Construction method and application of adriamycin-induced zebrafish osteoporosis-like model
By inducing osteoporosis in zebrafish embryos using doxorubicin, and combining staining and molecular detection, the limitations of rodent models were overcome, enabling a rapid and visualized platform for osteoporosis research and drug screening, suitable for research and drug evaluation of chemotherapy-related osteoporosis.
Patent Information
- Application Number
- CN202511838558.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-08
- Publication Date
- 2026-01-16
AI Technical Summary
Existing rodent models for constructing chemotherapy-related osteoporosis (CIOP) models suffer from problems such as long modeling cycles, low evaluation efficiency, significant differences in drug metabolism compared to humans, and difficulty in non-invasive real-time observation of early microstructural changes, which limit the effectiveness of CIOP pathogenesis research and drug screening.
Taking advantage of the transparency and rapid bone development of zebrafish embryos and juveniles, a visual zebrafish osteoporosis-like model was constructed by culturing 2-day-farming (dpf) zebrafish embryos to 6-day-farming (dpf) in 10-30 μM doxorubicin aqueous solution, combined with alizarin red staining, Alcian blue staining, and qPCR detection.
It enables real-time observation of visualized bone development changes, with a short model construction cycle, simple operation, and high reproducibility. It is suitable for studying the pathogenesis of chemotherapy-related osteoporosis and screening and evaluating the efficacy of anti-osteoporosis drugs.
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Figure CN121336743A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal model construction technology, and in particular to a method for constructing an doxorubicin-induced zebrafish osteoporosis-like model and its application. Background Technology
[0002] Osteoporosis (OP) is a global metabolic bone disease characterized by decreased bone mass, destruction of bone microstructure, and increased bone fragility, which easily leads to fractures and seriously threatens the health of the population. In addition to traditional risk factors (such as menopause, aging, endocrine disorders, and nutritional deficiencies), cancer patients are also prone to developing chemotherapy-induced osteoporosis (CIOP). CIOP not only has a high incidence rate, but also often lacks obvious symptoms in the early stages, allowing patients to still suffer fractures during treatment or even months or years after treatment, thus significantly increasing mortality and disability rates. Therefore, establishing suitable animal models of CIOP is crucial for revealing the pathogenesis, evaluating drug efficacy, and testing prevention strategies. Currently, rodents (such as mice and rats) are commonly used animal models of osteoporosis, but they have limitations: (1) the modeling period is long (weeks to months); (2) bone structure assessment relies on expensive micro-CT or tissue sections, which is inefficient; (3) drug metabolism, bone remodeling speed, and immune response differ significantly from those in humans, limiting clinical translation. Furthermore, early microstructural changes in CIOP are difficult to observe non-invasively and in real-time in rodent models. The lack of efficient and visualized animal models limits research into the pathogenesis of CIOP and the screening of potential drugs.
[0003] Zebrafish (Daniorerio) possess unique advantages in bone metabolism research due to their high genetic homology with humans (approximately 70%), transparent embryos and larvae, small size, ease of rearing, and rapid skeletal development. Especially during the 4-7 dpf (dayspost fertilization) larval stage, intramembranous and endochondral ossification processes in the vertebrae and skull can be directly assessed for bone density and mineralization using panoramic microscopy. However, a systematic CIOP model based on chemotherapy drugs has not yet been established. Therefore, there is an urgent need for a novel CIOP animal model with a short modeling cycle, high reproducibility, and direct visualization of bone structural changes. Summary of the Invention
[0004] Therefore, based on the above background, this invention utilizes the transparency and rapid bone development characteristics of zebrafish embryos and juveniles to construct an doxorubicin-induced chemotherapy-associated osteoporosis (CIOP) model. This zebrafish osteoporosis-like model constructed based on chemotherapy drugs not only provides a novel experimental platform for studying the pathogenesis of CIOP, but also provides a reliable basis for the early screening, efficacy evaluation, and clinical translation of anti-osteoporosis drugs.
[0005] The technical solution provided by this invention is as follows:
[0006] A method for constructing an doxorubicin-induced osteoporosis-like model in zebrafish includes the following steps:
[0007] 1) Zebrafish embryos were cultured in a 10-30 μM solution of doxorubicin until 6 days postpartum (dpf);
[0008] 2) The zebrafish fry cultivated in step 1) were stained with alizarin red, Alcian blue and detected by qPCR.
