Application of RPL3L overexpression vector in preparation of MIRI treatment medicine

Abstract

The invention relates to application of an RPL3L overexpression vector in preparation of an MIRI treatment medicine, and belongs to the technical field of biological medicine. In order to solve the problem that treatment of MIRI lacks an effective accurate intervention target, the invention provides application of an RPL3L overexpression vector in preparation of MIRI treatment drugs, the core value of RPL3L as a myocardial ischemia reperfusion injury accurate target is confirmed for the first time through an adeno-associated virus (AAV) mediated RPL3L overexpression system, and the application of the RPL3L overexpression vector in preparation of the MIRI treatment drugs is provided for the first time. The overexpression of the gene can realize targeted protection of myocardial ischemia reperfusion injury through three key dimensions of ribosome function improvement, injury mitochondrial autophagy inhibition and heart function improvement. The invention provides a new strategy taking RPL3L as a precise target spot for treatment of myocardial ischemia reperfusion injury, also lays a foundation for development of RPL3L-targeted myocardial protection drugs, and has outstanding clinical transformation potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of RPL3L overexpression vector in the preparation of MIRI therapeutic drugs. Background Technology

[0002] Myocardial ischemia-reperfusion (MI / R) is a crucial means of rescuing ischemic cardiomyocytes, but this process often induces myocardial ischemia-reperfusion injury (MIRI), characterized by progressively worsening myocardial tissue damage and even inducing arrhythmias, severely reducing the treatment efficacy of ischemic heart disease. The severity of MIRI is regulated by multiple factors, including ischemia duration and tissue oxygen demand. Despite continuous exploration of preventive strategies for MIRI in clinical practice, there remains a lack of effective programs that can significantly reduce myocardial infarction area and improve cardiac function. The core bottleneck lies in the absence of molecular targets that can precisely intervene in the occurrence and development of MIRI.

[0003] Ribosomes are core organelles for cellular protein translation; abnormal function leads to decreased cardiac protein synthesis, directly affecting myocardial repair and contributing significantly to poor prognosis of myocardial infarction (MIRI). Mitochondria, as the core of cellular energy metabolism, play a crucial regulatory role in MIRI through autophagy homeostasis imbalance; excessive autophagy can exacerbate cardiomyocyte damage. Cardiac ribosome-associated protein L3-like protein (RPL3L) is a conserved protein specifically expressed in the heart and skeletal muscle. Mutations or abnormal expression of RPL3L are closely related to the development of heart failure and cardiomyopathy, but the regulatory role and mechanism of RPL3L in MIRI have not yet been reported. Summary of the Invention

[0004] To address the lack of effective and precise intervention targets for the treatment of MIRI, this invention provides the application of the RPL3L overexpression vector in the preparation of MIRI therapeutic drugs.

[0005] The technical solution of the present invention: The application of RPL3L overexpression vector in the preparation of drugs for the treatment of myocardial ischemia-reperfusion injury, wherein the nucleotide sequence of RPL3L is shown in SEQ ID NO.1.

[0006] Furthermore, the RPL3L overexpression vector is an adeno-associated virus vector AAV9-RPL3L that overexpresses the RPL3L gene, containing a nucleotide sequence encoding RPL3L and a myocardial-specific promoter; the myocardial-specific promoter is the cardiac troponin T promoter.

[0007] Furthermore, the drug for treating myocardial ischemia-reperfusion injury has at least one of the following uses: (1) Improves cardiac function after reperfusion; (2) Increase the number and translational activity of myocardial ribosomal proteins after reperfusion; (3) Reduce post-reperfusion damage to mitophagy and improve mitochondrial function.

[0008] The beneficial effects of this invention are: This invention, through an adeno-associated virus (AAV)-mediated RPL3L overexpression system, demonstrates for the first time the core value of RPL3L as a precise target for myocardial ischemia-reperfusion injury. Its overexpression can achieve targeted protection against myocardial ischemia-reperfusion injury through three key dimensions: enhancing ribosome function, inhibiting damaging mitophagy, and improving cardiac function. This invention provides a novel strategy for the treatment of myocardial ischemia-reperfusion injury using RPL3L as a precise target, and lays the foundation for the development of cardioprotective drugs targeting RPL3L, demonstrating significant potential for clinical translation. Attached Figure Description

