Application of PTMA Short Peptide in the Preparation of Drugs for Treating Myocardial Infarction

Abstract

The present invention provides the use of PTMA short peptides in the preparation of drugs for treating myocardial infarction, and the PTMA short peptides are PTMA 2-29, PTMA 102-111, or a combination of PTMA 2-29 and PTMA 102-111. The PTMA short peptides provided by the present invention can reduce the area of myocardial infarction, promote the repair of the pumping function of the damaged heart, and have a long-term therapeutic effect on the cardiac function after myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and relates to the use of short peptide drugs for treating myocardial infarction, specifically to the application of PTMA short peptide in the preparation of drugs for treating myocardial infarction. Background Art

[0002] Myocardial infarction is one of the main factors leading to high mortality and morbidity globally. After a myocardial infarction occurs, a large number of myocardial cells in the infarcted area die, resulting in impaired cardiac function. Since the damaged heart of the patient cannot naturally recover its function, myocardial infarction progresses to heart failure. Most drug interventions for cardiovascular diseases such as acute myocardial infarction focus on improving hemodynamics (reducing afterload, controlling blood pressure and blood volume) or changing myocardial cell function, and can only repair cardiac function to a limited extent, restricting their clinical translation. Therefore, there is still a lack of effective drugs for treating myocardial infarction clinically.

[0003] Therefore, developing a drug that can promote cardiac function repair and be translated into clinical application has become an urgent problem to be solved. Summary of the Invention

[0004] To solve the above problems, the purpose of the present invention is to provide the application of PTMA short peptide in the preparation of drugs for treating myocardial infarction. This PTMA short peptide can reduce the area of myocardial infarction and promote the functional repair of damaged myocardial tissue.

[0005] To achieve the above purpose, the present invention provides the application of PTMA short peptide in the preparation of drugs for treating myocardial infarction. This PTMA short peptide is PTMA 2-29, PTMA 102-111, or a combination of PTMA 2-29 and PTMA 102-111; wherein, the amino acid sequence of PTMA 2-29 is as shown in Seq ID No.1, and the amino acid sequence of PTMA 102-111 is as shown in Seq ID No.2 or Seq ID No.3; the mass ratio of the combination of PTMA 2-29 and PTMA 102-111 is 1:1.

[0006] Preferably, the PTMA short peptide is of human or murine origin.

[0007] Preferably, the treatment of myocardial infarction is to reduce the area of myocardial infarction and promote the functional repair of damaged myocardial tissue.

[0008] The beneficial effects of the present invention are as follows:

[0009] The present invention provides the application of PTMA short peptide in the preparation of drugs for treating myocardial infarction. By injecting PTMA short peptide after myocardial infarction, it can reduce the area of myocardial infarction and promote the functional repair of damaged myocardial tissue, and has a long-term therapeutic effect on cardiac function after myocardial infarction. Description of the Drawings

[0010] Figure 1 Flow chart for preparing a myocardial infarction surgery model for adult mice and performing PTMA peptide treatment.

[0011] Figure 2 Pharmacodynamic fluorescence photographs of PTMA peptide in cardiomyocytes detected by immunofluorescence staining of cardiomyocytes in the hearts of adult mice 2 h and 3 d after myocardial infarction surgery.

[0012] Figure 3 Survival curves of adult mice injected with PTMA peptide and saline negative control group after myocardial infarction.

[0013] Figure 4 Typical echocardiogram of cardiac function detected by a small animal ultrasound instrument after 8 weeks of combined treatment with PTMA2-29, PTMA102-111 and PTMA2-29 and PTMA102-111 peptides alone in adult mice after myocardial infarction.

[0014] Figure 5 Result graphs of cardiac function detected by a small animal ultrasound instrument at different time points after combined treatment with PTMA2-29, PTMA102-111 and PTMA2-29 and PTMA102-111 peptides in adult mice after myocardial infarction.

[0015] Figure 6 Result graphs of cardiac function minus 4 days after myocardial infarction at different time points after combined treatment with PTMA2-29, PTMA102-111 and PTMA2-29 and PTMA102-111 peptides in adult mice after myocardial infarction.

[0016] Figure 7 Result graph of ejection fraction of the heart of adult mice at 2 months after myocardial infarction minus the ejection fraction at 3 days after myocardial infarction.

[0017] Figure 8 Result graph of ventricular wall thickness of the heart of adult mice at 2 months after myocardial infarction minus the ventricular wall thickness of the heart at 3 days after myocardial infarction.

