A method for constructing a mouse myocardial fibrosis model based on isoprenaline and angiotensin II composite sustained-release microspheres

Abstract

The application provides a composite sustained-release microsphere, which comprises: an active ingredient, and a sustained-release material for loading the active ingredient; wherein the active ingredient comprises isoprenaline and angiotensin II. The technical scheme of the application also comprises a method for constructing a myocardial fibrosis animal model, and the administration mode is injection administration; preferably, subcutaneous injection or intramuscular injection; the administration dose of the composite sustained-release microsphere is: 0.5-5 mg / kg body weight / day of isoprenaline; 0.3-3 mg / kg body weight / day of angiotensin II; and / or, the administration frequency is once every 5-10 days; and / or, the administration cycle is 2-4 weeks; by the sustained-release microsphere technology, the administration frequency is reduced to once every 7 days, and only 3-4 injections are needed in the whole modeling cycle (such as 3 weeks), so that the experimental operation is significantly simplified, and manpower and material resources are saved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and experimental animal model construction technology, specifically involving a sustained-release microsphere based on isoproterenol (ISO) and angiotensin II (AngII) and its application in constructing a mouse myocardial fibrosis model. Background Technology

[0002] Myocardial fibrosis (MF) refers to the excessive proliferation of myocardial fibroblasts, the accumulation of secreted collagen fibers in the extracellular matrix (ECM), which causes myocardial interstitial expansion, leading to myocardial remodeling and symptoms such as structural disorder, abnormal movement, restricted myocardial electrical signal conduction, and systolic and diastolic dysfunction.

[0003] Among the many methods for constructing mouse models of myocardial fibrosis, the isoproterenol (ISO) modeling method significantly reduces the difficulty and cost of modeling while ensuring a high success rate. However, the ISO modeling method still has many drawbacks, such as frequent drug administration, a single pathological mechanism, and high animal mortality.

[0004] Angiotensin II (AngII), as the core effector peptide of the renin-angiotensin system (RAS), is a classic target for inducing animal models of myocardial fibrosis. However, single-dose AngII administration often focuses on hypertension-related myocardial fibrosis, making it difficult to simulate the pathological features of multifactorial synergistic pathogenesis in clinical practice. AngII has a drastic effect on raising blood pressure, and a single high-dose injection can lead to death in the model animals, making combination therapy challenging.

[0005] While existing technologies include drug-release microsphere formulations, there are no specifically designed sustained-release formulations for inducing myocardial fibrosis models, nor are there microsphere products capable of simultaneously achieving the synergistic release of two drugs. Therefore, developing a simple, stable, and low-mortality tool for inducing myocardial fibrosis has significant application value. Summary of the Invention

[0006] This invention aims to provide a composite sustained-release microsphere for constructing an animal model of myocardial fibrosis and its manufacturing method. It addresses the technical problems of high animal mortality, cumbersome operation, and difficulty in simulating complex pathological states associated with existing isoproterenol-based modeling methods.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A composite sustained-release microsphere, the composite sustained-release microsphere comprising: an active ingredient and a sustained-release material for encapsulating the active ingredient; The active ingredients include isoproterenol and angiotensin II.

[0008] The sustained-release material is a biodegradable polymer; preferably, the biodegradable polymer is selected from one or more of polylactic acid-glycolic acid copolymer, polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer and trimethyl[3-(trimethoxysilyl)propyl]ammonium chloride crosslinking modifier.

[0009] The drug loading ratio of isoproterenol to angiotensin II is (1-3):(0.5-2); preferably, the drug loading ratio is (1.25-2.5):(0.75-1.5).

[0010] The composite sustained-release microspheres have a volume average particle size of 0.2-0.4 μm and a polydispersity index of less than 0.5. And / or, the composite sustained-release microspheres have no burst release effect during the in vitro release cycle, and the cumulative release rate reaches more than 80% of the drug loading within 7 days.

[0011] The present invention also provides a method for preparing the composite sustained-release microspheres, comprising encapsulating the active ingredient in the sustained-release material using an emulsification-solvent evaporation method; including the following steps: (1) Dissolve the sustained-release material in an organic solvent to form a polymer solution; (2) Isoproterenol solution and angiotensin II solution are added to the polymer solution, and after a first dispersion treatment, a colostrum is formed; (3) The primary emulsion is added to a polyvinyl alcohol aqueous solution and subjected to a second dispersion treatment to form a secondary emulsion; (4) Remove the organic solvent, and the solidified microspheres are separated, washed and dried to obtain the final product.

