Application of Nexilin overexpression recombinant vector in preparation of MIRI treatment medicine

Abstract

The invention relates to application of a Nexilin overexpression recombinant vector in preparation of a medicine for treating MIRI, and belongs to the technical field of biological medicine. In order to solve the problems of insufficient intervention of a core pathological mechanism and limited treatment effect of the existing MIRI treatment means, the invention provides application of a Nexilin overexpression recombinant vector in preparation of MIRI treatment medicines. The invention proves that the Nexilin overexpression vector can effectively improve myocardial ischemia-reperfusion injury for the first time, can remarkably improve the cardiac function of a mouse suffering from myocardial ischemia-reperfusion injury, reduce the content of myocardial injury markers, reduce the myocardial infarction area, restore the expression balance of Bcl-2 family proteins and inhibit myocardial cell apoptosis by up-regulating Bcl2 expression and down-regulating Bax expression, and has the advantages of being capable of improving myocardial ischemia-reperfusion injury, and being used for treating myocardial infarction. The protection effect on ischemia reperfusion myocardium is realized from multiple levels, and a brand new technical means and experimental basis are provided for clinically developing targeted therapeutic drugs for myocardial ischemia reperfusion injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of Nexilin overexpression recombinant vectors in the preparation of MIRI therapeutic drugs. Background Technology

[0002] Acute myocardial infarction is one of the leading causes of cardiovascular disease death worldwide, and timely restoration of blood perfusion to ischemic myocardium is crucial for saving lives and improving prognosis. However, after blood supply is restored to ischemic myocardium, secondary myocardial ischemia-reperfusion injury (MIRI) often occurs. This injury exacerbates cardiomyocyte necrosis and apoptosis, expands the infarct area, severely affects the effectiveness of reperfusion therapy, and can even lead to adverse outcomes such as heart failure and malignant arrhythmias, becoming a significant bottleneck restricting the improvement of ischemic heart disease treatment outcomes.

[0003] Numerous studies have confirmed that cardiomyocyte apoptosis is the core pathological step in the development of myocardial infarction (MIRI), and its regulatory process is closely related to the imbalance in the expression of Bcl-2 family proteins. In normal cardiomyocytes, the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 is in dynamic equilibrium, jointly maintaining normal cell survival. However, when MIRI occurs, this balance is completely disrupted, specifically manifested as a significant increase in the expression level of pro-apoptotic protein Bax and a significant decrease in the expression level of anti-apoptotic protein Bcl-2. This imbalance in their ratio directly accelerates the initiation and progression of the cardiomyocyte apoptosis program.

[0004] Currently, clinical treatments for myocardial infarction (MIRI) mainly include ischemic preconditioning and pharmacological interventions, such as the use of antioxidants, anti-inflammatory drugs, calcium channel blockers, and beta-blockers. These methods alleviate MIRI by inhibiting oxidative stress, reducing inflammatory infiltration, stabilizing myocardial cell membrane potential, and improving myocardial metabolism. However, these methods only alleviate the surface symptoms of myocardial injury without addressing the core pathological mechanisms, resulting in limited therapeutic efficacy and failing to meet clinical needs. Therefore, there is an urgent clinical need to identify and define core targets with clear regulatory effects, develop novel intervention methods, improve the overall treatment efficacy of MIRI, and enhance patients' quality of life. Summary of the Invention

[0005] To address the issues of insufficient intervention in the core pathological mechanisms and limited therapeutic effects of existing MIRI treatments, this invention provides the application of Nexilin overexpression recombinant vectors in the preparation of MIRI therapeutic drugs.

[0006] The technical solution of the present invention:

[0007] The application of the Nexilin overexpression recombinant vector in the preparation of MIRI therapeutic drugs, the nucleotide sequence encoding Nexilin is shown in SEQ ID NO.1.

[0008] Furthermore, the Nexilin overexpression recombinant vector is an adeno-associated virus vector AAV9-Nexilin, which contains a myocardial-specific promoter; the myocardial-specific promoter is the cardiac troponin T promoter.

[0009] Furthermore, the MIRI therapeutic drug is in the form of an injection.

