A method for establishing a zebrafish comorbidity model using aluminum ion induction and its application in the medical field.

Abstract

This invention discloses a method for establishing a zebrafish comorbidity model using aluminum ion induction. Using 3dpf zebrafish as the model, aluminum trichloride solution was administered for 4 days to obtain a zebrafish model of Alzheimer's disease combined with osteoporosis. This invention also discloses the use of active ingredients in the preparation of drugs for treating Alzheimer's disease combined with osteoporosis. The active ingredients are combinations of betaine, dermal resin alcohol, osthol, and notopterygium alcohol, and combinations of osthol and ginsenoside Rb3. Compared with the model group, the combinations of betaine, dermal resin alcohol, osthol, and notopterygium alcohol, and the combinations of osthol and ginsenoside Rb3 showed significantly increased alizarin red staining area, cumulative light intensity, behavioral mean velocity, velocity change, and alkaline phosphatase activity, and significantly decreased acetylcholinesterase activity.

Description

Technical Field

[0001] This invention belongs to the field of medicine and relates to a method using aluminum ions (Al) 3+ The invention relates to a method for inducing a zebrafish comorbidity model, particularly a method for inducing a zebrafish model of Alzheimer's disease combined with osteoporosis using aluminum ions, and a method for screening active drugs for treating Alzheimer's disease combined with osteoporosis based on the zebrafish comorbidity model. Background Technology

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease characterized by senile plaques formed by excessive deposition of Aβ amyloid protein, neurofibrillary tangles formed by hyperphosphorylation of microtubule-associated protein Tau, and neuronal apoptosis. Osteoporosis (OP) is a systemic metabolic skeletal disease characterized by decreased bone mass, reduced bone density, and increased bone fragility. The incidence of AD and OP is increasing year by year, seriously affecting the quality of life and physical health of middle-aged and elderly people. Traditional Chinese medicine describes "bone atrophy" as similar to OP. The main pathogenesis of bone atrophy is "kidney deficiency and marrow reduction," and "the kidney meridian produces marrow, which connects to the brain." The brain is the master of all marrow, thus indicating a close connection between AD and OP.

[0003] Common zebrafish models of Alzheimer's disease include: intraventricular injection of Aβ protein, APPsw transgenic zebrafish AD model, and chemically induced AD models (metal ions, okadaic acid, etc.). Common drugs for modeling osteoporosis include glucocorticoids, ferric ammonium citrate, and phosphorus-deficient diets.

[0004] Zebrafish are bony fish whose skeletons, like those of mammals, originate from three embryonic stem cell lines. Skeletal formation occurs through intramembranous and endochondral ossification, a process regulated by signaling pathways such as Wnt / β-catenin and related genes like runx2 and osterix. The zebrafish genome shares high similarity with the human genome. Zebrafish offer advantages such as high reproductive capacity, transparent and easily observable larvae, short experimental cycles, and low cost. As a model organism, zebrafish are widely used in genetics and developmental biology research. With the continuous improvement of evaluation indicators for zebrafish skeletal models, zebrafish have been widely applied in research on skeletal-related diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a method using aluminum ions (Al) 3+ Methods for inducing zebrafish comorbidity models and screening potential drugs and combinations for anti-AD combined with OP.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for establishing a zebrafish comorbidity model using aluminum ion induction was proposed. 3dpf zebrafish were treated with aluminum trichloride solution for 4 days to obtain a zebrafish model of Alzheimer's disease combined with osteoporosis.

[0008] Specifically, a method for establishing a zebrafish comorbidity model using aluminum ion induction includes the following steps:

[0009] Step (1): Zebrafish embryos were cultured in embryo culture medium to obtain 3dpf zebrafish;

[0010] Step (2): Prepare aluminum trichloride solution using embryo culture medium. Culture 3dpf zebrafish juveniles in aluminum trichloride solution for 4 days, and change half of the solution every 12 hours to obtain a zebrafish model of Alzheimer's disease combined with osteoporosis.

[0011] In step (1), the embryo culture medium is: 16.5 mL of LE3 culture medium is added to every 1 L of pure water; every 1 LE3 culture medium contains 17532 mg NaCl, 769.49 mg KCl, 2197.80 mg CaCl2 and 2383.30 mg MgSO4.

