A selenium-modified myocardial protective polypeptide and its preparation method and application

Abstract

The present invention belongs to the field of active peptides, and specifically relates to a selenium-modified myocardial protective polypeptide, a preparation method and an application thereof. The selenium-modified myocardial protective polypeptide is prepared by chelating the Elabela-Apelin-TAT fusion protein shown in SEQ ID NO: 6 with sodium selenite (Na2SeO3); the selenium peptide chelate has the effects of significantly reducing myocardial damage, reducing the area of ​​myocardial infarction, improving antioxidant capacity, inhibiting inflammatory response and long-term anti-myocardial fibrosis, and can be used to treat and / or prevent acute myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the field of active peptides, and in particular relates to a selenium-modified myocardial protective polypeptide and a preparation method and application thereof. Background Art

[0002] Cardiovascular disease, especially acute myocardial infarction (AMI), is one of the leading causes of sudden death worldwide. Myocardial infarction is mainly caused by coronary artery obstruction, which leads to ischemia and hypoxia of myocardial cells, and then triggers cell apoptosis, inflammatory response and fibrosis. Currently, the treatment methods for AMI mainly include thrombolytic therapy, interventional therapy and drug therapy. However, while these treatments restore myocardial blood supply, they may cause ischemia-reperfusion injury, aggravate oxidative stress response and inflammatory response, and ultimately affect myocardial repair and functional recovery. Therefore, the development of new treatment strategies that can effectively protect the myocardium and reduce ischemia-reperfusion injury is of great clinical value.

[0003] Active peptides show promising application prospects in the treatment of cardiovascular diseases. Among them, Elabela and Apelin, as endogenous peptides, have been shown to play an important role in regulating cardiovascular function, protecting cardiomyocytes, and alleviating myocardial fibrosis. In addition, TAT (trans-activator of transcription) peptides can improve the cell penetration ability of peptides, enhance their stability and efficiency in vivo. However, the use of these peptides alone still has certain limitations, such as rapid metabolic rate, poor in vivo stability, and insufficient activity. Therefore, how to improve their biological activity and clinical application value through chemical modification or structural optimization is one of the focuses of current research.

[0004] Selenium (Se) is an essential trace element with strong antioxidant and anti-inflammatory properties. It can reduce oxidative stress damage by regulating the activity of antioxidant enzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). In addition, selenium can also exert a cardioprotective effect by inhibiting the release of proinflammatory factors and reducing inflammatory responses. In recent years, selenium modification technology has been widely used in the functional optimization of proteins and peptides to enhance their biological activity and stability. Therefore, the introduction of selenium into the structure of cardioprotective peptides is expected to synergistically exert antioxidant, anti-inflammatory, anti-fibrotic and other cardioprotective effects, further enhancing their therapeutic effects.

[0005] Based on the above background, the present invention provides a selenium-modified myocardial protective polypeptide and its preparation method and application. The polypeptide is prepared by chelating Elabela-Apelin-TAT fusion protein with sodium selenite (Na2SeO3), combining the synergistic effect of Elabela and Apelin in cardiovascular protection, while utilizing selenium modification technology to enhance its antioxidant and anti-inflammatory properties. The selenium peptide chelate of the present invention can significantly reduce myocardial damage, reduce the area of ​​myocardial infarction, and improve antioxidant capacity, providing an innovative strategy for the adjuvant treatment and prevention of acute myocardial infarction. Summary of the Invention

[0006] To address these issues, the present invention first provides a selenium-modified cardioprotective peptide, prepared by chelating an Elabela-Apelin-TAT fusion protein with inorganic selenium. This peptide exhibits significant cardioprotective effects, alleviating myocardial damage, reducing the area of ​​myocardial infarction, enhancing antioxidant capacity, and inhibiting inflammatory responses.

[0007] In certain embodiments, the Elabela-Apelin-TAT fusion protein is selected from the polypeptide shown in SEQ ID NO: 6. The inorganic selenium is preferably sodium selenite (Na2SeO3), and the selenium element is combined with the Elabela-Apelin-TAT fusion protein through a chelation reaction to obtain a selenium-modified myocardial protective polypeptide chelate.