[0010] Furthermore, the drug acts on at least one aspect of myocardial ischemia-reperfusion injury:

[0011] (1) Restore the balance between pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 after myocardial ischemia-reperfusion, and reduce myocardial cell apoptosis;

[0012] (2) Reduce the area of ​​myocardial infarction after myocardial ischemia-reperfusion;

[0013] (3) Improve cardiac function after myocardial ischemia-reperfusion.

[0014] The beneficial effects of this invention are:

[0015] This invention is the first to demonstrate that a recombinant vector overexpressing the myocardial actin-binding protein Nexilin can effectively improve myocardial ischemia-reperfusion injury. It significantly improves cardiac function indicators such as ejection fraction and fractional shortening in mice with myocardial ischemia-reperfusion injury, reduces the levels of myocardial injury markers such as CK-MB and LDH in peripheral blood, and simultaneously reduces myocardial infarction area. By upregulating the expression of the anti-apoptotic protein Bcl2 and downregulating the expression of the pro-apoptotic protein Bax, it restores the expression balance of the Bcl-2 family of proteins, thereby inhibiting cardiomyocyte apoptosis. It achieves protective effects on ischemic-reperfused myocardium from multiple levels, including cardiac function, biochemical indicators, tissue morphology, and molecular mechanisms. This invention not only clarifies the application potential of Nexilin as a novel therapeutic target for myocardial ischemia-reperfusion injury but also breaks through the traditional intervention approach for myocardial protection, providing a new technical means and experimental basis for the clinical development of targeted therapies for myocardial ischemia-reperfusion injury. Attached Figure Description

[0016] Figure 1 The image shows a comparison of Western Blot results of Nexilin in the myocardial tissue of mice in the control group and the myocardial ischemia-reperfusion injury group in Example 1. A is a Western Blot result image, and B is a comparison of Nexilin protein expression levels.

[0017] Figure 2This is a comparison of the mRNA expression levels of Nexilin in the myocardial tissue of mice in the control group and the myocardial ischemia-reperfusion injury group in Example 1.

[0018] Figure 3 The image shows a comparison of immunofluorescence staining of myocardial tissue from mice in the control group and the myocardial ischemia-reperfusion injury group in Example 1. A represents the control group, and B represents the ischemia-reperfusion injury group.

[0019] Figure 4 Echocardiograms of four groups of mice in Example 2: A is the control group, B is the ischemia-reperfusion injury group, C is the Nexilin overexpression + myocardial ischemia-reperfusion injury group, and D is the control virus + myocardial ischemia-reperfusion injury group.

[0020] Figure 5 This is a comparison chart of cardiac ejection fraction and fractional shortening of four groups of mice in Example 2. A represents ejection fraction, and B represents fractional shortening.

[0021] Figure 6 This is a comparison chart of the levels of creatine kinase isoenzymes and lactate dehydrogenase in the blood pressure of four groups of mice in Example 3. A represents CK-MB, and B represents LDH.

[0022] Figure 7 The image shows a comparison of TTC staining of heart sections from mice in Example 3, which were in the Nexilin overexpression + myocardial ischemia-reperfusion injury group and the control virus + myocardial ischemia-reperfusion injury group. A is the control virus + myocardial ischemia-reperfusion injury group, and B is the Nexilin overexpression + myocardial ischemia-reperfusion injury group.

[0023] Figure 8 This is a comparison of the myocardial infarction area in mice in Example 3, specifically the Nexilin overexpression + myocardial ischemia-reperfusion injury group and the control virus + myocardial ischemia-reperfusion injury group.

[0024] Figure 9 The image shows a comparison of TUNEL staining results of four groups of mouse myocardial tissue in Example 4. A is the control group, B is the myocardial ischemia-reperfusion injury group, C is the Nexilin overexpression + myocardial ischemia-reperfusion injury group, and D is the control virus + myocardial ischemia-reperfusion injury group.