[0009] Preferably, the concentration of the doxorubicin aqueous solution in step 1) is 20 μM.
[0010] Preferably, the zebrafish embryo in step 1) is a 2dpf zebrafish embryo.
[0011] Based on the same inventive concept, this invention provides the application of the zebrafish osteoporosis-like model constructed by the above-mentioned method for constructing an doxorubicin-induced zebrafish osteoporosis-like model in any of the following, characterized in that the application includes:
[0012] ① Application in studying the pathogenesis of chemotherapy-related osteoporosis;
[0013] ② Application in screening anti-osteoporosis drugs or anti-osteoporosis drug dosage;
[0014] ③ Application in evaluating the efficacy of drugs for combating osteoporosis.
[0015] Preferably, the osteoporosis drug targets osteoporosis caused by chemotherapy drugs.
[0016] Based on the same inventive concept, this invention provides a method for screening or evaluating the efficacy of anti-osteoporosis drugs, comprising the following steps:
[0017] ① Zebrafish embryos were cultured in an aqueous solution containing 10-30 μM doxorubicin and an anti-osteoporosis drug until 6 days post-fertilization (dpf);
[0018] ② The zebrafish fry cultivated in step ① were stained with alizarin red, Alcian blue and detected by qPCR.
[0019] Preferably, the zebrafish embryo in step ① is a 2dpf zebrafish embryo.
[0020] Preferably, the concentration of doxorubicin in step ① is 20 μM.
[0021] Preferably, the anti-osteoporosis drug is alendronate sodium.
[0022] Preferably, the concentration of the anti-osteoporosis drug in step ① is 10 mg / ml.
[0023] The beneficial effects achieved by this invention are as follows:
[0024] This invention utilizes the transparency and rapid bone development characteristics of zebrafish embryos and juveniles. By selecting 2dpf zebrafish embryos and culturing them to 6dpf in a 20μM doxorubicin aqueous solution, a visual zebrafish osteoporosis-like model is efficiently constructed. The juvenile fish are transparent and the imaging is intuitive, allowing for real-time observation of bone development changes in vivo, thus realizing a visual assessment of skeletal development damage.
[0025] The zebrafish osteoporosis-like model constructed in this invention not only inhibits the development of bone in juvenile zebrafish, but also interferes with cartilage development by inhibiting the expression of key genes, further affecting the normal formation of bone tissue. It can clearly demonstrate the systemic inhibitory effect of chemotherapy drugs on bone formation and mineralization. Moreover, the zebrafish osteoporosis-like model of this invention has the characteristics of short construction cycle, simple operation, high reproducibility and drug sensitivity, and can be used for rapid dose screening and intervention experiments. It has great application potential in the study of the pathogenesis of CIOP and the screening and efficacy evaluation of anti-osteoporosis drugs. Attached Figure Description
[0026] Appendix Figure 1 This invention illustrates the effect of different concentrations of doxorubicin on the mortality rate of zebrafish embryos. Zebrafish embryos were treated with different concentrations of doxorubicin, and their mortality rate was recorded 3 days post-fertilization (3 dpf). The results showed that the embryo mortality rate increased in a dose-dependent manner with increasing DOX concentration (n=50).
[0027] Appendix Figure 2 The effect of doxorubicin on zebrafish skeletal development, as illustrated in this embodiment of the invention: Figure 2 A is a schematic diagram of the experimental procedure for treating zebrafish juveniles with doxorubicin (DOX); Figure 2 B shows the results of alizarin red staining of the head of the control group (Ctr) and the DOX group (ventral view). The arrows point to the Mack cartilage (mk), notochord (nc), ceratohyoid bone (ch), cleithrum (cl), occipital bone (oc), and branchial ray bone (bs). Figure 2 C is Figure 2Statistical analysis results of head bone density of juvenile fish in group B (n=10, ***P<0.001); Figure 2 D shows the Alcian blue staining results (ventral view) of the head of the control group (Ctr) and the DOX group. The arrows point to the McCormick cartilage (mk), parasphenoid bone (ps), quadratus palatine bone (pq), hornyohyoid bone (ch), hyoid mandibular arch (hm), operculum (op), and horny parotid bone (cbr). Figure 2 E is Figure 2 Statistical analysis results of head cartilage area of juvenile fish in group D (n=10, ***P<0.001); Figure 2 Expression changes of bone formation-related genes bmp2b, sp7 and bglap in juvenile fish in the control group (Ctr) and DOX group when F is 6 dpf (n=6, *P<0.05, **P<0.01, ***P<0.001). The scale bars in the figure are all 200 μM.
