Application of thymine in preparing medicine for treating or preventing adriamycin-induced cardiac damage

Abstract

The present invention provides the use of thymine in preparing a drug for treating or preventing adriamycin-induced cardiac damage, belonging to the technical field of drugs for resisting adriamycin-induced cardiac damage. The present invention is the first to discover that thymine has a significant effect of resisting adriamycin-induced cardiac damage. The present invention provides a theoretical basis and experimental basis for the research and development of new drugs for resisting adriamycin-induced cardiac damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of drugs for resisting cardiac damage caused by adriamycin, and particularly relates to the application of thymine in preparing drugs for treating or preventing cardiac damage caused by adriamycin. Background Art

[0002] Doxorubicin (DOX) is a cancer chemotherapy drug with a broad antitumor spectrum. However, treatment with doxorubicin can cause adverse drug reactions, including significant cardiotoxicity, which can lead to heart damage. This cardiotoxicity significantly limits its clinical use. Dilated cardiomyopathy (DCM) is one of the most common side effects of doxorubicin and is a difficult-to-treat cardiovascular disease. The main clinical features of DCM are ventricular dilatation, systolic dysfunction, arrhythmias, myocardial ischemia, and cardiomyocyte apoptosis. Studies have shown that patients with DCM experience gradual thinning of the ventricular wall, and electrocardiography and ultrasound techniques can detect symptoms such as arrhythmias and atrioventricular block. DCM has a poor prognosis. Dexrazoxane (DEX) is currently the only FDA-approved drug to reduce the risk of doxorubicin-induced cardiomyopathy. However, dexrazoxane has a short plasma half-life and is associated with adverse reactions such as allergic reactions and liver damage, limiting its clinical application. Therefore, innovative drugs for the treatment of doxorubicin-induced cardiomyopathy are urgently needed.

[0003] The zebrafish genome has a high homology with the human genome and is highly similar to that of mammals in cardiac structure and function. The heart is composed of atria and ventricles, and its relaxation and contraction mechanisms are highly conserved. Compared with mice, the zebrafish heart rate (120-140 beats / minute) is closer to that of humans. Because zebrafish larvae are transparent, transgenic zebrafish with green fluorescently labeled hearts can be used to directly observe the beating of the zebrafish heart and blood flow through a microscope, and calculate indicators such as the zebrafish's heart rate, pericardial area, ejection fraction, fractional shortening, stroke volume, and blood flow velocity to quickly assess cardiac function. Moreover, zebrafish behave similarly to humans in response to cardiotoxic drugs. Cardiotoxic drugs can cause impaired cardiac function and changes in cardiac tissue morphology in zebrafish, but zebrafish can still survive for a period of time even with damaged hearts. These advantages make the zebrafish model an advantageous model for studying cardiac function.

[0004] Thymine (Thy), a pyrimidine base isolated from the thymus, is the raw material for the anti-nucleic acid metabolism anti-tumor drug trifluorothymidine. Trifluorothymidine is commonly used in the chemotherapy of malignant hydatidiform mole, choriocarcinoma, breast cancer, gastrointestinal tumors, ovarian cancer, and primary bronchogenic carcinoma. Thymine's cardioprotective and anti-cardiomyopathy activities have not yet been reported. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of thymine in the preparation of a drug for treating or preventing cardiac damage caused by doxorubicin.

[0006] In this study, we used the zebrafish model of doxorubicin-induced cardiac injury and evaluated the anti-doxorubicin effect of thymidine on pericardial area, SV-BA distance, heart rate, stroke volume, fractional shortening, ejection fraction, blood flow velocity, cardiac erythrocyte area, myocardial cell apoptosis, and cardiac tissue morphology.

[0007] Application of thymine in the preparation of medicines for treating or preventing cardiac damage caused by adriamycin.

[0008] Preferably, according to the present invention, the doxorubicin-induced cardiac damage includes: cardiomyopathy, arrhythmia, and heart failure.

[0009] More preferably, the cardiomyopathy is dilated cardiomyopathy.

[0010] According to the present invention, preferably, the drug contains one or more pharmaceutically acceptable carriers or adjuvants.

[0011] More preferably, the auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.

[0012] According to the present invention, preferably, the dosage form of the drug is capsule, pill, tablet, oral liquid, granule, tincture or injection.

[0013] A drug for combating doxorubicin-induced cardiac damage, the active ingredient of which contains thymidine.

[0014] According to the present invention, preferably, the drug contains one or more pharmaceutically acceptable carriers or adjuvants.

