CN116602976B - Composition, and establishing method and application of doxorubicin-related zebra fish heart toxicity injury model - Google Patents
Composition, and establishing method and application of doxorubicin-related zebra fish heart toxicity injury model Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
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Abstract
The invention provides a composition, a method for establishing a zebra fish heart toxicity injury model related to doxorubicin and application thereof, belongs to the technical field of biological medicine and animal model construction, and particularly relates to a composition comprising doxorubicin and forsythin; the invention discovers that the combined use of the doxorubicin and the forsythin can be used for constructing a doxorubicin-related zebra fish heart toxicity injury model for the first time; according to the zebra fish heart toxicity model established by the invention, only heart rate reduction appears, and the zebra fish of each treatment group does not have local or systemic edema and other abnormalities, so that the occurrence of possible nephrotoxic injury caused by doxorubicin is eliminated; the zebra fish heart toxicity injury model provided by the invention can be used for screening drugs with the activity of preventing/treating heart toxicity injury caused by doxorubicin.
Description
Technical Field
The invention belongs to the technical field of biological medicine and animal model construction, and particularly relates to a composition, a method for constructing a zebra fish heart toxicity injury model related to doxorubicin and application thereof.
Background
Doxorubicin belongs to the anthracycline family, is a clinically common broad-spectrum antitumor drug, and can effectively act on tumor cells in various growth cycles. In application, the doxorubicin has cardiotoxicity, is easy to damage myocardial cells, and causes arrhythmia and ST-T change to cause congestive heart failure, and the severe heart failure occurs in 1-6 months after drug withdrawal. The cardiotoxicity of doxorubicin is closely related to the drug dosage and the combined use condition. Clinically, doxorubicin is generally used as the second line drug, and is considered for use when drug resistance is the first choice. The cardiac toxic side effects of doxorubicin greatly limit its application in clinical antitumor therapy, and therefore, research on the cardiac toxicity mechanism of doxorubicin is urgently needed.
The animal model is an important tool for researching human diseases, and can intuitively reflect the pathophysiological changes of human diseases by using the animal model to research the disease mechanism. Meanwhile, the animal model is also an important tool for screening active drugs and developing the drugs. The biological characteristics of rodents are similar to those of human beings, and the experimental results are representative, but the rodents have the defects of large size, high cost and long period. Zebra fish is a small tropical freshwater fish, and belongs to the carp family. The adult fish is only about 3-4 cm long, the spawning quantity is large, the embryo is transparent and develops in vitro, and the operation and the observation are very convenient. The embryo development period is short, many researches can be carried out in embryo period, and the dosage of the medicine is very small. The genome sequence of the zebra fish is highly similar to that of human genome, and a plurality of important tissues and organs including a cardiovascular system and a nervous system are similar to human in morphological structure, physiological function and pathological response, so that the zebra fish is widely applied in the fields of development biology, compound toxicity evaluation, human disease model research and the like, and is an ideal vertebra model organism.
The juvenile zebra fish is transparent, can clearly observe the heart and systemic blood circulation conditions under a microscope, has unique biological advantages in the aspects of drug cardiotoxicity research and active drug research and development, and is widely applied. Zhao Hui et al treat 36hpf zebra fish with doxorubicin, induce and build a myocardial injury model, and the zebra fish develops pericardial edema and increases the distance between venous sinus and arterial bulb (SV-BA). Xu Zhuoran et al treat developing 24hpf zebra fish with doxorubicin, obtain a zebra fish cardiotoxic injury model, and study the mechanism of doxorubicin toxicity injury. The zebra fish is treated by water-soluble administration, so that the experimental method is convenient and easy to carry out in high flux. However, the current research is mostly carried out in the embryo period (< 72 hpf) of the zebra fish, the normal zebra fish is completed when fertilization of 24hpf blood vessel development begins and 72hpf is carried out, at this time, most organs are mature, normal functions are achieved, young fishes hatch out membranes, so that the zebra fish with the length greater than 72hpf is more suitable for researching the toxic effect of medicines on mature organisms and organ functions, and the result has reference significance. Doxorubicin was microinjected by Rider et al into the 72hpf zebra fish blood circulation system and the zebra fish exhibited heart failure, but this method was relatively high in technical requirements and unsuitable for large-scale development. Therefore, a method which is easy to operate and reliable in effect is needed to be found, an doxorubicin cardiotoxicity damage model aiming at the juvenile fish of the zebra fish is induced and established, and an experimental basis is provided for the prevention and treatment research of cardiac function damage caused by clinical doxorubicin. And there are few reports in the prior art on the use of animal models for screening drugs having activity in the prevention/treatment of cardiotoxic damage caused by doxorubicin.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a composition, a method for establishing a zebra fish heart toxicity injury model related to doxorubicin and application thereof.
