Application of doxorubicin and lapatinib in constructing zebrafish cardiotoxicity injury model and drug screening method

Abstract

The application provides doxorubicin and lapatinib in constructing a zebrafish cardiotoxicity damage model and a drug screening method, and belongs to the technical field of biological medicine and animal model construction. The application discloses that doxorubicin and lapatinib can be used in combination to construct a doxorubicin-related zebrafish cardiotoxicity damage model for the first time, and provides a method for establishing a zebrafish cardiotoxicity damage model. The heart index of the application is very comprehensive, and the zebrafish cardiotoxicity damage model constructed by the application can be used to screen drugs of various different action mechanisms which have an improving effect on heart damage.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine and animal model construction, and particularly relates to application of doxorubicin and lapatinib in construction of a zebrafish cardiotoxicity damage model and a drug screening method. BACKGROUND

[0002] Doxorubicin (DOX) belongs to the anthracycline family and is a commonly used broad-spectrum anti-tumor drug in clinical practice, which is effective against various cancers such as leukemia and breast cancer. It is estimated that about 50-60% of cancer patients use at least one anthracycline drug during treatment. Lapatinib (LAP) is a tyrosine kinase inhibitor, which is usually used in combination with the anthracycline doxorubicin, can significantly enhance the anti-tumor effect, and improve the survival rate of patients with advanced HER2-positive breast cancer. Currently, there are relatively few studies on the potential cardiotoxicity risk of the fetus of breast cancer patients during pregnancy when using the combination chemotherapy drug.

[0003] Existing studies mainly focus on the cardiotoxicity of LAP and DOX combination therapy for adult, especially elderly, female patients with metastatic breast cancer. However, about 7% to 10% of breast cancer diagnoses occur in women under the age of 40, and some of these women are pregnant. The incidence of pregnancy-related breast cancer is between 1:3,000 and 1:10,000, accounting for about 3% of all breast cancer cases. Unfortunately, it is expected that the incidence of pregnancy-related breast cancer will increase in the coming years, and terminating the pregnancy does not seem to improve the prognosis of the patient. Therefore, it is particularly important to evaluate the potential toxic side effects of LAP and DOX combination chemotherapy on the fetal heart. However, due to ethical and clinical sample collection limitations, there are very limited studies on the potential fetal cardiotoxicity induced by LAP and DOX in humans. Therefore, developing a corresponding animal model system becomes an effective means to address this medical need.

[0004] Animal models are important tools for human disease research. Using animal models can directly reflect the pathophysiological changes of human diseases and conduct disease mechanism research. At the same time, animal models are also important tools for screening active drugs and drug development. Rodent organisms are close to humans, and the experimental results are representative. However, rodents generally have the disadvantages of large size, high cost, and long cycle. Zebrafish (Danio rerio) is a small tropical vertebrate, and its genome has a high degree of similarity to humans, up to 87%. In terms of cardiac electrophysiology and heart rate, zebrafish and humans exhibit a high degree of conservation. The average heart rate of zebrafish is about 100 beats per minute (bpm), which is closer to the average human heart rate of 60-90 bpm, and much lower than the average mouse heart rate of about 500 bpm. This makes zebrafish more widely used in cardiac toxicity research. Wang et al. selected 3-day post-fertilization (3dpf) zebrafish larvae and found that the combination of doxorubicin and forsythoside A could be used to construct a doxorubicin-related zebrafish cardiac toxicity damage model, and a phenotype of heart rate slowing was found. Cai et al. selected 2-day post-fertilization (2dpf) transgenic blood fluorescent zebrafish and found a method for establishing a doxorubicin-induced zebrafish vascular endothelial damage model. These studies used water-soluble drug administration to treat zebrafish, and the experimental method is convenient and easy to perform in high throughput. However, the heart phenotypes of these inventions are relatively single, and the present invention provides comprehensive cardiac indicators, including heart rate, ejection fraction, sarcomere structure, myocardial cell number, myocardial apoptosis, and electron microscope images, thereby better proving the successful construction of the model and various aspects of cardiac toxicity.

[0005] The p38 subunit of mitogen-activated protein kinase (MAPK) is a serine / threonine protein kinase activated by cellular stress. Since p38 MAPK plays a key role in the response of cells to external stress signals and inflammatory cytokines, it has become an attractive target in medical research. Typically, p38 MAPK inhibitors are mainly used for the treatment of pro-inflammatory diseases, for the inhaled treatment of obstructive or inflammatory lung diseases, such as chronic obstructive pulmonary disease and asthma. In addition, p38 MAPK inhibitors have some new applications for the prevention and treatment of degenerative diseases of the nervous system, such as dementia, Alzheimer's disease and Huntington's disease, and for the field of gastrointestinal diseases, such as ulcerative colitis. Currently, there is no application of p38 MAPK inhibitors in the field of cardiac toxicity. SUMMARY

[0006] In view of the shortcomings of the prior art, the present application provides the application of doxorubicin and lapatinib in constructing a zebrafish cardiac toxicity damage model and a drug screening method.

[0007] The present application adopts zebrafish embryo system to establish a fetal cardiotoxicity model induced by the combination of LAP (lapatinib) and DOX (doxorubicin), which exhibits dose dependence and further explores the pathological mechanism behind it. The present application reveals that the increase of oxidative stress and the activation of MAPK signaling pathway are the key pathological events in the combination of LAP and DOX, which provides a potential way for the development of fetal heart protection targets.

[0008] The present application first discovers that the combination of DOX and LAP can be used to construct a doxorubicin-related zebrafish cardiotoxicity injury model and screen a drug capable of relieving cardiotoxicity.

[0009] Term explanation:

[0010] hpf (hours post fertilization): refers to the number of hours after fertilization.

[0011] DOX (doxorubicin): doxorubicin.

[0012] LAP (lapatinib): lapatinib.

