Use of rpl3l overexpression vector in preparation of miri treatment drugs

Abstract

The application relates to application of an RPL3L overexpression vector in preparation of MIRI treatment drugs and belongs to the technical field of biological medicines. In order to solve the problem that treatment of MIRI lacks effective and precise intervention targets, the application provides application of the RPL3L overexpression vector in preparation of MIRI treatment drugs, through an adeno-associated virus (AAV) mediated RPL3L overexpression system, it is first confirmed that RPL3L is a core value of a precise target point of myocardial ischemia reperfusion injury, overexpression of the RPL3L can realize targeted protection of myocardial ischemia reperfusion injury through three key dimensions of improving ribosome function, inhibiting damage autophagy of mitochondria and improving heart function. The application provides a new strategy taking RPL3L as a precise target point for treatment of myocardial ischemia reperfusion injury, lays a foundation for development of myocardial protection drugs targeting RPL3L, and has outstanding clinical conversion potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of an RPL3L overexpression vector in preparation of a MIRI treatment drug. BACKGROUND

[0002] Myocardial ischemia reperfusion (MI / R) is an important means to save ischemic myocardial cells, but this process often induces myocardial ischemia reperfusion injury (MIRI), which is manifested as progressive aggravation of myocardial tissue damage, and even induces arrhythmia, thereby seriously reducing the treatment effect of ischemic heart disease. The degree of MIRI is regulated by various factors such as ischemia duration and tissue oxygen demand, although the prevention strategies for MIRI are continuously explored in the clinic, there is still a lack of effective solutions that can significantly reduce the myocardial infarction area and improve the damage of cardiac function, and the core bottleneck is that no molecular target that can precisely intervene in the occurrence and development of MIRI has been found.

[0003] Ribosomes are the core organelles of cell protein translation, and their dysfunction can lead to a decrease in myocardial protein synthesis capacity, directly affecting the myocardial repair process, and is an important inducement for poor prognosis of MIRI. Mitochondria, as the core of cellular energy metabolism, play a key regulatory role in MIRI when the autophagy homeostasis is imbalanced, and excessive autophagy can even exacerbate myocardial cell damage. Myocardial ribosome-related protein L3-like (RPL3L) is a conserved protein specifically expressed in the heart and skeletal muscle, and its mutation or abnormal expression is closely related to the occurrence and development of heart failure and cardiomyopathy, but there is no relevant research report on the regulatory role and mechanism of RPL3L in MIRI. SUMMARY

[0004] To solve the problem that there is a lack of effective and precise intervention targets for MIRI treatment, the application provides application of an RPL3L overexpression vector in preparation of a MIRI treatment drug.

[0005] The technical scheme of the application is as follows:

[0006] The application of the RPL3L overexpression vector in preparation of a myocardial ischemia reperfusion injury treatment drug, wherein the nucleotide sequence of the RPL3L is shown in SEQ ID NO. 1.

[0007] Further, the RPL3L overexpression vector is an adeno-associated virus vector AAV9-RPL3L overexpressing the RPL3L gene, which comprises a nucleotide sequence encoding the RPL3L and a myocardial specific promoter; the myocardial specific promoter is a cardiac troponin T promoter.

[0008] Further, the myocardial ischemia reperfusion injury treatment drug has at least one of the following uses:

[0009] (1) improving cardiac function after reperfusion;

[0010] (2) increasing the number of myocardial ribosomes and translation activity after reperfusion;

[0011] (3) reducing autophagy of damaged mitochondria after reperfusion and improving mitochondrial function.