[0025] Figure 10 This is a comparison of the number of TUNEL-positive cells in the myocardial tissue of four groups of mice in Example 4;

[0026] Figure 11 This is a comparison of Bcl2 protein expression levels in four groups of mouse cardiomyocytes in Example 4. A is a Western Blot result, and B is a comparison of Bcl2 protein expression levels.

[0027] Figure 12The figures show a comparison of Bax protein expression levels in four groups of mouse cardiomyocytes in Example 4. A is a Western Blot result, and B is a comparison of Bax protein expression levels. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0029] Example 1

[0030] This example confirms that Nexilin expression is reduced in myocardial tissue after myocardial ischemia-reperfusion.

[0031] I. Methods for grouping experimental animals and establishing experimental models:

[0032] Eight-week-old male C57BL / 6J mice (weighing 22-25g) were randomly divided into two groups:

[0033] (1) Control group: only open-chest surgery and suture threading were performed, without ligation of the coronary arteries;

[0034] (2) Myocardial ischemia-reperfusion injury group: Blood flow was restored 45 minutes after ligation of the left anterior descending coronary artery, and reperfusion was performed for 24 hours.

[0035] 2. Mice in the control group and the myocardial ischemia-reperfusion injury group were sacrificed 24 hours after surgery and 24 hours after reperfusion. The heart was quickly removed, the left ventricular myocardium was separated along the interventricular septum, and the right ventricle and atrium were removed on ice. The expression of Nexilin in the myocardial tissue was detected.

[0036] (1) Western Blot detection:

[0037] 1. Protein extraction: Homogenize left ventricular myocardial tissue with RIPA lysis buffer (containing PMSF), centrifuge at 13500 rpm for 15 minutes, and collect the supernatant;

[0038] 2. Electrophoretic transfer: Proteins were separated by 10% SDS-PAGE and transferred to an NC membrane;

[0039] 3. Antibody incubation: anti-Nexilin antibody (1:1000, Proteintech), anti-β-actin antibody (1:1000, Affinity).

[0040] 4. Results Analysis: Image J quantifies gray values, and the Nexilin / β-actin ratio represents the relative expression level.

[0041] The results are as follows Figure 1 As shown, compared with the control group, the expression of Nexilin protein in the myocardial tissue of mice in the myocardial ischemia-reperfusion injury group was significantly reduced (P<0.05).

[0042] (2) qRT-PCR detection:

[0043] 1. RNA extraction: Total RNA was extracted from the left ventricle using the Trizol method and quantified using NanoDrop.

[0044] 2. Reverse transcription: 1 μg of RNA was used to synthesize cDNA using the PrimeScript RT kit;

[0045] 3. Quantitative PCR: SYBR Green assay for Detection of Nexilin mRNA.

[0046] 4. Results Analysis: 2- ΔΔCt The relative expression level is calculated using this method.

[0047] The results are as follows Figure 2 As shown, the qRT-PCR results were consistent with the Western Blot results, and the content of Nexilin mRNA in the myocardial tissue of mice in the myocardial ischemia-reperfusion injury group was significantly reduced (P<0.05).

[0048] III. Immunofluorescence detection

[0049] Immunofluorescence technique employs multicolor immunofluorescence staining combined with confocal microscopy imaging. The specific method is as follows:

[0050] Two groups of mouse cardiomyocytes were fixed, permeabilized, and blocked using standard methods. Primary antibody incubation: rabbit anti-Nexilin antibody labeled Nexilin protein was added. Secondary antibody incubation: fluorescently labeled secondary antibody (Nexilin red) was added. Nuclear staining: DAPI staining solution was added, resulting in blue staining of the nuclei. The slides were mounted, and images were acquired using a confocal microscope.

[0051] Immunofluorescence results as follows Figure 3 As shown, compared with the control group, the red fluorescence signal in the myocardial ischemia-reperfusion injury group was significantly weakened, indicating that the expression level of Nexilin in cardiomyocytes of myocardial tissue was significantly reduced after MIRI.

[0052] The results of this embodiment indicate that Nexilin expression is reduced in mouse myocardial tissue after myocardial ischemia-reperfusion injury, suggesting that changes in its expression level are closely related to the occurrence and development of MIRI. Based on the results of this embodiment, it can be inferred that Nexilin, as a differentially expressed molecule in the MIRI process, is a targeted intervention point involved in the pathological regulation of MIRI.