[0009] Figure 1 The electrocardiograms of mice in the sham-operated group and the IR group before and after reperfusion in Example 1 are shown. Figure 2 The image shows a comparison of Western Blot results of RPL3L in the myocardial tissue of mice in the sham-operated group and the IR group in Example 1. A is a Western Blot result image, and B is a comparison of RPL3L protein expression levels. Figure 3 This is a comparison of the mRNA expression levels of RPL3L in the myocardial tissue of mice in the sham-operated group and the IR group in Example 1. Figure 4 Echocardiograms of four groups of mice in Example 2: A is the sham-operated group, B is the IR group, C is the RPL3L-IR group, and D is the NC-IR group. Figure 5 This is a comparison chart of cardiac ejection fraction and fractional shortening of four groups of mice in Example 2. A is ejection fraction EF and B is fractional shortening of the minor axis FS. Figure 6 This is a comparison of the left ventricular end-diastolic diameter and left ventricular end-systolic diameter in four groups of mice in Example 2. A is the left ventricular end-diastolic diameter LVIDd, and B is the left ventricular end-systolic diameter LVIDs. Figure 7 The following is a comparison of puromycin incorporation levels in four groups of mouse cardiomyocytes overexpressing RPL3L in Example 3: A is a Western blot diagram of the NC-IR group and the RPL3L-IR group; B is a statistical graph of incorporation levels in the NC-IR group and the RPL3L-IR group; C is a Western blot diagram of the NC-HR group and the RPL3L-HR group; and D is a statistical graph of incorporation levels in the NC-HR group and the RPL3L-HR group. Figure 8The following is a comparison of the puromycin incorporation levels in the four groups of RPL3L knockdown mouse cardiomyocytes in Example 3: A is a Western blot diagram of the si-NC group and the si-RPL3L group; B is a statistical diagram of the incorporation levels in the si-NC group and the si-RPL3L group; C is a Western blot diagram of the sh-NC group and the sh-RPL3L group; and D is a statistical diagram of the incorporation levels in the sh-NC group and the sh-RPL3L group. Figure 9 The images show the ribosomal sucrose density gradient centrifugation spectra of the eight groups of mouse myocardial tissue in Example 3. A represents the NC-IR group and the RPL3L-IR group, B represents the NC-HR group and the RPL3L-HR group, C represents the si-NC group and the si-RPL3L group, and D represents the sh-NC group and the sh-RPL3L group. Figure 10 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. Figure 11 This is a comparative diagram of the respiratory chain complex activity of mitochondria in four groups of mouse cardiomyocytes in Example 4; Figure 12 The images show a comparison of DHE fluorescence staining detection in four groups of mouse myocardial tissues in Example 4. A is a DHE staining image, and B is a fluorescence intensity statistical graph. Figure 13 The image shows a comparison of the expression levels of Drp1 and VDAC1 proteins in the myocardial tissues of four groups of mice in Example 5. A is a Western blot image, B is a statistical graph of Drp1 protein expression, and C is a statistical graph of VDAC1 protein expression. Figure 14 The image shows a comparison of LC3 and P62 protein expression levels in four groups of mouse cardiomyocytes in Example 5. A is a Western blot image, B is a statistical graph of the LC3Ⅰ / LC3-Ⅱ ratio, and C is a statistical graph of P62 protein expression levels. Detailed Implementation

[0010] 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.

[0011] Example 1 This example demonstrates that RPL3L expression is reduced in myocardial tissue after myocardial ischemia-reperfusion.

[0012] I. Methods for grouping experimental animals and establishing experimental models: Eight-week-old male C57BL / 6J mice (weighing 22-25g) were randomly divided into two groups: (1) Sham group: only open-chest surgery and suture threading were performed, without ligation of the coronary arteries; (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.

[0013] 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 rises after ligation of ischemia and falls after reperfusion, indicating that the IR group model was successfully constructed.

[0014] 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 removed on ice. The expression of RPL3L in the myocardial tissue was detected.

[0015] (1) Western Blot detection: 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; 2. Electrophoretic transfer: Proteins were separated by 10% SDS-PAGE and transferred to an NC membrane; 3. Antibody incubation: anti-RPL3L antibody (1:1000, Immunoway), anti-β-actin antibody (1:3000, CST).

[0016] 4. Results Analysis: Image J quantifies grayscale values, and the RPL3L / β-actin ratio represents the relative expression level.