[0008] The present invention also provides a preparation method, which comprises the following steps:

[0009] 1) Providing an Elabela-Apelin-TAT fusion protein as shown in SEQ ID NO: 6;

[0010] 2) Mixing Elabela-Apelin-TAT fusion protein with sodium selenite (Na2SeO3);

[0011] 3) Under specific reaction conditions, the Elabela-Apelin-TAT fusion protein is subjected to a chelation reaction with sodium selenite to prepare a selenium-modified myocardial protective polypeptide chelate.

[0012] The present invention finally provides an application: the selenium-modified cardioprotective polypeptide can be used to prepare a medicament for treating and / or preventing acute myocardial infarction. By regulating antioxidant stress and inflammatory responses, the selenium-modified polypeptide can effectively improve myocardial damage and reduce the area of ​​myocardial infarction, demonstrating a favorable myocardial protective effect.

[0013] In certain embodiments, the selenium-modified cardioprotective polypeptide can be used as a single drug preparation or in combination with other drugs to further enhance the therapeutic effect.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects:

[0015] Significantly improve myocardial damage: Selenium-modified cardioprotective peptides can significantly reduce myocardial damage caused by acute myocardial infarction.

[0016] Reducing the area of ​​myocardial infarction: The polypeptide can effectively reduce the area of ​​myocardial infarction and improve cardiac function.

[0017] Improve antioxidant capacity: Through the introduction of selenium, the peptide chelate can improve antioxidant capacity, thereby reducing the damage of oxidative stress to myocardial cells.

[0018] Inhibit inflammatory response: The polypeptide of the present invention can significantly inhibit the inflammatory response triggered during myocardial infarction, further protecting myocardial tissue.

[0019] Anti-myocardial fibrosis: The polypeptide of the present invention can significantly reduce myocardial collagen deposition, inhibit excessive production of type I collagen, and improve myocardial fibrosis.

[0020] Safety and stability: The selenium-modified polypeptide has good biocompatibility and stability and is suitable for clinical application.

[0021] The present invention is the first to chelate the Elabela-Apelin-TAT fusion protein with selenium to prepare a polypeptide with myocardial protective function, providing a new and effective auxiliary treatment for the treatment and prevention of acute myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Ni-NTA column peak diagram and SDS-PAGE detection of Elabela-Apelin-TAT fusion protein.

[0023] Figure 2 Potentiometric analysis of Elabela-Apelin-TAT fusion protein and its selenium chelate.

[0024] Figure 3 Evaluation of cardiac function in MI / RI mouse model by Elabela-Apelin-TAT fusion protein and its selenium chelate.

[0025] Figure 4 Analysis of the effects of Elabela-Apelin-TAT fusion protein and its selenium chelate on the determination of myocardial infarction size in the MI / RI mouse model.

[0026] Figure 5 Analysis of the antioxidant and anti-inflammatory abilities of Elabela-Apelin-TAT fusion protein and its selenium chelate in MI / RI mouse model. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0028] Example 1 Preparation of Elabela-Apelin-TAT Fusion Protein

[0029] According to the sequence recorded by NCBI ELABELA [Homo sapiens] (GenBank: AHW47894.1): MRFQQFLFAFFIFIMSLLLISGQRPVNLTMRRKLRKHNCLQRRC MPLHSRVPFP (SEQ ID NO: 1);

[0030] According to the sequence described in NCBI apelin [Homo sapiens] (GenBank: AAF25815.1): MNLRLCVQALLLLWLSLTAVCGGSLMPLPDGNGLEDGNVRHLVQPR GSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF (SEQ ID NO: 2);

[0031] TAT membrane-penetrating peptide: YGRKKRRQRRR (SEQ ID NO: 3);

[0032] Design of Elabela-Apelin-TAT fusion protein: Construct an Elabela-linker-Apelin-linker-TAT module, wherein the TAT transmembrane peptide is YGRKKRRQRRR (SEQ ID NO: 4); the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 5), and its amino acid sequence is as follows:

[0033] MRFQQFLFAFFIFIMSLLLISGQRPVNLTMRRKLRKHNCLQRRCPLHSRVPFPGGGGSGGGGSGGGGSGGGGSMNLRLCVQALLLLWLSLTAVCGGSLMPLPDGNGLEDGNVRHLVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPFGGGGSGGGGSGGGGSGGGGSYGRKKRRQRRR (SEQ ID NO: 6).

