Polypeptide for preventing or treating myocardial ischemia-reperfusion injury and application thereof

Abstract

This invention relates to the field of biomedicine, specifically a polypeptide for the prevention or treatment of myocardial ischemia-reperfusion injury and its application. This invention obtained an exercise-induced lncRNA, MSTRG 66788.1, through high-throughput screening. Functional studies showed that its encoded SEPs 66788.1-aa effectively inhibited cardiomyocyte ferroptosis and improved cardiac function in mice with myocardial ischemia-reperfusion injury, suggesting that 66788.1-aa has the potential to replace exercise as a specific drug for improving myocardial ischemia-reperfusion injury, eliminating the risks associated with excessive exercise.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a polypeptide for the prevention or treatment of myocardial ischemia-reperfusion injury and its applications. Background Technology

[0002] Myocardial ischemia-reperfusion injury primarily occurs during revascularization after acute myocardial infarction. While reperfusion therapy is crucial for saving ischemic cardiomyocytes in the post-infarction area, paradoxically, reperfusion itself can cause additional damage. Sometimes, the severity of myocardial ischemia-reperfusion injury is even comparable to that caused by ischemia. Currently, the clinical efficacy of most treatment options remains limited. Evidence suggests that appropriate exercise can improve myocardial ischemia-reperfusion injury after revascularization in patients with acute myocardial infarction and significantly improve patient prognosis. However, exercise therapy is not without risks; excessive exercise can lead to cardiac overload, increased heart rate, elevated blood pressure, and a sharp increase in cardiac workload and myocardial oxygen consumption, thereby inducing more severe myocardial ischemia. Therefore, replacing appropriate exercise with precise drug therapy may become an effective means of improving myocardial ischemia-reperfusion injury after revascularization in patients with myocardial infarction.

[0003] Cardiomyocyte death is the direct cause of cardiac dysfunction ultimately resulting from myocardial ischemia-reperfusion injury. Ferropreservation, a redefined form of cell death in recent years, is characterized by the accumulation of lipid peroxides and an imbalance in the oxidative and antioxidant systems. Multiple studies have confirmed that myocardial ischemia-reperfusion injury can induce cardiomyocyte ferroptosis. Although ferroptosis has become a hot topic in myocardial ischemia-reperfusion injury research in recent years, its underlying mechanisms still require further investigation. The development of novel ferroptosis inhibitors targeting cardiomyocytes is considered an effective way to improve myocardial ischemia-reperfusion injury in the short term. Notably, exercise can reduce ROS accumulation and lipid peroxidation by regulating iron metabolism and enhancing antioxidant defense, suggesting that ferroptosis may be a potential mechanism by which exercise improves myocardial ischemia-reperfusion injury.

[0004] Studies have shown that long non-coding RNAs (lncRNAs) embed small open reading frames (smORFs), which can encode polypeptides (smORF-encoded polypeptides, SEPs). As endogenous polypeptides, SEPs have low toxicity, high specificity, and good biocompatibility, showing potential for treating various diseases. Given the vast size of lncRNAs, this suggests that a large number of SEPs remain to be discovered. Currently, research on SEPs' involvement in tumor regulation and their use as tumor screening biomarkers is booming; however, research on SEPs in myocardial ischemia-related diseases is still lacking. Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide (66788.1-aa) for the prevention or treatment of myocardial ischemia-reperfusion injury and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] 1. Eight-week-old adult male C57BL / 6J mice were randomly divided into a control group (sitting) and an experimental group (running). The experimental group mice ran at a speed of 12 m / min for 1 hour / day for 8 weeks.

[0008] 2. LncRNA transcriptome sequencing was performed on myocardial tissue from both groups of mice to identify run-induced differentially expressed lncRNAs. The coding potential of these differentially expressed lncRNAs was assessed using online tools such as NCBIORF and CPAT, and the target lncRNA MSTRG 66788.1 was determined based on coding capacity score and ORF length.

[0009] 3. The encoding capability of the target lncRNA MSTRG 66788.1 embedded with smORF was verified by constructing a fusion expression vector. After transfecting cells with the smORF-enhanced green fluorescent protein (eGFP) fusion expression vector, the expression of the fusion protein was observed by eGFP fluorescence signal; simultaneously, cells were collected, and the fusion protein was detected by eGFP antibody, and then the expression of the fusion protein was analyzed based on molecular weight. LncRNA MSTRG 66788.1 can indeed encode a polypeptide, and the encoded polypeptide was named 66788.1-aa.