[0015] More preferably, the auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.

[0016] According to the present invention, preferably, the dosage form of the drug is capsule, pill, tablet, oral liquid, granule, tincture or injection.

[0017] Preferably, according to the present invention, the doxorubicin-induced cardiac damage includes: cardiomyopathy, arrhythmia, and heart failure.

[0018] The beneficial effects of the present invention include at least the following:

[0019] The present invention is the first to discover that thymine has a significant effect of resisting doxorubicin-induced cardiac damage, such as resisting doxorubicin-induced cardiomyopathy. The present invention provides a theoretical basis and experimental basis for the research and development of new drugs to resist doxorubicin-induced cardiac damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The diagram shows the changes in cardiac morphology of zebrafish in each group after 24 hours of treatment;

[0021] In the figure: A is the morphological diagram of zebrafish in each group after 24 hours of drug treatment, in which the red dotted box represents the heart; B is the statistical diagram of SV-BA distance of zebrafish in each group after 24 hours of drug treatment; C is the statistical diagram of pericardial area of ​​zebrafish in each group after 24 hours of drug treatment;

[0022] DOX stands for doxorubicin, DEX stands for dexrazoxane, and Thy stands for thymidine;

[0023] Compared with the blank group, ****P<0.0001; compared with the model group, # p<0.05, ## p<0.01, ### P<0.001, #### P<0.0001.

[0024] Figure 2 The graphs showing changes in cardiac function indicators of zebrafish in each group after 24 hours of treatment;

[0025] In the figures: Figure A shows the cardiac morphology of zebrafish in each group after 24 hours of drug treatment; Figure B shows the heart rate statistics of zebrafish in each group after 24 hours of drug treatment; Figure C shows the ejection fraction statistics of zebrafish in each group after 24 hours of drug treatment; Figure D shows the short axis shortening rate statistics of zebrafish in each group after 24 hours of drug treatment; Figure E shows the stroke volume statistics of zebrafish in each group after 24 hours of drug treatment;

[0026] DOX stands for doxorubicin, DEX stands for dexrazoxane, and Thy stands for thymidine;

[0027] Compared with the blank group, ****P<0.0001; compared with the model group, # p<0.05, ## p<0.01,

[0028] ### P<0.001, #### P<0.0001.

[0029] Figure 3 The graphs showing the changes in blood flow velocity of zebrafish in each group after 24 hours of treatment are shown;

[0030] In the figure: DOX represents doxorubicin, DEX represents dexrazoxane, and Thy represents thymidine;

[0031] Compared with the blank group, ****P<0.0001; compared with the model group, #### P<0.0001.

[0032] Figure 4 The red blood cell staining images of zebrafish in each group after 24 hours of treatment;

[0033] In the figure: Figure A shows the erythrocyte staining of zebrafish hearts in each group after 24 hours of drug treatment, and the red dotted box indicates the erythrocyte staining area; Figure B shows the statistical diagram of the erythrocyte staining area of ​​zebrafish hearts in each group after 24 hours of drug treatment;

[0034] DOX stands for doxorubicin, DEX stands for dexrazoxane, and Thy stands for thymidine;

[0035] Compared with the blank group, ****P<0.0001; compared with the model group, #### P<0.0001.

[0036] Figure 5 Figure 2 shows the cardiomyocyte apoptosis (AO staining) of zebrafish in each group after 24 hours of treatment;

[0037] In the figure: the red box indicates the pericardial area, and the fluorescent spots pointed by white arrows indicate apoptotic cells;

[0038] DOX stands for doxorubicin, DEX stands for dexrazoxane, and Thy stands for thymidine.

[0039] Figure 6 Figure 3 is the cardiomyocyte apoptosis (TUNEL staining) of zebrafish in each group after 24 hours of treatment;

[0040] In the figure: the green dotted box represents the zebra pericardium area, the fluorescent spots pointed by white arrows represent apoptotic cells; DOX represents doxorubicin, DEX represents dexrazoxane, and Thy represents thymidine.

[0041] Figure 7 This is a picture of the heart tissue pathology of zebrafish treated with DOX+Thy for 24 hours;

[0042] In the figure: blue arrows indicate normal intercellular spaces and closely arranged cardiomyocytes; green arrows indicate larger intercellular spaces and disordered arrangement of cardiomyocytes;

[0043] DOX stands for doxorubicin, and Thy stands for thymidine.