[0017] The results are as follows Figure 2 As shown, compared with the Sham group, the expression of RPL3L protein in the myocardial tissue of IR group mice was significantly reduced (P<0.05).

[0018] (2) qRT-PCR detection: 1. RNA extraction: Total RNA was extracted from the left ventricle using the Trizol method and quantified using NanoDrop. 2. Reverse transcription: 1 μg of RNA was used to synthesize cDNA using the PrimeScript RT kit; 3. Quantitative PCR: SYBR Green assay for RPL3L mRNA detection. 4. Results analysis: The relative expression level was calculated using the 2-ΔΔCt method.

[0019] The results are as follows Figure 3 As shown, the qRT-PCR results were consistent with the Western Blot results, and the RPL3L mRNA content in the myocardial tissue of IR group mice was significantly reduced (P<0.05).

[0020] The results of this embodiment indicate that RPL3L expression is reduced in myocardial tissue after myocardial ischemia-reperfusion injury.

[0021] Example 2 This embodiment demonstrates that overexpression of RPL3L can improve cardiac function after ischemia-reperfusion.

[0022] I. Construction of overexpression vectors (1) Carrier selection: This embodiment uses an adeno-associated virus type 9 (AAV9) vector system with a vector backbone of ssAAV-cTnT-RPL3L-SV40pA, containing the following elements: cardiac troponin T (cTnT) promoter, target gene RPL3L coding sequence, and SV40pA which helps improve expression efficiency and stability. The nucleotide sequence encoding RPL3L is shown in SEQ ID NO.1.

[0023] (2) RPL3L gene cloning and viral packaging RPL3L gene cloning and viral packaging were performed by Guangzhou Aizhe Biotechnology Co., Ltd. using a gene cloning-viral packaging-purification and concentration method, yielding purified AAV9-RPL3L virus particles with a titer of 1×10⁻⁶. 13 vg / mL.

[0024] (3) Control vector The RPL3L sequence was replaced with a meaningless sequence, while the remaining elements were completely identical to AAV9-RPL3L. 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.

[0025] II. Animal Experiment Grouping and Model Establishment Methods: Eight-week-old male C57BL / 6J mice (weighing 22-25g) were randomly divided into four groups: (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; (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. (3) RPL3L-IR group: AAV9-RPL3L (100μL, 1×10) was injected into the tail vein. 11(vg), blood flow was restored 30 minutes after ligation of the left anterior descending coronary artery 4 weeks later, followed by reperfusion for 48 hours; (4) NC-IR group: AAV9-NC (100μL, 1×10) was injected into the tail vein. 11 (vg), blood flow was restored 30 minutes after ligation of the left anterior descending coronary artery 4 weeks later, followed by reperfusion for 48 hours; 3. Monitor cardiac function through echocardiography.

[0026] Echocardiograms of the four groups of mice are as follows: Figure 4 As shown, cardiac ejection fraction (EF) is compared to, for example... Figure 5 As shown in A, shortening the fraction FS is like... Figure 5 As shown in B, the left ventricular end-diastolic diameter (LVIDd) is compared to... Figure 6 As shown in Figure A, the left ventricular end-systolic diameter (LVIDs) is compared to... 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 RPL3L-IR group mice showed restored ventricular wall motion amplitude, with significantly increased EF% and FS% (P<0.05); no significant changes were observed in LVIDd and LVIDs.

[0027] This result demonstrates that RPL3L 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 therapeutic drug.

[0028] Example 3 This embodiment demonstrates that overexpression of RPL3L can improve the number of myocardial ribosomes and translational function in mice after ischemia-reperfusion.