[0034] The recombinant expression plasmid pET-28a(+)-Elabela-Apelin-TAT was synthesized by GenScript Biotech Co., Ltd. Competent Escherichia coli BL21(DE3) was prepared and co-cultured with the recombinant expression plasmid pET-28a(+)-Elabela-Apelin-TAT. Transformation was performed using the heat shock method. After transformation, the bacteria were inoculated into LB medium containing ampicillin and cultured with shaking at 37°C overnight. Successfully transformed E. coli colonies were selected with ampicillin. Positive clones were selected and cultured on a small scale. The plasmid was extracted using a plasmid extraction kit for verification. Positive clones screened were inoculated into LB medium containing ampicillin (50 μg / mL) and cultured in a shaking incubator at 37°C until the optical density (OD600) of the culture reached 0.6-0.8, which is the optimal induction time for protein expression. When the culture reached an appropriate OD600 value, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce expression of the fusion protein and cultured for 2-6 hours. After induction, the culture was continued at 37°C, ensuring that the shaker speed remained within a reasonable range to ensure uniform bacterial growth. After induction, protein-expressing bacteria were harvested. The specific procedure was to centrifuge the culture at 4000 × g for 10 minutes. The supernatant was discarded, and the cell pellet was retained. The cell pellet was resuspended in lysis buffer containing PBS (pH 7.4). The cells were lysed by ultrasonication. The ultrasonication power and duration were adjusted according to the bacterial cell size, and the temperature was generally 0-4°C to minimize protein degradation. After the lysis is completed, the cell debris is removed by centrifugation, and the supernatant is taken for subsequent purification. Based on the fusion protein with a 6×His tag, affinity chromatography is used for purification, and the operation is as follows: Affinity chromatography is performed using a Ni-NTA column. First, wash the column with PBS buffer (pH 7.4) to remove impurities. Pre-wash with PBS buffer containing 20mM imidazole to remove unbound miscellaneous proteins. The cell lysate containing the fusion protein is loaded onto the Ni-NTA affinity column and incubated for 30-60 minutes to allow the His tag to bind to the Ni-NTA resin. Non-specifically bound miscellaneous proteins are removed by drainage elution. Gradient elution is performed with PBS buffer containing 20mM, 50mM, 100mM, and 250mM imidazole. Each elution fraction is collected and analyzed using SDS-PAGE to confirm the amount of protein in each fraction. The Elabela-Apelin-TAT fusion protein in the fraction is further separated and purified by an AKTA purifier to meet the requirements of subsequent experiments. See. Figure 1 .

[0035] Figure 1The results showed that the molecular weight of the Elabela-Apelin-TAT fusion protein was approximately 19.24 kDa, which was in line with expectations, and the protein had a high purity and could be used for subsequent biological activity experiments.

[0036] Example 2 Preparation and Identification of Elabela-Apelin-TAT Fusion Protein Selenium Chelate

[0037] The purified Elabela-Apelin-TAT fusion protein was dissolved in PBS buffer at a concentration of 6 mg / mL, maintaining a pH of 7.0 to ensure protein stability. Next, an appropriate amount of sodium selenite (Na2SeO3) was added to a 2 mM sodium selenite solution using deionized water, and the pH was adjusted to 7.0 to enhance its solubility and reactivity. The sodium selenite solution was slowly added dropwise to the Elabela-Apelin-TAT fusion protein solution with gentle stirring to ensure uniform distribution and reaction with the protein. The reaction was carried out at 20-25°C for 24 hours. After the reaction, the mixed solution was transferred to a dialysis bag (800 Da molecular weight cutoff) and dialyzed against a container containing buffer to remove unreacted sodium selenite. The dialysis process lasted for 24 hours, with the dialysate replaced every 4 hours with deionized water. After dialysis, the obtained protein-selenium complex was freeze-dried to obtain Elabela-Apelin-TAT fusion protein selenium chelate powder, which was stored at -20°C.