[0013] The technical scheme of the present application is as follows:

[0014] A composition for establishing a zebrafish cardiotoxicity injury model, comprising doxorubicin and lapatinib.

[0015] According to the present application, the concentration of components in the composition is (20-100) μM of doxorubicin and (5-30) μM of lapatinib.

[0016] Further preferably, the concentration of components in the composition is 20 μM of doxorubicin and 20 μM of lapatinib.

[0017] According to the present application, the zebrafish used for establishing a zebrafish cardiotoxicity injury model is a 24hpf zebrafish.

[0018] According to the present application, the zebrafish is an AB strain wild type zebrafish.

[0019] Application of doxorubicin and lapatinib in establishing a zebrafish cardiotoxicity injury model.

[0020] According to the present application, in the application, the concentration of doxorubicin is (20-100) μM and the concentration of lapatinib is (5-30) μM.

[0021] Further preferably, in the application, the concentration of doxorubicin is 20 μM and the concentration of lapatinib is 20 μM.

[0022] In a preferred embodiment of the present invention, the zebrafish used to establish the zebrafish cardiotoxicity injury model is a 24 hpf zebrafish.

[0023] According to a preferred embodiment of the present invention, the zebrafish used is the AB lineage wild-type zebrafish.

[0024] A method for establishing a zebrafish cardiotoxicity injury model includes the following steps:

[0025] Zebrafish were randomly divided into a model group and a control group. The model group was cultured in an aqueous solution containing doxorubicin and lapatinib, while the control group was the same as the model group but without doxorubicin and lapatinib. The zebrafish were cultured for 40-48 hours, and the following parameters were measured: ejection fraction, heart rate, and end-diastolic ventricular cross-sectional area. Compared with the control group, the model group showed a significant decrease in ejection fraction, a significant decrease in heart rate, and a significant reduction in end-diastolic ventricular cross-sectional area. The zebrafish cardiotoxicity injury model was successfully established if these parameters were not present.

[0026] According to a preferred embodiment of the present invention, the zebrafish used in the method is a 24hpf zebrafish.

[0027] According to a preferred embodiment of the present invention, the zebrafish used in the method is the AB lineage wild-type zebrafish.

[0028] According to a preferred embodiment of the present invention, in the method, the culture solution containing doxorubicin and lapatinib contains doxorubicin at a concentration of (20-100) μM and lapatinib at a concentration of (5-30) μM.

[0029] More preferably, in the method, the culture aqueous solution containing doxorubicin and lapatinib contains doxorubicin at a concentration of 20 μM and lapatinib at a concentration of 20 μM.

[0030] According to a preferred embodiment of the present invention, the culture aqueous solution contains: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and pH 7.2.

[0031] According to a preferred embodiment of the present invention, in the method, the number of zebrafish in each group is 8-20.

[0032] According to a preferred embodiment of the present invention, the cultivation conditions in the method are 28.5°C and the light / dark cycle is 14h / 10h.

[0033] According to a preferred embodiment of the present invention, in the method, ejection fraction, heart rate, and ventricular cross-sectional area are detected by video imaging.

[0034] According to a preferred embodiment of the present invention, the statistical basis for significant ejection fraction, heart rate, and end-diastolic ventricular cross-sectional area is one-way ANOVA.

[0035] According to a preferred embodiment of the present invention, the detection indicators in the method further include cardiomyocyte nuclei, cardiomyocyte apoptosis, cardiomyocyte sarcomeres, and / or mitochondria.

[0036] Further preferred models, compared with the control group, showed that the sarcomere structure was damaged, the number of cardiomyocyte nuclei was reduced, myocardial apoptosis was increased, and mitochondrial damage was observed.

[0037] Application of the zebrafish cardiotoxicity injury model established by the above method in screening drugs with activity in preventing / treating doxorubicin-induced cardiotoxicity injury.

[0038] A method for screening drugs with activity in preventing / treating doxorubicin-induced cardiotoxicity using a zebrafish cardiotoxicity injury model established by the above method includes the following steps:

[0039] Zebrafish were randomly divided into a control group, a model group, and a treatment group. The control group was cultured in aqueous solution without doxorubicin and lapatinib; the model group was cultured in aqueous solution containing doxorubicin and lapatinib; and the treatment group was cultured in aqueous solution containing doxorubicin, lapatinib, and the test sample. After 40-48 hours of culture, ejection fraction, heart rate, and end-diastolic ventricular cross-sectional area were measured. A significantly decreased ejection fraction, significantly slower heart rate, and significantly smaller end-diastolic ventricular cross-sectional area in the model group compared to the control group indicated successful establishment of the zebrafish cardiotoxicity model; otherwise, it was considered unsuccessful. To confirm successful model establishment, a significantly increased ejection fraction, significantly higher heart rate, and significantly larger end-diastolic ventricular cross-sectional area in the treatment group compared to the model group indicated that the zebrafish cardiotoxicity was alleviated, and the test drug had preventative / therapeutic activity against doxorubicin-induced cardiotoxicity; otherwise, it had no activity.

[0040] According to a preferred embodiment of the present invention, the zebrafish used in the method is a 24hpf zebrafish.

[0041] According to a preferred embodiment of the present invention, in the method, the number of zebrafish in each group is 8-20.

[0042] According to a preferred embodiment of the present invention, the cultivation conditions in the method are 28.5°C and the light / dark cycle is 14h / 10h.

[0043] According to a preferred embodiment of the present invention, the zebrafish used in the method is the AB lineage wild-type zebrafish.

[0044] According to a preferred embodiment of the present invention, in the method, the culture solution containing doxorubicin and lapatinib contains doxorubicin at a concentration of (20-100) μM and lapatinib at a concentration of (5-30) μM.