The invention discovers for the first time that the combined use of the doxorubicin and the forsythin can be used for constructing a doxorubicin-related zebra fish heart toxicity injury model.
The invention aims to provide a method for establishing a doxorubicin-related zebra fish heart toxicity injury model, which is used for researching a doxorubicin heart toxicity injury mechanism and screening active drugs for preventing or treating myocardial injury caused by doxorubicin.
Description of the terminology:
dpf (days post fertilization): refers to the number of days after fertilization.
The technical scheme of the invention is as follows:
a composition for modeling cardiac toxicity injury in zebra fish, comprising doxorubicin and forsythin.
According to a preferred embodiment of the invention, the components of the composition are used in a concentration of doxorubicin (20-40) μg/mL and forsythin (200-700) μg/mL.
Further preferably, the components of the composition are used at a concentration of 30 μg/mL doxorubicin and 200-700 μg/mL forsythin.
According to the invention, the zebra fish used for establishing the zebra fish heart toxicity damage model is preferably zebra fish with the zebra fish width of more than 3 dpf.
Further preferably, the zebra fish used to model the cardiac toxicity damage of the zebra fish is a zebra fish with a 3dpf or more and less than 6 dpf.
According to a preferred embodiment of the invention, the zebra fish is an AB wild type zebra fish.
The application of doxorubicin and forsythin in establishing a zebra fish heart toxicity injury model.
According to a preferred aspect of the invention, the doxorubicin is used at a concentration of (20-40) μg/mL and the forsythin is used at a concentration of (200-700) μg/mL.
Further preferred, in said application, doxorubicin is used at a concentration of 30 μg/mL and forsythin is used at a concentration of (200-700) μg/mL.
According to a preferred aspect of the invention, in the application, the zebra fish used for establishing the zebra fish heart toxicity damage model is zebra fish with a length of 3dpf or more.
Further preferred, in the application, the zebra fish used to model the cardiac toxicity injury of the zebra fish is a zebra fish with a 3dpf or more and less than 6 dpf.
According to a preferred embodiment of the invention, the zebra fish is an AB wild type zebra fish.
A method for establishing a zebra fish heart toxicity injury model comprises the following steps:
the method comprises the steps of randomly grouping zebra fish, namely a model group and a normal control group, wherein the model group is added into a culture water solution containing doxorubicin and forsythin, the normal control group is added into a culture water solution without doxorubicin and forsythin, culturing the model group and the normal control group for 20-28h, observing the heartbeat condition of each fish, counting the heart rate, and compared with the normal control group, the model group has obvious heart rate reduction, and the zebra fish cardiotoxicity injury model is successfully established, or else is unsuccessful.
According to a preferred embodiment of the invention, the zebra fish is a wild type zebra fish of the AB-line.
According to a preferred embodiment of the present invention, the zebra fish used in the method is a zebra fish having a 3dpf or higher.
Further preferred, in the method, the zebra fish used is one of 3dpf or more and less than 6 dpf.
According to a preferred embodiment of the present invention, in the method, the aqueous culture solution containing doxorubicin and forsythin has a concentration of doxorubicin of (20-40) μg/mL and a concentration of forsythin of (200-700) μg/mL.
Further preferably, in the method, the aqueous culture solution containing doxorubicin and forsythin has a concentration of doxorubicin of 30. Mu.g/mL and a concentration of forsythin of (200-700). Mu.g/mL.
According to a preferred embodiment of the present invention, the aqueous culture solution contains: containing 5mM NaCl,0.17mM KCl,0.33mM CaCl 2 、0.33mM MgSO 4 ，pH 7.2。
According to a preferred embodiment of the invention, the number of zebra fish per group is 15-30.
According to a preferred embodiment of the invention, the cultivation is carried out at a temperature of 27-28℃and a light/dark cycle of 14h/10h.
According to a preferred embodiment of the invention, the statistical basis for a significant heart rate reduction is the independent sample t-test.
