Polypeptide and application thereof in medicine for treating myocardial fibrosis

Abstract

The invention provides a polypeptide. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. Specifically, the sequence of the polypeptide is as follows: MLSVRVAAAVARALPRRAGLVSTGA. The invention also provides a DNA (deoxyribonucleic acid) molecule for coding the polypeptide according to the claim 1. The invention also provides a recombinant vector containing the DNA molecule. The invention also provides application of the polypeptide in preparation of medicines for treating myocardial fibrosis. The invention also provides a pharmaceutical composition which is characterized in that the effective component of the pharmaceutical composition is the polypeptide. The traditional Chinese medicine composition can relieve myocardial fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a polypeptide, specifically a polypeptide and its use in a drug for treating myocardial fibrosis. Background Art

[0002] Myocardial fibrosis is a common feature of the end-stage of various heart diseases. The accumulation of extracellular matrix in the myocardium can lead to an increased risk of arrhythmias and impaired cardiac function, and eventually develop into heart failure. With the increasing aging of the global population, the total number of heart failure patients continues to increase, posing a major challenge to global health. Currently, the clinical treatment of heart failure mainly includes drug therapy, interventional therapy and heart transplantation, but these methods all have limitations to varying degrees. Therefore, there is an urgent need to slow down or reverse the development of myocardial fibrosis to maintain cardiac function and find safer and more effective strategies for the prevention and treatment of heart failure.

[0003] Myocardial fibrosis begins with the activation of cardiac fibroblasts and leads to systolic and diastolic dysfunction under many cardiac pathological and physiological conditions. Existing studies have found that ① miR-384-5p can simultaneously target key receptors of the TGF-β / Wnt pathway, significantly attenuating TGF-β-induced cardiac fibroblast activation and myocardial fibrosis; ② high-dose SSA can inhibit the TGFβ / smad pathway in fibroblasts, inducing a protective effect on fibroblasts, thereby improving myocardial fibrosis; ③ PI (peptide inhibitor) reduces the phosphorylation of PKC-θ in the heart, reduces the infiltration of T cells, and increases the expression of ZO-1, thereby improving the cardiac function of diabetic mice and reducing cardiac fibrosis.

[0004] Resistance training (RT) can improve or maintain muscle mass and strength, and has beneficial physiological and clinical effects on cardiovascular disease (CVD) and CVD risk factors. Epidemiological evidence shows that RT is associated with a lower risk of all-cause mortality and CVD morbidity and mortality. Compared with adults who did not receive RT, adults who received RT had an approximately 15% lower all-cause mortality and a 17% lower CVD risk. In recent years, with the development of relevant clinical research, the good preventive and therapeutic effects of exercise on heart failure have received increasing attention. Studies have shown that regular and moderate exercise can reduce the risk of cardiovascular disease and total mortality by lowering blood pressure, improving cholesterol, and improving insulin sensitivity, and significantly improve patient prognosis. Currently, more and more studies have found that exercise, as a safe physiological stimulus, has a good preventive and therapeutic effect on heart failure.

[0005] Research has shown that peptide drugs, often synthetic analogs of endogenous peptides, are currently a hot topic in drug development, offering advantages in terms of safety and tolerability. First, compared to full-length proteins and antibodies, short peptides are less immunogenic, more stable in vitro (allowing for longer storage at room temperature), and have better tumor or organ penetration. Furthermore, their production is more cost-effective. Second, compared to small organic molecules, peptides exhibit higher efficacy and specificity. Finally, because amino acids are the primary products of peptide metabolism, these drugs are generally less toxic. Short peptides exhibit highly selective mechanisms of action, leading to low toxicity. Furthermore, with the incorporation of various stabilizing structural modifications and novel drug delivery systems, peptide-based drugs are proving promising as single or multi-agent therapies for treating diseases. Numerous preclinical and clinical studies of peptide-based therapies are currently underway. Therefore, we have explored and interpreted exercise at the peptide level, identifying peptides with ameliorative effects on myocardial fibrosis. Summary of the Invention

[0006] In response to the above-mentioned technical problems in the prior art, the present invention provides a polypeptide and its use in a drug for treating myocardial fibrosis. The polypeptide and its use in a drug for treating myocardial fibrosis are 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, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Specifically, the sequence of the polypeptide is: MLSVRVAAAVARALPRRAGLVSTGA.