[0012] The organic solvent is a mixture of dichloromethane and ethyl acetate, preferably, the volume ratio of dichloromethane to ethyl acetate is (8-10):1; And / or, the mass concentration of the polyvinyl alcohol aqueous solution is 0.5%-1%; And / or, the first dispersion treatment and / or the second dispersion treatment are independently selected from vortex oscillation or ultrasonic treatment.

[0013] In some preferred embodiments, the preparation of the isoproterenol and angiotensin II composite sustained-release microspheres is specifically as follows: 400 mg of polylactic acid-glycolic acid copolymer (PLGA) is accurately weighed and dissolved in 20 ml of a 9:1 mixture of dichloromethane and ethyl acetate. 1 ml of 10 mg / ml isoproterenol hydrochloride solution and 1 ml of 6 mg / ml angiotensin II acetate solution are added to the PLGA solution. The mixture is vortexed and sonicated for 5 min to uniformly disperse the drug in the PLGA solution, forming a homogeneous suspension. 40 ml of 0.5% polyvinyl alcohol (PVA) solution is added to the suspension, and the mixture is vortexed and sonicated for 10 min to form a homogeneous suspension. The suspension is placed in a fume hood and magnetically stirred for 24 h to remove the organic solvent. The suspension is then transferred to a centrifuge tube and centrifuged at 4°C and 6000 rpm for 10 min. The supernatant is removed, and the mixture is washed three times with pure water, vortexed, and freeze-dried.

[0014] The present invention also provides a method for constructing an animal model of myocardial fibrosis, comprising: administering the aforementioned composite sustained-release microspheres, or composite sustained-release microspheres prepared according to the method, to the animal.

[0015] The administration method is injection; preferably, subcutaneous injection or intramuscular injection. The dosage of the composite sustained-release microspheres is as follows: 0.5-5 mg / kg body weight / day based on isoproterenol; 0.3-3 mg / kg body weight / day based on angiotensin II. And / or, the frequency of application is once every 5-10 days; And / or, the application cycle is 2-4 weeks; The animal is a mammal; preferably, the mammal is a mouse, rat, or rabbit; more preferably, the mammal is a mouse.

[0016] Furthermore, the dosing regimen is as follows: Thirty-six C57BL / 6j mice were randomly divided into 6 groups, with 6 mice in each group: (1) Blank control group: Blank microspheres were injected subcutaneously once every 7 days for 3 consecutive weeks; (2) Isoproterenol subcutaneous injection group: daily subcutaneous injection of ISO 2.5 mg / kg for 3 consecutive weeks; (3) Isoproterenol microsphere group: Subcutaneous injection of ISO microspheres (equivalent to ISO dose 2.5 mg / kg / d), once every 7 days for 3 consecutive weeks; (4) Angiotensin II microsphere group: Ang II microspheres (equivalent to Ang II dose of 1.5 mg / kg / d) were injected subcutaneously once every 7 days for 3 consecutive weeks; (5) Low-dose composite microsphere group (LISO+Ang II): Subcutaneous injection of composite microspheres (equivalent to ISO 1.25mg / kg / d + Ang II 0.75mg / kg / d), once every 7 days for 3 consecutive weeks; (6) High-dose composite microsphere group (LISO+AngⅡ): Subcutaneous injection of composite microspheres (equivalent to ISO 2.5mg / kg / d + AngⅡ 1.5mg / kg / d), once every 7 days for 3 consecutive weeks.

[0017] The application of the composite sustained-release microspheres or the composite sustained-release microspheres prepared by the method in the preparation of reagents or kits for constructing animal models of myocardial fibrosis.

[0018] The animal model of myocardial fibrosis was used to screen drugs for myocardial fibrosis.

[0019] The beneficial effects of this invention: 1. Traditional ISO modeling requires daily injections. This invention reduces the dosing frequency to once every 7 days through sustained-release microsphere technology. The entire modeling cycle (e.g., 3 weeks) only requires 3-4 injections, which significantly simplifies the experimental operation and saves manpower and resources.

[0020] 2. Traditional ISO daily injections can easily lead to drastic fluctuations in blood drug concentration, causing acute cardiotoxicity and even death. The microspheres of this invention achieve stable and continuous drug release, avoiding the fatal risk caused by excessively high peak concentrations. Data from the examples show that no animals died in the composite microsphere group, while the mortality rate was significantly higher in the traditional ISO subcutaneous injection group.

