Use of ogn overexpression vector in preparation of a drug for preventing reperfusion myocardial injury

Abstract

The application relates to application of an OGN overexpression vector in preparation of a reperfusion myocardial injury prevention drug and belongs to the technical field of biological medicines. In view of the problem that existing mitochondrial targeting strategies are insufficient in multi-target point cooperation in MI / RI prevention, the application provides application of an OGN overexpression vector in preparation of a reperfusion myocardial injury prevention drug. The OGN overexpression vector is a recombinant adeno-associated virus vector and comprises a nucleotide sequence coding OGN. The application first realizes three-dimensional mitochondrial steady-state regulation of myocardial ischemia-reperfusion injury, significantly improves myocardial energy metabolism and reduces cell death by enhancing mitochondrial respiratory chain activity, stabilizing membrane potential and inhibiting ROS explosion, thereby effectively reversing heart function failure caused by MI / RI. Experimental data prove that AAV-mediated OGN overexpression has a significant clinical conversion potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of OGN overexpression vector in the preparation of drugs for the prevention of reperfusion myocardial injury. Background Technology

[0002] Myocardial ischemia-reperfusion injury (MI / RI) is a common complication following revascularization after acute myocardial infarction. Its core pathological contradiction lies in the dual impact of "energy metabolism disorder during ischemia" and "oxidative stress burst during reperfusion." Although timely restoration of blood flow can save some dying myocardium, the reperfusion process itself further exacerbates myocardial damage through mechanisms such as calcium overload, mitochondrial dysfunction, and reactive oxygen species (ROS) bursts, leading to myocardial stunning, arrhythmias, and even heart failure.

[0003] Mitochondrial homeostasis plays a crucial driving role in the pathological process of MI / RI, and its disruption mediates cell death through a triple cascade: First, damage to the electron transport chain complex triggers a burst of reactive oxygen species (ROS), leading to oxidative stress damage; second, calcium overload induces irreversible opening of the mitochondrial membrane permeability transition pore (mPTP), disrupting the inner membrane potential gradient; finally, it triggers cytochrome c efflux, activating the caspase cascade and inducing apoptosis. Recent studies have confirmed that the maintenance of mitochondrial homeostasis depends on the synergistic effects of kinetic regulation (fusion / fission), autophagy clearance, and biosynthesis, providing a theoretical basis for improving MI / RI by targeting mitochondrial quality regulation. However, current intervention strategies targeting mitochondria (such as regulating kinetics, activating biosynthesis, and promoting autophagy) have limited effectiveness in clinical translation, suggesting that traditional pathways have insufficient mechanistic coverage, necessitating the exploration of novel regulatory targets at the molecular level.

[0004] Osteoglycin (OGN), a member of the leucine-rich small proteoglycan family, exhibits high expression in cardiac tissue, particularly involved in the regulation of left ventricular development and compensatory hypertrophy. Although the regulatory role of OGN in cardiovascular diseases such as heart failure and ventricular remodeling has been preliminarily verified, its functional role in MI / RI remains unexplored, and no studies have yet found its application in the prevention of MI / RI. Summary of the Invention

[0005] To address the issue of insufficient multi-target synergy in existing mitochondrial targeting strategies for the prevention of myocardial infarction / reperfusion injury, this invention provides the application of OGN overexpression vectors in the preparation of drugs for the prevention of myocardial infarction / reperfusion injury.

[0006] The technical solution of the present invention:

[0007] Application of OGN overexpression vector in the preparation of drugs for the prevention of reperfusion myocardial injury.

[0008] Furthermore, the OGN overexpression vector is a recombinant adeno-associated virus vector containing a nucleotide sequence encoding OGN and a myocardial-specific promoter.

[0009] Furthermore, the nucleotide sequence encoding OGN is shown in SEQ ID NO. 1.

[0010] Furthermore, the myocardial-specific promoter is the cardiac troponin T (cTnT) promoter.

[0011] Furthermore, the serotype of the recombinant adeno-associated virus vector is AAV9.

[0012] Furthermore, the OGN overexpression vector is an adeno-associated virus vector AAV9-OGN that overexpresses the OGN gene.