The zebra fish heart toxicity injury model established by the method is applied to screening of drugs with the activity of preventing/treating heart toxicity injury caused by doxorubicin.
The method for screening the drug with the activity of preventing/treating the cardiotoxic injury caused by the doxorubicin by using the zebra fish cardiotoxic injury model established by the method comprises the following steps of:
the method comprises the steps of randomly grouping zebra fish into a normal control group, a model group and a treatment group respectively, wherein the normal control group is added into a culture water solution without doxorubicin and forsythin, the model group is added into a culture water solution with doxorubicin and forsythin, the treatment group is added into a culture water solution with a sample to be tested in advance for pretreatment for 2-5h, then doxorubicin and forsythin are added to the required concentration, or the treatment group is added into a culture water solution with doxorubicin, forsythin and the sample to be tested, the normal control group, the model group and the treatment group are cultivated for 20-28h, the heartbeat condition of each fish is observed, and heart rate is counted, and compared with the model group, the heart rate is obviously increased, so that the heart toxicity damage of the zebra fish is relieved, and the drug to be tested has the activity of preventing/treating the heart toxicity damage caused by the doxorubicin, or no activity is obtained.
Advantageous effects
1. The invention discovers for the first time that the combined use of the doxorubicin and the forsythin can be used for constructing a doxorubicin-related zebra fish heart toxicity injury model.
2. According to the invention, the zebra fish juvenile fish (more than or equal to 3 dpf) is treated by the combination of the doxorubicin and the forsythin, the zebra fish heart toxicity damage model is induced and established, and as each organ of the zebra fish is developed, the toxicity influence of the doxorubicin on the heart function under normal conditions can be reflected more accurately, and the obtained result has better reference significance and reference value for the clinical study of the heart toxic and side effects of the doxorubicin.
3. The zebra fish heart toxicity model established by the invention only shows heart rate reduction, and the zebra fish of each treatment group does not have local or systemic edema and other abnormalities, so that the occurrence of possible nephrotoxicity damage caused by doxorubicin is eliminated, the model can be specifically used for the related research of doxorubicin heart toxicity, and the interference of other viscera function changes is eliminated.
4. The method for treating the juvenile zebra fish by the water-soluble administration route is convenient, short in treatment period, small in medicine dosage, obvious in effect, and suitable for high-flux medicine screening, and experimental efficiency is effectively improved.
5. The zebra fish heart toxicity injury model provided by the invention can be used for screening drugs with the activity of preventing/treating heart toxicity injury caused by doxorubicin.
Drawings
FIG. 1 is a graph showing the effect of doxorubicin combined with forsythin on zebra fish morphology.
Detailed Description
The invention is further illustrated by the following specific examples, without limiting the scope of protection.
The drugs and reagents used in the examples are, unless otherwise specified, commercially available products and examples
What is not described in detail in the foregoing is known in the art.
Materials and reagents
The AB-series wild zebra fish used for the test is derived from the biological institute of the university of Qilu industry (Shandong national academy of sciences) and can also be purchased from the national zebra fish resource center. Raising at 28deg.C under the condition of light/dark cycle of 14/10 h. The brine shrimp is fed twice daily. In the evening, male and female parent fishes are mixed according to a ratio of 1:1 are put into a spawning jar with a sieve plate and are placed in darkness, and the ovulation is stimulated by light irradiation in the next day. The collected roe is washed by 0.003% (v/v) sodium hypochlorite solution and then placed in culture water, and then placed in a 28 ℃ illumination incubator for continuous incubation, and belongs to a conventional culture method in the field.
Doxorubicin (Doxorubicin), available from Shanghai source leaf biotechnology limited, molecular weight 579.99, hplc > 98%, was dissolved in purified water to make 2mg/mL stock solution. Forsythin (Philliyrin), available from Shanghai Seiya Biotechnology Co., ltd., molecular weight 534.55, was dissolved in dimethyl sulfoxide (DMSO) to prepare a 0.2g/mL stock solution. Dexrazoxane (Dexrazoxane), available from Shanghai Ala Biochemical technologies Co., ltd., molecular weight 268.27, purity not less than 97%, was dissolved in dimethyl sulfoxide to prepare a 5mg/mL stock solution. Acetylcysteine (Acetylcysteine), available from Beijing Soy Bao technology Co., ltd., molecular weight 163.195, HPLC not less than 98%, was dissolved in culture water to give the desired concentration. Tricaine (MS-222) was purchased from Beijing Soy Bao technology Co. The stock solutions of each sample were stored at 4℃in the dark.