[0021] 3. This invention is the first to achieve synergistic sustained-release administration of ISO and AngII, which can simultaneously activate the sympathetic nervous system and the renin-angiotensin system. These two pathways are often co-activated in the clinical progression of heart failure and myocardial fibrosis. Results from the examples show that the composite microsphere group of mice exhibited significantly better results than the single-drug group in key indicators such as collagen deposition area in myocardial tissue, degree of cardiomyocyte hypertrophy, and serum LDH level, demonstrating that this model better simulates the complex pathological features of multifactorial clinical causes.

[0022] 4. The prepared microspheres have uniform particle size (PDI<0.5) and stable release curves, ensuring consistent pharmacokinetic exposure for each animal, thus obtaining a more stable and uniform model and reducing intragroup error. Attached Figure Description

[0023] Figure 1 The particle size distribution curve of the isoproterenol sustained-release microspheres prepared in Example 1 of this invention is shown.

[0024] Figure 2 The in vitro release curve of isoproterenol sustained-release microspheres prepared in Example 1 of this invention is shown.

[0025] Figure 3 The graphs show the weight changes of model mice under different dosing regimens in Examples 2 and 3 of this invention.

[0026] Figure 4 Charts showing the body weight (BW), heart weight (HW), and heart weight index (CWI) of mice in each group.

[0027] Figure 5 HE staining images of heart tissue from each group of mice (200×).

[0028] Figure 6 Masson staining images (200×) of heart tissue from each group of mice.

[0029] Figure 7 A statistical chart showing the serum lactate dehydrogenase (LDH) levels in mice from each group.

[0030] Figure 8 The in vitro release curve of isoproterenol sustained-release microspheres prepared in Example 4 of this invention is shown. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments.

[0032] Example 1 Preparation of isoproterenol sustained-release microspheres and their encapsulation efficiency, drug loading, particle size distribution, and in vitro release characteristics. Weigh 400 mg of PLGA and dissolve it in 20 ml of a 9:1 mixture of dichloromethane and ethyl acetate. Add 1 ml of 10 mg / ml isoproterenol hydrochloride solution to the PLGA solution, vortex and sonicate for 5 min to uniformly disperse the drug in the PLGA solution and form a homogeneous suspension. Add 40 ml of 0.5% PVA solution to the suspension, vortex and sonicate for 10 min to form a homogeneous suspension. Place the suspension in a fume hood and magnetically stir for 24 h to remove organic solvents. Transfer the suspension to a centrifuge tube and centrifuge at 4℃ and 6000 rpm for 10 min. Remove the supernatant, wash three times with pure water, vortex to mix, and freeze dry.

[0033] The particle size and particle size distribution of the microspheres were determined using a particle size analyzer. Deionized water was placed in the sample dispersion device of the particle size analyzer and stirred at 2200 rpm. Approximately 100 mg of microsphere sample was taken and poured into the sample cell. After the sample was evenly dispersed, its particle size was measured.

[0034] Each sample was measured in triplicate, and the average value was taken. The drug loading and encapsulation efficiency of the microspheres were determined by ultraviolet spectrophotometry. A 0.512 mg / mL isoproterenol hydrochloride solution was prepared, serially diluted, and the ISO absorbance standard curve at 280 nm was obtained. 100 mg of drug-loaded microspheres were accurately weighed, dissolved in 5 mL of DMSO, and the absorbance at 280 nm was measured. The drug loading of the microspheres was calculated according to the standard curve. The drug loading and encapsulation efficiency were calculated using the following formula: Drug loading (%) = (Weight of drug in microspheres / Total weight of microspheres) × 100% Encapsulation efficiency (%) = Actual drug loading of microspheres / Theoretical drug loading × 100% Accurately weigh an appropriate amount of microspheres into 5 mL of release medium, then pour it into a dialysis bag and place it in a centrifuge tube containing 15 mL of release medium. Under simulated in vivo conditions (37℃, rotation speed 100 r / min), take 1 mL of sample into the centrifuge tube at each time point (and add 1 mL of release medium at the same time), measure the absorbance with a UV-Vis spectrophotometer, calculate the isoproterenol at each time point, and then calculate the cumulative release rate (Q) of isoproterenol at each time point according to the following formula.