[0045] More preferably, in the method, the culture aqueous solution containing doxorubicin and lapatinib contains doxorubicin at a concentration of 20 μM and lapatinib at a concentration of 20 μM.

[0046] According to a preferred embodiment of the present invention, the culture aqueous solution contains: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and pH 7.2.

[0047] According to a preferred embodiment of the present invention, in the method, ejection fraction, heart rate, and ventricular cross-sectional area are detected by video imaging.

[0048] According to a preferred embodiment of the present invention, the statistical basis for the significance of ejection fraction, heart rate, and end-diastolic ventricular cross-sectional area is one-way ANOVA.

[0049] Application of SB203580 in the preparation of drugs to alleviate cardiotoxicity caused by the combined use of DOX and LAP.

[0050] Beneficial effects

[0051] 1. This invention is the first to discover that the combination of doxorubicin and lapatinib can be used to construct a zebrafish cardiotoxicity injury model related to doxorubicin. This invention provides a new zebrafish cardiotoxicity injury model, which is constructed by using the combined administration of doxorubicin and lapatinib. The model provided by this invention is of great significance for the study of cardiotoxicity, especially in the case of cardiotoxicity induced by the antitumor drug doxorubicin.

[0052] 2. This invention not only establishes a model, but also explores in depth the pathological mechanisms of cardiotoxicity, especially the two key pathological events of increased oxidative stress and activation of the MAPK signaling pathway, providing new insights for future treatment and prevention strategies.

[0053] 3. The zebrafish cardiotoxicity injury model provided by this invention includes multiple indicators of cardiac function, which can better illustrate the successful construction of the model and various aspects of cardiotoxicity.

[0054] 4. The zebrafish cardiotoxicity model established in this invention only showed signs of decreased cardiac function. No abnormalities such as body length or systemic edema were observed in the zebrafish of each treatment group, thus ruling out the possible nephrotoxic damage caused by doxorubicin. This model can be specifically used for doxorubicin-related cardiotoxicity studies, eliminating interference from changes in the function of other organs.

[0055] 5. This invention treats juvenile zebrafish via a water-soluble drug delivery route. The method is convenient, has a short treatment cycle, requires a small amount of drug, and has significant effects, effectively improving experimental efficiency and making it suitable for high-throughput drug screening.

[0056] 6. The model provided by this invention can screen for drugs that have the activity of preventing or treating cardiotoxic damage caused by doxorubicin, which provides a new tool for the treatment of cardiotoxicity. Attached Figure Description

[0057] Figure 1 This is a representative image of the morphology of zebrafish when doxorubicin is used in combination with lapatinib.

[0058] Figure 2 The effect of doxorubicin combined with lapatinib on the body length of zebrafish (72hpf);

[0059] In the figure: ***: Compared with the control group, P < 0.001.

[0060] Figure 3 A representative figure showing the effect of combined doxorubicin and lapatinib on the cross-sectional area of ​​the ventricle at end-diastole in zebrafish (72hpf);

[0061] In the figure: the dashed line marks the ventricle of zebrafish at the end of diastole. Representative images of the control group, low-dose group (10 μM LAP + 20 μM DOX), model group (20 μM LAP + 20 μM DOX), and high-dose group (30 μM LAP + 20 μM DOX) were selected. The scale bar is 200 μm.

[0062] Figure 4 The figure shows the effect of doxorubicin combined with lapatinib on ventricular cross-sectional area, ejection fraction and heart rate in zebrafish (72hpf);

[0063] In the figure: *: P < 0.05 compared with the control group; **: P < 0.01 compared with the control group; ***: P < 0.001 compared with the control group.

[0064] Figure 5 The effect of doxorubicin combined with lapatinib on the nuclei of zebrafish (72hpf) cardiomyocytes;

[0065] In the figure: (A,D) shows the distribution of cardiomyocytes in the atria and ventricles of the control group and the model group after treatment with the transgenic zebrafish strain Tg(cmlc2:dsRed); (B,E) shows the number of cardiomyocytes in the ventricles, and it was found that the number of cardiomyocytes in the model group was significantly reduced compared with the control group, and nuclear pyknosis was observed; (C,F) shows the number of cardiomyocytes in the atria, and it was found that the number of cardiomyocytes in the model group was reduced to zero compared with the control group; a: atrium, v: ventricle; scale bars for A and D are 50 μm; scale bars for B, C, E, and F are 20 μm.

[0066] Figure 6Figure showing the effect of doxorubicin combined with lapatinib on myocardial apoptosis in zebrafish (72hpf);

[0067] In the figure: A and D show the distribution of cardiomyocyte nuclei in the control and model groups by staining the heart at 72 hpf with Dapi reagent; B and E show the distribution of cardiomyocyte nuclei in the control and model groups by staining the heart at 72 hpf with TUNEL reagent, and compared with the control group, the model group showed an increase in apoptotic cells in both the atria and ventricles (indicated by white arrows); C and F are merge plots; a: atrium, v: ventricle; scale bar is 50 μm.

[0068] Figure 7 The effect of doxorubicin combined with lapatinib on the sarcomere structure of zebrafish heart (72hpf);

[0069] In the figure: α-Actinin antibody was used to stain the heart of a 72 hpf heart. Compared with the control group, the heart structure of the model group was changed, with both the atria and ventricles shrinking (A, D). Compared with the regularly distributed periodic Z-disc structure in the control group, the model group showed irregularly distributed myosarcoma structures and cavitation in the ventricles (B, E). C and F are the Merge plots of the control group and the model group, respectively; a: atrium, v: ventricle; scale bars of A and D are 50 μm; scale bars of B, C, E, and F are 20 μm.

[0070] Figure 8 Electron micrograph of the heart (72 hpf) of a zebrafish treated with a combination of doxorubicin and lapatinib;

[0071] In the figure: A and B were analyzed by transmission electron microscopy, which confirmed the myosarcoma destruction phenotype of zebrafish at 72 hpf (indicated by the red arrow), and also detected mitochondrial abnormalities in the heart region where LAP and DOX were used in combination, with vacuolization (yellow *).