[0018] Figure 9 Result graph of heart cavity size of adult mice at 2 months after myocardial infarction minus the heart cavity size at 3 days after myocardial infarction.

[0019] Figure 10 Statistical analysis graph showing cardiac fibrosis and myocardial infarction area in heart sections of adult mice 8 weeks after myocardial infarction surgery. Detailed implementation methods

[0020] The embodiments of the present invention will be described in detail and comprehensively below, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0021] The human Ptma gene is located on chromosome 2, and the mouse Ptma gene is located on chromosome 1. The protein encoded by the Ptma gene is called thymosin α. The proteins encoded by the Ptma genes of humans and mice are both composed of 111 amino acid sequences.

[0022] The short peptide PTMA 2-29 used in the present invention is composed of the N-terminal 2-29 amino acid sequences of thymosin α, and the amino acid sequence is as shown in Seq ID No.1: SDAAVDTSSEITTKDLKEKKEVVEEAEN. This sequence is completely conserved in humans and mice. This sequence is a commercial product, also known as thymosin α1 (PTa1), thymic 28 peptide. This sequence is clinically used to treat chronic hepatitis B and enhance immune system reactivity. In order to distinguish the synthetic short peptide from the commercial short peptide PTa1, the synthetic short peptide of the N-terminal 28 amino acids of PTMA in the present invention is named PTMA 2-29.

[0023] The PTMA 102-111 used in the present invention is a short peptide of 10 amino acids at the C-terminal 102-111 positions of thymosin α, with a difference of one amino acid between humans and mice. Among them, the amino acid sequence of mouse-derived PTMA 102-111 is as shown in Seq ID No.2: TKKQKTEEDD. The amino acid sequence of human-derived PTMA 102-111 is as shown in Seq ID No.3: TKKQKTDEDD.

[0024] Materials.

[0025] 1. C57BL / 6J adult mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0026] 2. Polypeptide synthesis was completed by Beijing Zexiyuan Biotechnology Co., Ltd.

[0027] 3. Sakura OTC embedding agent is of Sakura brand, and the product number is 4583.

[0028] 4. Sirius red staining kit was purchased from Solarbio, and the product number is G1472.

[0029] 5. Fast green staining solution was purchased from sigma, and the product number is 68724.

[0030] 6. Goat serum was purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd., and the product number is ZLI-9056.

[0031] 7. The Alexa Fluor 594 donkey anti-mouse immunofluorescent antibody was purchased from Invitrogen, with the product number A-32742.

[0032] 8. The cTNT antibody was purchased from the Developmental Studies Hybridoma Bank (DSHB) in the United States, with the product number ea5 / 26 / 61.

[0033] The ethical reviews of the animal experiments involved in the examples were all approved by the Laboratory Animal Ethics Committee of Fuwai Hospital, Chinese Academy of Medical Sciences, and complied with the relevant regulations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health (NIH) in the United States. The experimental animals were housed in the Animal Experiment Center of the State Key Laboratory of Cardiovascular Diseases, Fuwai Hospital, Chinese Academy of Medical Sciences.

[0034] Example 1 Synthesis of PTMA Peptide

[0035] The PTMA peptide PTMA 2-29 shown in Seq ID No.1 and PTMA 102-111 shown in Seq ID No.2 were entrusted to Beijing Zexiyuan Biotechnology Co., Ltd. for synthesis. To facilitate the pharmacodynamic detection of the PTMA peptide, FITC was labeled at the N-terminus of the PTMA peptide during synthesis, and amidation treatment was performed at the C-terminus. The purity of both polypeptides was >95%.

[0036] Before use, PTMA 2-29 and PTMA 102-111 were respectively dissolved in physiological saline to prepare a PTMA 2-29 solution with a concentration of 5 μg / μl and a PTMA 102-111 solution with a concentration of 5 μg / μl. The drug concentrations of the two short peptide drugs in the combined use of PTMA 2-29 and PTMA 102-111 were both 5 μg / μl (dissolved in physiological saline at a ratio of 1:1).

[0037] Example 2 Therapeutic Effect of PTMA Peptide on Myocardial Infarction in Adult Mice

[0038] Myocardial infarction surgery was performed on adult 8-week-old C57BL / 6J mice (P56), and PTMA peptide was simultaneously injected into the myocardial layer for cardiac treatment after myocardial infarction. The specific operation steps of the myocardial infarction surgery are as follows:

[0039] 1. Adult mice were intraperitoneally injected with tribromoethanol solution (200 μl / 20 g).