[0044] Figure 8 This is a diagram showing the clustering of differentially expressed genes in zebrafish after treatment with DOX+Thy for 24 hours;

[0045] In the figures: Figure A: Veen plot of DOX-vs-Control group and Thy-vs-DOX group; Figure B: Volcano plot of differentially expressed genes between DOX-vs-Control group and Thy-vs-DOX group;

[0046] DOX stands for doxorubicin, and Thy stands for thymidine.

[0047] Figure 9 RNA-Seq was used to detect the gene expression profile of zebrafish after treatment with DOX+Thy;

[0048] In the figure: Figure A: KEGG map of the DOX-vs-Control group; Figure B: KEGG map of the Thy-vs-DOX group; Figure C: GO map of the Thy-vs-DOX group; the red boxes in Figures AB indicate enriched pathways related to cardiac function;

[0049] DOX stands for doxorubicin, and Thy stands for thymidine.

[0050] Figure 10 This is a diagram showing the effect of thymidine on the expression levels of genes related to the PPAR signaling pathway in zebrafish with DOX-induced cardiac injury;

[0051] In the figure: DOX represents doxorubicin, Thy represents thymidine;

[0052] Compared with the blank group, *P<0.05, ***P<0.001, ****P<0.0001; compared with the DOX group, # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001.

[0053] Figure 11 This figure shows the effect of thymidine on the expression levels of genes related to the ferroptosis pathway in zebrafish with DOX-induced cardiac injury;

[0054] In the figure: DOX represents doxorubicin, Thy represents thymidine;

[0055] Compared with the blank group, *P<0.05, ***P<0.001, ****P<0.0001; compared with the DOX group, #P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001. DETAILED DESCRIPTION

[0056] The present invention will be further described in detail below with reference to the Examples, but the scope of the present invention is not limited thereto. Where specific conditions are not specified in the Examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used without manufacturer's indication are conventional products that can be purchased commercially.

[0057] The inventors screened and evaluated the activity of various compounds against doxorubicin-induced cardiac damage and found that the compound thymine had a significant effect against doxorubicin-induced cardiac damage.

[0058] 1 Experimental Materials

[0059] 1.1 Experimental Animals

[0060] Wild-type AB zebrafish and cardiac marker green fluorescent transgenic zebrafish (cmlc2:EGFP) were purchased from the National Zebrafish Resource Center. Commercially available products can also be used. Adult zebrafish were maintained at (28 ± 0.5)°C with a 14 h / 10 h light / dark cycle and fed twice daily. Healthy adult zebrafish were selected the previous evening and placed in an ovulation box at a 2:2 ratio of male to female. The next day, when the lights were on, eggs were removed from the ovulation box and collected within 2 hours. The eggs were cultured in a 28°C incubator with constant temperature and illumination until fertilization (48 hpf).

[0061] 1.2 Drugs and main reagents

[0062] Doxorubicin (CAS No.: 23214-92-8, Batch No.: A396641337769) was purchased from APE×BIO, USA; dexrazoxane (CAS No.: 24584-09-6, Batch No.: 0448942-18) was purchased from Cayman, USA; and thymidine (CAS No.: 65-71-4, Batch No.: C14851700) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. All three compounds were prepared into stock solutions with dimethyl sulfoxide and diluted into working solutions with fresh E3 culture medium before use. Methylcellulose (CAS No. 9004-67-5, Batch No. Q22J9F63939, 250 g) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., dimethyl sulfoxide (CAS No. 67-68-5, Batch No. F508BA0021, 500 mL) was purchased from Sangon Biotechnology Co., Ltd., and methylene blue (CAS No. 7220-79-3, Batch No. 20110520, 25 g) and acridine orange (AO) dye (Sigma A6014) were purchased from Sigma. E3 culture medium contained 5 mmol / L NaCl, 0.17 mmol / L KCl, 0.4 mmol / L CaCl2, and 0.16 mmol / L MgSO4.

[0063] 1.3 Experimental instruments

[0064] Zebrafish culture system (Beijing Aisheng Technology Co., Ltd.); HPG280BX illumination incubator (Harbin Donglian Electronic Technology Development Co., Ltd.); Zeiss AXIO zoom V16 stereo fluorescence microscope (Carl Zeiss, Germany); OLYMPUS inverted fluorescence microscope (Olympus (China) Co., Ltd.); electronic analytical balance (Hemetler-Toledo Instrument Co., Ltd.); LightCycler 96 fluorescence quantitative PCR instrument (Roche, Switzerland).