[0010] 4. Based on the lncRNA smORF sequence, 66788.1-aa was synthesized in vitro. The polypeptide sequence is MRWPLEKRNWCRYVLVSTEEIKTSHPKVCVLTGSSPMYFLLGLKPGANG.

[0011] Based on the above technical solution, in a first aspect, the present invention provides a polypeptide, the amino acid sequence of which is shown in SEQ ID No. 1:

[0012] MRWPLEKRNWCRYVLVSTEEIKTSHPKVCVLTGSSPMYFLLGLKPGAN G (SEQ ID No. 1).

[0013] Furthermore, the nucleotide sequence of the gene encoding the polypeptide is shown in SEQ ID No. 2:

[0014] Nucleotide sequence encoding the polypeptide (ORF sequence encoding the polypeptide above LncRNA MSTRG 66788.1):

[0015] ATGCGATGGCCTCTTGAGAAGAGGAATTGGTGTAGATACGTCTTGGTAAGCACTGAAGAAATAAAGACTTCTCACCCAAAGTGTGTGTCCTCACTGGCTCTTCTCCCATGTACTTCCTGTTAGGTCTCAAACCTGGGGCAAATGGTTAA (SEQ ID No. 2).

[0016] The LncRNA sequence encoding the polypeptide, MSTRG 66788.1 (MSTRG.66788.1 73300448-3301594):

[0017] (SEQ ID No.3).

[0018] In a second aspect, the present invention provides the use of the polypeptide described above in the preparation of a medicament for the prevention or treatment of myocardial ischemia-reperfusion injury.

[0019] Furthermore, the peptide improves myocardial ischemia-reperfusion injury in a dose-dependent manner.

[0020] Furthermore, the peptide is used in the preparation of drugs that effectively reduce the degree of myocardial fibrosis and decrease the area of ​​myocardial damage.

[0021] A third aspect of the present invention provides a medicament for preventing or treating myocardial ischemia-reperfusion injury, wherein the active ingredient is a polypeptide as described above.

[0022] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0023] Furthermore, the pharmaceutically acceptable carrier is a pharmaceutically acceptable excipient, suspending agent, filler, and / or diluent.

[0024] In a fourth aspect, the present invention provides a modified polypeptide having the amino acid sequence shown in SEQ ID No. 5:

[0025] MRWPLEKRNWCRYVLVSTEEIKTSHPKVCVLTGSSPMYFLLGLKPGANGWLSEAGPVVTVRALRGTGSW(SEQ ID No.5)

[0026] In a fifth aspect, the present invention provides the use of the modified polypeptide as described above in the preparation of a medicament for the prevention or treatment of myocardial ischemia-reperfusion injury.

[0027] The advantages of this invention are:

[0028] This invention obtained an exercise-induced lncRNA, MSTRG 66788.1, through high-throughput screening. Functional studies showed that its encoded SEPs 66788.1-aa could effectively inhibit cardiomyocyte ferroptosis and improve cardiac function in mice with myocardial ischemia-reperfusion injury, suggesting that 66788.1-aa has the potential to replace exercise as a specific drug for improving myocardial ischemia-reperfusion injury and eliminate the risks associated with excessive exercise. Attached Figure Description

[0029] Figure 1 The levels of cTNT and CK-MB in peripheral blood were measured 24 days after IR / I modeling in mice.

[0030] Labeling notes: (1) sham, sham surgery control group; (2) IR / I, myocardial ischemia-reperfusion injury control group; (3) 66788.1-aa+IR / I, myocardial ischemia-reperfusion injury 66788.1-aa group (multi-point myocardial injection). N = 10:18:19 (cTNT image). N = 3:15:10 (cTNT image).

[0031] Figure 2 TTC staining of frozen sections of the heart 24 days after mouse IR / I modeling;

[0032] Notes: (1) sham, sham surgery group; (2) IR / I, myocardial ischemia-reperfusion injury group; (3) 66788.1-aa+IR / I, myocardial ischemia-reperfusion injury 66788.1-aa (4μg / μl) group (multi-point injection of myocardium). Infarct area, infarct area = (infarct area of ​​5 cardiac cross sections) / (total area of ​​5 cardiac cross sections); N = 5:16:10; *P<0.05.