[0012] In step (2), the inventors previously administered aluminum trichloride solutions at concentrations of 75, 150, and 300 μM. Based on alizarin red staining (skull mineralization area and cumulative optical density (IOD)), alkaline phosphatase (ALP) activity, and acetylcholinesterase (AChE) activity, it was found that compared with the blank control group, treatment with 150 and 300 μM aluminum trichloride solutions significantly reduced the skull mineralization area and IOD, significantly decreased ALP activity, and significantly increased AChE activity. Considering the condition of the zebrafish juveniles, 150–300 μM aluminum trichloride, preferably 150 μM, was selected as the modeling agent. After four days of aluminum trichloride treatment, the skeletal development of the zebrafish skull was relatively complete, and the modeling effect was the most obvious and stable.

[0013] Therefore, the concentration of AlCl3 in the aluminum trichloride solution is 150–300 μM, and the pH is 5.8.

[0014] Preferably, the concentration of AlCl3 in the aluminum trichloride solution is 150 μM and the pH is 5.8.

[0015] Specifically, the aluminum trichloride solution is prepared using embryo culture medium and the pH is adjusted to 5.8 using dilute hydrochloric acid.

[0016] Excessive aluminum accumulation in zebrafish can induce memory decline, neuronal apoptosis, and oxidative stress. It also inhibits osteoblast activity and differentiation, causes calcium and phosphorus metabolism disorders, and exacerbates oxidative stress. This invention uses aluminum trichloride as a co-inducer of acute osteoarthritis (AD) and osteoproliferative disorders (OP) in zebrafish to induce dual diseases, resulting in simultaneous AD and OP-like responses, thus establishing a dual-disease model in zebrafish. This invention, using aluminum trichloride to induce AD ​​combined with OP in juvenile zebrafish, has the advantages of a short experimental cycle, low cost, and the ability to induce both AD and OP simultaneously.

[0017] The inventors collected 7-day-fed zebrafish and performed alizarin red staining, alkaline phosphatase (ALP) activity measurement, behavioral analysis, and acetylcholinesterase (AChE) activity measurement. Compared with the blank control group, the AlCl3-induced zebrafish showed significantly reduced skull mineralization area and density, and significantly decreased ALP activity, indicating the successful establishment of the AlCl3-induced osteoporosis model. Compared with the blank control group, the AlCl3-induced zebrafish showed significantly improved movement and significantly increased AChE activity, indicating the successful establishment of the AlCl3-induced Alzheimer's disease model. This demonstrates that the present invention successfully induced simultaneous Alzheimer's disease (AD) and osteoporosis (OP) in juvenile zebrafish using AlCl3, suggesting the feasibility of using this animal model to screen for dual-effect drugs for the simultaneous treatment of Alzheimer's disease and osteoporosis, and providing an excellent animal model for further elucidating the intrinsic link between Alzheimer's disease and osteoporosis.

[0018] Another object of the present invention is to provide a method for screening active ingredients for treating Alzheimer's disease comorbid with osteoporosis based on an established zebrafish comorbidity model, comprising:

[0019] Step (1): Zebrafish embryos were cultured in embryo culture medium to obtain 3dpf zebrafish;

[0020] Step (2): Set up a blank group, a model group and a drug treatment group respectively. The blank group was treated with 0.1% DMSO solution prepared with embryo culture medium for 4 days. The model group was treated with aluminum trichloride solution prepared with embryo culture medium for 4 days. The drug treatment group was treated with a mixed solution of aluminum trichloride prepared with embryo culture medium and the active ingredient to be screened for 4 days.

[0021] Step (3): Collect 7-day-old zebrafish from the blank group, model group, and drug-treated group, and perform alizarin red staining, alkaline phosphatase activity measurement, behavioral analysis, and acetylcholinesterase activity measurement, respectively. Compare the above four indicators between the model group and the blank group. The alizarin red staining area, cumulative light density, average speed, speed change, and alkaline phosphatase activity of the model group are significantly reduced, while the acetylcholinesterase activity is significantly increased, indicating that the AD combined with OP zebrafish model has been successfully established. Compare the above four indicators between the drug-treated group and the model group. If the alizarin red staining area, cumulative light density, average speed, speed change, and alkaline phosphatase activity of the drug-treated group are significantly increased, while the acetylcholinesterase activity is significantly reduced, it indicates that the active ingredient has the potential to treat Alzheimer's disease combined with osteoporosis.