[0065] 2 Experimental methods

[0066] 2.1 Drug Grouping

[0067] Normally developed Tg(cmlc2:EGFP) zebrafish at 48 hpf were selected and placed in 24-well plates, with 15 juveniles per well. Based on the pre-experimental data, a blank control group (with fresh E3 culture medium) was established, along with an adriamycin-induced model (60 μM DOX), a dexrazoxane-treated group (60 μM DOX + 10 μM DEX), and a thymidine-treated group (60 μM DOX + 10, 20, or 40 μM Thy). Each group was set up with three replicate wells and incubated in a lighted incubator at a constant temperature (28°C) for 24 hours.

[0068] 2.2 Effects of thymidine on cardiac morphology in zebrafish with DOX-induced cardiac injury

[0069] Twenty-four hours after administration, zebrafish were anesthetized with 0.3% tricaine and fixed in 4% methylcellulose. Cardiac morphology was recorded and photographed from the side under a stereoscopic inverted microscope. The sinus venosus-bulbus arteriosus (SV-BA) distance and pericardial area were measured using Image-Pro Plus 6.0 software.

[0070] 2.3 Effects of thymidine on cardiac function indices in zebrafish with DOX-induced cardiac injury

[0071] 24 hours after administration, the zebrafish were fixed with 4% methylcellulose. After fixation, the zebrafish prone heartbeat video was recorded under an inverted fluorescence microscope, and the ventricular end-diastolic and end-systolic images were extracted. The zebrafish end-diastolic and end-systolic long-axis and short-axis lengths were measured using Image-Pro Plus 6.0 software, and the ejection fraction, short-axis shortening rate, and stroke volume were calculated. The specific calculation formulas are shown in (1), (2), (3), and (4).

[0072] (1) Ventricular volume = 0.523 × minor axis length 2 × Major axis length

[0073] (2)

[0074] (3) Stroke volume = end-diastolic volume - end-systolic volume

[0075] (4)

[0076] 2.4 Effect of thymidine on blood flow velocity in zebrafish with DOX-induced cardiac injury

[0077] 24 hours after administration, the zebrafish were anesthetized with anesthetic and fixed with 4% methylcellulose. The blood flow velocity of the zebrafish artery was measured using a blood flow meter. TM Blood flow videos were analyzed using the ViewPoint software (v1.3.2, ViewPoint, Lyon, France). This software can detect changes in pixel density and combine them with vessel diameter to generate flow rate per frame in nl / s.

[0078] 2.5 Effects of thymidine on cardiac erythrocytes in zebrafish with DOX-induced cardiac injury

[0079] Twenty-four hours after drug administration, zebrafish larvae were stained with 1 mg / mL o-dianisidine for 20 minutes. Ten randomly selected larvae from each group were observed and photographed for erythrocytes in the cardiac region under a stereofluorescence microscope. Image-Pro Plus 6.0 software was used to quantitatively analyze the erythrocyte staining area in the heart to evaluate the anti-ischemic effect of the drug.

[0080] 2.6 Myocardial cell apoptosis detection (AO staining / TUNEL staining)

[0081] Acridine orange (AO) staining: 48 hours after fertilization of wild-type zebrafish (AB), normally developed juveniles were selected and placed in 24-well plates, with 15 juveniles per well. Twenty-four hours after administration, the prepared acridine orange (AO) stock solution was added to the corresponding wells of each zebrafish group to a final concentration of 5 μg / mL. The cells were incubated in a 28°C incubator for 30 minutes and then rinsed three times with PBS. The zebrafish were then photographed under a stereofluorescence microscope to observe the number of apoptotic cells in the heart.

[0082] TUNEL staining: 48 hours after fertilization of wild-type zebrafish AB, normally developed zebrafish larvae were selected and placed in a 24-well plate, with 15 larvae per well. 24 hours after administration, cells were fixed with 3 mL of 4% paraformaldehyde and refrigerated at 4°C overnight. 0.3% Triton X-100 and sodium citrate antigen retrieval solution (1X) were mixed at a ratio of 1:300, and 3 mL of the mixture was added to each well. The cells were incubated at 4°C for 10 minutes. After washing with PBS, Tdt enzyme and fluorescent labeling solution were mixed at a ratio of 1:10, and 50 μL of the mixture was added to each centrifuge tube. The cells were incubated in a 37°C incubator in the dark for 1.5 hours. Finally, the mixture was aspirated, and 1 mL of PBS was added. The cells were shaken in the dark at 90 rpm for 5 minutes, repeated three times. Zebrafish larvae were photographed under a stereofluorescence microscope to observe the number of apoptotic cells in the heart.