[0029] I. Grouping and Model Establishment Methods for Animal and Cell Experiments: Eight-week-old male C57BL / 6J mice (weighing 22-25g) were randomly divided into two groups: (1) RPL3L-IR group: AAV9-RPL3L (100μL, 1×10) was injected into the tail vein. 11 (vg), blood flow was restored 30 minutes after ligation of the left anterior descending coronary artery 4 weeks later, followed by reperfusion for 48 hours; (2) NC-IR group: AAV9-NC (100μL, 1×10) was injected into the tail vein. 11 (vg), blood flow was restored 30 minutes after ligation of the left anterior descending coronary artery 4 weeks later, followed by reperfusion for 48 hours; Eight-week-old male C57BL / 6J mice (weighing 22-25g) were randomly divided into two groups: (1) sh-RPL3L group: AAV9-sh-RPL3L (RPL3L targets short hairpin RNA adeno-associated virus sh-RPL3L, 100μL, 1×10) was injected into the tail vein. 11 vg), and subsequent experimental treatment will be carried out after 4 weeks; (2) sh-NC group: AAV9-sh-NC (negative control adeno-associated virus sh-NC, 100 μL, 1×10⁻⁶) was injected via tail vein. 11 vg), and subsequent experimental treatment will be carried out after 4 weeks; Primary mouse cardiomyocytes were isolated and randomly divided into two groups: (1) RPL3L-HR group: mouse primary cardiomyocytes were isolated, cultured for 48 hours and transfected with AAV9-RPL3L. After 24 hours, they were placed in a hypoxic incubator (95% N2 + 5% CO2) for 6 hours of hypoxia, and then transferred to a normoxic incubator (95% air + 5% CO2) for 12 hours of reoxygenation. (2) NC-HR group: mouse primary cardiomyocytes were isolated, cultured for 48 hours and transfected with AAV9-NC. After 24 hours, they were placed in a hypoxic incubator (95% N2 + 5% CO2) for 6 hours of hypoxia, and then transferred to a normoxic incubator (95% air + 5% CO2) for 12 hours of reoxygenation. Primary mouse cardiomyocytes were isolated and randomly divided into two groups: (1) si-RPL3L group: mouse primary cardiomyocytes were isolated, transfected with si-RPL3L (RPL3L targets small interfering RNA, siRNA-RPL3L sequence), and cultured for 48 hours before subsequent experimental treatment; (2) si-NC group: mouse primary cardiomyocytes were isolated, transfected with si-NC (negative control small interfering RNA, nucleotide sequence as shown in SEQ ID NO:5), and cultured for 48 hours before subsequent experimental treatment; The siRNA-RPL3L sequence in this embodiment is: Chain of Justice: 5'-GGUAGAAGCUGUGACAAUUTT-3'; Antonym chain: 5'-AAUUGUCACAGCUUCUACCTT-3'; sh-RPL3L sequence: GGUAGAAGCUGUGACAAUUTT.

[0030] To comply with WIPO ST.26 standards, as shown in SEQ ID NO:2, the nucleotide sequence of the siRNA-RPL3L sense strand uses T to replace the U at positions 3, 10, 12, 18, and 19 of the 5' end to represent uracil in RNA. Similarly, as shown in SEQ ID NO:3, the nucleotide sequence of the siRNA-RPL3L antisense strand uses T to replace the U at positions 3, 4, 6, 13, 14, and 16 of the 5' end to represent uracil in RNA. Likewise, as shown in SEQ ID NO:4, the nucleotide sequence of sh-RPL3L uses T to replace the U at positions 3, 10, 12, 18, and 19 of the 5' end to represent uracil in RNA.

[0031] II. Puromycin incorporation assay for detecting myocardial ribosome translational activity: Puromycin is a tRNA-mimicking antibiotic that competitively binds to the peptidyl transferase center of the ribosome, incorporating itself into the terminal end of a synthesizing peptide chain, causing premature termination and release from the ribosome. By detecting the amount of puromycin-incorporated peptide chain in cells / tissues, the efficiency of ongoing protein synthesis by the ribosome can be directly reflected; a higher incorporation amount indicates stronger ribosome translational activity.

[0032] 1. Puromycin treatment: Live mice were injected intraperitoneally with puromycin (final concentration 100 μM), and primary cardiomyocytes were incubated with puromycin (final concentration 50 μM) in the culture medium for 30 min. 2. Collect myocardial tissue / cells, extract total protein using RIPA lysis buffer, and quantify protein concentration using the BCA method; 3. Western Blot detection: Equal amounts of protein were subjected to SDS-PAGE electrophoresis, and after transfer to a membrane, the mixture was incubated with anti-purinemycin antibody and anti-β-actin antibody (internal control), followed by ECL staining. 4. Activity quantification: The ratio of the gray value of the puromycin band to the gray value of the β-actin band represents the ribosomal translation activity.

[0033] The amount of puromycin incorporation in the cardiomyocytes of four groups of mice overexpressing RPL3L was compared with that in other mice. Figure 7 As shown, the puromycin incorporation levels in both the RPL3L-IR and RPL3L-HR groups were significantly higher than those in the NC control group; indicating that overexpression of RPL3L can enhance myocardial ribosome translation activity in ischemia-reperfusion mice.