[0013] Furthermore, the reperfusion myocardial injury prophylaxis drug is administered 3 weeks prior to revascularization surgery.

[0014] Furthermore, the preventive medicine has at least one of the following uses:

[0015] (1) Enhance the mitochondrial respiration capacity of myocardial cells during myocardial ischemia-reperfusion;

[0016] (2) Increase the mitochondrial membrane potential of myocardial cells undergoing myocardial ischemia-reperfusion.

[0017] (3) Reduce the ROS level in myocardial tissue that is ischemic-reperfused.

[0018] The beneficial effects of this invention are:

[0019] This invention, through an adeno-associated virus (AAV) vector-mediated osteoglobin overexpression system, achieves for the first time three-dimensional mitochondrial homeostasis regulation of myocardial ischemia-reperfusion injury. By enhancing mitochondrial respiratory chain activity, stabilizing membrane potential, and inhibiting ROS bursts, it significantly improves myocardial energy metabolism and reduces cell death, thereby effectively reversing MI / RI-induced cardiac failure. Experimental data show that AAV9-OGN treatment significantly improved cardiac function indicators (EF% and FS%) in I / R mice, confirming the protective effect of AAV vector-mediated OGN overexpression on cardiac function.

[0020] This invention confirms that OGN delivered by an adeno-associated virus (AAV) vector effectively restores mitochondrial function in I / R-damaged myocardial tissue. In vitro experimental results show that AAV9-OGN treatment enhances mitochondrial respiration, stabilizes membrane potential, and reduces ROS levels. More importantly, OGN overexpression significantly regulates the expression of mitochondrial dynamics-related proteins: reducing the expression levels of the fission proteins Drp1 and Fis1, while increasing the expression levels of the fusion proteins OPA1, MFN1, and MFN2, thereby reshaping the mitochondrial fission / fusion balance. In vivo experiments further validate that AAV-mediated OGN overexpression in neonatal rat cardiomyocytes enhances mitochondrial respiration, increases membrane potential, and reduces ROS levels, highly consistent with in vitro experimental results.

[0021] This invention systematically demonstrates that adeno-associated virus (AAV) vector-mediated OGN overexpression can exert cardioprotective effects by regulating mitochondrial homeostasis. It not only improves mitochondrial function but also maintains mitochondrial homeostasis by regulating the expression of mitochondrial splitting / fusion proteins. This invention provides a novel AAV vector-based strategy for the treatment of myocardial ischemia-reperfusion injury and lays an important foundation for the development of drugs targeting OGN to regulate mitochondrial homeostasis, demonstrating significant clinical translational potential. Attached Figure Description

[0022] Figure 1 The electrocardiograms of mice in the sham-operated group and the IR group before and after reperfusion in Example 1 are shown.

[0023] Figure 2 This is a comparison of the Western Blot results of OGN in the myocardial tissue of mice in the sham-operated group and the IR group in Example 1;

[0024] Figure 3 This is a comparison of OGN expression levels in the myocardial tissue of mice in the sham-operated group and the IR group in Example 1.

[0025] Figure 4 This is a comparison of OGN mRNA expression levels in the myocardial tissue of mice in the sham-operated group and the IR group in Example 1;

[0026] Figure 5 Echocardiograms of the four groups of mice in Example 2;

[0027] Figure 6 This is a comparison chart of cardiac ejection fraction and shortening fraction in four groups of mice in Example 2. A represents ejection fraction and B represents shortening fraction.

[0028] Figure 7 The images show the DHE fluorescence staining detection of myocardial tissue from four groups of mice in Example 3.

[0029] Figure 8This is a comparison of the relative levels of reactive oxygen species in the myocardial tissues of four groups of mice in Example 3;

[0030] Figure 9 This is a graph showing the oxygen consumption curves of the stepwise substrate-inhibitor addition method in the mitochondria of four groups of mouse cardiomyocytes in Example 4.

[0031] Figure 10 This is a comparative diagram of the respiratory chain complex activity of mitochondria in four groups of mouse cardiomyocytes in Example 4;

[0032] Figure 11 This is a comparison of the mRNA expression levels of the respiratory chain complex genes ATP6, Cox1, Cytb, and ND1 in the mitochondria of four groups of mouse cardiomyocytes in Example 4.