[0022] Based on the established zebrafish comorbidity model, the inventors screened out the osthol-notopterygium alcohol combination, the osthol-ginsenoside Rb3 combination, betaine, and dermal resin alcohol, which have potential for treating Alzheimer's disease comorbid with osteoporosis.

[0023] Another object of the present invention is to provide the use of an active ingredient in the preparation of a medicament for treating Alzheimer's disease comorbid with osteoporosis, wherein the active ingredient is a combination of betaine, dermal resin alcohol, osthol and gentianol, or a combination of osthol and ginsenoside Rb3.

[0024] In the combination of osthol and notopterygium alcohol, the molar ratio of osthol to notopterygium alcohol is 1:1.5 to 1:9, preferably 1:1.5 to 1:2.5, and more preferably 1:1.5 or 3:7.

[0025] In the combination of osthol and ginsenoside Rb3, the molar ratio of osthol to ginsenoside Rb3 is 16:100.

[0026] Another object of the present invention is to provide a medicament for treating Alzheimer's disease complicated with osteoporosis, characterized in that: the active ingredients are a combination of betaine, corticosteroids, osthol, and notopterygium alcohol, or a combination of osthol and ginsenoside Rb3, and are formulated into a pharmaceutically acceptable formulation with pharmaceutically acceptable excipients. Detailed Implementation

[0027] The technical solution of the present invention will be further described below through specific embodiments.

[0028] Embryo culture medium: 16.5 mL of LE3 culture medium is added to 1 L of pure water to prepare the embryo culture medium; each 1 L of LE3 culture medium contains 17532 mg NaCl, 769.49 mg KCl, 2197.80 mg CaCl2 and 2383.30 mg MgSO4.

[0029] 1 Experimental Methods 1.1 Collection of Zebrafish Embryos

[0030] The night before egg collection, place the fish in a breeding tank (approximately 2 / 3 full of water) at a 1:1 ratio of female to male. Separate the males and females with a partition, keeping them in darkness. The next morning, remove the partition and allow the males and females to mate freely under light. Observe the spawning time. Two hours after spawning, collect the eggs in a petri dish, wash them twice with embryo culture medium, and incubate them in a 28°C light incubator for later use. Remove any white, dead eggs daily. If there are too many dead eggs, change the culture medium, replacing half of it.

[0031] 1.2 Experimental Grouping

[0032] Select zebrafish juveniles that are 3 days post-fertilization and in good condition (i.e., 3dpf zebrafish) and transfer them to a 6-well plate, with 30 fish per well and 1 well per group. Randomized groups were formed: Ctrl group (0.1% DMSO), low-concentration AlCl3 group (75 μM AlCl3, pH adjusted to 5.8 with dilute hydrochloric acid), medium-concentration AlCl3 group (150 μM AlCl3, pH adjusted to 5.8 with dilute hydrochloric acid), high-concentration AlCl3 group (300 μM AlCl3, pH adjusted to 5.8 with dilute hydrochloric acid), positive control group (150 μM AlCl3 + donepez hydrochloride, Dpz, 10 μM), and drug administration group (150 μM AlCl3 + different concentrations of drugs: *Qianghuo alcohol: osthol = 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 10:0, total drug concentration 20 μM; ginsenoside Rb3: osthol = 5:1; *Dermatitis resin alcohol 1.25, 2.5, 5, 10, 20 μM, denoted as 1.25). Med, 2.5 Med, 5 Med, 10 Med, 20 Med; betaine 2.5, 5, 10 μM, denoted as 2.5 Bet, 5 Bet, 10 Bet, respectively. All drugs were prepared from embryo culture medium, and half of the media in all treatment groups were changed every 12 h.