[0040] 2. The anesthetized mice were taken out and fixed on the operating table in a right lateral supine position with medical tape; first, the chest skin of the mice was disinfected from the inside to the outside with 5% iodophor cotton balls, and then disinfected with 75% alcohol cotton balls.

[0041] 3. Using an ophthalmic scissors, make an incision in the skin on the left 4th intercostal space of the mouse. Bluntly separate the subcutaneous tissue and muscle into the chest with a microsurgical scissors. Use an ophthalmic forceps to open the thoracic cavity, gently tear the pericardium to expose the heart.

[0042] 4. Ligate the left anterior descending coronary artery: At 2 mm below the lower edge of the left atrial appendage, a bright red vessel with an inverted Y-shaped course can be seen. The vessel running towards the apex of the heart is the left anterior descending coronary artery of the mouse. Precisely ligate the left anterior descending coronary artery with 7 / 0 polyester thread. Immediately, the ventricular muscle below the ligature becomes pale, indicating successful model establishment.

[0043] 5. After wiping the bloodstains with a sterilized cotton swab, suture the ribs and skin in a figure-eight pattern with 6 / 0 polyester thread, and wipe with a 75% alcohol cotton ball to prevent infection.

[0044] 6. Place the postoperative mouse on a warming table at 37 °C for rewarming; wait for the mouse to recover spontaneous movement, then put it back into the cage for feeding, and perform echocardiographic examination of cardiac function at appropriate time points.

[0045] 7. The sham operation group model completes the above steps without ligating the coronary artery.

[0046] On the day of surgery, different mice were respectively injected with 30 μL of PTMA 2 - 29 solution, PTMA 102 - 111 solution, or a mixed solution of PTMA 2 - 29 and PTMA 102 - 111 into the myocardium. The injection was evenly distributed at 5 points from the infarct periphery to the apex. The control group was injected with an equal volume of normal saline (Saline). The pharmacokinetics of different PTMA short peptides injected were analyzed by sampling at 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h after surgery. Cardiac function was detected at 3 d, 1 w, 2 w, 4 w, 6 w, and 8 w after myocardial infarction. Cardiac samples were taken 8 weeks after surgery. The schematic diagram of the experimental procedure is as Figure 1 shown: First, construct a myocardial infarction model (MI surgery), then inject PTMA short peptides (PTMA peptide injection), dynamically monitor the cardiac function of mice by echocardiography at multiple time points after myocardial infarction, and take cardiac samples 8 w after myocardial infarction. Quantify the myocardial cell area by immunofluorescence staining and detect myocardial remodeling and cardiac repair by Sirius red and fast green staining.

[0047] Figure 2Frozen sections were taken at 2 h (MI-2 h) and 3 d (MI-3 d) after myocardial infarction, and immunofluorescence staining of cardiomyocytes was performed to detect the pharmacokinetics of PTMA peptides in cardiomyocytes. It can be seen that the injected PTMA2-29 and PTMA102-111 were highly expressed in cardiomyocytes 2 hours after myocardial infarction and were still highly expressed 3 d (72 h) after myocardial infarction.

[0048] Figure 3 Survival curves of mice injected with PTMA peptides and saline negative control group after myocardial infarction. It can be seen that the injection groups of PTMA2-29, PTMA102-111 and the mixture of PTMA2-29 and PTMA102-111 could all improve the survival rate of adult mice with myocardial infarction.

[0049] Figure 4 Typical images of cardiac function detected by a small animal ultrasound instrument 8 weeks after treatment with PTMA2-29, PTMA102-111 or the mixture of PTMA2-29 and PTMA102-111 in adult mice after myocardial infarction. It can be seen that the cardiac function was well restored in the PTMA2-29 single treatment group 8 weeks after myocardial infarction, and the cardiac recovery effects of the PTMA102-111 and the mixture of PTMA2-29 and PTMA102-111 treatment groups were second.

[0050] Figure 5 Results of cardiac function detected by a small animal ultrasound instrument at 3 d, 1 w, 2 w, 4 w, 6 w and 8 w after treatment with PTMA2-29, PTMA102-111 or the mixture of PTMA2-29 and PTMA102-111 in adult mice after myocardial infarction. Figure 5 It can be seen that the injection of peptides in the sham operation group (sham) had no adverse effects on the heart. Compared with 4 days after myocardial infarction, the ejection fraction (EF) in the saline control group continued to decline to about 37%, while the ejection fraction in the PTMA2-29 treatment group increased to about 58%, the body blood fraction in the combined treatment group of PTMA2-29 and PTMA102-111 increased to about 50%, and the ejection fraction of PTMA102-111 remained at about 43%. It shows that PTMA2-29, PTMA102-111 and the mixture of PTMA2-29 and PTMA102-111 can all improve the cardiac function after adult myocardial infarction. The repair effect of PTMA2-29 peptide on cardiac function is the most obvious, while the repair effects of PTMA102-111 and the mixture of PTMA2-29 and PTMA102-111 are second.