[0033] Figure 12 This is a comparison of the mRNA expression levels of the splitting proteins Drp1 and Fis1, and the fusion proteins Mfn1, Mfn2, and OPA1 in the mitochondria of four groups of mouse cardiomyocytes in Example 5.

[0034] Figure 13 This is a comparison of Western Blot results of Drp1, the splitting protein of mitochondria in four groups of mouse cardiomyocytes in Example 5.

[0035] Figure 14 This is a comparison of the expression levels of the splitting protein Drp1 in the mitochondria of four groups of mouse cardiomyocytes in Example 5.

[0036] Figure 15 This is a fluorescence staining image of Drp1, a mitochondrial splitting protein, in four groups of mouse cardiomyocytes in Example 5.

[0037] Figure 16 This is a comparison of the relative fluorescence intensity of Drp1, a splitting protein in mitochondria of four groups of mouse cardiomyocytes in Example 5.

[0038] Figure 17 The graph shows the dynamic response curves of oxygen consumption rate of four groups of cardiomyocytes in Example 6.

[0039] Figure 18 This is a comparison chart of oxygen consumption rates for basal and maximal respiration in four groups of cardiomyocytes in Example 6. A represents basal respiration, and B represents maximal respiration.

[0040] Figure 19 The images show JC-1 fluorescence staining detection of four groups of cardiomyocytes in Example 6.

[0041] Figure 20 This is a comparison diagram of the mitochondrial membrane potential of four groups of cardiomyocytes in Example 6;

[0042] Figure 21The images show DCFH-DA fluorescence staining detection of four groups of cardiomyocytes in Example 6;

[0043] Figure 22 This is a comparison of the relative levels of reactive oxygen species in four groups of cardiomyocytes in Example 6. Detailed Implementation

[0044] 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.

[0045] Example 1

[0046] This embodiment demonstrates, through mouse experiments, that OGN expression is reduced in myocardial tissue after myocardial ischemia-reperfusion.

[0047] I. Methods for grouping experimental animals and establishing experimental models:

[0048] Eight-week-old male C57BL / 6J mice (weighing 22-25g) were randomly divided into two groups:

[0049] (1) Sham group: only open-chest surgery and suture threading were performed, without ligation of the coronary arteries;

[0050] (2) Ischemia-reperfusion (IR) group: Blood flow was restored 30 minutes after ligation of the left anterior descending coronary artery, and reperfusion was performed for 48 hours.

[0051] Surface electrocardiograms were monitored in the Sham and IR groups before and after reperfusion, and the results are as follows: Figure 1 As shown, the ST segment arched upward immediately after ligation, and the ST segment decreased by ≥50% after reperfusion, indicating that the IR group model was successfully constructed.

[0052] 2. Mice in the Sham group were sacrificed 48 hours after surgery and mice in the IR group were reperfused 48 hours after surgery. The heart was quickly removed, the left ventricular myocardium was separated along the interventricular septum, and the right ventricle and atrium were excised on ice. The expression of OGN in the myocardial tissue was detected.

[0053] (1) Western Blot detection:

[0054] 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;

[0055] 2. Electrophoretic transfer: Proteins were separated by 10% SDS-PAGE and transferred to an NC membrane;

[0056] 3. Antibody incubation: anti-OGN antibody (1:1000, Proteintech), anti-β-actin antibody (1:3000, CST).

[0057] 4. Results Analysis: Image J quantifies gray values, and the OGN / β-actin ratio represents the relative expression level.

[0058] The results are as follows Figure 2 and Figure 3 As shown, compared with the Sham group, the OGN content in the myocardial tissue of mice in the IR group was significantly reduced (P<0.05).

[0059] (2) qRT-PCR detection:

[0060] 1. RNA extraction: Total RNA was extracted from the left ventricle using the Trizol method and quantified using NanoDrop.