[0033] Table 1. Ratio of Notopterygium alcohol and Osthol

[0034]

[0035] Table 2. Ratio of Ginsenoside Rb3 and Osthol

[0036]

[0037] 1.3 Osteoporosis detection indicators

[0038] 1.3.1 Alizarin Red Staining

[0039] Four days after drug administration, zebrafish in each group were anesthetized with 0.02% tricaine and fixed with 4% paraformaldehyde for 2 hours. The zebrafish were then bleached with 3% H₂O₂ / 0.5% KOH for 30 minutes, and shaken until the fish were colorless. The zebrafish skulls were stained with 0.1% alizarin red for 30 minutes, the staining solution was removed, and the samples were permeated with 50% glycerol / 0.5% KOH for 30 minutes. The permeation solution was then replaced, and the samples were incubated overnight. Zebrafish samples were stored at 4°C in 50% glycerol. Finally, zebrafish larvae were placed and fixed on slides containing 50% glycerol, and stained images of the zebrafish in a prone position were captured using a stereomicroscope (Olympus ZX16). The alizarin red stained area (i.e., the mineralized area of ​​the skull) and cumulative optical density (IOD) were calculated using ImagePro Plus 6.0.

[0040] 1.3.2 Alkaline phosphatase activity assay

[0041] Collect 30 zebrafish juveniles per group into 1.5 mL Eppendorf tubes, aspirate as much of the reagent as possible, and weigh. Add pre-chilled physiological saline (4℃) at a weight-to-volume ratio of 1:9 (g / mL). Place the Eppendorf tubes on ice and homogenize using a tissue homogenizer to ensure complete tissue lysis. After complete tissue lysis, centrifuge at 2500 rpm for 10 min at 4℃ and collect the supernatant. Perform the remaining procedures according to the kit instructions to determine the zebrafish alkaline phosphatase (ALP) activity.

[0042] The results of Alizarin Red staining in different AlCl3 groups are shown in Table 3. Compared with the Ctrl group, there was no significant difference in the skull mineralization area and cumulative optical density (IOD) of zebrafish in the low-concentration AlCl3 group (75 μM AlCl3), while the skull mineralization area and cumulative optical density (IOD) of zebrafish in the medium-concentration AlCl3 group (150 μM AlCl3) and the high-concentration AlCl3 group (300 μM AlCl3) were significantly reduced (P<0.001).

[0043] Table 3. Effects of different AlCl3 concentrations on mineralized area and cumulative optical density in zebrafish

[0044]

[0045] The alkaline phosphatase activity results of different AlCl3 concentration groups are shown in Table 4. Compared with the Ctrl group, there was no significant difference in ALP activity in the low concentration AlCl3 group (75 μM AlCl3), while the ALP activity in the medium concentration AlCl3 group (150 μM AlCl3) and the high concentration AlCl3 group (300 μM AlCl3) was significantly reduced (P<0.001).

[0046] Table 4. Effects of different concentrations of AlCl3 on ALP activity in zebrafish

[0047]

[0048] Based on the results of alizarin red staining, alkaline phosphatase and acetylcholinesterase activities in different AlCl3 groups, as well as the condition of the zebrafish themselves, 150 μM aluminum trichloride was selected as the modeling agent for subsequent efficacy experiments. The concentration of AlCl3 in the AlCl3 model group was 150 μM.

[0049] The results of alizarin red staining in the osthol-notopterygium alcohol group are shown in Table 5. Compared with the AlCl3 model group, the mineralization area and mineralization density of the zebrafish skull in the treatment group were significantly increased (P<0.01, P<0.001), and QS7 was the drug ratio with the best efficacy, which was better than that of a single drug.

[0050] Table 5. Effects of osthol-notopterygol on mineralized area and cumulative optical density of zebrafish

[0051]

[0052] Note: Data are expressed as mean ± standard deviation (n = 8). Compared with the Ctrl group, ### P<0.001; compared with the AlCl3 model group, **P<0.01, ***P<0.001.

[0053] The results of alizarin red staining in the osthol-ginsenoside Rb3 treatment group are shown in Table 6. Compared with the AlCl3 model group, the mineralization area and mineralization density of the zebrafish skull in the treatment group were significantly increased (P<0.001), and the 100+16 group had the best efficacy, which was better than that of a single drug.