[0028] Appendix Figure 3 The repair effect of sodium alendronate on doxorubicin-induced bone development damage in zebrafish, as described in this embodiment of the invention: Figure 3 A is a schematic diagram of the experimental procedure for treating zebrafish juveniles with doxorubicin (DOX) and intervening with alendronate sodium (AL); Figure 3 B shows the results of alizarin red staining on the heads of juvenile fish in the control group (Ctr), DOX group, and AL group (ventral view); Figure 3 C is Figure 3 Statistical analysis results of head bone density of juvenile fish in group B (n=10, ***P<0.001); Figure 3 D shows the Alcian blue staining results (ventral view) of the heads of juvenile fish in the control group, DOX group, and AL group; Figure 3 E is Figure 3 Statistical analysis results of head cartilage area of juvenile fish in group D (n=10, ***P<0.001); Figure 3 F shows the lateral fluorescence imaging results of the head of juvenile fish in the control group, DOX group, and AL group (Tg(osx:mCherry)). The arrows point to the cleithrum (cl), McEl cartilage (mk), and mandibular arch (ma); G: Quantitative results of fluorescence intensity in Figure F (n=10, ***P<0.001). Figure 3 Expression changes of bone formation-related genes bmp2b, sp7 and bglap in juvenile fish of control group, DOX group and AL group when H is 6 dpf (n=6; *P<0.05,**P<0.01,***P<0.001), scale bar in figure is 200μM. Detailed Implementation
[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0030] (1) Laboratory animals
[0031] The experiment used wild-type AB zebrafish and Tg(osx:mCherry) transgenic zebrafish.
[0032] Zebrafish were cultured at 28.5℃ with a photoperiod of 14 hours light / 10 hours dark. All experiments complied with relevant animal protection and welfare regulations.
[0033] (2) Drug treatment
[0034] First, let's explore the effects of different concentrations of doxorubicin on zebrafish.
[0035] In the toxicity analysis of doxorubicin (Sangon Biotech, A603456), wild-type embryos at 0.75 hpf were exposed to 5, 10, 20, 30, and 40 μM doxorubicin aqueous solutions, with three replicates per group. Embryo mortality was calculated at 3 dpf. Results are as follows: Figure 1 As shown, DOX treatment increased embryo mortality in a concentration-dependent manner, with the mortality rate exceeding 50% in the 30 μM group and approximately 20% in the 20 μM group, and no significant cardiotoxicity was observed.
[0036] Therefore, this invention uses a 20 μM doxorubicin aqueous solution to culture 2 dpf embryos to construct a zebrafish osteoporosis-like model.
[0037] Embryos at 2dpf were treated with 20 μM doxorubicin, and juvenile fish were collected at 6dpf for alizarin red staining, alcian blue staining, and qPCR detection.
[0038] To verify the effectiveness of the doxorubicin-induced osteoporosis-like model, embryos at 2 days post-exposure (dpf) were treated with an aqueous solution containing doxorubicin (20 μM) and alendronate sodium (Fuyuan, H20059029) at 10 mg / ml. The repair effect of alendronate sodium on doxorubicin-induced bone development damage was then examined at 6 days post-exposure.
[0039] (3) Alizarin Red staining
[0040] Juvenile fish (6 dpf) were collected, cleaned to remove drug residues, and fixed overnight in 4% paraformaldehyde at 4°C. After fixation, they were washed three times with PBST (5 min each time). They were then bleached with 0.5% KOH / 3% H2O2 solution to remove melanin and yellow pigment, and washed three more times with PBST. After dehydration, they were sequentially passed through a methanol gradient (25%, 50%, 100%) and stored in 20% DMSO / 80% methanol. After rehydration to PBST, alizarin red staining solution (Solarbio, G1450) was added, and staining was performed at room temperature in the dark for 1 h. After staining, they were washed three times with PBST (5 min each time) and finally stored in 80% glycerol in the dark for microscopic observation and photography.