[0061] 2. Reverse transcription: 1 μg of RNA was used to synthesize cDNA using the PrimeScript RT kit;

[0062] 3. Quantitative PCR: SYBR Green assay for OGN mRNA detection.

[0063] 4. Results Analysis: 2 -ΔΔCt The relative expression level is calculated using this method.

[0064] The results are as follows Figure 4 As shown, the qRT-PCR results were consistent with the Western Blot results, and the OGN content in the myocardial tissue of IR group mice was significantly reduced (P<0.05).

[0065] The experimental results of this embodiment show that OGN expression is reduced in myocardial tissue after myocardial ischemia-reperfusion injury.

[0066] Example 2

[0067] This embodiment demonstrates that overexpression of OGN can improve cardiac function after ischemia-reperfusion.

[0068] I. Construction of overexpression vectors

[0069] (1) Carrier selection:

[0070] The adeno-associated virus type 9 (AAV9) vector system was selected, with a vector backbone of ssAAV-cTnT-OGN-SV40pA, containing the following elements: cardiac troponin T (cTnT) promoter, the target gene OGN coding sequence, and SV40pA, which helps improve expression efficiency and stability. The nucleotide sequence encoding OGN is shown in SEQ ID NO.1.

[0071] (2) OGN gene cloning and viral packaging

[0072] OGN gene cloning and viral packaging were performed by Guangzhou Aizhe Biotechnology Co., Ltd. using a gene cloning-virus packaging-purification and concentration method, yielding purified AAV9-OGN virus particles with a titer of 1×10⁻⁶. 13 vg / mL.

[0073] (3) Control vector

[0074] The OGN sequence was replaced with a meaningless sequence, while the remaining elements were completely identical to AAV9-OGN. The process was completed 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.

[0075] II. Animal Experiment Grouping and Model Establishment Methods:

[0076] Eight-week-old male C57BL / 6J mice (weighing 22-25g) were randomly divided into four groups:

[0077] (1) Sham 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;

[0078] (2) IR group: The same amount of normal saline was injected into the tail vein. After 4 weeks, the left anterior descending coronary artery was ligated for 30 minutes and blood flow was restored. The reperfusion lasted for 48 hours.

[0079] (3) OGN-IR group: AAV9-OGN (100μL, 1×10¹¹vg) was injected into the tail vein. After 4 weeks, blood flow was restored 30 minutes after ligation of the left anterior descending coronary artery, and then reperfused for 48 hours.

[0080] (4) NC-IR group: AAV9-NC (100μL, 1×10¹¹vg) was injected into the tail vein. After 4 weeks, blood flow was restored 30 minutes after ligation of the left anterior descending coronary artery, and then reperfused for 48 hours.

[0081] 3. Monitor cardiac function through echocardiography.

[0082] Echocardiograms of the four groups of mice are as follows: Figure 5 As shown in the figure, the cardiac ejection fraction (EF) comparison chart is as follows: Figure 6 As shown in Figure A, the comparison chart of shortened fraction FS is as follows. Figure 6 As shown in Figure B, compared with the Sham group, the IR group mice showed weakened ventricular wall motion, with significantly reduced EF% and FS%, indicating that ischemia-reperfusion caused weakened cardiac function in mice. In contrast, the OGN-IR group mice showed improved ventricular wall motion, with significantly increased EF% and FS%, which was statistically different from the NC-IR group (P<0.05).

[0083] This result demonstrates that OGN overexpression mediated by the AAV9 vector can significantly reverse cardiac systolic dysfunction caused by myocardial ischemia-reperfusion, providing direct functional evidence for its application as a preventive drug.

[0084] Example 3

[0085] This embodiment demonstrates that overexpression of OGN can reduce ROS levels in myocardial tissue after ischemia-reperfusion.

[0086] Based on the animal experiment grouping and model establishment in Example 2, mice in each group were sacrificed after 48 hours of reperfusion, and their hearts were quickly removed. Left ventricular myocardial tissue was harvested and cut into 5μm thick frozen sections. DHE fluorescent probe staining technology was used to detect changes in ROS in the myocardial tissue.