[0054] Table 6. Effects of osthol-ginsenoside Rb3 on mineralized area and cumulative optical density in zebrafish.

[0055]

[0056]

[0057] Note: Data are expressed as mean ± standard deviation (n = 8). Compared with the Ctrl group, ### P<0.001; compared with the AlCl3 model group, **P<0.01, ***P<0.001.

[0058] The results of alizarin red staining in the alizarin alcohol-treated group are shown in Table 7. Compared with the AlCl3 model group, the mineralization area and mineralization density of the zebrafish skull in the treated group were significantly increased.

[0059] Table 7. Effects of styrax resin alcohol on mineralized area and cumulative optical density of zebrafish

[0060]

[0061] Note: Data are expressed as mean ± standard deviation (n = 8). Compared with the Ctrl group, ### P<0.001; compared with the AlCl3 model group, *P<0.05, **P<0.01, ***P<0.001.

[0062] The alizarin red staining results of the betaine-treated group are shown in Table 8. Compared with the AlCl3 model group, the mineralization area and mineralization density of the zebrafish skull in the betaine-treated group were significantly increased.

[0063] Table 8. Effects of betaine on mineralized area and cumulative optical density in zebrafish

[0064]

[0065] Note: Data are expressed as mean ± standard deviation (n = 8). Compared with the Ctrl group, ### P<0.001; compared with the AlCl3 model group, **P<0.01, ***P<0.001.

[0066] The alkaline phosphatase activity results of the osthol-notopterygium alcohol treatment group are shown in Table 9. Compared with the AlCl3 model group, the ALP activity of zebrafish in the treatment group was significantly enhanced (P<0.05, P<0.01, P<0.001), and QS7 had the best efficacy, which was better than that of a single drug, consistent with the results of zebrafish alizarin red staining experiment.

[0067] Table 9. Effects of osthol-notopterygol on ALP activity in zebrafish.

[0068]

[0069] Note: Data are expressed as mean ± standard deviation (n=3). Comparison of the Ctrl group and the AlCl3 model group, ### P<0.001; Compared with the AlCl3 model group, *P<0.05, **P<0.01, ***P<0.001 in the drug-treated group.

[0070] The results of alkaline phosphatase activity assay in the osthol-ginsenoside Rb3 treatment group are shown in Table 10. Compared with the AlCl3 model group, the ALP activity in zebrafish in the treatment group was significantly enhanced, and the 100+16 group had the best efficacy, which was better than that of a single drug, consistent with the results of the zebrafish alizarin red staining experiment.

[0071] Table 10. Effects of osthol-ginsenoside Rb3 on ALP activity in zebrafish

[0072]

[0073]

[0074] Note: Data are expressed as mean ± standard deviation (n = 3). Compared with the AlCl3 model group, the Ctrl group... ### P<0.001; Compared with the AlCl3 model group, *P<0.05, **P<0.01, ***P<0.001 in the drug-treated group.

[0075] The results of alkaline phosphatase activity assay in the tannin-treated group are shown in Table 11. Compared with the AlCl3 model group, the ALP activity in the zebrafish treated with tannin-treated group was significantly enhanced.

[0076] Table 11. Effects of dermal resin alcohol on ALP activity in zebrafish

[0077]

[0078] Note: Data are expressed as mean ± standard deviation (n = 3). Compared with the AlCl3 model group, the Ctrl group... ### P<0.001; Compared with the AlCl3 model group, *P<0.05, **P<0.01.

[0079] The results of alkaline phosphatase activity assay in the betaine-treated group are shown in Table 12. Compared with the AlCl3 model group, the ALP activity in the zebrafish treated with betaine was significantly enhanced.

[0080] Table 12. Effects of betaine on ALP activity in zebrafish

[0081]

[0082] Note: Data are expressed as mean ± standard deviation (n = 3). Compared with the AlCl3 model group, the Ctrl group... ### P<0.001; Compared with the AlCl3 model group, *P<0.05, **P<0.01.