[0034] The amount of puromycin incorporation in four groups of RPL3L knockout cardiomyocytes was compared to, for example... Figure 8 As shown, the amount of puromycin incorporated in both the si-RPL3L group and the sh-RPL3L group was significantly lower than that in the NC control group; indicating that silencing RPL3L in cells reduces ribosomal translation activity.

[0035] This indicates that, in both animal and cell models, the expression level of RPL3L is positively correlated with myocardial ribosome translational activity: overexpression of RPL3L enhances translational activity, while interference with RPL3L inhibits this activity, confirming the regulatory role of RPL3L in ribosome function.

[0036] III. Ribosome mapping analysis method for detecting changes in myocardial ribosome count: Four groups of myocardial tissue were minced and added to pre-chilled ribosome lysis buffer. After homogenization on ice, the tissue was centrifuged and the supernatant was collected. Four other groups of cell basins were washed with PBS, added to pre-chilled ribosome lysis buffer, incubated on ice, and then centrifuged and the supernatant was collected. In ultracentrifuge tubes, 10%, 20%, 30%, 40%, and 50% sucrose solutions were added layer by layer to form a continuous density gradient, and the mixture was pre-chilled at 4°C. The supernatants from the above samples were slowly loaded onto the top layer of the sucrose gradient solution and ultracentrifuged at 35,000 rpm for 3 hours (tissue samples) or 2.5 hours (cell samples) at 4°C. The centrifuged sucrose gradient components were collected using a continuous fractionator, and the absorbance at 254 nm was simultaneously measured. A ribosome-sucrose density gradient centrifugation spectrum was plotted with absorbance as the ordinate and collection location as the abscissa.

[0037] Cellular ribosome sucrose gradient centrifugation spectrum as shown in the figure Figure 9 As shown, the polyribosome peaks in the RPL3L-IR and RPL3L-HR groups were higher than those in the NC control group, indicating that overexpression of RPL3L can increase the number of polyribosomes actively translating. The polyribosome peaks in the si-RPL3L and sh-RPL3L groups were lower than those in the NC control group, while monomeric and subunit peaks increased, indicating that silencing RPL3L disrupts the ribosome assembly balance and reduces the number of polyribosomes. This spectrum, from the perspective of ribosome assembly and function, confirms that "RPL3L expression level is positively correlated with ribosome number / activity": overexpression of RPL3L promotes ribosome assembly into actively translating polyribosomes, while interference with RPL3L inhibits ribosome assembly, further supporting the regulatory role of RPL3L in ribosome function.

[0038] Example 4 This embodiment demonstrates that overexpression of RPL3L can enhance myocardial mitochondrial respiratory function and reduce reactive oxygen species content.

[0039] Based on the animal experiment grouping and model establishment in Example 2, the mice in each group were sacrificed after 48 hours of reperfusion, and their hearts were quickly removed.

[0040] I. The Orbororos O2k technique was used to detect changes in myocardial mitochondrial respiratory function after in vivo overexpression of RPL3L.

[0041] Left ventricular myocardial tissue from each group of mice was cut into 5mg samples, placed in a manual glass homogenizer, and 200μL of pre-cooled mitochondrial respiratory medium (MiR05) was added. The homogenizer was homogenized on ice, and large debris was filtered out after homogenization to obtain the homogenized sample to be tested.

[0042] (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).

[0043] (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: Pyruvate (5mM), malic acid (2mM), glutamate (10mM), and ADP (2.5mM) were used as substrates for complex I (NADH dehydrogenase), and rotenone (1μM) was used as an inhibitor of complex I. Succinic acid (10 mM) 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. Ascorbic acid (2 mM) and TMPD (0.5 mM) were used as substrates for complex IV (cytochrome c oxidase).

[0044] The results of collecting and measuring the respiratory curve are as follows: Figure 10 As shown, compared with the Sham group, the oxygen consumption rate of the IR group was significantly reduced, indicating that IR damage led to a decline in mitochondrial respiratory function; the oxygen consumption rate of the RPL3L-IR group significantly rebounded, approaching the Sham level. Changes in respiratory chain complex activity are shown in... Figure 11 As shown, compared with the Sham group, the activity of each complex in the IR group was significantly reduced, while the activity of each complex in the RPL3L-IR group was significantly increased.