[0072] Figure 9 A quantitative diagram of the effects of doxorubicin combined with lapatinib on the nucleus and myocardial apoptosis of zebrafish (72hpf) cardiomyocytes;

[0073] In the figure: *: P < 0.05 compared with the control group; **: P < 0.01 compared with the control group.

[0074] Figure 10 ROS levels in a cardiotoxicity model induced by the combined use of doxorubicin and lapatinib;

[0075] In the figure: (AD) Zebrafish at 72 hpf were stained with the oxidant sensing probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The results showed that compared with embryos treated with LAP or DOX alone or the control group (A,B,C), the fluorescence intensity in the model group was stronger and the ROS level was significantly higher (D); the scale bar is 200 μm.

[0076] Figure 11 Biochemical indicators of oxidative damage in a zebrafish model of cardiotoxicity induced by the combined use of doxorubicin and lapatinib;

[0077] **: Compared with the blank control group, P < 0.01; ***: Compared with the blank control group, P < 0.001.

[0078] Figure 12 This is a diagram showing the detection of the MAPK signaling pathway in a cardiotoxicity model induced by the combined use of doxorubicin and lapatinib.

[0079] Figure 13 A graph showing the quantitative analysis of MAPK signaling pathway dysregulation in a cardiotoxicity model induced by the combined use of doxorubicin and lapatinib.

[0080] In the figure: *: P < 0.05 compared with the blank control group; **: P < 0.01 compared with the blank control group; ***: P < 0.001 compared with the model group.

[0081] Figure 14 A diagram showing the ventricular cross-sectional area for screening cardioprotective drugs using a cardiotoxicity model induced by doxorubicin and lapatinib.

[0082] In the figure: the dotted line indicates the size of the zebrafish ventricle. Treatment group 1 was treated with doxorubicin and lapatinib at a final concentration of 20 μM, followed by the addition of SB203580 5 μM. Treatment group 2 was treated with doxorubicin and lapatinib at a final concentration of 20 μM, followed by the addition of PD0325901 10 μM. Treatment group 3 was treated with doxorubicin and lapatinib at a final concentration of 20 μM, followed by the addition of long pepperamide 50 nM. Treatment group 4 was treated with doxorubicin and lapatinib at a final concentration of 20 μM, followed by the addition of astaxanthin 10 nM. The scale bar is 200 μm.

[0083] Figure 15 Figure 1 shows the ventricular cross-sectional area analysis of SB203580 after combined treatment with doxorubicin and lapatinib.

[0084] In the figure: *: P < 0.05; **: P < 0.01.

[0085] Figure 16This is a graph showing the ejection fraction analysis of SB203580 after combined treatment with doxorubicin and lapatinib.

[0086] In the figure: **: P < 0.01; ***: P < 0.001.

[0087] Figure 17 Heart rate analysis of SB203580 after combined treatment with doxorubicin and lapatinib;

[0088] In the figure: *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns is not significant. Detailed Implementation

[0089] The present invention will be further illustrated below with specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0090] Unless otherwise specified, the drugs and reagents used in the examples are ordinary commercially available products. All contents not described in detail in the examples are based on the prior art.

[0091] Materials and reagents

[0092] Zebrafish used in the experiment, including wild-type AB strain zebrafish and transgenic Tg(cmlc2-nls-DsRed) / Tg(cmlc2-EGFP) strains, were obtained from the Biomedical Research Institute of Qingdao University, or could be purchased from the National Zebrafish Resource Center. They were reared at 28℃ under a 14 / 10h light / dark cycle. They were fed brine shrimp twice daily. In the evening, adult males and females were placed in a spawning tank with a sieve plate at a 1:1 ratio and kept in darkness. The next day, they were stimulated by light to spawn. The collected eggs were rinsed with 0.003% (v / v) sodium hypochlorite solution and then placed in culture water and incubated at 28℃ in a light-controlled incubator, a standard culture method in this field.

[0093] Doxorubicin (DOX) (purity ≥99.0%) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China), molecular weight 579.99, and dissolved in purified water to prepare a 10 mM stock solution. Lapatinib (LAP) (purity ≥99.0%), molecular weight 581.06, was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. SB203580 (purity = 99.9%), molecular weight 377.43, was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. PD0325901 (purity ≥99.0%) was purchased from Albisi (Shanghai) Biotechnology Co., Ltd., molecular weight 482.19, and dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. Astaxanthin (purity ≥98.7%) was purchased from MedChemEpress (Shanghai, China), molecular weight 536.87, and dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 mM stock solution. Piperazine (purity = 99.9%) was purchased from MedChemEpress (Shanghai, China), molecular weight 317.34, and dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. Tricaine (MS-222) was purchased from Beijing Solarbio Science & Technology Co., Ltd. All sample stock solutions were stored at 4°C protected from light.

[0094] The zebrafish culture water was an aqueous solution containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 and 0.33 mM MgSO4, with a pH of 7.2.

[0095] Experimental Example 1

[0096] Effects of doxorubicin and lapatinib on body length of zebrafish (72 hpf)

[0097] Wild-type AB zebrafish at 24 hpf were randomly divided into groups of eight and placed in 12-well cell culture plates. Treatment groups were prepared by adding culture water containing 20 μM doxorubicin and 0, 5, 10, 15, 20, 25, and 30 μM lapatinib, with a final volume of 2 mL per well. Control groups were prepared by adding culture water containing the same volume ratio of DMSO as the treatment groups, with a final volume of 2 mL per well. The plates were then capped and placed in a 28°C light-controlled incubator with a 14 / 10 h light / dark cycle for 48 h. After treatment, the zebrafish were removed from the wells and placed under a microscope to observe the phenotype and microscopic imaging of each group. The body length of each group (n=8) was recorded using ImageJ.