The zebra fish culture water contains 5mM NaCl,0.17mM KCl,0.33mM CaCl 2 ,and 0.33mM MgSO 4 Is at a pH of 7.2.
Experimental example 1
Effects of Adriamycin and Forsythian combination on cardiac function of zebra fish (3 dpf)
Zebra fish of 3dpf are randomly grouped into 30 groups and added into 24-well cell culture plates. Adding 30 mug/mL of doxorubicin and 0, 100, 200, 400, 700 and 1000 mug/mL of forsythin in culture water solution into the wells, wherein the final volume of each well solution is 2mL, adding culture water containing DMSO (dimethyl sulfoxide) in the same volume ratio as that of the treatment group into the control group, adding 2mL of culture water into each well solution, capping, placing into a 28 ℃ light-controlled incubator, and carrying out light/dark period of 14/10h and 24h treatment. After the treatment, zebra fish in the wells are taken out and placed under a microscope, the phenotype and heartbeat condition of each group of fish are observed, and the heart rate is counted.
TABLE 1 Effect of Adriamycin in combination with Forsythian on the heart rate of zebra fish (3 dpf)
* P is less than 0.05 compared with a blank control group; #: p < 0.05 compared with doxorubicin 30 μg/mL group.
The results show that after the 3dpf zebra fish is treated by 30 mug/mL of the mixed solution of the doxorubicin and the forsythin with different concentrations for 24 hours, the heart rate of the zebra fish in the control group is 163+/-3.2 times/min, and the heart rate of the zebra fish in the 30 mug/mL of the zebra fish in the control group is 160+/-5.1 times/min, and the ratio of the zebra fish to the control group is not obviously changed. The heart rate differences among groups are compared by adopting independent sample t test, and the heart rate of the zebra fish in the group treated by combining 30 mug/mL of doxorubicin and 200-700 mug/mL of forsythin is reduced and significantly different from that of the zebra fish in the normal control group (P is less than 0.05), the zebra fish in the group treated by 30 mug/mL of doxorubicin and 1000 mug/mL of forsythin die more, and a few surviving zebra fish show arrhythmia, which indicates that the heart function of the zebra fish is damaged by the combined application of doxorubicin and forsythin, and the damage degree and the forsythin are dose-dependent (table 1). No apparent local or systemic edema was seen in each of the treatment groups of zebra fish (fig. 1), indicating that no impairment of renal function was caused.
Experimental example 2
Effects of Adriamycin and Forsythian combination on cardiac function of zebra fish (5 dpf)
Zebra fish of 5dpf were randomly grouped into 20 groups and added to 24 well cell culture plates. Adding culture water solution containing doxorubicin 40 μg/mL and forsythin 200, 400, 700 μg/mL into the wells, wherein the final volume of each well solution is 2mL, adding culture water containing DMSO with the same volume ratio as that of the treatment group into the control group, and adding the culture water into the control group, wherein the final volume of each well solution is 2mL, capping, placing the control group into a light-operated incubator at 28 ℃, wherein the light/dark period is 14/10h, and treating for 24h. After the treatment, zebra fish in the wells were removed, treated with 30 μg/mL MS-222 for 1min, then placed under a microscope, the phenotype, heartbeat condition, and heart rate of each group of fish were observed.
The result shows that after the 5dpf zebra fish is treated by 40 mug/mL of the mixed solution of the doxorubicin and the forsythin with different concentrations for 24 hours, the heart rate of the zebra fish in the control group is 163+/-3.2 times/min, and the heart rate of the zebra fish in the 40 mug/mL of the zebra fish in the control group is 160+/-5.1 times/min, and the ratio of the zebra fish to the control group is not obviously changed. The heart rate differences between groups were compared using independent sample t-test, and it was found that the heart rate of zebra fish in the group treated with 40. Mu.g/mL doxorubicin combined with 200-700. Mu.g/mL forsythin was reduced and significantly different (P < 0.05) from that in the normal control group (Table 2). No apparent local or systemic edema was seen in each treatment group of zebra fish, indicating no impairment of renal function was caused.