[0012] Advantages of the present application:

[0013] The present application proves for the first time that RPL3L is a core value as a precise target of myocardial ischemia-reperfusion injury, and its overexpression can achieve targeted protection of myocardial ischemia-reperfusion injury through three key dimensions of improving ribosome function, inhibiting autophagy of damaged mitochondria, and improving cardiac function. The present application provides a new strategy for the treatment of myocardial ischemia-reperfusion injury with RPL3L as a precise target, and lays a foundation for the development of myocardial protection drugs targeting RPL3L, which has outstanding clinical translation potential. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The body surface electrocardiogram of the mice in the sham operation group and the IR group in Example 1 before and after reperfusion is shown in the figure;

[0015] Figure 2 The Western Blot results of RPL3L in the myocardial tissues of the mice in the sham operation group and the IR group in Example 1 are shown in the figure, A is the Western Blot result figure, and B is the comparison figure of the expression level of RPL3L protein;

[0016] Figure 3 The comparison figure of the mRNA expression amount of RPL3L in the myocardial tissues of the mice in the sham operation group and the IR group in Example 1 is shown in the figure;

[0017] Figure 4 The echocardiogram of the four groups of mice in Example 2 is shown in the figure, A is the sham operation group, B is the IR group, C is the RPL3L-IR group, and D is the NC-IR group;

[0018] Figure 5 The comparison figure of the cardiac ejection fraction and the fractional shortening of the four groups of mice in Example 2 is shown in the figure, A is the ejection fraction EF, and B is the fractional shortening FS;

[0019] Figure 6 The comparison figure of the left ventricular end-diastolic diameter and the left ventricular end-systolic diameter of the four groups of mice in Example 2 is shown in the figure, A is the left ventricular end-diastolic diameter LVIDd, and B is the left ventricular end-systolic diameter LVIDs;

[0020] Figure 7Figure 6 is a comparison chart of the amount of puromycin incorporation in four groups of mouse cardiomyocytes overexpressing RPL3L in Example 3, A is a Western blot chart of the NC-IR group and the RPL3L-IR group, B is a statistical chart of the amount of incorporation of the NC-IR group and the RPL3L-IR group; C is a Western blot chart of the NC-HR group and the RPL3L-HR group, D is a statistical chart of the amount of incorporation of the NC-HR group and the RPL3L-HR group;

[0021] Figure 8 Figure 7 is a comparison chart of the amount of puromycin incorporation in four groups of mouse cardiomyocytes with RPL3L knockdown in Example 3, A is a Western blot chart of the si-NC group and the si-RPL3L group, B is a statistical chart of the amount of incorporation of the si-NC group and the si-RPL3L group; C is a Western blot chart of the sh-NC group and the sh-RPL3L group, D is a statistical chart of the amount of incorporation of the sh-NC group and the sh-RPL3L group;

[0022] Figure 9 Figure 8 is a ribosome sucrose density gradient centrifugation spectrum of eight groups of mouse myocardial tissues in Example 3, A is the NC-IR group and the RPL3L-IR group, B is the NC-HR group and the RPL3L-HR group, C is the si-NC group and the si-RPL3L group, D is the sh-NC group and the sh-RPL3L group;

[0023] Figure 10 Figure 9 is a stepwise substrate-inhibitor addition method oxygen consumption curve of four groups of mouse cardiomyocyte mitochondria in Example 4;

[0024] Figure 11 Figure 10 is a comparison chart of the respiratory chain complex activity of four groups of mouse cardiomyocyte mitochondria in Example 4;

[0025] Figure 12 Figure 11 is a comparison chart of the DHE fluorescence staining detection of four groups of mouse myocardial tissues in Example 4, A is a DHE staining picture, B is a fluorescence intensity statistical chart;

[0026] Figure 13 Figure 12 is a comparison chart of the expression levels of Drp1 and VDAC1 proteins in four groups of mouse myocardial tissues in Example 5, A is a Western blot chart, B is a statistical chart of the expression amount of Drp1 protein, C is a statistical chart of the expression amount of VDAC1 protein;

[0027] Figure 14 Figure 13 is a comparison chart of the expression levels of LC3 and P62 proteins in four groups of mouse cardiomyocytes in Example 5, A is a Western blot chart, B is a statistical chart of the LC3 I / LC3- II ratio, C is a statistical chart of the expression amount of P62 protein. DETAILED DESCRIPTION

[0028] The technical solutions of the present application are further described below in combination with examples, but are not limited thereto. Any modification or equivalent replacement to the technical solutions of the present application without departing from the spirit and scope of the present application shall be encompassed in the protection scope of the present application. The process equipment or device not specifically mentioned in the following examples is the conventional equipment or device in the art. If not specifically mentioned, the raw materials used in the examples of the present application are commercially available. If not specifically mentioned, the technical means used in the examples of the present application are the conventional means known to those skilled in the art.