[0038] The selenium content is determined using the 3,3′-diaminobenzidine colorimetric method: the chelate sample is mixed with a 3,3′-diaminobenzidine (DAB) solution and reacted in an acidic environment. Under acidic conditions, selenium reacts with DAB to form a dark blue complex that has a distinct absorption peak at a specific wavelength. By measuring the absorbance of this absorption peak, the selenium content in the sample can be calculated based on the standard curve. The selenium chelation rate is calculated according to formula (1):

[0039] Selenium chelation rate (%) = (A1 / A2) × 100% (1)

[0040] Wherein: A1 is the total amount of selenium in the Elabela-Apelin-TAT fusion protein selenium chelate g; A2 is the total amount of selenium in the reaction system g; the results show that the selenium chelation rate is 73+0.32%.

[0041] Potential analysis:

[0042] The potential changes of Elabela-Apelin-TAT fusion protein and its selenium chelate were measured by Nano-2sZEN3600 nanoparticle size potentiometry to evaluate whether the chelation reaction was successful. 1.0 mg of Elabela-Apelin-TAT fusion protein and Elabela-Apelin-TAT fusion protein selenium chelate were dissolved in 1 mL of deionized water at a concentration of 1.0 mg / mL. After reacting at 37°C for 10 minutes, the potential was measured using a potentiometry instrument. Each sample was measured 3 times and the average value was taken to compare the potential difference. If the potential change is significant, it indicates that selenium has successfully chelated with the Elabela-Apelin-TAT fusion protein; if the change is small, the chelation reaction is not complete, see Figure 2 .

[0043] Figure 2 The results showed that the zeta potential value of the Elabela-Apelin-TAT fusion protein selenium chelate was higher than that of the Elabela-Apelin-TAT fusion protein, indicating that after chelation, the negative charge on the surface of the fusion protein was significantly reduced. That is, the potential measurement results showed that the potential value of the selenium chelate was significantly different from that of the Elabela-Apelin-TAT fusion protein solution, indicating that selenium was successfully chelated with the Elabela-Apelin-TAT fusion protein.

[0044] Example 3 Experimental study of Elabela-Apelin-TAT fusion protein and its selenium chelate in a mouse acute myocardial infarction reperfusion injury (MI / RI) model

[0045] Forty 8-week-old male C57 mice weighing 18.5 ± 1.5 g were purchased from Xi'an Zhongkai Experimental Animal Co., Ltd. (Xi'an, China). Normal mouse chow was purchased from Jiangsu Collaborative Biological Co., Ltd. The animals were housed in the SPF-grade experimental animal room of Hubei University of Medicine. This experiment was approved by the Animal Ethics Committee of Hubei University of Medicine. To prevent the influence of selenium from drinking water, all mice drank ddH2O (deionized water) during the experiment.

[0046] Animal grouping: Mice that had been adaptively fed for one week were randomly divided into four groups with free access to water, namely the Sham group, MI / RI model group, fusion protein group, and protein selenium chelate group.

[0047] Sham group: 4 weeks before modeling, 300 mg / kg normal mouse diet was administered daily; MI / RI model was established for 30 minutes, and samples were collected for testing 24 hours after myocardial ischemia-reperfusion injury. After breathing stabilized, the heart was exposed, and 6-0 silk suture was threaded below the junction of the pulmonary artery cone and the left atrial appendage without ligating the anterior descending coronary artery. This was to exclude the influence of the surgical operation (such as thoracotomy, heart exposure, threading, etc.) itself on the experimental results and ensure that the observed effects were due to ischemia-reperfusion injury or drug intervention, rather than surgical trauma.