TABLE 2 Effect of Adriamycin in combination with Forsythian on the heart rate of zebra fish (5 dpf)
* P is less than 0.05 compared with a blank control group; #: p < 0.05 in the group ratio of 40 mug/mL with doxorubicin.
In total, under the condition of the ambient temperature of 23-28 ℃, the normal heart rate range of the zebra fish fertilized for 3-5 days is 160-168 times/min, and after the heart function is subjected to toxic damage by the treatment method, the heart rate can be reduced to below 120-130 times/min, and the heart rate is seriously reduced to 60 times/min, so that death occurs.
Zebra fish with the swimming capability of less than 6dpf has low, transparent fish body, is suitable for under-lens observation, can survive in a pore plate without feeding, is suitable for experiments, has the advantages of quick growth of zebra fish with the swimming capability of more than 6dpf, needs to feed paramecium, and is not suitable for drug treatment and observation.
Experimental example 3
Protection of cardiotoxic injury caused by doxorubicin by dexrazoxane
Zebra fish of 3dpf are randomly grouped into 20 groups and added into 24-well cell culture plates. Taking a dexrazoxane stock solution, diluting with culture water to obtain a working concentration of 10, 50 and 100 mug/mL, adding into a plate hole, setting two holes in a control group, adding a culture water solution containing the same volume of DMSO, capping, placing into a 28 ℃ light-operated incubator, and treating for 4 hours at a light/dark period of 14/10 h. Taking out the culture plate, adding doxorubicin and forsythin into a control group, wherein the doxorubicin and forsythin are added into a culture aqueous solution, the final concentration of the doxorubicin is 30 mug/mL, the final concentration of the forsythin is 400 mug/mL, the control group is a damage group, the doxorubicin and forsythin are added into a rightward propyl imine treatment group, the final concentration of the doxorubicin is 30 mug/mL, the final concentration of the forsythin is 400 mug/mL, the rightward propyl imine concentration is unchanged, the control group is a treatment group, the blank control group is a culture aqueous solution containing DMSO with the same volume, and the final volume of each hole is 2mL. Capping, placing in a light-operated incubator at 28 ℃ for treatment for 20 hours with an illumination/darkness period of 14/10 hours. After the treatment, zebra fish in the wells are taken out and placed under a microscope, the phenotype and heartbeat condition of each group of fish are observed, and the heart rate is counted.
The results show that the heart rate of the zebra fish in the blank group is 165+/-2.5 times/min, the heart rate of the zebra fish in the damaged group after being treated by doxorubicin (30 mug/mL) and forsythin (400 mug/mL) is 128+/-4.5 times/min, and the heart rate difference between the groups is compared by adopting independent sample t test, so that the heart rate of the damaged group is obviously reduced (P is less than 0.05) compared with that of the blank group. Zebra fish were pretreated with a solution containing 100. Mu.g/mL of dexrazoxane for 4h, then treated with doxorubicin (30. Mu.g/mL) and forsythin (400. Mu.g/mL), and zebra fish heart rates were 152.+ -. 5.6 times/min, respectively, higher than the injury group and significantly different (P < 0.05), but still significantly lower than the blank control group, indicating that 100. Mu.g/mL of dexrazoxane can alleviate to some extent the cardiotoxic injury caused by the combination of doxorubicin and forsythin (Table 3).
TABLE 3 Effect of dexrazoxane pretreatment on cardiotoxic effects of doxorubicin and forsythin
* P is less than 0.05 compared with the control group; #: p < 0.05 compared with the injury group.
Example 4
Use of acetylcysteine in cardiotoxic damage caused by doxorubicin
Zebra fish of 4dpf were randomly grouped and 15 pieces each were added to 24-well cell culture plates. The culture aqueous solution containing 30 mug/mL of doxorubicin and 400 mug/mL of forsythin is added into the wells of the injury group, the newly prepared acetylcysteine culture aqueous solution with three concentrations of 5, 10 and 50mg/mL is added into the treatment group on the basis of the injury group solution, the culture aqueous solution containing the same volume of DMSO is added into the control group, the final volume of each well is 2mL, the culture aqueous solution is capped and then placed into a 28 ℃ light-operated incubator, the light/dark period is 14/10h, and the treatment is carried out for 26h. After the treatment, zebra fish in the wells are taken out and placed under a microscope, the phenotype and heartbeat condition of each group of fish are observed, and the heart rate is counted.