[0029] Example 1

[0030] This example demonstrates that the expression of RPL3L in myocardial tissue is reduced after cardiac ischemia reperfusion.

[0031] I. Experimental animal grouping and model establishment method:

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

[0033] (1) Sham group: only thoracotomy and threading operation were performed, and the coronary artery was not ligated;

[0034] (2) Ischemia-reperfusion IR group: the left anterior descending coronary artery was ligated for 30 minutes and then the blood flow was restored, and reperfusion was performed for 48 hours.

[0035] The body surface electrocardiogram of the Sham group and the IR group before and after reperfusion was monitored, and the results are shown in Figure 1 The ST segment was elevated after ligation ischemia, and the ST segment was decreased after reperfusion, indicating that the IR group model was successfully constructed.

[0036] II. The mice were sacrificed 48 hours after the Sham group operation and 48 hours after reperfusion of the IR group, and the heart was quickly removed. The left ventricular myocardium was separated along the interventricular septum, and the right ventricle and atrium were cut off on ice. The expression of RPL3L in myocardial tissue was detected.

[0037] (1) Western Blot detection:

[0038] 1. Protein extraction: homogenate the left ventricular myocardial tissue with RIPA lysis buffer (containing PMSF), centrifuge at 13500 rpm for 15 minutes, and take the supernatant;

[0039] 2. Electrophoresis and membrane transfer: separate the protein by 10% SDS-PAGE and transfer the NC membrane;

[0040] 3. Antibody incubation: anti-RPL3L antibody (1:1000, Immunoway), anti-β-actin antibody (1:3000, CST).

[0041] 4. Result analysis: Image J quantified gray value, and RPL3L / β-actin ratio represented relative expression.

[0042] Results as shown in Figure 2 compared with Sham group, RPL3L protein expression in myocardial tissue of IR group mice was significantly reduced, P<0.05.

[0043] (2) qRT-PCR detection:

[0044] 1. RNA extraction: total RNA of left ventricle was extracted by Trizol method, and quantified by NanoDrop;

[0045] 2. Reverse transcription: 1 μg RNA was synthesized into cDNA by PrimeScript RT kit;

[0046] 3. Quantitative PCR: RPL3L mRNA was detected by SYBR Green method,

[0047] 4. Result analysis: relative expression was calculated by 2-ΔΔCt method.

[0048] Results as shown in Figure 3 qRT-PCR results were consistent with Western Blot results, and RPL3L mRNA content in myocardial tissue of IR group mice was significantly reduced, P<0.05.

[0049] The results of this example showed that RPL3L expression was reduced in myocardial tissue after myocardial ischemia-reperfusion injury.

[0050] Example 2

[0051] This example demonstrated that overexpression of RPL3L could improve cardiac function after ischemia-reperfusion.

[0052] I. Construction of overexpression vector

[0053] (1) Vector selection:

[0054] In this example, adeno-associated virus type 9 (AAV9) vector system was selected, and the vector skeleton was ssAAV-cTnT-RPL3L-SV40pA, which contained the following elements: cardiac troponin T (cTnT) promoter, target gene RPL3L coding sequence, and SV40pA which helped to improve expression efficiency and stability. The nucleotide sequence encoding RPL3L is shown in SEQ ID NO. 1.

[0055] (2) RPL3L gene cloning and virus packaging

[0056] The RPL3L gene cloning and virus packaging were completed by Guangzhou Aizhe Biotechnology Co., Ltd. according to the gene cloning-virus packaging-purification and concentration method, and AAV9-RPL3L purified virus particles were obtained, with a titer of 1×10 13 vg / mL.