[0083] 2.7 Cardiac histopathology

[0084] 24 hours after administration, 10 zebrafish larvae were randomly selected from the blank control group, the doxorubicin model group (60 μM), and the drug-treated group (20 μM Thy + 60 μM DOX), fixed with 4% paraformaldehyde fixative, and then dehydrated with ethanol gradient and immersed in xylene until transparent. They were then paraffin-embedded and sectioned. The sections were dewaxed in xylene, anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol to water, and then stained with hematoxylin-eosin (HE staining). After dehydration and sealing, the tissue sections were observed under a microscope and the heart tissue was photographed and recorded.

[0085] 2.8 RNA-Seq sequencing to detect differential gene changes in zebrafish

[0086] 24 hours after administration, 90 fish were randomly selected from each of the blank control group, the doxorubicin model group, and the drug-treated group (20 μM Thy), washed three times with enzyme-free water, and then collected. Total RNA was extracted using TRIzol reagent according to the manufacturer's protocol. RNA purity and quantification were tested, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries were constructed according to the instructions of the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA). The entire RNA-Seq sequencing and analysis process was completed by Ouyi Biotechnology Co., Ltd. (Shanghai, China).

[0087] 2.9 RT-qPCR detection of changes in related gene expression levels

[0088] 24 hours after administration, 30 zebrafish were randomly selected from each of the blank control group, doxorubicin model group, and drug treatment group (DOX+20μM Thy), washed three times with enzyme-free water, and then collected. Total RNA of each zebrafish sample was extracted using an RNA extraction kit according to the instructions, and reverse transcription was performed using a reverse transcription kit and a gradient PCR instrument to obtain cDNA of each zebrafish sample. RT-qPCR detection kit was used in Quantitative real-time RT-qPCR was performed on a 96-well plate. The program was: 95°C denaturation for 1 minute, followed by 45 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 30 seconds. β-actin was used as an internal reference gene. The primer sequences used in this article are shown in Table 1.

[0089] Table 1 RT-qPCR related primer sequences

[0090]

[0091]

[0092] 2.10 Data Analysis

[0093] Experimental data are expressed as mean ± SD, and statistical differences were analyzed using the Student's Student T test. P < 0.05 indicated a significant difference, and P < 0.01 indicated a highly significant difference. Statistical analysis was performed using GraphPad Prism 8.0 and Image-Pro Plus 6.0 software.

[0094] 3 Experimental results

[0095] 3.1 Effects of thymidine on cardiac morphology in zebrafish with DOX-induced cardiac injury

[0096] Changes in the size of the zebrafish pericardium can directly reflect abnormalities in the zebrafish heart; the stretching of the heart's S-ring causes the distance between the bulbus arteriosus and the sinus venosus (SV-BA) to lengthen. Therefore, the inventors can use the pericardial area and SV-BA distance to directly observe whether abnormalities occur in the zebrafish heart. Figure 1 As shown in middle A, compared with the control group, pericardial edema was obvious in the doxorubicin model group (indicated by the red dotted line), and the SV-BA distance was significantly longer, indicating that the model was successful. Compared with the doxorubicin model group, the pericardial area of ​​the dexrazoxane positive drug group was reduced and the SV-BA distance was shortened. 10, 20, and 40 μM Thy all had an effect on pericardial edema caused by doxorubicin ( Figure 1 Middle B), SV-BA growth has an improving effect ( Figure 1 C), both can reduce the pericardial area and shorten the SV-BA distance. The effect of resisting doxorubicin-induced pericardial edema and increasing the SV-BA distance is most significant at a concentration of 20 μM.

[0097] 3.2 Effects of thymidine on cardiac function in zebrafish with DOX-induced cardiac injury

[0098] The clinical manifestations of dilated cardiomyopathy are arrhythmia, ventricular dilatation, and cardiac systolic dysfunction. The heart rate, ejection fraction, fractional shortening, and stroke volume of zebrafish reflect the heart rhythm, ventricular dilatation, and cardiac systolic function. These indicators can be used to evaluate the effect of thymidine on the cardiac function of zebrafish with DOX-induced cardiac injury.