[0083] 1.4 Alzheimer's Disease Detection Indicators

[0084] 1.4.1 Behavioral Analysis

[0085] Seven-day-old (dpf) juvenile zebrafish were placed in 96-well plates after modeling / drug administration, with 200 μL of embryo culture medium added to each well. One fish was placed in each well, and the culture temperature was maintained at 28°C. All experiments lasted 60 min, including three light / dark cycles (10 min each). The zebrafish's movement distance D and velocity change ΔS were recorded using a viewpoint behavior analyzer for light-dark and dark-light cycles for further analysis. The kinetic recovery rate (DRR, %) and response efficiency (RE, %) were used as evaluation indicators for the kinetic disturbances in zebrafish after drug administration.

[0086] The behavioral analysis results of the osthol-notopterygium alcohol treatment group are shown in Table 13. The movement of zebrafish in both the positive control group (Dpz) and the treatment group was significantly improved. QS6 had the best efficacy, which was better than that of a single drug.

[0087] Table 13. Experimental results of the effects of osthol-notopterygol on DRR (%) and RE (%) in zebrafish.

[0088]

[0089] Note: Data are expressed as mean ± standard deviation (n = 10).

[0090] The behavioral analysis of the osthol-ginsenoside Rb3 treatment group is shown in Table 14. The movement of zebrafish in both the positive control group (Dpz) and the treatment group was significantly improved. The 100+16 group had the best efficacy, which was better than that of a single drug (Table 12).

[0091] Table 14. Effects of osthol-ginsenoside Rb3 on DRR (%) and RE (%) in zebrafish

[0092]

[0093]

[0094] Note: Data are expressed as mean ± standard deviation (n = 10).

[0095] The behavioral analysis results of the dermal resin alcohol treatment group are shown in Table 15. The movement of zebrafish in both the positive control group (Dpz) and the treatment group was significantly improved.

[0096] Table 15. Effects of dermal resin alcohol on DRR (%) and RE (%) in zebrafish

[0097]

[0098] Note: Data are expressed as mean ± standard deviation (n = 10).

[0099] The behavioral analysis results of the betaine-treated group are shown in Table 16. The movement of zebrafish in the positive control group (Dpz) and the treated group showed significant improvement.

[0100] Table 16 Effects of betaine on DRR (%) and RE (%) in zebrafish

[0101]

[0102]

[0103] Note: Data are expressed as mean ± standard deviation (n = 10).

[0104] 1.4.2 Acetylcholinesterase activity assay

[0105] Collect 30 zebrafish juveniles per group into 1.5 mL Eppendorf tubes, aspirate the drug solution as dry as possible, and weigh. Add pre-chilled physiological saline at a weight-to-volume ratio of zebrafish to pre-chilled physiological saline of 1:9 (g / mL). Place the Eppendorf tubes on ice and homogenize using a tissue homogenizer (pre-chilled) to ensure complete tissue lysis. After complete tissue lysis, centrifuge at 2500 rpm for 10 min at 4°C and collect the supernatant. Perform the remaining procedures according to the kit instructions to determine the zebrafish acetylcholinesterase (AChE) activity.

[0106] The results of acetylcholinesterase activity assays in different AlCl3 groups are shown in Table 17. Compared with the Ctrl group, there was no significant difference in AChE activity in the low-concentration AlCl3 group (75 μM AlCl3), while the AChE activity in the AlCl3 model group (150 μM AlCl3) and the high-concentration AlCl3 group (300 μM AlCl3) was significantly increased (P<0.001).

[0107] Table 17. Effects of different concentrations of AlCl3 on AChE activity

[0108]

[0109] The results of acetylcholinesterase activity assay in the osthol-notopterygium alcohol group are shown in Table 18. Compared with the AlCl3 model group, the AChE activity of zebrafish in both the positive control group (Dpz) and the drug administration group was significantly decreased (P<0.001). QS6 showed the best efficacy, which was better than that of a single drug, consistent with the results of the behavioral analysis of zebrafish juveniles.

[0110] Table 18. Effects of osthol-notopterygol on AChE activity in zebrafish

[0111]

[0112]

[0113] Note: Data are expressed as mean ± standard deviation (n = 3). Compared with the AlCl3 model group, the Ctrl group... ### P<0.001; Compared with the AlCl3 model group, the drug-treated group showed ***P<0.001.