[0098] The results showed that after 48 hours of treatment with a mixed solution of 20 μM doxorubicin and different concentrations of lapatinib, the body length of zebrafish in the control group was 8.2 mm, while the body length of zebrafish in the group treated with the combined 20 μM doxorubicin and 20 μM lapatinib was not significantly different from that in the control group. Figure 1 and Figure 2One-way ANOVA was used to compare the differences in body length among the groups. It was found that the zebrafish treated with the combination of 20 μM doxorubicin and 25 μM lapatinib had significantly shorter body lengths than the normal control group (P < 0.001). The 20 μM doxorubicin and 30 μM lapatinib group showed higher mortality rates, significantly shorter body lengths, and some individuals exhibited systemic edema. Figure 1 and Figure 2 The results indicate that this concentration may cause kidney damage. Therefore, the combined use of 20 μM doxorubicin and 20 μM lapatinib does not lead to shortening of body length or systemic edema, indicating that it does not cause kidney damage and ruling out interference with other systems.

[0099] The inventors' experiments showed that when doxorubicin was used at a concentration of less than 20 μM, it did not cause significant damage to the zebrafish hearts. However, when doxorubicin was used in combination with lapatinib at a concentration of greater than 20 μM, the number of embryonic deaths was excessive.

[0100] Experiment Example 2

[0101] Effects of doxorubicin and lapatinib on cardiac function in zebrafish (72 hpf)

[0102] Wild-type AB zebrafish at 24 hpf were randomly divided into groups of eight and placed in 12-well cell culture plates. Treatment groups were prepared by adding culture water containing 20 μM doxorubicin and 0, 5, 10, 15, 20, 25, and 30 μM lapatinib, with a final volume of 2 mL per well. Control groups were prepared by adding culture water containing the same volume ratio of DMSO as the treatment groups, with a final volume of 2 mL per well. The plates were then capped and placed in a 28°C light-controlled incubator with a 14 / 10 h light / dark cycle for 48 h. After treatment, the zebrafish were removed from the wells and observed under a microscope to assess their phenotype and perform video imaging to observe ventricular cross-sectional area, ejection fraction, and heart rate.

[0103] The results showed that after 48 hours of treatment with a mixture of 20 μM doxorubicin and different concentrations of lapatinib in zebrafish at 24 hpf, the end-diastolic ventricular cross-sectional area of ​​the control group zebrafish was 0.24 mm. 2 The heart rate was 149 beats / minute and the ejection fraction was 35%. One-way ANOVA was used to compare the differences in end-diastolic ventricular cross-sectional area, ejection fraction, and heart rate among the groups. (See attached table). Figure 3 , Figure 4In zebrafish treated with a combination of 20 μM doxorubicin and 0-15 μM lapatinib, there were no significant changes in end-diastolic ventricular cross-sectional area, heart rate, and ejection fraction compared to the control group. However, in the group treated with a combination of 20 μM doxorubicin and 20 μM lapatinib, the heart rate and ejection fraction were significantly lower than those in the normal control group (P < 0.001, P < 0.05, P < 0.01). Although the changes in end-diastolic cross-sectional area, ejection fraction, and heart rate were more pronounced when 20 μM doxorubicin was used in combination with 25-30 μM lapatinib, considering the effects on other systems at this concentration, such as body length and generalized edema, in conclusion, the combined use of 20 μM doxorubicin and 20 μM lapatinib did not cause changes in zebrafish body length but did alter cardiac function, thus successfully establishing a zebrafish cardiotoxicity injury model.

[0104] Experimental Example 3

[0105] The inventors investigated other indicators in the zebrafish cardiotoxicity injury model constructed using the combined use of 20μMLAP and 20μMDOX, as detailed below:

[0106] Wild-type AB zebrafish and transgenic zebrafish strain Tg(cmlc2-nls-dsRed) at 24 hpf were randomly divided into a control group and a model group, with 4 zebrafish in each group. The zebrafish were added to 6-well cell culture plates. For the model group, culture water containing 20 μM doxorubicin and 20 μM lapatinib was added to each well, with a final volume of 2 mL per well. For the control group, culture water containing the same volume ratio of DMSO as the treatment group was added to each well, with a final volume of 2 mL per well. The plates were then capped and placed in a 28℃ light-controlled incubator with a light / dark cycle of 14 / 10 h for 48 h. After treatment, the embryonic hearts of 72 hpf zebrafish of the Tg(cmlc2-nls-dsRed) strain were dissected under a microscope using two U-40 insulin syringes. The dissected embryonic hearts were then transferred to a glass slide using a 10 μl pipette and fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min. The fixed heart samples were then embedded in a mounting medium, flattened with a glass slide and cover plate, and confocal images were captured from the flattened heart to count the orange-yellow cell nuclei. AB wild-type zebrafish were then subjected to microscopic examination using… Two U-40 insulin syringes were used to dissect the hearts of 72 hpf embryos. The samples were then transferred to glass slides using 10 μl pipettes, fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min, washed twice with PBS for 5 minutes each time, stained with an in situ TUNEL kit (Elabscience, E-CK-A320), embedded in a mounting medium, sealed with a DAPI-containing mounting solution, and the slides and coverslips were flattened before confocal microscopy (Carl Fibre) imaging. Zeiss), Image J image analysis software analysis showed obvious bright spots after TUNEL staining, and apoptosis was manifested by obvious yellow-green granules; the dissected 72 hpf AB wild-type zebrafish heart was fixed with 4% paraformaldehyde PBS for 20 min at room temperature, washed 3 times with PBS containing 0.1% Triton X-100 (PBST), and then incubated in PBST containing 10% sheep serum for 30 min, and then incubated overnight at 4°C with anti-α-Actinin (1:200, Sigma, Cat#: A7811).Subsequently, the hearts were washed three times with PBST for 10 min each time, and then incubated with secondary antibody Alexa Fluor 488 (1:200, Beyotime, Cat#: A0428). Finally, the immunostained embryonic hearts were mounted with mounting media containing DAPI (Beyotime) and imaged and analyzed using a Zeiss microscope. Dissected 72 hpf AB wild-type zebrafish hearts were fixed at room temperature in Trump fixation solution (4% paraformaldehyde and 1% glutaraldehyde 0.1M phosphate buffer [pH 7.2]) for 1 h at room temperature, and then incubated overnight at 4°C. The fixed samples were then processed and imaged using a HITACHI HT7700 transmission electron microscope.