[0057] (3) Control vector

[0058] The RPL3L sequence was replaced with a nonsense sequence, and the remaining elements were completely consistent with AAV9-RPL3L. The AAV9-NC purified virus particles were obtained by Guangzhou Aizhe Biotechnology Co., Ltd. according to the gene cloning-virus packaging-purification and concentration method, with a titer of 1×10 13 vg / mL.

[0059] II. Animal experiment grouping and model establishment method:

[0060] 8-week-old male C57BL / 6J mice (body weight 22-25 g) were randomly divided into 4 groups:

[0061] (1) Sham group: tail vein injection of equal amount of normal saline, only open chest without ligation of left anterior descending branch of coronary artery;

[0062] (2) IR group: tail vein injection of equal amount of normal saline, 4 weeks later, ligation of left anterior descending branch of coronary artery for 30 minutes, then recovery of blood flow, and reperfusion for 48 hours;

[0063] (3) RPL3L-IR group: tail vein injection of AAV9-RPL3L (100 μL, 1×10 11 vg), 4 weeks later, ligation of left anterior descending branch of coronary artery for 30 minutes, then recovery of blood flow, and reperfusion for 48 hours;

[0064] (4) NC-IR group: tail vein injection of AAV9-NC (100 μL, 1×10 11 vg), 4 weeks later, ligation of left anterior descending branch of coronary artery for 30 minutes, then recovery of blood flow, and reperfusion for 48 hours;

[0065] III. Monitoring of cardiac function by echocardiography.

[0066] The echocardiograms of the 4 groups of mice are shown in Figure 4 , the cardiac ejection fraction EF is shown in Figure 5 A, the fractional shortening FS is shown in Figure 5 B, the left ventricular end diastolic diameter LVIDd is shown in Figure 6 A, and the left ventricular end systolic diameter LVIDs is shown in Figure 6As shown in B; compared with the Sham group, the ventricular wall movement of the IR group mice was weakened, and both EF% and FS% were significantly reduced, indicating that ischemia-reperfusion caused the weakening of the heart function of the mice. The amplitude of the ventricular wall movement of the RPL3L-IR group mice was restored, and both EF% and FS% were significantly increased (P < 0.05); no significant change was observed in LVIDd and LVIDs.

[0067] This result shows that overexpression of RPL3L mediated by an AAV9 vector can significantly reverse the cardiac systolic dysfunction caused by myocardial ischemia-reperfusion, thereby providing direct functional evidence for its application as a therapeutic drug.

[0068] Example 3

[0069] This example demonstrates that overexpression of RPL3L can improve the number and translation function of ribosomes in the myocardium of mice after ischemia-reperfusion.

[0070] I. Grouping and model establishment method of animal experiments and cell experiments:

[0071] Eight-week-old male C57BL / 6J mice (weight 22-25 g) were randomly divided into two groups:

[0072] (1) RPL3L-IR group: tail vein injection of AAV9-RPL3L (100 μL, 1 x 10 11 vg), 4 weeks later, the left anterior descending coronary artery was ligated for 30 minutes and then the blood flow was restored, and reperfusion was performed for 48 hours;

[0073] (2) NC-IR group: tail vein injection of AAV9-NC (100 μL, 1 x 10 11 vg), 4 weeks later, the left anterior descending coronary artery was ligated for 30 minutes and then the blood flow was restored, and reperfusion was performed for 48 hours;

[0074] Eight-week-old male C57BL / 6J mice (weight 22-25 g) were randomly divided into two groups:

[0075] (1) sh-RPL3L group: tail vein injection of AAV9-sh-RPL3L (RPL3L-targeted short hairpin RNA adenovirus sh-RPL3L, 100 μL, 1 x 10 11 vg), 4 weeks later, the subsequent experimental treatment was performed;