[0087] DHE staining steps:

[0088] (1) Probe incubation: 10 μM DHE (Dihydroethidium, MCE) working solution was added to the slice and incubated at 37°C in the dark for 30 minutes;

[0089] (2) Washing: Rinse with PBS 3 times (5 minutes each time) to remove unbound probes.

[0090] (3) Mounting: Mount the slide with an anti-fluorescence quenching agent (containing DAPI) and store at 4°C away from light.

[0091] The confocal microscopy imaging results of each group of stained samples are as follows: Figure 7 As shown, compared with the Sham group, the IR group showed strong red fluorescence, indicating the presence of ROS burst, while the red fluorescence of the OGN-IR group was significantly weakened; Figure 8 The relative levels of reactive oxygen species (ROS) in the myocardial tissue of mice in each group showed that the ROS level in the IR group was significantly increased, while the ROS level in the OGN-IR group was significantly decreased after AAV9-OGN treatment. Furthermore, the decrease in ROS level in the OGN-IR group compared to the NC-IR group was statistically significant (P<0.05).

[0092] This result indicates that OGN overexpression can reduce oxidative stress-induced cardiomyocyte damage and improve cardiac function by inhibiting reactive oxygen species (ROS) generation following myocardial ischemia-reperfusion injury. The experimental results of this embodiment corroborate the cardiac function improvement results shown by echocardiography in Example 2, jointly supporting the close relationship between the protective effect of OGN overexpression in myocardial ischemia-reperfusion injury and its regulation of oxidative stress levels.

[0093] Example 4

[0094] This embodiment demonstrates that overexpression of OGN can regulate mitochondrial homeostasis in cardiomyocytes and improve mitochondrial respiration.

[0095] Based on the animal experiment grouping and model establishment in Example 2, the mice in each group were sacrificed after 48 hours of reperfusion, the heart was quickly removed, the left ventricular myocardial tissue was taken, cut into 5mg small samples, placed in a manual glass homogenizer, 200 ml of pre-cooled mitochondrial respiratory medium (MiR05) was added, and homogenized on ice. After homogenization, large debris was filtered out to obtain the homogenized sample to be tested.

[0096] I. Mitochondrial respiratory capacity was detected using a high-resolution respiratory measurement method.

[0097] The Oxygraph-2k high-resolution respirometer was used for detection, and the detection procedure is as follows:

[0098] (1) Basal respiration: Add tissue homogenate to the respiration chamber, adjust the volume to 2 mL, seal the respiration chamber, and record the basal oxygen consumption rate (OCR).

[0099] (2) Once the respiratory curve has stabilized, the substrate and inhibitor of each electron transfer chain (ETC) complex are placed into the breathing chamber in sequence:

[0100] Pyruvate (5 mM), malic acid (2 mM), glutamate (10 mM), and ADP (2.5 mM) were used as substrates for complex I (NADH dehydrogenase), and rotenone (1 μM) was used as an inhibitor of complex I.

[0101] Succinic acid (10 M) was used as the substrate for complex II+III (succinate dehydrogenase + cytochrome c reductase), and antimycin A (1 μM) was used as the inhibitor of complex II+III.

[0102] Ascorbic acid (2 mM) and TMPD (0.5 mM) were used as substrates for complex IV (cytochrome c oxidase).

[0103] The results of collecting and measuring the respiratory curve are as follows: Figure 9 As shown, the changes in respiratory chain complex activity are as follows: Figure 10As shown, compared with the Sham group, the oxygen consumption rate in the IR group was significantly reduced (P<0.01), indicating that ischemia-reperfusion injury led to impaired overall mitochondrial respiratory function. The oxygen consumption rate in the OGN-IR group recovered to 80% of that in the Sham group, while there was no significant difference between the NC-IR group and the IR group (P>0.05). This suggests that OGN overexpression improves respiratory function through the following pathways: 1. Repairing respiratory chain complex activity and restoring the complete electron transport steps; 2. Reducing ROS accumulation and protecting mitochondrial structural integrity.

[0104] II. The expression changes of mitochondrial respiratory chain complex genes ATP6, Cox1, Cytb and ND1 were detected by conventional qRT-PCR.