[0107] The results showed that after 48 hours of treatment with a mixed solution of 20 μM doxorubicin and 20 μM lapatinib, 24 hpf zebrafish showed [results]. Figure 5 , Figure 6 , Figure 9 As shown, compared with the control group, the model group resulted in a significant decrease in the number of cardiomyocyte nuclei in the atria and ventricles of zebrafish (P < 0.05) and an increase in myocardial apoptosis (P < 0.01), and through... Figure 7 , Figure 8 It can be determined that, compared with the control group which exhibits a regularly distributed periodic Z-disc structure, the model group shows irregularly distributed sarcomere structures and cavitation in the ventricular region, as well as mitochondrial abnormalities and vacuolation.

[0108] Therefore, these indicators further demonstrate that the zebrafish heart injury model constructed in this application can be used to screen for drugs with different mechanisms of action that have a beneficial effect on heart injury.

[0109] Experiment Example 4

[0110] Oxidative damage induced in a zebrafish cardiotoxicity model by the combined use of doxorubicin and lapatinib

[0111] Wild-type AB strain zebrafish at 24 hpf were randomly divided into three groups: a normal control group, a single-drug treatment group, and a model group. The normal control group was placed in culture water without doxorubicin and lapatinib. The single-drug treatment groups were given culture water containing only 20 μM doxorubicin and only 20 μM lapatinib, respectively. The model group was placed in culture water containing both 20 μM doxorubicin and 20 μM lapatinib. All groups were cultured for 48 hours. At 72 hpf, 50 embryos were homogenized in physiological saline to form a 10% homogenate. The supernatant was collected by centrifugation for protein concentration determination (502 nm). Subsequently, the contents of malondialdehyde (MDA), an oxidant, and catalase and superoxide dismutase (SOD) were determined according to the manufacturer's instructions. After treatment, spectral analysis was performed. iD3 multimode was used to measure the absorbance of malondialdehyde at 532 nm; catalase content was measured at 405 nm; and superoxide dismutase content was measured at 550 nm. Furthermore, zebrafish from different treatment groups at 72 hpf were stained with the oxidant-sensing probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) under the following conditions: 28.5°C for 2 hours. After staining, the zebrafish were washed three times with PBS and then imaged and analyzed using a Zeiss microscope.

[0112] See Figure 10 , Figure 11 The results showed that after 48 h of treatment with a mixture of 20 μM doxorubicin and / or 20 μM lapatinib in 24 hpf zebrafish, one-way ANOVA was used to compare the differences in the activities of the oxidant malondialdehyde (MDA) and the antioxidants catalase and superoxide dismutase among the groups. Compared with the control group, the MDA content in the 20 μM doxorubicin and 20 μM lapatinib combined treatment group was significantly increased (P < 0.01), and the increase was more significant than that in the 20 μM doxorubicin alone treatment group and the 20 μM lapatinib alone treatment group. Regardless of whether it was the 20 μM doxorubicin alone treatment group or the 20 μM doxorubicin and 20 μM lapatinib combined treatment group, the activities of the antioxidants catalase and superoxide dismutase were significantly reduced compared with the normal control group (P < 0.01). Therefore, the biochemical indicators and reactive oxygen species (ROS) staining together indicate that oxidative damage is a pathological factor in the cardiotoxicity induced by the combined treatment of doxorubicin and lapatinib.

[0113] Experimental Example 5

[0114] Expression of MAPK signaling pathway proteins in a zebrafish model of cardiotoxicity induced by doxorubicin and lapatinib.