[0076] (2) sh-NC group: tail vein injection of AAV9-sh-NC (negative control adenovirus sh-NC, 100 μL, 1 x 10 11 vg), 4 weeks later, the subsequent experimental treatment was performed;

[0077] Mouse primary myocardial cells were isolated and randomly divided into two groups:

[0078] (1) RPL3L-HR group: mouse primary cardiomyocytes were isolated and cultured for 48 hours, then transfected with AAV9-RPL3L, and placed in a hypoxic incubator (95% N2+5% CO2) for 6 hours, and then transferred to a normoxic incubator (95% air+5% CO2) for 12 hours of reoxygenation;

[0079] (2) NC-HR group: mouse primary cardiomyocytes were isolated and cultured for 48 hours, then transfected with AAV9-NC, and placed in a hypoxic incubator (95% N2+5% CO2) for 6 hours, and then transferred to a normoxic incubator (95% air+5% CO2) for 12 hours of reoxygenation;

[0080] Mouse primary cardiomyocytes were isolated and randomly divided into 2 groups:

[0081] (1) si-RPL3L group: mouse primary cardiomyocytes were isolated and transfected with si-RPL3L (RPL3L-targeted small interfering RNA, siRNA-RPL3L sequence), and cultured for 48 hours before subsequent experimental treatment;

[0082] (2) si-NC group: mouse primary cardiomyocytes were isolated and 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;

[0083] The siRNA-RPL3L sequence in this embodiment is:

[0084] Sense strand: 5'-GGUAGAAGCUGUGACAAUUTT-3';

[0085] Antisense strand: 5'-AAUUGUCACAGCUUCUACCTT-3';

[0086] sh-RPL3L sequence: GGUAGAAGCUGUGACAAUUTT.

[0087] To meet the WIPO ST.26 standard requirements, the U at positions 3, 10, 12, 18, and 19 of the 5' end of the nucleotide sequence of the siRNA-RPL3L sense strand shown in SEQ ID NO: 2 is replaced by T to represent uracil in RNA, the U at positions 3, 4, 6, 13, 14, and 16 of the 5' end of the nucleotide sequence of the siRNA-RPL3L antisense strand shown in SEQ ID NO: 3 is replaced by T to represent uracil in RNA. The U at positions 3, 10, 12, 18, and 19 of the 5' end of the sh-RPL3L nucleotide sequence shown in SEQ ID NO: 4 is replaced by T to represent uracil in RNA.

[0088] II. Puromycin incorporation assay to detect myocardial ribosome translation activity:

[0089] Puromycin is an antibiotic that mimics tRNA, which can competitively bind to the peptidyl transferase center of ribosomes, incorporate into the end of the peptide chain being synthesized, and make the peptide chain prematurely terminate and release from the ribosome. By detecting the amount of peptide chain incorporated by puromycin in cells / tissues, the efficiency of ongoing protein synthesis by ribosomes can be directly reflected, and the more the amount of incorporation, the stronger the ribosome translation activity.

[0090] 1. Puromycin treatment: intraperitoneal injection of puromycin in live mice (final concentration 100 μM), and addition of puromycin in the culture medium of primary myocardial cells (final concentration 50 μM), incubation for 30 min;

[0091] 2. Collection of myocardial tissues / cells, extraction of total protein with RIPA lysis buffer, and quantification of protein concentration by BCA method;

[0092] 3. Western Blot detection: equal amount of protein was subjected to SDS-PAGE electrophoresis, and after membrane transfer, incubation with anti-puromycin antibody and anti-β-actin antibody (internal reference), ECL color development;

[0093] 4. Activity quantification: the ratio of the gray value of the puromycin band to the gray value of the β-actin band represents the ribosome translation activity.

[0094] The amount of puromycin incorporation in four groups of myocardial cells overexpressing RPL3L was compared as shown in Figure 7 The amount of puromycin incorporation in RPL3L-IR and RPL3L-HR groups was significantly higher than that in the NC control group; it was shown that in ischemia-reperfusion mice, overexpression of RPL3L could enhance myocardial ribosome translation activity.