[0041] (4) Alcian blue staining
[0042] Juvenile fish (6 dpf) were collected and fixed overnight in 4% paraformaldehyde at 4°C. They were washed twice with 95% ethanol (5 min each time), followed by staining overnight with Alcian blue (Savillex, G1027). After staining, the fish were destained sequentially with a gradient of ethanol (60%, 50%, 30%) and washed twice with PBST. Treatment with 0.05% trypsin dissolved in saturated sodium tetraborate for 1-3 h softened the epidermis, making the blue skeleton clearly visible. The fish were then bleached with 1% KOH / 3% H₂O₂ solution for 30 min, followed by elution with a gradient of 1% KOH and glycerol in ratios (3:1, 1:1, 1:3) until the skeleton was clearly visible. The final samples were stored in 70% glycerol for microscopic observation and photography.
[0043] (5) RNA extraction and qPCR analysis
[0044] After collecting zebrafish juvenile samples, the samples were homogenized, and total RNA was extracted and purified using RNA-easy Isolation Reagent (Vazyme, R701). Subsequently, [the following was used]... III RT SuperMix (Vazyme, R323) was used for reverse transcription to synthesize cDNA. qPCR was performed using... qPCR was performed using SYBR Green Master Mix (Vazyme, R701). The expression of osteogenic-related genes (sp7, bglap) and bone-inducing signaling-related genes (bmp2b) was analyzed, and 2... -ΔΔCt The method calculates the relative expression level, using eef1a1l1 as an internal reference.
[0045] (6) Statistical analysis
[0046] All statistical graphs of the experimental results in this paper were generated using GraphPad software. Bar and scatter plots represent experimental data as mean ± standard deviation (mean ± SD), and statistical analysis was performed using GraphPad software. Between-group comparisons were performed using Student's t-test or one-way ANOVA, depending on the experimental design. The criteria for statistical significance were: *P < 0.05, **P < 0.01, ***P < 0.001.
[0047] result:
[0048] (1) To investigate the effect of doxorubicin on bone development, 2-day-fed zebrafish juveniles were treated with DOX, and samples were collected at 6 days post-fed for analysis using alizarin red and alcine blue staining. Figure 2 A). Alizarin red staining results showed that the mineralization of the head bones in zebrafish treated with DOX was significantly reduced, specifically manifested in a significant decrease in the staining intensity of bone structures such as Macole cartilage (MK), notochord (NC), cerulohyoid bone (CH), cleithrum (CL), occipital bone (OC), and branchial rays (BS). Figure 2 BC staining suggests that DOX significantly inhibits bone development. Regarding cartilage, Alcian blue staining results show that DOX significantly reduces the stained area in the cartilaginous regions of the zebrafish head, particularly in the areas of the Macole cartilage (MK), parasphenoid bone (PS), quadratus palatinatus (PQ), cerhyoid bone (CH), hyoid mandibular arch (HM), operculum (OP), and cerebral cartilage (CBR). Figure 2 DE indicates that it interferes with cartilage development.
[0049] To further explore the molecular mechanism of DOX on bone development, qPCR results showed that DOX treatment significantly downregulated the expression of bone formation-related genes bmp2b, sp7, and bglap. Figure 2 F). This result indicates that DOX not only significantly inhibits bone development in juvenile zebrafish, but may also interfere with cartilage development by inhibiting the expression of key genes, thereby further affecting the normal formation of bone tissue.
[0050] (2) Alendronate sodium can improve doxorubicin-induced bone development defects.
[0051] To verify the applicability of the DOX-induced zebrafish bone development injury model of this invention to drug screening, the following experiment selected alendronate sodium (AL) as a candidate drug for intervention. Figure 3A). The responsiveness of this model in drug screening was evaluated by comparing the bone development damage in juvenile fish after DOX treatment with the improvement after AL (10 mg / ml) intervention. Alizarin red staining results showed that AL could significantly improve the decrease in ossification induced by DOX, especially in the head bone tissue, manifested as a recovery of mineralization. Figure 3 BC). Furthermore, Alcian blue staining showed that AL treatment effectively alleviated DOX-induced cartilage area reduction, indicating that the drug has a protective effect on cartilage development. Figure 3 DE). To further verify the repair effect of AL on bone developmental damage, the experiment also used Tg(osx:mCherry) transgenic zebrafish to observe osteoblast differentiation. The results showed that DOX treatment significantly inhibited the fluorescence signal of osteoblasts, while AL intervention significantly restored the fluorescence intensity, suggesting that osteoblast differentiation was improved. Figure 3 FG). Furthermore, qPCR analysis showed that AL significantly upregulated the expression of bone formation-related genes bmp2b, sp7, and bglap, which were downregulated after DOX treatment. Figure 3 (H) This further supports the role of AL in repairing bone developmental damage at the molecular level. Intervention experiments using alendronate sodium showed that the drug significantly improved doxorubicin-induced bone developmental defects, increased ossification and cartilage area, and restored the expression of osteogenic genes, validating the model's sensitivity to anti-osteoporosis drugs. This indicates that this model can not only simulate chemotherapy-related osteoporosis but also serve as a platform for early drug screening and efficacy evaluation, providing feasible experimental evidence for the development of chemotherapy-protective drugs against bone damage.