[0105] qRT-PCR results are as follows Figure 11 As shown, compared with the Sham group, the mRNA expression levels of mitochondrial-encoded respiratory chain complex genes ATP6, Cox1, Cytb and ND1 in the myocardial tissue of IR group mice were significantly reduced, while OGN overexpression significantly upregulated the mRNA expression of respiratory chain complex genes ATP6, Cox1, Cytb and ND1.

[0106] This result indicates that OGN overexpression regulates the expression of mitochondrial respiratory chain complex genes at the transcriptional level, further consolidating the improvement in mitochondrial respiratory function and confirming the direct regulatory effect of OGN on the respiratory capacity of mitochondria themselves.

[0107] Example 5

[0108] This embodiment demonstrates that overexpression of OGN can regulate the expression of mitochondrial splitting / fusion proteins in cardiomyocytes and maintain mitochondrial morphological homeostasis.

[0109] Based on the animal experiment grouping and model establishment in Example 2, mice in each group were sacrificed after 48 hours of reperfusion, and their hearts were quickly removed. Left ventricular myocardial tissue was taken to detect the expression of splitting / fusion proteins in the myocardial tissue.

[0110] I. The expression changes of mitochondrial splitting proteins Drp1 (Dynamin-related protein 1), Fis1 (Fission protein 1), and fusion proteins Mfn1 (Mitofusin 1), Mfn2 (Mitofusin 2), and OPA1 (Optic atrophy 1) in mouse cardiomyocytes were detected by conventional qRT-PCR.

[0111] qRT-PCR results are as follows Figure 12As shown, compared with the Sham group, the expression levels of mitochondrial splitting proteins Drp1 and Fis1 in cardiomyocytes of mice in the IR group were significantly increased, while the expression levels of Drp1 and Fis1 were significantly decreased after AAV9-OGN treatment. Compared with the Sham group, the expression levels of mitochondrial fusion proteins OPA1, MFN1, and MFN2 in cardiomyocytes of mice in the IR group were significantly decreased, while the expression levels of OPA1, MFN1, and MFN2 were significantly increased after AAV9-OGN treatment.

[0112] II. The expression level of mitochondrial splitting protein Drp1 was detected by Western blotting and immunofluorescence techniques.

[0113] Immunofluorescence technique employs multicolor immunofluorescence staining combined with confocal microscopy imaging. The specific method is as follows:

[0114] Four groups of mouse cardiomyocytes were fixed, permeabilized, and blocked using standard methods. They were incubated with primary antibodies: Drp1 (rabbit anti-Drp1 antibody) and α-actinin (mouse anti-α-actinin antibody, which labels sarcomere structures). They were also incubated with secondary antibodies: fluorescently labeled secondary antibodies: Drp1 red, α-actinin green, and nuclear staining blue. The cells were then mounted and images were acquired using a confocal microscope.

[0115] Western Blot results are as follows Figure 13 , Figure 14 As shown, the expression level of Drp1, a mitochondrial splitting protein in mouse cardiomyocytes, was significantly increased, while the expression level of Drp1 was significantly decreased after AAV9-OGN treatment.

[0116] Immunofluorescence results as follows Figures 15-16 As shown, compared with the Sham group, the IR group showed high Drp1 expression, indicating active mitochondrial division. In the OGN-IR group, Drp1 expression was significantly decreased, and mitochondrial division activity was significantly inhibited.

[0117] This result indicates that ischemia-reperfusion injury leads to enhanced mitochondrial division and weakened fusion, resulting in disruption of the mitochondrial network structure. OGN overexpression can inhibit the expression of mitochondrial division proteins Drp1 and Fis1, while upregulating the expression of fusion proteins Mfn1, Mfn2, and OPA1. By balancing the mitochondrial division and fusion processes, it maintains the integrity of the mitochondrial network structure and thus improves mitochondrial function to alleviate myocardial ischemia-reperfusion injury.

[0118] Example 6

[0119] This embodiment demonstrates through cell experiments that overexpression of OGN can enhance the respiratory capacity of cardiomyocytes, increase mitochondrial membrane potential, and reduce ROS levels.