[0115] Wild-type AB zebrafish at 24 hpf were randomly divided into three groups: a normal control group, a single-drug treatment group, and a model group. The normal control group received culture water containing neither doxorubicin nor lapatinib. The single-drug treatment groups received culture water containing only 20 μM doxorubicin and only 20 μM lapatinib, respectively. The model group zebrafish were cultured in culture water containing both 20 μM doxorubicin and 20 μM lapatinib. After 48 h, at 72 hpf, the treated zebrafish embryos were homogenized in a tissue protein extraction reagent containing protease inhibitors and phosphatase inhibitors, and then electrolyzed twice at 60 Hz for 15 seconds each time. After centrifugation at 10000 g for 20 min at 4 °C, the supernatant was collected for protein concentration determination (502 nm). Based on the measured protein concentration, each group was adjusted to an equal concentration. 30 mg of protein was loaded and separated using a 12% SDS-PAGE gel. The separation conditions were 70V for 50 min, then 120V for 2 h until bromophenol blue reached the bottom of the gel. Electrophoresis was then stopped and the power was turned off. For membrane transfer: First, PVDF was activated by soaking in methanol for at least 1 min and then transferred to pre-cooled transfer buffer at 4°C. Simultaneously, six sheets of filter paper were soaked in the pre-cooled transfer buffer at 4°C. Next, the electrophoresis plate was removed, the glass short plate was pried open with a pry bar, and excess material from the stacking gel was cut off. The separating gel was transferred to the pre-cooled transfer buffer using a pry bar. The black negative electrode of the transfer clamp was laid flat in the water tank containing transfer buffer, and then the sandwich structure was sequentially laid out. "Sandwich Structure": (Negative electrode) Blackboard + sponge + 3 layers of filter paper (soaked in transfer solution). Use a glass rod to remove air bubbles + gel + PVDF membrane (to avoid air bubbles between the gel and PVDF membrane, add several drops of transfer solution to the gel, take the PVDF membrane and let one side touch the edge of the gel first, then slowly lower it to adhere it completely) + 3 layers of filter paper + sponge + white board. Finally, close the clamp and place it in the transfer tank (black side corresponds to the negative electrode, red side corresponds to the positive electrode). Place the sandwich structure in the transfer tank (positive and negative electrodes aligned), add the rotor and ice pack, or place it in a 4°C freezer under constant current of 400mA in an ice-water bath for 50 minutes for transfer. After the transfer is complete, turn off the power, collect the filter paper, soak the PVDF membrane in transfer solution, place it in a pre-cut plastic bag, and cut the target strip. The molecular weight of Erk protein is 44 kDa, the molecular weight of p-ERK Erk protein is 44 kDa, and the molecular weight of internal control β-actin protein is 41 kDa. The PVDF membrane was completely dissolved in 5% skim milk and then blocked by shaking at room temperature for 1 hour. The blocked PVDF membrane was then washed three times with TBST washing buffer for 5 min each time. The primary antibodies ERK (1:1000 Cat#: 9102, CST), p-ERK (1:1000; Cat#: 9106, CST), and β-actin (1:10000; Cat# AC026, Abclonal) were diluted with 5% BSA according to this dilution ratio and incubated overnight at 4°C. After recovering the primary antibodies, the membrane was washed four times with TBST for 10 min each time.Secondary antibody incubation: Dilute the HRP-labeled secondary antibody with 5% BSA as described above and incubate at room temperature with shaking for 1 hour. Washing: After discarding the secondary antibody, wash the membrane 4 times with TBST for 10 minutes each time, and finally develop. Analyze the protein bands using Image J (see image). Figure 12 .

[0116] This invention delves into the molecular mechanisms of cardiotoxicity induced by the combined use of doxorubicin and lapatinib. Given that oxidative stress (ROS) can trigger MAPK signaling, this invention focuses particularly on changes in the expression of downstream effector factors in the MAPK signaling pathway. In a cardiotoxicity model using the combination of doxorubicin and lapatinib, decreased expression of phosphorylated Erk protein and significantly increased expression of phosphorylated P38 and Jnk proteins were observed, with the combined treatment showing a more pronounced effect compared to embryos treated with either doxorubicin or lapatinib alone. These findings suggest that lapatinib may enhance doxorubicin-induced cardiotoxicity by further activating the P38 and Jnk branches of the MAPK signaling pathway.

[0117] See Figure 13 The results showed that after 48 h of treatment with a mixture of 20 μM doxorubicin and / or 20 μM lapatinib in 24 hpf zebrafish, one-way ANOVA was used to compare the differences in erk, p38, and jnk protein phosphorylation among the groups. Compared with the control group, the phosphorylation levels of p38 and jnk proteins were significantly increased in the 20 μM doxorubicin and 20 μM lapatinib combined treatment group (P < 0.001), and the increase was more pronounced in the combined treatment group than in the single treatment group (P < 0.01). Regardless of whether 20 μM doxorubicin or 20 μM lapatinib was used alone, or in the 20 μM doxorubicin and 20 μM lapatinib combined treatment group, the phosphorylation level of erk protein was significantly decreased compared with the control group (P < 0.05). Therefore, both Western blotting and quantitative analysis indicate that dysregulation of the MAPK signaling pathway is one of the pathological events leading to cardiotoxicity caused by the combined use of doxorubicin and lapatinib.

[0118] Experimental Example 6

[0119] Screening of cardioprotective drugs using cardiotoxicity models induced by doxorubicin and lapatinib.

[0120] Wild-type AB zebrafish at 24 hpf were randomly divided into three groups of 20 each and added to 12-well cell culture plates. The groups were divided into a control group, a model group, and a treatment group. Prepared drug solutions were used, and astaxanthin (10 nM), long peppermint (50 nM), SB203580 (5 μM), and PD0325901 (10 μM) were diluted with culture water and added to the wells. The blank control group was treated with culture water containing the same volume of DMSO. The model group was treated with doxorubicin and lapatinib until the final concentrations of doxorubicin and lapatinib in the culture water reached 20 μM each. Treatment group 1 was treated with doxorubicin and lapatinib until the final concentrations of doxorubicin and lapatinib in the culture water reached 20 μM each, and SB203580 was added simultaneously. Treatment group 2 received 5 μM of doxorubicin and lapatinib, with the final concentrations of doxorubicin and lapatinib in the culture solution reaching 20 μM and 10 μM respectively, followed by the addition of PD0325901. Treatment group 3 received doxorubicin and lapatinib, with the final concentrations of doxorubicin and lapatinib in the culture solution reaching 20 μM and 50 nM respectively, followed by the addition of long peppermint amide. Treatment group 4 received doxorubicin and lapatinib, with the final concentrations of doxorubicin and lapatinib in the culture solution reaching 20 μM and 10 nM respectively, followed by the addition of astaxanthin. The final volume of the solution in each well was 2 mL. After capping, the wells were placed in a light-controlled incubator at 28°C with a light / dark cycle of 14 / 10 h for 48 h. After treatment, the zebrafish in the wells were removed and placed under a microscope to observe the phenotype of each group of fish and to perform microscopic imaging to observe the ventricular cross-sectional area, ejection fraction, and heart rate. Figure 14 , Figure 15 , Figure 16 , Figure 17 .