[0099] like Figure 2 As shown in Figure A, 24 hours after administration, the heart morphology of zebrafish in the adriamycin model group changed. Compared with the blank control group, the heart rate, ejection fraction, short axis shortening rate, and stroke volume of zebrafish in the adriamycin model group were significantly decreased. Compared with the model group, the heart rate, ejection fraction, short axis shortening rate, and stroke volume of the dexrazoxane positive drug group were significantly increased. 10μM Thy had a significant effect on the heart rate of zebrafish ( Figure 2 Middle B), ejection fraction ( Figure 2 Middle C) had no significant effect, the short axis shortening rate ( Figure 2 D) and stroke volume ( Figure 2 (E) increased; at a Thy concentration of 20 μM, the zebrafish's heart rate, ejection fraction, fractional shortening, and stroke volume all increased significantly; at a Thy concentration of 40 μM, Thy increased the zebrafish's heart rate, ejection fraction, and fractional shortening, but had no effect on heart rate and stroke volume. Therefore, Thy at a concentration of 20 μM has a significant ameliorative effect on DOX-induced cardiac function damage.

[0100] 3.3 Effect of thymidine on blood flow velocity in zebrafish with DOX-induced cardiac injury

[0101] Patients with cardiomyopathy often suffer from heart failure, which leads to cardiac pump dysfunction. In this experiment, the effect of thymidine on the blood flow velocity of zebrafish with DOX-induced cardiac injury was evaluated by detecting blood flow velocity.

[0102] like Figure 3 As shown in the figure, compared with the blank control group, the blood flow velocity of zebrafish in the DOX group was significantly decreased. Compared with the model group, the blood flow velocity in the dexrazoxane-positive group increased. After co-treatment with DOX and Thy, the blood flow velocity recovered compared with the model group. The recovery effect of blood flow velocity was most significant when the Thy concentration was 20 μM. There was no significant change at Thy concentrations of 10 and 40 μM.

[0103] 3.4 Effects of thymidine on cardiac erythrocytes in zebrafish with DOX-induced cardiac injury

[0104] Patients with cardiomyopathy present with weakened cardiac function, often accompanied by myocardial ischemia. This study used o-dianisidine staining to observe red blood cell counts to assess whether doxorubicin causes myocardial ischemia and whether thymidine has anti-ischemic activity.

[0105] like Figure 4 As shown, 24 hours after drug administration, the erythrocyte staining area in the zebrafish larvae model group was significantly smaller than that in the control group. Compared with the model group, the erythrocyte staining area in the dexrazoxane-positive group was larger. Co-treatment with Thy and DOX increased the erythrocyte staining area compared with the doxorubicin-treated group. Thy at a Thy concentration of 20 μM showed the greatest efficacy in preventing doxorubicin-induced myocardial ischemia.

[0106] 3.5 Effects of thymidine on cardiomyocyte apoptosis (AO / TUNEL) in zebrafish with DOX-induced cardiac injury

[0107] Acridine orange (AO) is a fluorescent dye that can penetrate the cell membrane. After staining the nucleus of apoptotic cells, the chromatin appears yellow-green and concentrated on the inner side of the nuclear membrane. The cell membrane is visible as bulging bubbles and apoptotic bodies. As shown in the AO results ( Figure 5 Compared with the control group, the zebrafish treated with doxorubicin showed a large number of fluorescent spots in the heart, suggesting that 60 μM DOX treatment can induce cell apoptosis in the zebrafish heart region (indicated by white arrows) and pericardial edema (indicated by red boxes). Compared with the doxorubicin group, the fluorescent spots in the dexrazoxane-positive group were significantly reduced. The fluorescent spots in the heart region of the groups treated with Thy at different concentrations were significantly reduced compared with the model group, with the most significant effect at 20 μM. This suggests that thymidine can attenuate doxorubicin-induced cardiomyocyte apoptosis.

[0108] TUNEL staining can mark DNA breaks caused by activation of DNA endonucleases during cell apoptosis, thereby detecting cell apoptosis. As shown in the TUNEL staining results ( Figure 6 Compared with the control group, the doxorubicin-treated zebrafish showed a large number of fluorescent spots in the heart (indicated by white arrows), indicating that 60μM DOX treatment can cause cell apoptosis in the zebrafish heart region and pericardial edema. Compared with the doxorubicin-treated group, the dexrazoxane-positive group showed a significant decrease in fluorescent spots in the heart region. Thymidine treatment at different concentrations showed a significant decrease in fluorescent spots in the heart region compared with the model group, with the most significant effect at 20μM. This suggests that thymidine can attenuate doxorubicin-induced cardiomyocyte apoptosis.