[0120] I. Cell grouping and model establishment methods

[0121] Primary cardiomyocytes from neonatal mice were randomly divided into 4 groups:

[0122] (1) Blank control Ctl group: normal culture, no transfection, no hypoxia / reoxygenation;

[0123] (2) HR group: normal culture, hypoxia / reoxygenation treatment;

[0124] (3) OGN-HR group: transfected with OE-OGN, hypoxia / reoxygenation treatment;

[0125] (4) NC-HR group: transfected with OE-NC, hypoxia / reoxygenation treatment;

[0126] OE-OGN is a recombinant plasmid that overexpresses the mouse OGN gene, and OE-NC is an empty vector NC control plasmid.

[0127] The methods for cardiomyocyte culture and plasmid transfection are as follows:

[0128] 1. Primary cardiomyocyte culture of suckling rats:

[0129] Surgical instruments were sterilized at high temperature two hours in advance. Newborn Kunming mice (1-3 days old) were sterilized with 75% ethanol in a laminar flow hood and then euthanized by decapitation. After decapitation, the chest was opened with ophthalmic scissors, and the heart was removed with curved forceps. The heart was washed away with pre-cooled PBS buffer containing a mixture of penicillin, streptomycin, and amphotericin B (triple antibody) to remove blood. All hearts were collected and placed in 50ml centrifuge tubes. The hearts were rinsed three times with an appropriate amount of D-Hanks buffer, and the remaining D-Hanks buffer was aspirated. Trypsin digestion solution (40% trypsin + 60% D-Hanks) was added, and digestion was carried out overnight at 4°C in a shaker. The next day, DMEM complete culture medium containing 10% fetal bovine serum was added to terminate digestion. The digestion solution was discarded, and type II collagenase digestion solution (0.8 mg type II collagenase per ml of DMEM) was added. Digestion was repeated three times at 37°C in a shaker, 10 minutes each time. After each round of digestion, the supernatant of the digestion solution was collected, centrifuged at 1000 rpm for 5 minutes, and the supernatant was slowly discarded. The precipitate was retained, and an appropriate amount of DMEM culture medium containing 10% fetal bovine serum was added to resuspend the cell pellet and distribute it evenly. The cells were pre-cultured in an incubator (5% CO2, 37℃) for 1 hour to allow the cardiomyocytes to adhere to the culture plate. The cardiomyocytes were gently blown off, and the suspension containing the cardiomyocytes was collected and evenly seeded into culture plates. The cells were cultured in a cell culture incubator for 48 hours. After the cardiomyocytes adhered and grew, subsequent experiments were performed.

[0130] 2. Plasmid transfection:

[0131] According to the instructions for the transfection reagent Lipofectamine 2000, mix 1 μg of OE-OGN or OE-NC with 3 μL of transfection reagent, add to 50 μL of Opti-MEM medium and incubate for 15 minutes; add the resulting complex dropwise to cell culture wells, shake gently to mix, and incubate at 37°C, 5% CO2 for 6 hours. Then replace with complete medium and continue culturing for 24-48 hours.

[0132] 3. Construction of Hypoxia / Reoxygenation (H / R) Model

[0133] Hypoxia treatment: After 48 hours of transfection of OGN-HR and NC-HR groups, the original culture medium was discarded and replaced with serum-free D-Hanks buffer containing phenol red. The cells were then transferred to a three-gas incubator and the conditions were adjusted to 1% O2, 5% CO2, and 94% N2 for 6 hours.

[0134] Reoxygenation treatment: After 6 hours of hypoxia, the cells in the OGN-HR group and NC-HR group were replaced with reoxygenation medium (DMEM + 10% fetal bovine serum) and placed back in a conventional incubator (37℃, 5% CO2) for 12 hours of recovery culture.

[0135] II. The changes in oxygen consumption rate (OCR) of four groups of myocardial cells were detected using the Seahorse XFe24 Analyzer and the XF Cell Mito Stress Test kit.