[0121] The results showed that, compared with the control group, the model group had a decrease in ventricular cross-sectional area, ejection fraction, and heart rate with significant differences (P < 0.01). After the addition of SB203580, PD0325901, astaxanthin, and long pepper, only the treatment group 1 (SB203580 group) showed that the ventricular cross-sectional area, ejection fraction, and heart rate simultaneously recovered to levels similar to the blank control group, and there were significant differences compared with the model control group (P < 0.01). This indicates that SB203580 has therapeutic activity against cardiotoxicity caused by the combined use of doxorubicin and lapatinib.

[0122] This invention is the first to discover that the combination of doxorubicin and lapatinib can be used to construct a zebrafish model of doxorubicin-related cardiotoxicity injury. This invention utilizes the combined treatment of zebrafish embryos (24 hpf) with doxorubicin and lapatinib to induce a zebrafish cardiotoxicity injury model. Since the zebrafish heart formation and circulation begin at this stage, interference with early cardiogenesis is avoided, and the results obtained have greater reference value and significance for clinical research on the cardiotoxic side effects of doxorubicin. The cardiac indicators of this invention are very comprehensive, and the zebrafish cardiotoxicity injury model constructed by this invention can be used to screen for drugs with various mechanisms of action that have ameliorative effects on cardiac injury.

[0123] The zebrafish cardiotoxicity model established in this invention only shows manifestations related to cardiac function. No abnormalities such as local or systemic edema were observed in zebrafish in each treatment group, ruling out the possibility of nephrotoxic damage caused by doxorubicin. This makes the model specifically applicable to doxorubicin-related cardiotoxicity studies, eliminating interference from changes in other organ functions. The zebrafish cardiotoxicity damage model provided by this invention can be used to screen for drugs with activity in preventing / treating doxorubicin-induced cardiotoxic damage.

Claims

1. A method for establishing a zebrafish cardiotoxicity injury model, characterized in that, Includes the following steps: Zebrafish were randomly divided into a model group and a control group. The model group was cultured in an aqueous solution containing doxorubicin and lapatinib, while the control group was the same as the model group but without doxorubicin and lapatinib. The zebrafish were cultured for 40-48 hours, and the following parameters were measured: ejection fraction, heart rate, and end-diastolic ventricular cross-sectional area. A significantly lower ejection fraction, significantly slower heart rate, and significantly smaller end-diastolic ventricular cross-sectional area compared to the control group indicated a successful zebrafish cardiotoxicity model; otherwise, the model was considered unsuccessful. The zebrafish is a 24hpf zebrafish; The zebrafish is a wild-type zebrafish belonging to the AB lineage; In the method, the culture aqueous solution containing doxorubicin and lapatinib contains doxorubicin at a concentration of 20 μM and lapatinib at a concentration of 20 μM.

2. The method as described in claim 1, characterized in that, The culture solution contains: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, pH 7.

2.

3. The method as described in claim 1, characterized in that, Each group of zebrafish consists of 8-20 fish; The cultivation conditions were 28.5℃, with a light / dark cycle of 14h / 10h.

4. The method as described in claim 1, characterized in that, Ejection fraction, heart rate, and ventricular cross-sectional area are detected using video imaging methods; In the aforementioned method, the statistical basis for significant ejection fraction, heart rate, and end-diastolic ventricular cross-sectional area is one-way ANOVA.

5. The method as described in claim 1, characterized in that, The detection indicators also include cardiomyocyte nuclei, cardiomyocyte apoptosis, cardiomyocyte sarcomeres and / or mitochondria; Compared with the control group, the model group showed damage to sarcomere structure, reduction of cardiomyocyte nuclei, increase of myocardial apoptosis, and mitochondrial damage.

6. The use of the zebrafish cardiotoxicity injury model established by the method of any one of claims 1-5 in screening drugs with activity in preventing / treating cardiotoxicity injury caused by doxorubicin.

7. A method for screening drugs with activity in preventing / treating doxorubicin-induced cardiotoxicity using a zebrafish cardiotoxicity injury model established by the method of any one of claims 1-5, comprising the following steps: Zebrafish were randomly divided into a control group, a model group, and a treatment group. The control group was cultured in aqueous solution without doxorubicin and lapatinib; the model group was cultured in aqueous solution containing doxorubicin and lapatinib; and the treatment group was cultured in aqueous solution containing doxorubicin, lapatinib, and the test sample. After 40-48 hours of culture, ejection fraction, heart rate, and end-diastolic ventricular cross-sectional area were measured. A significantly decreased ejection fraction, significantly slower heart rate, and significantly smaller end-diastolic ventricular cross-sectional area in the model group compared to the control group indicated successful establishment of the zebrafish cardiotoxicity model; otherwise, it was considered unsuccessful. To confirm successful model establishment, a significantly increased ejection fraction, significantly higher heart rate, and significantly larger end-diastolic ventricular cross-sectional area in the treatment group compared to the model group indicated that the zebrafish cardiotoxicity was alleviated, and the test drug had preventative / therapeutic activity against doxorubicin-induced cardiotoxicity; otherwise, it had no activity. The zebrafish is a 24hpf zebrafish; The zebrafish is a wild-type zebrafish belonging to the AB lineage; In the method, the culture aqueous solution containing doxorubicin and lapatinib contains doxorubicin at a concentration of 20 μM and lapatinib at a concentration of 20 μM.

8. The method as described in claim 7, characterized in that, Each group of zebrafish consists of 8-20 fish; The cultivation conditions were 28.5℃, with a light / dark cycle of 14h / 10h; The culture solution contains: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, pH 7.

2.

9. The method as described in claim 7, characterized in that, Ejection fraction, heart rate, and ventricular cross-sectional area are detected using video imaging methods.

10. The method as described in claim 7, characterized in that, The statistical basis for the significance of ejection fraction, heart rate, and end-diastolic ventricular cross-sectional area is one-way ANOVA.

Images