[0095] The amount of puromycin incorporation in four groups of myocardial cells with RPL3L knockdown was compared as shown in Figure 8 The amount of puromycin incorporation in si-RPL3L and sh-RPL3L groups was significantly lower than that in the NC control group; it was shown that silencing RPL3L in cells could reduce ribosome translation activity.

[0096] This shows that whether in animal models or cell models, the expression level of RPL3L is positively correlated with myocardial ribosome translation activity: overexpression of RPL3L can enhance the translation activity, and interference with RPL3L can inhibit the activity, which confirms the regulatory effect of RPL3L on ribosome function.

[0097] III. Ribosome profiling method to detect changes in myocardial ribosome quantity:

[0098] Take 4 groups of myocardial tissue after cutting and adding pre-cooled ribosome lysis buffer, homogenate on ice and centrifuge to take the supernatant; another 4 groups of cell homogenate with PBS and add pre-cooled ribosome lysis buffer, incubate on ice and centrifuge to take the supernatant; in the ultracentrifuge tube, add 10%, 20%, 30%, 40%, 50% sucrose solution layer by layer, and form a continuous density gradient at 4°C. Slowly load the supernatant of the above samples into the top layer of the sucrose gradient, and ultracentrifuge at 35000 rpm for 3h (tissue samples) or 2.5h (cell samples) at 4°C. Use a continuous fraction collector to collect the sucrose gradient components after centrifugation, and simultaneously detect the absorbance value at 254nm wavelength. Plot the ribosome sucrose density gradient centrifugation spectrum with "absorbance" as the vertical coordinate and "collection position" as the horizontal coordinate.

[0099] The ribosome sucrose gradient centrifugation spectrum of the cells is shown in Figure 9 As shown in the figure, the polyribosome peaks of the RPL3L-IR group and the RPL3L-HR group are higher than those of the NC control group, indicating that overexpression of RPL3L can increase the number of active translation polyribosomes. The polyribosome peaks of the si-RPL3L group and the sh-RPL3L group are lower than those of the NC control group, and the monomer peak and the subunit peak increase, indicating that silencing RPL3L can disrupt the assembly balance of ribosomes and reduce the number of polyribosomes. This spectrum from the perspective of ribosome assembly and functional form confirms that "the expression level of RPL3L is positively correlated with the number / activity of ribosomes": overexpression of RPL3L can promote ribosome assembly into active translation polyribosomes, and interference with RPL3L can inhibit ribosome assembly, further supporting the regulatory effect of RPL3L on ribosome function.

[0100] Example 4

[0101] This example demonstrates that overexpression of RPL3L can enhance the mitochondrial respiratory function of myocardial mitochondria and reduce the content of reactive oxygen species.

[0102] On the basis of the animal experiment grouping and model establishment of Example 2, the mice in each group were sacrificed 48 hours after reperfusion, and the hearts were quickly removed.

[0103] I. Detecting the change of myocardial mitochondrial respiratory function after overexpression of RPL3L at the in vivo level by Oroboros O2k technology.

[0104] Take the left ventricular myocardial tissue of each group of mice, cut into 5mg small samples, and put into a manual glass homogenizer. Add 200μL pre-cooled mitochondrial respiratory medium (MiR05) and homogenize on ice. After homogenization, filter out the large debris to obtain the homogenate sample for detection.

[0105] (1) Basal respiration: add tissue homogenate to the respiration chamber, adjust the volume to 2mL, seal the respiration chamber, and record the basal oxygen consumption rate (OCR).

[0106] (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:

[0107] 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.

[0108] 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.

[0109] Ascorbic acid (2 mM) and TMPD (0.5 mM) were used as substrates for complex IV (cytochrome c oxidase).

[0110] 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.

[0111] 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.

[0112] II. Immunofluorescence technique was used to detect changes in myocardial mitochondrial respiratory function and myocardial reactive oxygen species content.