The results show that the heart rate of the zebra fish in the control group is 168+/-5.5 times/min, the heart rate of the zebra fish in the damaged group after being treated by doxorubicin (30 mug/mL) and forsythin (400 mug/mL) is 126+/-3.6 times/min, and the heart rate difference between groups is compared by adopting independent sample t test, so that the heart rate of the damaged group is obviously reduced (P is less than 0.05) compared with that of the blank control group. After adding three concentrations of 5, 10 and 50mg/mL acetylcysteine, the zebra fish heart rate was not significantly different from the heart rate of the injury group, the zebra fish heart rate was still lower than that of the blank control group, and the difference was significant (P < 0.05), which indicates that the acetylcysteine has no therapeutic activity on cardiotoxic injury caused by the combined administration of doxorubicin and forsythin (Table 4).
TABLE 4 Effect of acetylcysteine on cardiotoxic effects of doxorubicin and forsythin
* P is less than 0.05 compared with the control group.
Comparative example 1
Effect of doxorubicin on cardiac function in zebra fish
Zebra fish of 3dpf are randomly grouped into 30 groups and added into 24-well cell culture plates. To the wells, an aqueous culture solution containing 10, 20, 30, 40, 50. Mu.g/mL doxorubicin was added, and the control group was added with the same volume of the aqueous culture solution, with a final volume of 2mL per well. Capping, placing in a light-operated incubator at 28deg.C, and continuously treating for 72 hr with light/dark period of 14/10 hr, and changing 1/2 of the medicinal liquid every day. Zebra fish in the wells were taken out every 24 hours under a microscope, morphological changes, heartbeat conditions were observed, and heart rates were counted. After treatment with 10-40 mug/mL doxorubicin for 24, 48, 72 hours, the juvenile fish is found to have no obvious abnormality in morphology, no local or systemic edema, and no significant difference in heart rate from the control group. After 24h of doxorubicin treatment at 50. Mu.g/mL, all the young fish in the wells died.
Comparative example 2
Influence of forsythin on cardiac function of zebra fish
Zebra fish of 3dpf are randomly grouped, 25 zebra fish of each group are added into 24-hole cell culture plates. And (3) taking a forsythin stock solution, diluting with culture water to obtain a concentration group of 100, 200, 400, 700 and 1000 mug/mL, adding the concentration group into a plate hole, adding a culture aqueous solution containing DMSO (dimethyl sulfoxide) in the same volume ratio as that of a forsythin treatment group into a control group, and enabling the final volume of the solution per hole to be 2mL. Capping, placing in a light-operated incubator at 28deg.C, and processing for 24 hr with light/dark cycle of 14/10 hr. After the treatment, the zebra fish in the wells are taken out and placed under a microscope, the phenotype and heartbeat condition of each fish are observed, and the heart rate is counted. It was found that,
the result shows that the juvenile fish of the treatment group with 100-1000 mug/mL forsythin has no obvious abnormality in morphology, no local or systemic edema and no obvious difference in heart rate from the control group.
According to the experiment, the zebra fish is treated by the doxorubicin or the forsythin respectively and independently, the treatment time is prolonged, compared with a control group, the zebra fish has no heart rate reduction and no abnormal heart function, which indicates that no toxic damage is caused to the zebra fish heart, and the technical scheme of combining the doxorubicin and the forsythin can realize the effect of causing the toxic damage to the zebra fish heart.
The invention discovers for the first time that the combined use of the doxorubicin and the forsythin can be used for constructing a doxorubicin-related zebra fish heart toxicity injury model. According to the invention, the zebra fish juvenile fish (more than or equal to 3 dpf) is treated by the combination of the doxorubicin and the forsythin, the zebra fish heart toxicity damage model is induced and established, and as each organ of the zebra fish is developed, the toxicity influence of the doxorubicin on the heart function under normal conditions can be reflected more accurately, and the obtained result has better reference significance and reference value for the clinical study of the heart toxic and side effects of the doxorubicin.
The zebra fish heart toxicity model established by the invention only shows heart rate reduction, and the zebra fish of each treatment group does not have local or systemic edema and other abnormalities, so that the occurrence of possible nephrotoxicity damage caused by doxorubicin is eliminated, the model can be specifically used for the related research of doxorubicin heart toxicity, and the interference of other viscera function changes is eliminated.
The zebra fish heart toxicity injury model provided by the invention can be used for screening drugs with the activity of preventing/treating heart toxicity injury caused by doxorubicin.