[0035] ; In the formula, Cn is the mass fraction of the sample at the nth time point, mg / mL; V is the total volume of the release medium, mL; Vi is the sampling volume at the ith time point, mL; Ci is the mass fraction of the sample at the ith time point, mg / mL; W is the total mass of the microspheres, mg; and DL is the drug loading of the microspheres, g / g.

[0036] result: After testing ( Figure 1 The prepared drug-loaded microspheres exhibited a uniform particle size distribution. The volume average particle size of the microspheres was 0.282 μm, and the length average particle size was 0.237 μm. The cumulative median of the volume average particle size distribution, D50, was 0.262 μm, indicating that the overall particle size of the microspheres was small and the distribution was concentrated. The polydispersity index (PDI) was 0.27, significantly less than 0.5, fully demonstrating that the microspheres possessed good uniformity and dispersion stability, with no obvious large particle aggregation.

[0037] Based on the in vitro release curve ( Figure 2 The results showed that the microspheres did not have a significant burst release effect in the early stage, and the drug release process was stable and continuous. After 7 days of microsphere release, the cumulative release rate of the microspheres increased slowly with the release time and eventually stabilized. During the monitoring period, the cumulative drug release of the microspheres eventually reached 80% of the drug loading.

[0038] Example 2: Construction of a myocardial fibrosis model using isoproterenol microspheres and verification of its effects Thirty-six male SPF-grade C57BL / 6j mice (6 weeks old, weighing 20±2g) were randomly divided into 6 groups of 6 mice each. The mice were administered the drug according to the above-described regimen for 21 days.

[0039] (1) Modeling observation: The body weight changes and mortality of mice in each group were recorded during the modeling period. The results showed that ( Figure 3 In the blank control group, the isoproterenol microsphere group, and the angiotensin II microsphere group, the body weight of mice increased steadily. The body weight of the isoproterenol subcutaneous injection group increased significantly five days after administration. The isoproterenol subcutaneous injection group experienced animal death on the 15th day of modeling, while no animal death occurred in the blank control group and the isoproterenol microsphere group.

[0040] (2) Mouse Cardiac Weight Index (CWI) Statistical Analysis: The mice were weighed before sampling, the heart was removed, blood vessels and connective tissue were removed, blood was absorbed, and the absolute weight of the heart was measured. The CWI was calculated using the following formula: Heart weight HW = Wet weight of mouse heart (mg) Cardiac Body Weight Index (CWI) = Body weight (g) / Heart weight (mg) × 100% The results show ( Figure 4 The heart weight (HW) and body weight (BW) of mice in each treatment group were significantly greater than those in the blank control group; in contrast, there was no significant difference in CWI between the ISO subcutaneous group and the ISO microsphere group.

[0041] (3) Results of HE staining of mouse heart: Results of HE staining of mouse heart as follows Figure 5 As shown, the myocardial tissue structure in the blank control group was normal, with myocardial cells arranged neatly and densely, relatively uniform in size, and with clear cell outlines; no obvious fibrosis was observed. In contrast, the myocardial tissue in the ISO subcutaneous injection group, ISO microsphere group, and Ang II microsphere group all showed abnormalities. The ISO subcutaneous injection group showed tissue fibrosis, condensed cell nuclei with vacuoles (indicated by red arrows), and hypertrophic peripheral myocardial cells (indicated by black arrows). The ISO microsphere group showed significant myocardial tissue abnormalities, condensed cell nuclei (indicated by red arrows), blurred cell outlines, significantly hypertrophic myocardial cells, and obvious homogeneous red staining (indicated by black arrows); a small number of inflammatory cells proliferated (indicated by yellow arrows). The Ang II microsphere group showed myocardial tissue fibrosis, condensed cell nuclei with vacuoles, and hypertrophic peripheral myocardial cells.

[0042] (4) Masson staining results of mouse heart: Masson staining results of mouse hearts as follows Figure 6As shown, compared with the blank control group, the blue staining area of ​​the myocardial tissue of mice in the ISO subcutaneous group, ISO microsphere group, and Ang II microsphere group was significantly increased; the collagen area in the Ang II microsphere group was lower than that in the ISO microsphere group and ISO subcutaneous injection group; the collagen deposition area in the ISO microsphere group was 23.4%, which was higher than that in the ISO subcutaneous group (14.8%).

