activated the proliferation of fibroblasts ( Figure 2 A) and increased the levels of myofibroblast-related markers (COL1A1, α-SMA), while the EICMP1 polypeptide of Example 1 significantly reduced these indicators ( Figure 2 BC). After Ang Ⅱ stimulation, the expression levels of SRF and Calcineurin increased significantly, but the intervention of peptides inhibited the expression of both ( Figure 2 D).

[0054] 2. EICMP1 antagonizes myocardial fibrosis induced by hypertension in mice

[0055] EICMP1 adeno-associated virus carrying the cardiac fibroblast-specific promoter Postn and control virus were injected into the tail vein of 8-week-old C57BL / 6 male mice, and the myocardial fibrosis model was established by subcutaneous implantation of 1.5 mg / kg Ang Ⅱ mini-osmotic pump in the back of the neck. The mice were divided into control group, EICMP1 group, Ang Ⅱ+control group and Ang Ⅱ+EICMP1 group. Four weeks after modeling, the body weight and heart weight of the mice were measured, and the ratio of heart weight to body weight of the mice was calculated. The results showed that EICMP1 could reduce the heart-to-body weight ratio of the mice ( Figure 3 A); H&E staining results of heart tissue pathology sections showed that EICMP1 intervention can reduce the thickness of the heart wall and increase the size of the heart cavity ( Figure 3B); Masson staining showed that EICMP1 could reduce the level of myocardial fibrosis ( Figure 3 C).

Claims

1. A polypeptide, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. A DNA molecule encoding the polypeptide of claim 1.

3. A recombinant vector, characterized in that: It contains the DNA molecule according to claim 2.

4. A host cell, characterized in that Comprising the recombinant vector according to claim 3.

5. An expression system, characterized in that The expression system contains the recombinant vector of claim 3, or the exogenous DNA molecule of claim 2 integrated into the genome.

6. Use of the polypeptide according to claim 1 in the preparation of a drug for treating myocardial fibrosis.

7. A pharmaceutical composition, characterized in that The active ingredient is the polypeptide according to claim 1.

8. A pharmaceutical composition according to claim 7, characterized in that: The composition also contains pharmaceutical excipients.

9. A pharmaceutical composition according to claim 7, characterized in that: The dosage form of the composition is tablet, capsule or injection.

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