[0052] In summary, the zebrafish osteoporosis-like model constructed based on DOX in this invention can not only simulate the changes in chemotherapy-related osteoporosis, but also shows a good response to anti-osteoporosis drugs such as AL. This model provides a powerful platform for drug screening, evaluation of bone developmental toxicity, and research on the mechanisms of chemotherapy-related bone injury, and is particularly suitable for early bone development intervention research.
[0053] The doxorubicin-induced bone development and injury model in zebrafish constructed in this study is simple to operate, highly responsive, and reproducible. It is the first time that the bone toxicity of chemotherapy drugs and their intervention effects have been systematically presented in non-mammalian vertebrates, providing a reliable platform for mechanism research and drug screening.
[0054] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.
Claims
1. A method for constructing a doxorubicin-induced zebrafish osteoporosis-like model, characterized by, The method comprises the following steps: 1) adding zebra fish embryos into an aqueous doxorubicin solution with a concentration of 10-30 μM to cultivate to 6 dpf; 2) using alizarin red staining, al new blue staining and qPCR detection on the zebra fish seedlings cultivated in step 1).
2. The method of claim 1, wherein the doxorubicin-induced zebrafish osteoporosis-like model is constructed by administering doxorubicin to the zebrafish at a concentration of 0.5 mg / L for 3 days. The concentration of the aqueous doxorubicin solution in step 1) is 20 μM.
3. The method of claim 1, wherein the doxorubicin-induced zebrafish osteoporosis-like model is constructed by administering doxorubicin to the zebrafish at a concentration of 0.5 mg / L for 3 days. The zebra fish embryos in step 1) are 2 dpf zebra fish embryos.
4. Use of a zebrafish osteoporosis-like model constructed by the method of any one of claims 1 to 3 in any one of the following, characterized in that, The application comprises: ① application in researching the pathogenesis of chemotherapy-related osteoporosis; ② application in screening anti-osteoporosis drugs or anti-osteoporosis drug doses; ③ application in evaluating the curative effect of anti-osteoporosis drugs.
5. The use according to claim 1, characterized in that, The disease targeted by the anti-osteoporosis drug is chemotherapy drug-induced osteoporosis.
6. A method for screening or evaluating the efficacy of an anti-osteoporosis drug, characterized by, The method comprises the following steps: ① adding zebra fish embryos into an aqueous solution containing doxorubicin and an anti-osteoporosis drug with a mass concentration of 10-30 μM to cultivate to 6 dpf; ② using alizarin red staining, al new blue staining and qPCR detection on the zebra fish seedlings cultivated in step ①.
7. The method for screening or evaluating therapeutic effect of anti-osteoporosis drug according to claim 6, wherein, The zebra fish embryos in step ① are 2 dpf zebra fish embryos.
8. The method for screening or evaluating therapeutic effect of anti-osteoporosis drug according to claim 6, wherein the bone mineral density is measured by dual energy X-ray absorptiometry. The concentration of the doxorubicin in step ① is 20 μM.
9. The method for screening or evaluating the therapeutic effect of an anti-osteoporosis drug according to claim 8, wherein the bone mineral density is measured by the method of measuring the bone mineral density of the lumbar vertebrae. The anti-osteoporosis drug is alendronate sodium.
10. The method for screening or evaluating the therapeutic effect of an anti-osteoporosis drug according to claim 9, wherein the bone mineral density is measured by the method of measuring the bone mineral density according to claim 8. The concentration of the anti-osteoporosis drug in step ① is 10 mg / ml.