[0033] Figure 3 Echocardiographic results of mice with IR / I model 30 days after induction;

[0034] Notes: (1) sham, sham surgery group; (2) IR / I, myocardial ischemia-reperfusion group; (3) 66788.1-aa+IR / I, myocardial ischemia-reperfusion 66788.1-aa (4μg / μl) group (multi-point injection of myocardium). EF, left ventricular ejection fraction; FS, left ventricular fractional shortening; N=15:39:28; *P<0.05, **P<0.01, ***P<0.001, ****P<0.001.

[0035] Figure 4 Results of masson staining on paraffin sections of mouse hearts 30 days after IR / I model;

[0036] Notes: (1) sham, sham surgery group; (2) IR / I, myocardial ischemia-reperfusion injury group; (3) 66788.1-aa+IR / I, myocardial ischemia-reperfusion injury 66788.1-aa (4μg / μl) group (multiple myocardial injection); N=5.

[0037] Figure 5 The levels of cTNT and CK-MB in peripheral blood were measured 24 days after IR / I modeling in mice.

[0038] Labeling notes: (1) sham, sham surgery control group; (2) IR / I, myocardial ischemia-reperfusion injury control group; (3) 66788.1-aa (myocardial multipoint injection) + IR / I, myocardial ischemia-reperfusion injury 66788.1-aa group (myocardial multipoint injection); (4) 66788.1-aa (tai vein) + IRI, myocardial ischemia-reperfusion injury + 66788.1-aa-targeting group (tail vein injection); N = 10:18:19 (cTNT diagram). N = 10:18:18:23. Detailed Implementation

[0039] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0040] Example 1:

[0041] To simulate human myocardial ischemia-reperfusion injury, an experimental protocol was adopted to establish a myocardial ischemia-reperfusion model at the left anterior descending artery (LAD) of C57BL / 6J mice. Left ventricular ischemia was induced for 40 minutes by ligating the proximal LAD, followed by unligation to achieve LAD reperfusion for 24 hours, thus establishing the myocardial ischemia-reperfusion injury model (IR / I model).

[0042] To evaluate the effects of 66788.1-aa on cardiac function in IR / I mice, the levels of myocardial injury markers cTnT and CK-MB in peripheral blood of mice were measured 24 hours after modeling. Figure 1 Compared with the sham group, the IR / I group showed significantly increased levels of cTnT and CK-MB in peripheral blood. Compared with the IR / I group, the 66788.1-aa+IR / I group showed a decreasing trend in peripheral blood levels of cTnT and CK-MB, and this decreasing trend became more pronounced with increasing 66788.1-aa concentration. This indicates that in the mouse IR / I model, 66788.1-aa can improve myocardial ischemia-reperfusion injury in a dose-dependent manner.

[0043] Example 2:

[0044] To simulate human myocardial ischemia-reperfusion injury, an experimental protocol was adopted to establish a myocardial ischemia-reperfusion model in the left anterior descending artery (LAD) of C57BL / 6J mice. Left ventricular ischemia was induced for 40 minutes by ligating the proximal LAD, followed by unligation to achieve LAD reperfusion for 24 hours, thus generating reperfusion injury.

[0045] To more intuitively observe cardiac damage, this invention performed triphenyltetrazolium chloride (TTC) staining analysis on five sections of the heart to assess the size and extent of the infarct area. Compared with the IR / I group, the infarct area was significantly reduced in the 66788.1-aa (concentration 4 μg / μl) + IR / I group. Figure 2 This indicates that drugs 66788.1-aa can effectively improve myocardial ischemia-reperfusion injury.

[0046] Thirty days after IR / I modeling, changes in cardiac function were assessed using echocardiography. The results showed ( Figure 3 Compared with the IR / I group, the EF and FS of the 66788.1-aa (concentration of 4ug / μl) + IR / I group also showed an upward trend, indicating that timely revascularization after myocardial infarction combined with 66788.1-aa treatment can further salvage cardiac function.

[0047] The results of masson staining 30 days after modeling showed ( Figure 4 Compared with the IR / I group, the area of ​​collagen fibers in the heart of mice in the 66788.1-aa (concentration of 4ug / μl) + IR / I group was significantly reduced and the location was more concentrated. This indicates that 66788.1-aa can effectively reduce the degree of myocardial fibrosis and reduce the area of ​​myocardial damage.