[0109] 3.6 Effects of thymidine on the pathological structure of zebrafish hearts following DOX-induced cardiac injury

[0110] The ventricular wall of patients with cardiomyopathy gradually becomes thinner and the arrangement of myocardial cells becomes disordered. HE staining can be used to observe morphological changes under a microscope to diagnose whether cardiomyopathy has occurred. Figure 7 As shown, the normal control group zebrafish had normal atria and ventricles, normal intercellular spaces, and densely packed cardiomyocytes (indicated by blue arrows). The model group zebrafish had significantly enlarged atria and ventricles, larger intercellular spaces, and disordered cardiomyocyte arrangement (indicated by green arrows). After treatment with 20 μM Thy, pericardial edema was significantly improved compared to the model group, and cells were more densely packed.

[0111] 3.7 Effects of thymidine on gene expression profiles in zebrafish with DOX-induced cardiac injury

[0112] To investigate the effects of thymidine on gene expression in zebrafish exposed to DOX-induced cardiac injury, we compared gene expression profiles between the adriamycin-induced model (DOX 60 μM) and the drug-treated group (DOX + Thyme 20 μM). Genes with a p < 0.05 and a fold difference in gene expression greater than 1.5 were defined as differentially expressed genes. The results of the differentially expressed gene detection are as follows: Figure 8 A in the middle is a veen diagram of differentially expressed genes. It can be seen from the diagram that there are 1242 differentially expressed genes between the blank group and the doxorubicin model group, and 350 differentially expressed genes between the doxorubicin model group and the drug-treated group, of which 156 are common differentially expressed genes. Figure 8 Middle B is the volcano plot of differentially expressed genes. The results show that compared with the blank group, the doxorubicin model group had 806 upregulated differential genes and 436 downregulated differential genes. Compared with the doxorubicin model group, the drug-treated group had 271 upregulated differential genes and 79 downregulated differential genes.

[0113] (1) KEGG enrichment analysis results The horizontal axis of the KEGG enrichment pathway analysis bubble chart is the enrichment score. The larger the bubble, the more differential genes the pathway contains; the smaller the P value, the higher the significance. The KEGG enrichment pathway analysis bubble chart shows that the differential genes between the blank group and the doxorubicin model group are mainly enriched in ferroptosis, necroptosis, P53 signaling pathway, PPAR signaling pathway, myocardial contraction pathway ( Figure 9 Middle A), the differentially expressed genes between the doxorubicin model group and the drug treatment group were mainly enriched in focal adhesion, necroptosis, cell adhesion molecule steroid biosynthesis, and PPAR signaling pathway ( Figure 9 Middle B). The common differentially expressed genes in the doxorubicin model group, blank group, drug-treated group, and doxorubicin model group were highly enriched in PPAR, ferroptosis, and necroptosis pathways.

[0114] (2) GO enrichment analysis results Figure 9 Middle C is a bar chart of GO function enrichment analysis of differentially expressed genes in the adriamycin model group and the drug treatment group. Figure 9Figure C shows that differentially expressed genes between the doxorubicin-treated and drug-treated groups are primarily clustered within the three GO terms: biological process, cellular composition, and molecular function. These biological processes include neutrophil activation, inflammatory response, and prostaglandin biosynthesis. Cellular components include nucleosomes, lysosomes, and the extracellular domain. Molecular function encompasses hydrolase activity, methylase activity, and cytokine activity.

[0115] 3.8 Effects of thymidine on the expression levels of genes related to the PPAR signaling pathway in zebrafish with DOX-induced cardiac injury

[0116] PPARs are widely expressed in cardiomyocytes and participate in the regulation of cardiomyocyte energy metabolism, proliferation, differentiation, development, and cell death. Studies have found that DOX can cause mitochondrial dysfunction by acting on PPARs, activating oxidative stress and inflammation and ultimately inducing apoptosis. In addition, PPARs affect cardiac energy metabolism in cardiomyocytes, thereby affecting cardiomyopathy.

[0117] RT-qPCR was used to examine the mRNA expression levels of genes involved in the PPAR signaling pathway. The results showed that compared with the blank control group, the DOX group showed an increased expression of cpt1ab, while the mRNA expression levels of pparg, apoa1a, acsl5, pltp, fabp1b.1, slc27a2a, and lpl decreased, consistent with the transcriptome analysis. When Thy was added at a concentration of 10 μM, compared with the DOX group, the mRNA expression levels of pparg, apoa1a, acsl5, pltp, fabp1b.1, slc27a2a, and lpl increased, while the mRNA expression level of cpt1ab decreased. When Thy was added at a concentration of 20 μM, compared with the DOX group, the mRNA expression level of cpt1ab decreased, while the mRNA expression levels of apoa1a, acsl5, pltp, slc27a2a, and lpl increased; the other mRNA expressions were not significant. When the concentration of Thy was 40 μM, compared with the DOX group, the mRNA expression levels of acsl5, pltp, and lpl increased, and the mRNA expression level of cpt1ab decreased, while the others had no significant difference ( Figure 10 ).