[0114] The acetylcholinesterase activity of the osthol-ginsenoside Rb3 treatment group is shown in Table 19. Compared with the AlCl3 model group, the AChE activity of zebrafish in both the positive control group and the treatment group was significantly decreased (P<0.001). The 100+16 group had the best efficacy, which was better than that of a single drug, and was consistent with the results of the behavioral analysis of zebrafish juveniles (Table 16).

[0115] Table 19. Effects of osthol-ginsenoside Rb3 on AChE activity in zebrafish

[0116]

[0117] Note: Data are expressed as mean ± standard deviation (n = 3). Compared with the AlCl3 model group, the Ctrl group... ### P<0.001; Compared with the AlCl3 model group, the drug-treated group showed ***P<0.001.

[0118] The results of acetylcholinesterase activity assay in the tannin-treated group are shown in Table 20. Compared with the AlCl3 model group, the AChE activity of zebrafish in both the positive control group and the treated group was significantly decreased (P<0.001) (Table 17).

[0119] Table 20. Effects of corticosteroids on AChE activity in zebrafish

[0120]

[0121]

[0122] Note: Data are expressed as mean ± standard deviation (n = 3). Compared with the AlCl3 model group, the Ctrl group... ### P<0.001; Compared with the AlCl3 model group, the drug-treated group showed ***P<0.001.

[0123] The results of acetylcholinesterase activity assay in the betaine-treated group are shown in Table 21. Compared with the AlCl3 model group, the AChE activity of zebrafish in the positive control group and the betaine-treated group was significantly decreased (P<0.001).

[0124] Table 21. Effects of betaine on AChE activity in zebrafish

[0125]

[0126] Note: Data are expressed as mean ± standard deviation (n = 3). Compared with the AlCl3 model group, the Ctrl group... ### P<0.001; Compared with the AlCl3 model group, the drug-treated group showed ***P<0.001.

[0127] Based on the behavioral analysis and acetylcholinesterase experiment results of the zebrafish Ctrl group and the model group, it is suggested that the 150μM AlCl3-induced Alzheimer's disease model was successfully established.

[0128] This invention demonstrates the successful simultaneous induction of Alzheimer's disease (AD) and osteoporosis (OP) in juvenile zebrafish using 150 μM AlCl3, and its successful application in screening the optimal concentration ratios of betaine, dermal resin alcohol, osthol-forsythiaol, and osthol-ginsenoside Rb3 for treating AD and OP. This suggests the feasibility of using this animal model to screen dual-effect drugs for the simultaneous treatment of Alzheimer's disease and osteoporosis, providing an excellent animal model for further elucidating the intrinsic link between Alzheimer's disease and osteoporosis.

Claims

1. The use of the active ingredient in the preparation of a medicament for treating Alzheimer's disease complicated with osteoporosis, characterized in that: The active ingredients are a combination of betaine, dermal resin alcohol, osthol, and notopterygium alcohol, or a combination of osthol and ginsenoside Rb3; in the combination of osthol and notopterygium alcohol, the molar ratio of notopterygium alcohol to osthol is 1:1.5 to 1:9; in the combination of osthol and ginsenoside Rb3, the molar ratio of osthol to ginsenoside Rb3 is 16:

100.

2. The use according to claim 1, characterized in that... In the combination of osthol and notopterygium alcohol, the molar ratio of notopterygium alcohol to osthol is 1:1.5 to 1:2.

5.

3. The use according to claim 2, characterized in that... In the combination of osthol and notopterygium alcohol, the molar ratio of notopterygium alcohol to osthol is 1:1.5 and 3:

7.

4. A drug for treating Alzheimer's disease complicated with osteoporosis, characterized in that: A pharmaceutically acceptable formulation is prepared with a combination of betaine, styraxol, osthol, and notopterygium alcohol, or a combination of osthol and ginsenoside Rb3, as the active ingredients, and pharmaceutically acceptable excipients; wherein, in the combination of osthol and notopterygium alcohol, the molar ratio of notopterygium alcohol to osthol is 1:1.5 to 1:9; and in the combination of osthol and ginsenoside Rb3, the molar ratio of osthol to ginsenoside Rb3 is 16:100.

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