[0045] This indicates that IR damage significantly inhibits mitochondrial oxygen consumption and respiratory chain complex activity, while RPL3L overexpression can effectively improve mitochondrial respiratory function and restore respiratory chain complex activity in IR mice, which is one of the important mechanisms by which it exerts its cardioprotective effect.

[0046] II. Immunofluorescence technique was used to detect changes in myocardial mitochondrial respiratory function and myocardial reactive oxygen species content.

[0047] Left ventricular myocardial tissue was collected from each group of mice and cut into 5 μm thick frozen sections. DHE fluorescent probe staining was used to detect changes in ROS in the myocardial tissue.

[0048] DHE staining steps: (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; (2) Washing: Rinse with PBS 3 times (5 minutes each time) to remove unbound probes.

[0049] (3) Mounting: Mount the slide with an anti-fluorescence quenching agent (containing DAPI) and store at 4°C away from light.

[0050] The confocal microscopy imaging results of each group of stained samples are as follows: Figure 12 As shown, compared with the Sham group, the IR group exhibited strong red fluorescence, indicating that IR damage induced the generation of a large amount of ROS, while the red fluorescence in the RPL3L-IR group was significantly weakened, indicating a decrease in ROS levels. This suggests that RPL3L overexpression can effectively inhibit IR-induced ROS generation and reduce damaged mitophagy, indicating that RPL3L has a regulatory role in cardiomyocyte mitophagy.

[0051] Example 5 This embodiment demonstrates that overexpression of RPL3L can maintain normal levels of mitophagy and improve mitochondrial function.

[0052] 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 from each group of mice, and Western blotting was used to detect changes in the expression of Drp1 (mitochondrial fission protein), VDAC1 (mitochondrial autophagy recognition marker), LC3 (autophagy activity marker), and P62 (autophagy substrate protein).

[0053] The comparison results of Drp1 and VDAC1 protein expression levels in the myocardial tissues of four groups of mice are as follows: Figure 13 As shown, compared with the Sham group, Drp1 expression was significantly increased in the IR group, indicating that IR induces enhanced mitochondrial division. Drp1 expression in the RPL3L-IR group was lower than that in the IR group, indicating that RPL3L overexpression can inhibit excessive mitochondrial division. Compared with the Sham group, VDAC1 expression was significantly decreased in the IR group, indicating that IR damages mitophagy-related proteins. VDAC1 expression in the RPL3L-IR group was higher than that in the IR group, indicating that RPL3L overexpression can restore VDAC1 levels.

[0054] The expression levels of LC3 and P62 proteins in four groups of mouse cardiomyocytes were compared as follows: Figure 14 As shown, the LC3-II / LC3-I ratio reflects autophagy activity. The ratio in the IR group was higher than that in the Sham group, indicating that IR induces excessive autophagy. However, the ratio in the RPL3L-IR group was lower than that in the IR group, demonstrating that RPL3L overexpression can inhibit excessive autophagy. The P62 level in the IR group was lower than that in the Sham group, indicating that IR leads to excessive degradation of autophagy substrates. Conversely, the P62 expression in the RPL3L-IR group was higher than that in the IR group, indicating that RPL3L overexpression can restore P62 levels.

[0055] The above experimental results indicate that IR injury induces excessive mitochondrial division and abnormal autophagy in the myocardium, while RPL3L overexpression can precisely regulate the expression of autophagy-related proteins, inhibit excessive mitochondrial division, restore autophagy balance, thereby improving mitochondrial function and exerting a cardioprotective effect.

Claims

1. The application of RPL3L overexpression vector in the preparation of drugs for the treatment of myocardial ischemia-reperfusion injury, characterized in that, The nucleotide sequence of RPL3L is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The RPL3L overexpression vector is an adeno-associated virus vector AAV9-RPL3L that overexpresses the RPL3L gene, containing a nucleotide sequence encoding RPL3L and a myocardial-specific promoter; the myocardial-specific promoter is the cardiac troponin T promoter.

3. The application according to claim 2, characterized in that, The drug for treating myocardial ischemia-reperfusion injury has at least one of the following uses: (1) Improves cardiac function after reperfusion; (2) Increase the number and translational activity of myocardial ribosomal proteins after reperfusion; (3) Reduce post-reperfusion damage to mitophagy and improve mitochondrial function.

Images