[0053] Example 2

[0054] This embodiment demonstrates that overexpression of Nexilin can improve cardiac function after myocardial ischemia-reperfusion.

[0055] I. Construction of overexpression vectors

[0056] (1) Carrier selection:

[0057] This embodiment uses an adeno-associated virus type 9 (AAV9) vector system with a vector backbone of ssAAV-cTnT-Nexilin-SV40pA, containing the following elements: cardiac troponin T (cTnT) promoter, target gene Nexilin coding sequence, and SV40pA which helps improve expression efficiency and stability. The nucleotide sequence encoding Nexilin is shown in SEQ ID NO.1.

[0058] (2) Nexilin gene cloning and viral packaging

[0059] Nexilin gene cloning and viral packaging were performed by Shanghai Jikai Gene Biotechnology Co., Ltd. using a gene cloning-viral packaging-purification and concentration method, yielding purified AAV9-Nexilin virus particles with a titer of 1×10⁻⁶. 13 vg / mL.

[0060] (3) Control virus

[0061] The Nexilin sequence was replaced with a meaningless sequence, while the remaining elements were completely identical to those of AAV9-Nexilin. The process was performed by Guangzhou Aizhe Biotechnology Co., Ltd. using a gene cloning-virus packaging-purification and concentration method to obtain purified AAV9-NC virus particles with a titer of 1×10⁻⁶. 13 vg / mL.

[0062] II. Animal Experiment Grouping and Model Establishment Methods:

[0063] Eight-week-old male C57BL / 6J mice (weighing 22-25g) were randomly divided into four groups:

[0064] (1) Control group: The same amount of normal saline was injected into the tail vein, and the left anterior descending coronary artery was opened but not ligated;

[0065] (2) Myocardial ischemia-reperfusion injury group: equal volume of normal saline was injected into the tail vein. After 4 weeks, the left anterior descending coronary artery was ligated for 45 minutes and blood flow was restored. Reperfusion was carried out for 24 hours.

[0066] (3) Overexpression of Nexilin + myocardial ischemia-reperfusion injury group: AAV9-Nexilin (100μL, 1×10) was injected via tail vein. 11 (vg), blood flow was restored 45 minutes after ligation of the left anterior descending coronary artery 4 weeks later, followed by reperfusion for 24 hours;

[0067] (4) Control group of virus + myocardial ischemia-reperfusion injury: AAV9-NC (100μL, 1×10) was injected into the tail vein. 11 (vg), blood flow was restored 45 minutes after ligation of the left anterior descending coronary artery 4 weeks later, followed by reperfusion for 24 hours;

[0068] 3. Monitor cardiac function through echocardiography.

[0069] Echocardiograms of the four groups of mice are as follows: Figure 4 As shown, the control group exhibited regular myocardial structure and regular movement, indicating normal myocardial contraction / diastolic rhythm. The myocardial structure in the myocardial ischemia-reperfusion injury group was disordered, and the amplitude of movement was weakened, indicating that MIRI led to a decline in myocardial contractile function. The myocardial structure and movement in the Nexilin overexpression + myocardial ischemia-reperfusion injury group were similar to those in the control group, showing significant improvement compared to the myocardial ischemia-reperfusion injury group. The structure / movement of the control virus + myocardial ischemia-reperfusion injury group showed no significant difference from that of the myocardial ischemia-reperfusion injury group.

[0070] The cardiac ejection fraction and fractional shortening of the four groups of mice were compared to, for example... Figure 5 As shown in the figure, compared with the control group, the left ventricular ejection fraction and left ventricular short-axis shortening rate were significantly reduced in the myocardial ischemia-reperfusion injury group, indicating that myocardial ischemia-reperfusion caused weakened cardiac function in mice. However, the left ventricular ejection fraction and left ventricular short-axis shortening rate were significantly restored in the Nexilin+ myocardial ischemia-reperfusion injury group (P<0.0001).