[0009] The present invention also provides a DNA molecule encoding the polypeptide according to claim 1.

[0010] The present invention also provides a recombinant vector containing the above DNA molecule.

[0011] The present invention also provides a host cell comprising the above-mentioned recombinant vector.

[0012] 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.

[0013] The present invention also provides use of the above polypeptide in preparing a drug for treating myocardial fibrosis.

[0014] The present invention also provides a pharmaceutical composition, characterized in that its active ingredient is the above-mentioned polypeptide.

[0015] Furthermore, the pharmaceutical composition also contains pharmaceutical excipients.

[0016] Furthermore, the pharmaceutical composition is in the form of tablets, capsules or injections.

[0017] Compared to existing technologies, the present invention offers significant and positive technical benefits. Unlike existing polypeptides for treating myocardial fibrosis, the polypeptide is secreted physiologically after exercise, resulting in no significant adverse reactions in the organism. Exercise can alleviate myocardial fibrosis in normal organisms and those in the early stages of the disease. In the middle and late stages of the disease, in vitro administration of the polypeptide can also alleviate myocardial fibrosis, offering additional benefits for specific populations at high risk for cardiovascular disease. Therefore, the present invention can alleviate myocardial fibrosis.

[0018] Compared with existing drugs for treating myocardial fibrosis, the polypeptide of the present invention has fewer side effects, low immunogenicity, and low energy consumption for production. In addition, the polypeptide can cover patients with multiple risk factors and can better manage and treat patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown are: A. left ventricular ejection fraction of mouse heart; B. fractional shortening of mouse heart; C. left ventricular end-systolic diameter of mouse heart; D. left ventricular end-diastolic diameter of mouse heart; E. left ventricular mass of mouse heart; F. systolic blood pressure level of mouse; G. diastolic blood pressure level of mouse.

[0020] Figure 2 The homology of the polypeptide of the present invention in vertebrates is shown. DETAILED DESCRIPTION

[0021] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0022] Example 1 Screening of polypeptides

[0023] We constructed a mouse model of exercise endurance using 8-week-old C57BL / 6 male mice as research subjects. The mice first underwent adaptive exercise training by running at a speed of 10 meters / minute for 5 minutes per day for 2 days. Then, the mice started running exercise training every day from an initial speed of 10 meters / minute, and the speed was increased by 1 meter / minute every 20 minutes until exhaustion. The exercise training lasted for 14 days. Normally raised, non-exercised mice served as the control group.

[0024] After modeling, the heart tissues of mice in each group were collected for peptide biopsy. We performed bioinformatics analysis and screening on the sequencing results based on Log2(foldchange)>1 and P<0.05, and found a peptide (Mus_musculus / 1-25) whose expression was significantly increased after exercise, and which had high homology in different species (such as Figure 2 There is currently no report on the function and mechanism of this polypeptide.

[0025] The sequence of the polypeptide is: MLSVRVAAAVARALPRRAGLVSTGA (SEQ ID NO. 1).

[0026] Example 2 Preparation method of polypeptide

[0027] The polypeptide described in Example 1 was prepared using conventional methods, specifically as follows:

[0028] Peptides are synthesized from the C-terminus to the N-terminus.

[0029] 1. Weigh 3 g of RINK resin (degree of substitution 0.3 mmol / g) into a 150 ml reactor and soak it with 50 ml of dichloromethane (DCM).

[0030] After 2.2 hours, the resin was washed with nitrogen-dimethylformamide (DMF) 3 times the volume of the resin, and then dried. This was repeated four times. The resin was dried and set aside for use.

[0031] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake on a decolorization shaker for 20 minutes to remove the Fmoc protecting group from the resin. After deprotection, wash the resin four times with DMF (3 times the resin volume) and then drain.

[0032] 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.

[0033] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxybenzotriazole (HOBT) into a 50ml centrifuge tube, add 20ml of DMF to dissolve them, then add 3ml of N,N-diisopropylcarbodiimide (DIC) and shake for 1min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction.

[0034] After 6.2 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.

[0035] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake on a decolorization shaker for 20 minutes to remove the Fmoc protecting group from the resin. After deprotection, wash the resin four times with DMF and then drain.

[0036] 8. Take a small amount of resin and test it using 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.