[0113] 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.

[0114] DHE staining steps:

[0115] (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;

[0116] (2) Washing: Rinse with PBS 3 times (5 minutes each time) to remove unbound probes.

[0117] (3) Mounting: Mount the slide with an anti-fluorescence quencher (containing DAPI) and store at 4°C protected from light.

[0118] The imaging results of confocal microscopy of each group of dyed samples are shown in FIG. 2. Figure 12 As shown in FIG. 2, compared with the Sham group, the IR group showed strong red fluorescence, indicating that IR injury induced a large amount of ROS generation, while the red fluorescence of the RPL3L-IR group was significantly weakened, and the ROS level was reduced. This shows that overexpression of RPL3L can effectively inhibit the generation of ROS induced by IR, reduce injury-induced mitochondrial autophagy, and indicates that RPL3L has a regulatory effect on mitochondrial autophagy of cardiomyocytes.

[0119] Example 5

[0120] This example demonstrates that overexpression of RPL3L can maintain normal mitochondrial autophagy levels and improve mitochondrial function.

[0121] On the basis of the grouping and model establishment of the animal experiment of Example 2, the mice in each group were sacrificed 48 hours after reperfusion, and the hearts were quickly removed. The left ventricular myocardial tissue of the mice in each group was taken, and Western Blot was used to detect the expression changes of Drp1 (mitochondrial fission protein), VDAC1 (mitochondrial autophagy recognition marker), LC3 (autophagy activity marker), and P62 (autophagy substrate protein).

[0122] The comparison results of the expression levels of Drp1 and VDAC1 proteins in the myocardial tissues of the four groups of mice are shown in FIG. 4. Figure 13 As shown in FIG. 4, compared with the Sham group, the expression of Drp1 in the IR group was significantly increased, indicating that IR induced enhanced mitochondrial fission, and the expression of Drp1 in the RPL3L-IR group was lower than that in the IR group, indicating that overexpression of RPL3L can inhibit excessive mitochondrial fission. Compared with the Sham group, the expression of VDAC1 in the IR group was significantly reduced, indicating that IR damaged mitochondrial autophagy-related proteins, and the expression of VDAC1 in the RPL3L-IR group was higher than that in the IR group, indicating that overexpression of RPL3L can restore the level of VDAC1.

[0123] The comparison of the expression levels of LC3 and P62 proteins in the myocardial cells of the four groups of mice is shown in FIG. 5. Figure 14 As shown in FIG. 5, the LC3-II / LC3-I ratio reflects autophagy activity, and the ratio in the IR group was higher than that in the Sham group, indicating that IR induced excessive autophagy, while the ratio in the RPL3L-IR group was lower than that in the IR group, indicating that overexpression of RPL3L can inhibit excessive autophagy. The P62 in the IR group was lower than that in the Sham group, indicating that IR caused excessive degradation of autophagy substrates, and the expression of P62 in the RPL3L-IR group was higher than that in the IR group, indicating that overexpression of RPL3L can restore the level of P62.

[0124] The above experimental results show that IR injury can induce excessive mitochondrial fission and abnormal autophagy in myocardial mitochondria, and overexpression of RPL3L can precisely regulate the expression of autophagy-related proteins, inhibit excessive mitochondrial fission, restore autophagy balance, thereby improve mitochondrial function, and play a myocardial protective role.

Claims

1. Use of an RPL3L overexpression vector in the preparation of a medicament for treating myocardial ischemia-reperfusion injury, characterized in that, The nucleotide sequence of the RPL3L is shown as SEQ ID NO. 1, and the drug is used for treating myocardial ischemia-reperfusion injury in mice.

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

3. Use according to claim 2, characterized in that, The myocardial ischemia-reperfusion injury treatment drug has at least one of the following uses: (1) improving cardiac function after reperfusion; (2) increasing the number of myocardial ribosomes and translation activity after reperfusion; (3) reducing injury autophagy of mitochondria after reperfusion and improving mitochondrial function.

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