[0136] The resulting dynamic response curve of oxygen consumption rate is shown in the figure below. Figure 17 As shown, the oxygen consumption rates of basal respiration and maximal respiration are compared, for example... Figure 18 As shown, compared with the blank control group, the mitochondrial respiration capacity of cardiomyocytes in the HR group was significantly reduced, while the respiration capacity of cardiomyocytes overexpressing OGN was significantly improved, indicating that OGN overexpression treatment can improve the mitochondrial respiration capacity of cardiomyocytes.

[0137] III. JC-1 staining to detect changes in mitochondrial membrane potential in cardiomyocytes

[0138] Four groups of cardiomyocytes were seeded into confocal culture dishes and cultured routinely for 48 hours. After the cardiomyocytes adhered and grew, the experimental and control groups were simultaneously cultured according to the plasmid transfection and hypoxia-reoxygenation construction method described in step one. Then, the culture medium was removed, JC-1 working solution was added, and the cells were incubated at 37°C for 15-30 minutes. The cells were washed twice with pre-warmed buffer, and live-cell imaging and fluorescence intensity analysis were performed. JC-1 formed multimers in the mitochondrial matrix, indicating an increased membrane potential; JC-1 existed in monomeric form, suggesting a decreased membrane potential.

[0139] JC-1 staining results are as follows Figure 19 As shown, the membrane potential is as follows: Figure 20As shown, compared with the Ctl group, the mitochondrial membrane potential of cardiomyocytes in the HR group was significantly reduced, indicating that hypoxia / reoxygenation treatment caused mitochondrial damage; while OGN overexpression treatment significantly increased the mitochondrial membrane potential of cardiomyocytes. This shows that OGN overexpression can effectively reverse the hypoxia / reoxygenation-induced decrease in mitochondrial membrane potential of cardiomyocytes, maintain the stability of mitochondrial membrane structure, and thus reduce cardiomyocyte damage.

[0140] IV. Detection of ROS Changes using DCFH-DA Fluorescent Probe Staining Technology

[0141] Four groups of cardiomyocytes were seeded into confocal culture dishes and cultured routinely for 48 hours. After the cardiomyocytes adhered and grew, the experimental and control groups were simultaneously cultured according to the plasmid transfection and hypoxia-reoxygenation construction method described in step one. Then, the culture medium was removed, and 10 μM DCFH-DA working solution was added. The cells were incubated in a 37°C cell culture incubator in the dark for 20-30 minutes to allow the probe to fully penetrate the cells and be hydrolyzed into DCFH by esterase. The cells were washed 1-2 times with serum-free cell culture medium to thoroughly remove any DCFH-DA that had not entered the cells. Fluorescence was observed under a fluorescence microscope using a FITC filter.

[0142] The results of DCFH-DA fluorescence staining are as follows: Figure 21 As shown, the relative levels of reactive oxygen species are as follows: Figure 22 As shown, compared with the Ctl group, the ROS level of cardiomyocytes in the HR group was significantly increased, and the OGN overexpression treatment significantly reduced the ROS level of cardiomyocytes.

[0143] In summary, this embodiment demonstrates that OGN overexpression can enhance the respiratory capacity of neonatal mouse cardiomyocytes, increase mitochondrial membrane potential, and reduce ROS levels, thus regulating mitochondrial homeostasis in cardiomyocytes. This is consistent with the results of animal experiments showing that OGN overexpression inhibits ROS generation in myocardial tissue, suggesting that OGN may reduce oxidative stress damage at the cellular level by regulating mitochondrial function-related pathways, providing more direct cellular-level evidence for its protective effect against myocardial ischemia-reperfusion injury.

Claims

1. Application of OGN overexpression vector in the preparation of drugs for the prevention of reperfusion myocardial injury, wherein the nucleotide sequence encoding OGN is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The OGN overexpression vector is a recombinant adeno-associated virus vector containing a nucleotide sequence encoding OGN and a myocardial-specific promoter.

3. The application according to claim 2, characterized in that, The myocardial-specific promoter is the cardiac troponin T promoter.

4. The application according to claim 3, characterized in that, The serotype of the recombinant adeno-associated virus vector is AAV9.

5. The application according to claim 4, characterized in that, The OGN overexpression vector is an adeno-associated virus vector AAV9-OGN that overexpresses the OGN gene.

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