[0118] 3.9 Effects of thymidine on the expression levels of genes related to the ferroptosis pathway in zebrafish with DOX-induced cardiac injury

[0119] Ferroptosis is a non-apoptotic form of regulated cell death that results from the excessive production of phospholipid hydroperoxides in an iron-dependent manner, inducing lipid peroxidation and oxidative damage. Studies have shown that doxorubicin-induced cardiomyopathy is closely related to the ferroptosis pathway.

[0120] The expression levels of mRNA of genes related to the ferroptosis pathway were detected by RT-qPCR. The results showed that compared with the blank control group, the expression levels of zgc:198419 and zgc:92006 in the DOX group showed an upward trend, and the mRNA expression levels of tfa and si:ch211-254p-10.2 decreased, which was consistent with the transcriptome results. When the concentration of Thy was 10 μM, compared with the DOX group, the mRNA expression level of si:ch211-254p-10.2 increased, and the mRNA expression levels of zgc:198419 and zgc:92006 decreased. When the concentration of Thy was 20 and 40 μM, compared with the DOX group, the mRNA expression levels of zgc:198419 and zgc:92006 decreased, and the mRNA expression levels of tfa and si:ch211-254p-10.2 increased ( Figure 11 ).

[0121] This study used transgenic zebrafish (cmlc2:EGFP) with green fluorescent markers as the research subjects, and observed the morphology and function of the zebrafish heart with the help of a fluorescence microscope. A zebrafish heart injury model induced by doxorubicin was established by intervention with 60 μM doxorubicin. This heart injury model showed symptoms of dilated cardiomyopathy, and further verified the effect of thymidine on doxorubicin-induced heart injury. The results showed that compared with the control group, the zebrafish in the doxorubicin model group had significant pericardial edema, enlarged SV-BA, and significantly decreased heart rate, ejection fraction, fractional shortening, and stroke volume. The blood flow rate slowed down, the number of apoptotic cardiomyocytes increased, the number of red blood cells in the heart area decreased, and the arrangement of cardiomyocytes was disordered. Compared with the doxorubicin model group, the addition of thymidine alleviated the cardiotoxicity caused by doxorubicin, and the effect was consistent with that of positive drugs. In addition, this study used RNA-Seq to identify differentially expressed genes and pathways associated with thymidine resistance to doxorubicin-induced cardiac injury in zebrafish. RT-qPCR was also used to examine the expression of genes involved in the PPAR signaling pathway and ferroptosis pathway to elucidate the mechanism of thymidine's activity against doxorubicin-induced cardiac injury in zebrafish. These results provide a theoretical and experimental basis for further investigation of the mechanism of thymidine's activity against doxorubicin-induced cardiac injury in zebrafish.

[0122] The present invention discovers for the first time that thymidine has the effect of resisting doxorubicin-induced cardiac damage and further has the effect of resisting doxorubicin-induced cardiomyopathy. Thymidine can resist doxorubicin-induced pericardial edema. Compared with the doxorubicin model group, thymidine has an effect of improving heart rate, ejection fraction, fractional shortening, stroke volume, and blood flow velocity. It can resist doxorubicin-induced myocardial ischemia, reduce doxorubicin-induced myocardial cell apoptosis, and improve doxorubicin-induced cardiac tissue pathology. The present invention provides a theoretical basis and experimental basis for the development of new drugs to resist doxorubicin-induced cardiac damage.

Claims

1. The use of thymine in the preparation of drugs for treating or preventing doxorubicin-induced cardiac damage.

2. The use according to claim 1, characterized in that The adriamycin-induced cardiac damage is selected from the group consisting of cardiomyopathy, arrhythmia, and heart failure.

3. The use according to claim 2, characterized in that The cardiomyopathy is dilated cardiomyopathy.

4. The use according to claim 1, wherein The medicine contains one or more pharmaceutically acceptable carriers or adjuvants.

5. The use according to claim 4, characterized in that The auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant or a lubricant.

6. The use according to claim 1, wherein The dosage form of the medicine is capsule, pill, tablet, oral liquid, granule, tincture or injection.

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