[0043] (5) Mouse serum LDH detection: The results showed ( Figure 7 Serum LDH levels in all treatment groups were higher than those in the blank control group; among them, the serum LDH level in the ISO microsphere group was the highest.

[0044] Example 3: Construction of a myocardial fibrosis model using ISO-Ang II composite microspheres and verification of its effects Thirty-six male SPF-grade C57BL / 6j mice (6 weeks old, weighing 20±2g) were randomly divided into 6 groups of 6 mice each. The mice were administered the drug according to the above-described regimen for 21 days.

[0045] (1) Modeling observation: The results showed that ( Figure 3 In the blank control group and the low-dose composite microsphere group (LISO+Ang II), the body weight increased steadily, while the body weight of the high-dose microsphere group (HISO+Ang II) changed more significantly and the time point of weight loss was after administration; no model animals died in the composite microsphere group.

[0046] (2) Mouse cardiac weight index (CWI) statistics: The results showed ( Figure 4 The heart weight (HW) and body weight (BW) of mice in each treatment group were significantly greater than those in the blank control group; in contrast, the heart weight of mice in the low-dose composite microsphere group was significantly lower than that of mice in the high-dose composite microsphere group.

[0047]

[0048] (3) Results of HE staining of mouse heart: Results of HE staining of mouse heart as follows Figure 5 As shown, the blank control group had normal myocardial tissue structure, with cardiomyocytes arranged neatly and densely, relatively uniform in size, and with clear cell outlines; no obvious fibrosis was observed. In the single-drug model groups (ISO subcutaneous group, ISO microsphere group, and Ang II microsphere group), cardiomyocyte hypertrophy and nuclear pyknosis / vacuolation were observed. Compared with the single-drug model groups, the composite microsphere group showed more pronounced myocardial tissue abnormalities, with nuclear pyknosis, blurred cell outlines, significant cardiomyocyte hypertrophy, obvious homogeneous red staining, and more severe nuclear pyknosis. Compared with the low-dose composite microsphere group, the high-dose composite microsphere group showed more pronounced cardiomyocyte hypertrophy, more blurred cell outlines, and more severe fibrosis.

[0049] (4) Masson staining results of mouse heart: Masson staining results of mouse hearts as follows Figure 6 As shown, compared with the blank control group, the collagen deposition area and blue staining area of ​​the model groups were significantly increased. Among them, the collagen deposition area of ​​the single-drug model groups (ISO subcutaneous group, ISO microsphere group, Ang II microsphere group) was significantly smaller than that of the composite microsphere group; the high-dose composite microsphere group showed thickening of the cardiac vascular wall and collagen fiber deposition, with a collagen deposition area of ​​30.1%, the largest among all drug administration groups.

[0050] Example 4: Preparation of isoproterenol sustained-release microspheres using trimethyl[3-(trimethoxysilyl)propyl]ammonium chloride as a stabilizer and determination of their in vitro release characteristics. Weigh 400 mg of PLGA and dissolve it in 20 ml of a 9:1 mixture of dichloromethane and ethyl acetate. Add 1 ml of 10 mg / ml isoproterenol hydrochloride solution to the PLGA solution and vortex and sonicate for 5 min to uniformly disperse the drug in the PLGA solution and form a homogeneous suspension. Prepare 40 ml of 0.3% trimethyl[3-(trimethoxysilyl)propyl]ammonium chloride solution (adjust the pH of the aqueous phase to 4.0-5.0 with glacial acetic acid) and stir magnetically for 30 min at room temperature. Add 40 ml of 0.3% trimethyl[3-(trimethoxysilyl)propyl]ammonium chloride solution to the suspension and vortex and sonicate for 10 min to form a homogeneous suspension. Place the suspension in a fume hood and stir magnetically for 24 h to remove the organic solvent. Transfer the suspension to a centrifuge tube and centrifuge at 4℃ and 6000 rpm for 10 min. Remove the supernatant, wash three times with pure water, vortex to mix, and freeze-dry.

[0051] Accurately weigh an appropriate amount of microspheres into 5 mL of release medium, then pour it into a dialysis bag and place it in a centrifuge tube containing 15 mL of release medium. Under simulated in vivo conditions (37℃, rotation speed 100 r / min), take 1 mL of sample into the centrifuge tube at each time point (and add 1 mL of release medium at the same time), measure the absorbance with a UV-Vis spectrophotometer, calculate the isoproterenol at each time point, and then calculate the cumulative release rate (Q) of isoproterenol at each time point according to the following formula.