Claims (16)
1. A composition for establishing a zebra fish heart toxicity injury model, which is characterized by comprising doxorubicin and forsythin;
the concentration of the components in the composition is 20-40 mug/mL of doxorubicin and 200-700 mug/mL of forsythin;
zebra fish used for establishing the zebra fish heart toxicity injury model is zebra fish with more than 3 dpf.
2. The composition of claim 1, wherein the concentration of the components in the composition is 30 μg/mL of doxorubicin and 200-700 μg/mL of forsythin.
3. A composition according to claim 1, wherein the zebra fish used to model the cardiac toxicity injury of the zebra fish is greater than 3dpf and less than 6 dpf.
4. The composition of claim 1, wherein the zebra fish used to model the heart toxicity injury of the zebra fish is wild-type zebra fish of the AB system.
5. The application of the doxorubicin and the forsythin in the establishment of the zebra fish heart toxicity injury model is characterized in that the concentration of the doxorubicin is 20-40 mug/mL, and the concentration of the forsythin is 200-700 mug/mL; zebra fish used to model the cardiac toxicity damage of zebra fish are those above 3dpf and less than 6 dpf.
6. The use according to claim 5, wherein the concentration of doxorubicin is 30 μg/mL and the concentration of forsythin is 200-700 μg/mL.
7. The use according to claim 5, wherein the zebra fish used to model the heart toxicity damage of zebra fish is wild type zebra fish of the AB line.
8. The method for establishing the zebra fish heart toxicity injury model is characterized by comprising the following steps of:
randomly grouping zebra fish to form a model group and a normal control group respectively, wherein the model group is added into a culture water solution containing doxorubicin and forsythin, the normal control group is added into a culture water solution without doxorubicin and forsythin, the model group and the normal control group are cultivated for 20-28h, the heartbeat condition of each fish is observed, and the heart rate is counted, so that the heart rate is remarkably reduced compared with the normal control group, and the zebra fish cardiotoxicity injury model is successfully established, otherwise, the zebra fish cardiotoxicity injury model is unsuccessful;
the zebra fish is more than 3dpf and less than 6 dpf;
in the culture water solution containing doxorubicin and forsythin, the concentration of doxorubicin is 20-40 mug/mL and the concentration of forsythin is 200-700 mug/mL.
9. The method of claim 8, wherein the zebra fish is an AB wild type zebra fish.
10. The method according to claim 8, wherein in the method, the concentration of doxorubicin in the aqueous culture solution containing doxorubicin and forsythin is 30 μg/mL and the concentration of forsythin is 200-700 μg/mL.
11. The method according to claim 8, wherein the aqueous culture solution contains: containing 5mM NaCl,0.17mM KCl,0.33mM CaCl 2 、0.33 mM MgSO 4 The pH of the aqueous culture solution was 7.2.
12. The method of claim 8, wherein the number of zebra fish per set is 15-30.
13. The method according to claim 8, wherein the culturing conditions are 27-28℃and the light/dark period is 14h/10h.
14. The method of claim 8, wherein the statistical basis for significant heart rate reduction is a separate sample t-test.
15. Use of a model of cardiotoxic injury in zebra fish established by the method of any one of claims 8-14 for screening for drugs having cardiotoxic injury activity caused by doxorubicin.
16. A method for screening a drug having cardiotoxic injury activity caused by doxorubicin using the zebra fish cardiotoxic injury model established by the method of any one of claims 8-14, comprising the steps of:
the method comprises the steps of randomly grouping zebra fish into a normal control group, a model group and a treatment group respectively, wherein the normal control group is added into a culture water solution without doxorubicin and forsythin, the model group is added into a culture water solution with doxorubicin and forsythin, the treatment group is added into a culture water solution with a sample to be tested in advance for pretreatment for 2-5h, then doxorubicin and forsythin are added to the required concentration, or the treatment group is added into a culture water solution with doxorubicin, forsythin and the sample to be tested, the normal control group, the model group and the treatment group are cultivated for 20-28h, the heartbeat condition of each fish is observed, and heart rate is counted, and compared with the model group, the heart rate is obviously increased, so that the heart toxicity damage of the zebra fish is relieved, and the drug to be tested has the activity of preventing/treating the heart toxicity damage caused by the doxorubicin, or no activity is obtained.
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