[0051] Figure 6The results of subtracting the cardiac function at 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks, and 8 weeks after treatment with PTMA2-29, PTMA102-111, or a mixture of PTMA2-29 and PTMA102-111 from that at 3 days after myocardial infarction in adult mice were used to reflect the recovery of cardiac function after myocardial infarction. From Figure 6 Analyzing the repair of cardiac function after postoperative injection of PTMA short peptides, it can be seen that the cardiac function of the PTMA2-29 single-treatment group continued to be repaired, and the ejection fraction of the heart continued to increase. The PTMA102-111 single-treatment group mainly maintained the cardiac function from deteriorating, while the cardiac function of the PTMA2-29 and PTMA102-111 mixture treatment group was also continuously repaired, but the repair effect was inferior to that of the PTMA2-29 single-treatment group.

[0052] From Figures 7 to 9 It can be seen that by 8 weeks after myocardial infarction, the ejection fraction of the PTMA2-29 treatment group recovered by about 17% compared with that at 3 days after myocardial infarction, the ejection fraction of the PTMA2-29 and PTMA102-111 mixture treatment group recovered by about 8%, and the ejection fraction of PTMA102-111 could maintain the ejection fraction of the heart unchanged after myocardial infarction. The ejection fraction of the normal saline control group decreased by about 8%. This further illustrates the role of PTMA2-29 in repairing cardiac function after myocardial infarction at 8 weeks.

[0053] The left ventricular posterior wall thickness (LVPW) of the heart increased and the left ventricular internal diameter (LVID) decreased significantly in the PTMA2-29 single-treatment group and the PTMA2-29 and PTMA102-111 mixture treatment group. While PTMA102-111 maintained the left ventricular posterior wall thickness and the size of the cardiac cavity. It shows that both the PTMA2-29 single-treatment group and the PTMA2-29 and PTMA102-111 mixture treatment group can significantly improve the functional repair of the infarcted heart, but in terms of effect, the PTMA2-29 single-treatment group is better than the PTMA2-29 and PTMA102-111 mixture treatment group. The PTMA102-111 treatment group can maintain the cardiac function after myocardial infarction and prevent the damaged heart from developing heart failure.

[0054] Sirius red and fast green staining maps of cardiac sections taken at 200-μm intervals from the ligation site towards the direction of myocardial infarction in adult mice 8 weeks after myocardial infarction. Statistical results of cardiac fibrosis and myocardial infarction area after treatment with PTMA2-29, PTMA102-111, and a mixture of PTMA2-29 and PTMA102-111 are as Figure 10 shown. From Figure 10It can be seen that after myocardial infarction surgery in adult mice, the myocardial tissue was repaired after 8 weeks of treatment with PTMA2-29, PTMA102-111, and the mixture of PTMA2-29 and PTMA102-111, the occurrence of cardiac fibrosis was alleviated, and the cardiac infarction area was significantly reduced.

[0055] As can be seen from the above examples, after treatment with PTMA2-29 provided by the present invention, the myocardial tissue was effectively repaired, the cardiac infarction area was reduced, and the cardiac function was significantly improved. After treatment with PTMA102-111, the cardiac function after myocardial infarction could be retained, and the damaged heart was prevented from developing into heart failure. When the subject is a human, PTMA102-111 is replaced with the sequence shown in human-derived Seq ID No. 3. The PTMA short peptide provided by the present invention has a long-term therapeutic effect on the cardiac function after myocardial infarction.

[0056] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. Application of PTMA short peptide in preparing a drug for treating myocardial infarction, characterized in that, The PTMA short peptide is PTMA 2-29, PTMA 102-111, or a combination of PTMA 2-29 and PTMA 102-111; Among them, the amino acid sequence of PTMA 2-29 is shown in Seq ID No.1, and the amino acid sequence of PTMA 102-111 is shown in Seq ID No.2 or Seq ID No.3; The mass ratio of the combination of PTMA 2-29 and PTMA 102-111 is 1:

1.

2. The application according to claim 1, wherein, The PTMA short peptide is of human or murine origin.

3. The application according to claim 1, characterized in that The treatment of myocardial infarction is to reduce the area of myocardial infarction and promote the recovery of cardiac pumping function after myocardial infarction.

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