[0048] MI / RI model group: 4 weeks before modeling, mice were fed 300 mg / kg of normal mouse chow daily. 30 minutes after the MI / RI model was established, and 24 hours after myocardial ischemia-reperfusion injury, the heart was exposed after breathing stabilized, and the anterior descending coronary artery was ligated with 6-0 silk suture below the junction of the pulmonary artery cone and the left atrial appendage. The ligature was then released 30 minutes later to restore blood flow.

[0049] Fusion protein group: 4 weeks before model establishment, mice were fed 300 mg / kg normal mouse diet plus 5 mg / kg (intraperitoneal injection) of Elabela-Apelin-TAT fusion protein daily. MI / RI model was established for 30 minutes, and myocardial ischemia-reperfusion injury was detected 24 hours after model establishment. The model establishment technique and evaluation criteria were the same as those of the MI / RI model group.

[0050] Protein selenium chelate group: 4 weeks before modeling, mice were fed 300 mg / kg normal mouse diet + 5 mg / kg (intraperitoneal injection) of Elabela-Apelin-TAT fusion protein selenium chelate daily. The MI / RI model was established for 30 minutes, and samples were collected for testing 24 hours after myocardial ischemia-reperfusion injury. The modeling technique and evaluation criteria were the same as those of the MI / RI model group.

[0051] An acute myocardial infarction (MI / RI) model was established: C57 mice were anesthetized with isoflurane gas, immobilized, skin prepared, and disinfected. A longitudinal cervical incision was performed, and the mice were connected to a small animal ventilator (SA430) at a respiratory rate of 80 breaths / min and a 1:1 respiratory-hour ratio. After stable breathing, a 1-cm longitudinal incision was made at the left sternal border where the pulse was palpable. The heart was then exposed layer by layer. The descending anterior coronary artery was quickly ligated with 6-0 silk suture just below the junction of the pulmonary artery conus and the left atrial appendage. Successful acute myocardial infarction (AMI) model was confirmed when the local myocardium showed blanching, decreased activity, and an ST-segment elevation greater than 0.1 mV or pathological Q waves were present on the electrocardiogram (ECG). The heart was then returned to the chest cavity, and the ligature was released 30 minutes later. The myocardium was allowed to redden, indicating restoration of myocardial blood supply. The sutures were then applied layer by layer, maintaining negative chest pressure.

[0052] Cardiac function assessment: After 6 hours of recovery, mice in each group underwent color Doppler echocardiography. Echocardiography was performed using a Vivid7 Echocardiography PRO system equipped with a water-mediated I13 L14-MHz linear array probe. The biplane Simpson method (biplane biventricular volume method) was used to calculate LVEF by measuring left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV).

[0053]

[0054] The short-axis cardiac image is obtained through a chest wall probe, and the left ventricular end-diastolic (LVIDd) and end-systolic internal dimensions (LVIDs) are measured to calculate the LVFS:

[0055]

[0056] The results of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) of each group are shown in Figure 3 .

[0057] Figure 3 The results showed that compared with the Sham group, the left ventricular ejection fraction (LVEF) (P<0.01) and left ventricular fractional shortening (LVFS) in the MI / RI model group decreased more significantly (P<0.05); compared with the MI / RI model group, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in the fusion protein group and protein selenium chelate group were significantly improved; and the improvement effect of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in the protein selenium chelate group was better than that in the fusion protein group (P<0.01), indicating that the Elabela-Apelin-TAT fusion protein selenium chelate can significantly reduce myocardial damage.