[0052] ; In the formula, Cn is the mass fraction of the sample at the nth time point, mg / mL; V is the total volume of the release medium, mL; Vi is the sampling volume at the ith time point, mL; Ci is the mass fraction of the sample at the ith time point, mg / mL; W is the total mass of the microspheres, mg; and DL is the drug loading of the microspheres, g / g.

[0053] result: like Figure 8As shown, after emulsification and cross-linking modification with trimethyl[3-(trimethoxysilyl)propyl]ammonium chloride, a siloxane cross-linking barrier is formed on the surface of the microspheres, which significantly inhibits the initial burst release of the drug. Compared with microspheres prepared by PVA (which take about 8 days to fully release), the overall sustained-release period of the composite drug can be extended to 10-12 days, which is more long-lasting and stable than that prepared by PVA. After 10 days of release, the cumulative release rate of the microspheres increases slowly with the release time and eventually tends to stabilize. During the monitoring period, the cumulative drug release of the microspheres eventually reached 88.9% of the drug loading.

Claims

1. A composite sustained-release microsphere, characterized in that, The composite sustained-release microspheres comprise: an active ingredient and a sustained-release material for encapsulating the active ingredient; The active ingredients include isoproterenol and angiotensin II.

2. The composite sustained-release microspheres according to claim 1, characterized in that, The sustained-release material is a biodegradable polymer material; The biodegradable polymer material is selected from one or more of polylactic acid-glycolic acid copolymer, polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer and trimethyl[3-(trimethoxysilyl)propyl]ammonium chloride crosslinking modifier.

3. The composite sustained-release microspheres according to claim 2, characterized in that, The drug loading ratio of the isoproterenol to the angiotensin II is (1-3):(0.5-2).

4. The composite sustained-release microspheres according to any one of claims 1-3, characterized in that, The composite sustained-release microspheres have a volume average particle size of 0.2-0.4 μm and a polydispersity index of less than 0.

5. And / or, the composite sustained-release microspheres have no burst release effect during the in vitro release cycle, and the cumulative release rate reaches more than 80% of the drug loading within 7 days.

5. A method for preparing the composite sustained-release microspheres according to any one of claims 1-4, characterized in that, This includes encapsulating the active ingredient in the sustained-release material using an emulsification-solvent evaporation method; and includes the following steps: (1) Dissolve the sustained-release material in an organic solvent to form a polymer solution; (2) Isoproterenol solution and angiotensin II solution are added to the polymer solution, and after a first dispersion treatment, a colostrum is formed; (3) The primary emulsion is added to a polyvinyl alcohol aqueous solution and subjected to a second dispersion treatment to form a secondary emulsion; (4) Remove the organic solvent, and the solidified microspheres are separated, washed and dried to obtain the final product.

6. The method according to claim 5, characterized in that, The organic solvent is a mixture of dichloromethane and ethyl acetate, with a volume ratio of dichloromethane to ethyl acetate of (8-10):

1. And / or, the mass concentration of the polyvinyl alcohol aqueous solution is 0.5%-1%; And / or, the first dispersion treatment and / or the second dispersion treatment are independently selected from vortex oscillation or ultrasonic treatment.

7. A method for constructing an animal model of myocardial fibrosis, characterized in that, include: The composite sustained-release microspheres according to any one of claims 1-4, or the composite sustained-release microspheres prepared by the method according to any one of claims 5-6, are administered to animals.

8. The construction method according to claim 7, characterized in that, The administration method is injection; preferably, subcutaneous injection or intramuscular injection. The dosage of the composite sustained-release microspheres is as follows: 0.5-5 mg / kg body weight / day based on isoproterenol; 0.3-3 mg / kg body weight / day based on angiotensin II. And / or, the frequency of application is once every 5-10 days; And / or, the application cycle is 2-4 weeks; The animal is a mammal; the mammal is a mouse, rat, or rabbit.

9. The use of the composite sustained-release microspheres according to any one of claims 1-4 or the composite sustained-release microspheres prepared by the method according to any one of claims 5-6 in the preparation of reagents or kits for constructing animal models of myocardial fibrosis.

10. The application according to claim 9, characterized in that, The animal model of myocardial fibrosis was used to screen drugs for myocardial fibrosis.

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