[0071] This result indicates that overexpression of Nexilin mediated by the AAV9 vector can significantly improve myocardial dysfunction caused by MIRI. Combined with the structure of reduced Nexilin expression after MIRI in Example 1, this further verifies that Nexilin is a protective molecule against MIRI. Overexpression of Nexilin can alleviate cardiac dysfunction caused by myocardial ischemia-reperfusion, providing direct functional evidence for its application as a therapeutic drug.

[0072] Example 3

[0073] This embodiment demonstrates that overexpression of Nexilin can reverse myocardial damage caused by myocardial ischemia-reperfusion and reduce the area of ​​myocardial infarction.

[0074] I. Detection of CK-MB and LDH levels in blood

[0075] CK-MB (creatine kinase isoenzyme) is mainly found in cardiomyocytes and is a specific biochemical marker of myocardial injury; LDH (lactate dehydrogenase) is present in high amounts in cardiomyocytes and is a sensitive marker reflecting the degree of cardiomyocyte damage. When cardiomyocytes are damaged / necrotized, the cell membrane integrity is disrupted, and intracellular CK-MB and LDH are released into the bloodstream. The larger the infarct area and the more cardiomyocytes die, the higher the levels of these two substances in the blood. In this example, based on the animal experiment grouping and model establishment in Example 2, peripheral blood was collected from mice in each group. After centrifugation to separate serum / plasma, the concentrations of CK-MB and LDH were detected using CK-MB and LDH kits.

[0076] The results are as follows Figure 6 As shown, the levels of CK-MB and LDH in the control group were at a low baseline level; the levels of both enzymes in the myocardial ischemia-reperfusion injury group were significantly increased, indicating that after myocardial ischemia-reperfusion, myocardial cells die and cell membranes rupture, leading to the release of large amounts of intracellular CK-MB and LDH into the bloodstream; the levels of both enzymes in the Nexilin overexpression + myocardial ischemia-reperfusion injury group were significantly lower than those in the myocardial ischemia-reperfusion injury group, indicating that Nexilin overexpression can reduce myocardial cell necrosis and rupture, and has a cardioprotective effect.

[0077] II. In order to examine the effect of overexpressing Nexilin on myocardial injury from the perspective of tissue morphology changes, this example, based on the animal experimental grouping and model establishment in Example 2, performed TTC staining on heart sections of mice in the Nexilin overexpression + myocardial ischemia-reperfusion injury group and the control virus + myocardial ischemia-reperfusion injury group.

[0078] After TTC staining, living myocardial tissue appeared red, while necrotic infarct areas appeared pale white; mouse TTC staining results were as follows. Figure 7 As shown, in the sections of the control virus + myocardial ischemia-reperfusion injury group, the pale white infarct area was large; in the sections of the overexpressing Nexilin + myocardial ischemia-reperfusion injury group, the pale white area was significantly reduced, and the red survival area was significantly increased.

[0079] Quantified myocardial infarction area for example Figure 8As shown, the infarct area in the control virus + myocardial ischemia-reperfusion injury group was approximately 30%, while the infarct area in the Nexilin overexpression + myocardial ischemia-reperfusion injury group decreased to approximately 15%. This result further validates that Nexilin has a clear protective effect against myocardial ischemia-reperfusion injury.

[0080] Example 4

[0081] This embodiment demonstrates that overexpression of Nexilin can reduce cardiomyocyte apoptosis after myocardial ischemia-reperfusion.

[0082] The Bcl-2 protein family is a core regulatory family of apoptosis, primarily regulating cell survival and death through interactions among its members. Pro-apoptotic protein Bax and anti-apoptotic protein Bcl2, as classic members of this family, regulate apoptosis via the mitochondrial pathway through their interactions, and are key regulatory molecules in the apoptotic process during myocardial ischemia-reperfusion injury. Under normal conditions, Bax and Bcl2 maintain a balance, sustaining cell survival; after myocardial ischemia-reperfusion, injury signals induce increased Bax expression and decreased Bcl2 expression, disrupting the Bax / Bcl2 balance and ultimately initiating the apoptotic program.