[0037] 9. Weigh an appropriate amount of the second amino acid and HOBT into a 50ml centrifuge tube, add 25ml of DMF to dissolve them, then add 2.5ml of DIC and shake for 1min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction.

[0038] After 10.1 hours, take a small amount of resin for testing using the ninhydrin method (two drops each of Test A and Test B, react at 100°C for 1 minute). If the resin is colorless, the reaction is complete; if the resin has color, the condensation is incomplete and the reaction should be continued.

[0039] 11. After the reaction is complete, wash the resin four times with DMF and drain. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake on a decolorization shaker for 20 minutes to remove the Fmoc protecting group from the resin. After deprotection, wash the resin four times with DMF and drain to check for protection.

[0040] 12. Follow steps 9-11 to connect the following amino acids.

[0041] 13. After the last amino acid is attached, remove the protection and wash the resin four times with DMF. Drain the resin with methanol. Cleave the peptide from the resin using a 95% cleavage buffer (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) (10 ml of cleavage buffer per gram of resin). Centrifuge four times with icy ether (cleavage buffer: ether = 1:9). Finally, purify the peptide using HPLC and lyophilize to obtain a desired purity.

[0042] 14. Purification conditions are as follows:

[0043] Stationary phase: C18 column (commercially available);

[0044] Mobile phase configuration:

[0045] PumpA: V(tfa) / V(water)=1 / 1000;

[0046] PumpB: V(TFA) / v(acetonitrile)=1 / 1000;

[0047] Flow rate: 10 ml / min;

[0048] Retention time: between 20-30 minutes.

[0049] Example 3 Application of polypeptides

[0050] (1) Experimental animals and groups:

[0051] Eight-week-old C57BL / 6 male mice were randomly divided into four groups (n=3):

[0052] Control group: conventional feeding without intervention;

[0053] Peptide intervention group: peptide-related virus (prepared by the method in Example 2) was injected via tail vein;

[0054] AngII group: The model was established by subcutaneously implanting a 1.5 mg / kg / d AngII mini-osmotic pump in the back of the neck;

[0055] AngII+peptide intervention group: AngII modeling + tail vein injection of peptide-related virus;

[0056] (2) Cardiac ultrasound examination:

[0057] After the modeling, the mice were subjected to echocardiography to measure parameters such as left ventricular mass (LVMass), left ventricular end-systolic diameter (D;s), end-diastolic diameter (D;d), ejection fraction (EF%), and fractional shortening (FS%).

[0058] (3) Blood pressure measurement:

[0059] The systolic blood pressure and diastolic blood pressure of the tail artery of each group of mice were measured using an intelligent non-invasive blood pressure meter.

[0060] (4) Results:

[0061] Compared with the control group, the left ventricular ejection fraction (EF%) and fractional shortening (FS%) of the hearts of mice in the AngII group were significantly decreased ( Figure 1 A. Figure 1 B), both the end-systolic diameter (D;s) and the end-diastolic diameter (D;d) increased ( Figure 1 C. Figure 1 D), indicating that the left ventricular myocardial contractility was impaired; in terms of structural remodeling, the left ventricular mass (LVMass) increased significantly ( Figure 1 E); In terms of arterial blood pressure, both systolic and diastolic blood pressure increased significantly ( Figure 1 F. Figure 1G). However, there was no significant change in the peptide intervention group.

[0062] The ejection fraction (EF%) and fractional shortening (FS%) of the AngII+peptide intervention group were significantly improved compared with the AngII group ( Figure 1 A. Figure 1 B), the end-systolic diameter (D;s) and the end-diastolic diameter (D;d) were reduced ( Figure 1 C, 1D), indicating that left ventricular systolic function improved; in terms of structural remodeling, the left ventricular mass (LVMass) in the AngII+ peptide intervention group was significantly reduced ( Figure 1 E); In terms of arterial blood pressure, the systolic blood pressure of the AngII+ peptide intervention group ( Figure 1 F) and diastolic blood pressure ( Figure 1 G) were significantly lower than those in the AngII group ( Figure 1 ).

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 according to claim 3, or the exogenous DNA molecule according to claim 2 integrated into the genome.

6. Use of the polypeptide according to claim 1 in the preparation of a medicament 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 pharmaceutical composition also contains pharmaceutical excipients.

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

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