[0058] Determination of myocardial infarction area: After 24 hours of reperfusion, the model of each group of mice was completed. The hearts were removed, washed with PBS, and frozen at -80℃ for 30 minutes. Then, they were cut into thin slices of about 2 mm from the apex to the base of the heart. The slices were placed in a 2% TTC solution and preheated at 37℃. They were placed in a centrifuge tube containing 10% formaldehyde overnight, and the heart slices were photographed in sequence. The myocardial infarction area was analyzed using ImageJ software. The red area represents the non-infarcted myocardial tissue area, and the white area represents the infarcted myocardium. Myocardial infarction area (%) = (infarcted myocardial area / left ventricular area) × 100%, see Figure 4 ;

[0059] Figure 4The results showed that compared with the Sham group, the infarct area in the MI / RI model group was larger; compared with the MI / RI model group, the myocardial infarction area in the fusion protein group and the protein selenium chelate group was significantly reduced, and the myocardial infarction area in the protein selenium chelate group was the most significantly reduced (P<0.01).

[0060] Biochemical index detection: qPCR was used to detect the oxidative stress indicators SOD and GSH in serum to evaluate the oxidative damage and antioxidant capacity of myocardial cells; at the same time, the cytokine levels of TNF-α and IL-6 were detected to reflect the ability to inhibit the inflammatory response of the heart. Figure 5 .

[0061] Figure 5 The results showed that compared with the Sham group, the expression levels of SOD (P<0.01) and GSH (P<0.05) in the MI / RI model group were significantly decreased; compared with the MI / RI model group, the expression levels of SOD and GSH in the fusion protein group and the protein selenium chelate group were significantly increased, among which the protein selenium chelate group had the strongest antioxidant capacity; compared with the Sham group, the expression levels of TNF-α and IL-6 in the MI / RI model group were significantly increased, and compared with the MI / RI model group, the expression levels of TNF-α and IL-6 in the fusion protein group and the protein selenium chelate group were significantly decreased, among which the protein selenium chelate group had a significant ability to inhibit the inflammatory response of the heart.

[0062] Long-term anti-fibrosis assessment: Based on histopathological staining techniques, the myocardial infarction area and surrounding tissues of each group of mice were stained with Masson's trichrome staining 8 weeks after surgery. Collagen fibers (blue), cardiomyocytes (red), and nuclei (black) were distinguished by staining, and the percentage of collagen fibers in the myocardial tissue was quantified. At least five non-overlapping fields of view (per sample) were acquired from the infarct margin using a microscope (10× or 20× objective). Images were captured using a high-resolution color CCD camera (avoiding overexposure) and quantitatively analyzed using ImageJ software. Open Image → Image → Color → Split Channels to separate the blue channel (collagen fibers); select Process → Binary → Make Binary to binarize the image (collagen is black and background is white); and select Analyze → Measure to calculate the percentage of the blue area to the total field of view (CVF) (see Table 1).

[0063] formula:

[0064]

[0065] Table 1 Long-term anti-myocardial fibrosis test results of mice in each group

[0066] Group Collagen volume fraction (CVF,%) Sham Group 2.1±0.3 MI / RI model group 38.5±4.2** Fusion protein group <![CDATA[25.7±3.1 # ]]> Protein selenium chelate group <![CDATA[16.4±2.6 ## ]]>

[0067] Note: **p<0.01 vs Sham group; #p<0.05, ##p<0.01 vs MI / RI model group.

[0068] The results in Table 1 showed that both the fusion protein group and the protein selenium chelate group could significantly reduce the collagen volume fraction in the acute myocardial infarction reperfusion injury (MI / RI) model of mice, and the long-term anti-myocardial fibrosis effect of the protein selenium chelate group was better than that of the fusion protein group.

[0069] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A selenium-modified myocardial protective polypeptide, characterized in that: The polypeptide is prepared by chelating the Elabela-Apelin-TAT fusion protein with inorganic selenium; the Elabela-Apelin-TAT fusion protein is selected from the polypeptide shown in SEQ ID NO: 6; and the inorganic selenium is selected from sodium selenite (Na2SeO3).

2. A method for preparing a selenium-modified myocardial protective polypeptide, characterized in that: The Elabela-Apelin-TAT fusion protein shown in SEQ ID NO: 6 is subjected to a chelation reaction with selenium, wherein sodium selenite (Na2SeO3) is used as a selenium source.

3. Use of the polypeptide according to claim 1 in the preparation of a medicament for treating acute myocardial infarction.

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