[0083] To investigate whether overexpression of Nexilin reduces myocardial injury and decreases myocardial cell apoptosis, this example, based on the animal experiment grouping and model establishment in Example 2, detected apoptosis-related proteins in each group of mice.

[0084] I. TUNEL staining to detect cardiomyocyte apoptosis

[0085] Four groups of mouse myocardial tissue sections were fixed, permeabilized, and blocked. Terminal deoxynucleotidyl transferase (TdT) and fluorescently labeled dUTP were added for enzymatic labeling. Then, DAPI staining solution was added to label all cell nuclei, and the staining results were observed using a fluorescence microscope. The results of staining and comparison of the number of TUNEL-positive cells are shown below. Figure 9 and Figure 10 As shown, red fluorescence represents TUNEL-labeled apoptotic cells, blue represents DAPI-labeled total cell nuclei, and green represents cardiac muscle structure markers. Figure 9 The results showed that the control group had almost no red apoptosis signal, indicating that apoptosis of normal cardiomyocytes was minimal; the red apoptosis signal was significantly increased in the myocardial ischemia-reperfusion injury group, indicating that myocardial ischemia-reperfusion induced a large number of cardiomyocyte apoptosis; the red apoptosis signal was significantly reduced in the Nexilin overexpression + myocardial ischemia-reperfusion injury group, approaching the level of the control group. Figure 10The results also showed that overexpression of Nexilin significantly reduced apoptosis following myocardial ischemia-reperfusion. This indicates that overexpression of Nexilin can significantly inhibit myocardial ischemia-reperfusion-induced apoptosis, further validating the cardioprotective effect of Nexilin.

[0086] II. Western blot analysis to detect the effect of Nexilin overexpression on Bax / Bcl2.

[0087] Total protein was extracted from the myocardial tissue of mice in each group, and Western blot was used to detect the expression of BAX and Bal2 proteins. Results are as follows: Figure 11 and Figure 12 As shown, the control group had a higher Bcl2 level; the myocardial ischemia-reperfusion injury group had a significantly lower Bcl2 level; the Nexilin overexpression group had a significantly increased Bcl2 level; the control group had a lower Bax level; the myocardial ischemia-reperfusion injury group had a significantly increased Bax level; and the Nexilin overexpression group had a significantly decreased Bax level. This indicates that myocardial ischemia-reperfusion leads to an imbalance in the Bcl-2 family, with decreased expression of the anti-apoptotic protein Bcl2 and increased expression of the pro-apoptotic protein Bax. Overexpression of Nexilin can reverse this imbalance by upregulating Bcl2 and downregulating Bax, restoring the Bcl2 / Bax balance. Combined with previous results on apoptosis and injury, this suggests that Nexilin exerts its cardioprotective effect by regulating the expression balance of the Bcl-2 family and inhibiting cardiomyocyte apoptosis.

Claims

1. The application of Nexilin overexpression recombinant vector in the preparation of MIRI therapeutic drugs, characterized in that, The nucleotide sequence encoding Nexilin is shown in SEQ ID NO.

1.

2. The application of the Nexilin overexpression recombinant vector according to claim 1 in the preparation of MIRI therapeutic drugs, characterized in that, The Nexilin overexpression recombinant vector is the adeno-associated virus vector AAV9-Nexilin, which contains a myocardial-specific promoter; the myocardial-specific promoter is the cardiac troponin T promoter.

3. The application of the Nexilin overexpression recombinant vector according to claim 2 in the preparation of MIRI therapeutic drugs, characterized in that, The MIRI treatment drug is in the form of an injection.

4. The application of the Nexilin overexpression recombinant vector according to claim 3 in the preparation of MIRI therapeutic drugs, characterized in that, The drug has an effect on at least one aspect of myocardial ischemia-reperfusion injury: (1) Restore the balance between pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 after myocardial ischemia-reperfusion, and reduce myocardial cell apoptosis; (2) Reduce the area of ​​myocardial infarction after myocardial ischemia-reperfusion; (3) Improve cardiac function after myocardial ischemia-reperfusion.

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