[0048] The above results indicate that 66788.1-aa can improve myocardial ischemia-reperfusion injury in mice.

[0049] A new sequence is formed by combining a polypeptide sequence and a myocardial targeting peptide sequence:

[0050] Among them, the polypeptide sequence is:

[0051] MRWPLEKRNWCRYVLVSTEEIKTSHPKVCVLTGSSPMYFLLGLKPGAN G(SEQ ID No.1)

[0052] Cardiac-targeting peptide: WLSEAGPVVTVRALRGTGSW (SEQ ID No. 4)

[0053] Peptide + cardiac targeting peptide (66788.1-aa-targeting) sequence:

[0054] MRWPLEKRNWCRYVLVSTEEIKTSHPKVCVLTGSSPMYFLLGLKPGANGWLSEAGPVVTVRALRGTGSW(SEQ ID No.5)

[0055] Multi-site injection method of polypeptide into the myocardium:

[0056] Myocardial multi-point injection is a method of directly injecting drugs or viruses into the heart tissue. The main steps of this procedure are as follows:

[0057] 1. Preparation before the experiment: Anesthetize the mice and fix them on the operating table.

[0058] 2. Heart exposure and fixation: Trim chest hair, disinfect surgical area, open chest and expose heart, fix heart position with sutures or instruments.

[0059] 3. Peptide injection: Select multiple injection points (usually 3-5 points) on the surface of the myocardium, and inject an appropriate amount of peptide (4 μg / μl peptide solution, 25 μl in total) at each point. Take care to avoid backflow of blood during injection and ensure that the injection solution is evenly distributed.

[0060] 4. Animal resuscitation and follow-up care: After the injection, the chest cavity was closed and sutured. After the mouse regained consciousness, the endotracheal tube was removed, artificial respiration was stopped, and follow-up observation and care were provided.

[0061] Tail vein injection:

[0062] The main steps of tail vein injection in mice are as follows:

[0063] 1. Preparation and Fixation: Prepare a 1ml syringe, alcohol swabs, mouse restraint device, etc. Place the mouse in the restraint device, ensuring that the mouse cannot move freely but does not affect its breathing.

[0064] 2. Vasodilation: Wipe the mouse's tail with an alcohol swab, or soak it in warm water or heat it with an infrared lamp to dilate the blood vessels.

[0065] 3. Needle insertion and injection: Locate the mouse's tail vein, generally selecting the left and right tail veins for injection. The needle should be at approximately a 30° angle to the blood vessel, with the bevel of the needle pointing upwards. Gently prick the skin, and immediately align the needle tip parallel to the blood vessel at a 45° angle. After confirming the needle is inside the blood vessel, slowly inject the medication.

[0066] 4. Hemostasis and Recovery: After injection, apply pressure to the injection site with medical cotton to stop the bleeding. Remove the mouse from the restraint and return it to its cage for recovery.

[0067] Figure 5 The results showed that tail vein administration of targeted peptide-modified peptides and multi-point administration of targeted peptides to the myocardium had comparable effects.

[0068] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

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

1.

2. The polypeptide according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the polypeptide is shown in SEQ ID No.

2.

3. The use of the polypeptide as described in claim 1 in the preparation of a medicament for the prevention or treatment of myocardial ischemia-reperfusion injury.

4. The use of the polypeptide according to claim 3 in the preparation of a medicament for the prevention or treatment of myocardial ischemia-reperfusion injury, characterized in that, The application of the described polypeptide in the preparation of drugs that effectively reduce the degree of myocardial fibrosis and decrease the area of ​​myocardial damage.

5. A drug for preventing or treating myocardial ischemia-reperfusion injury, characterized in that, Its active ingredient is the polypeptide as described in claim 1.

6. The drug according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable carriers.

7. The drug according to claim 6, characterized in that, The pharmaceutically acceptable carrier is a pharmaceutically acceptable excipient, suspending agent, filler, and / or diluent.

8. A modified polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID No.

5.

9. The use of the modified polypeptide as described in claim 8 in the preparation of a medicament for the prevention or treatment of myocardial ischemia-reperfusion injury.

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