A method for constructing a myocardial fibrosis organoid model based on natural toxin induction

Abstract

The application discloses a kind of myocardial fibrosis organoid model construction methods based on natural toxin induction, belong to biomedical engineering and drug screening technical field.The application aims at solving the problems of existing two-dimensional in vitro model pathological mechanism not true, microenvironment is missing and not friendly to multi-target natural product screening.The model is formed by human pluripotent stem cell-derived cardiomyocytes and cardiac fibroblasts are co-cultured in three-dimensional matrigel according to a certain proportion.The application uses natural toxin that can induce endoplasmic reticulum stress of cardiomyocyte, in a chronic, low-concentration manner stimulates organoid, simulates the gradual fibrosis process initiated by myocardial cell injury, mediated by paracrine signal, to provide a new in vitro drug screening platform with more real pathological mechanism, more complex microenvironment, more suitable for screening natural product with multi-target mechanism.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and drug screening technology, specifically to a method for constructing an organoid model of myocardial fibrosis based on natural toxin induction. Background Technology

[0002] Myocardial fibrosis is a key pathological step in the progression of various cardiovascular diseases (such as myocardial infarction, hypertensive heart disease, and diabetic cardiomyopathy) to heart failure. Its core characteristic is the abnormal activation of cardiac fibroblasts into myofibroblasts, leading to excessive deposition and abnormal cross-linking of extracellular matrix components such as collagen I / III. This results in myocardial tissue stiffness, decreased compliance, and electrical conduction disorders, ultimately impairing the heart's pumping function. Currently, there are no effective drugs in clinical practice to reverse or halt the progression of myocardial fibrosis. This makes screening lead compounds with novel chemical structures and multi-target mechanisms of action from abundant natural product libraries a highly promising strategy for new drug development.

[0003] However, existing in vitro drug screening models have significant limitations in predictability and physiological relevance: ① Oversimplified cell models: Most mainstream models are based on two-dimensional monolayer cultured cardiac fibroblasts and use potent pro-fibrotic factors such as transforming growth factor-β for acute, direct stimulation. This model completely ignores the core role of cardiomyocyte injury in the initiation of fibrosis and cannot simulate the fibroblast activation cascade mediated by paracrine signals (such as ROS, TGF-β, CTGF, IL-6, etc.) released after cardiomyocyte stress / death in vivo, leading to a disconnect between the model's pathological mechanism and the actual in vivo situation. ② Unrealistic pathological processes: Acute, high-concentration TGF-β stimulation simulates a violent tissue damage repair response, while myocardial fibrosis in many cardiovascular diseases (such as metabolic syndrome-related cardiomyopathy) is a slow process driven by chronic, low-level stress (such as endoplasmic reticulum stress, oxidative stress, and metabolic disorders). Existing models struggle to reproduce this chronicity characteristic; screened compounds may be effective against acute injury models but cannot address the chronic pathological environment in vivo. ③ Lack of Microenvironment: Two-dimensional static culture systems lack three-dimensional tissue structure and mechanical microenvironment. The interaction between cardiomyocytes and fibroblasts in three-dimensional space and the dynamic remodeling of the extracellular matrix have a decisive impact on cell phenotype and drug response. In addition, natural products often exert their effects through multiple pathways and multiple targets, regulating the microenvironment as a whole. Simplified models are not sensitive to such effects and are prone to missing screenings. ④ Unfriendly to Natural Product Screening: Traditional models are usually optimized for a single, well-defined target (such as the TGF-β receptor). However, many natural products have complex mechanisms of action, unknown targets, or pleiotropic effects. Under traditional models, they may be deemed ineffective due to off-target effects or weak multi-target synergistic effects.

[0004] Therefore, there is an urgent need in this field for an in vitro model that can more realistically simulate the entire process of cardiomyocyte injury initiation, paracrine signal transduction, chronic fibroblast activation, and three-dimensional matrix reconstruction, in order to establish a drug discovery platform that is more predictive and more suitable for screening multi-target natural products. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention aims to provide a method for constructing a myocardial fibrosis organoid model based on natural toxin-induced stress. This model simulates the chronic, progressive fibrotic pathological process caused by myocardial cell homeostasis imbalance in a three-dimensional myocardial organoid formed by co-culturing myocardial cells and cardiac fibroblasts derived from human pluripotent stem cells using natural small-molecule compounds capable of selectively inducing endoplasmic reticulum stress in cardiomyocytes. This model abandons the traditional approach of directly and strongly stimulating fibroblasts, instead focusing on myocardial cell stress and dysfunction, thereby more realistically reproducing the initiation and development mechanisms of fibrosis in vivo.

[0006] As one aspect of this invention, this invention provides a method for constructing an organoid model of myocardial fibrosis based on natural toxin-induced myocardial fibrosis:

[0007] Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and cardiac fibroblasts (hCFs) were mixed in a specific ratio to obtain a cell suspension. The cell suspension was then mixed with pre-chilled ice-cold matrix gel in a specific ratio. The mixed cell suspension was then seeded into low-attachment 96-well plates at a certain volume and incubated at 37°C for 30 min to allow gel polymerization. Subsequently, 200 μL of cardiac organoid culture medium containing 10% FBS and 1% penicillin-streptomycin was added to each culture well. The cells were pre-cultured for 5 days to establish stable intercellular interactions and spontaneous beating function. From day 6, a chronic induction phase was initiated, with the culture medium containing natural toxins replaced every 3 days for 28 days to finally obtain a cardiac fibrosis organoid model. The natural toxins were compounds that could induce endoplasmic reticulum stress in cardiomyocytes.

[0008] As a preferred embodiment of the method for constructing an organoid model of myocardial fibrosis according to the present invention, the cardiomyocytes derived from human pluripotent stem cells and cardiac fibroblasts are mixed at a cell ratio of 7:3.

[0009] As a preferred embodiment of the method for constructing an organoid model of myocardial fibrosis according to the present invention: the cell density of the cell suspension is 5 × 10⁻⁶. 6 cells / mL.

[0010] As a preferred embodiment of the method for constructing a myocardial fibrosis organoid model according to the present invention: the cell suspension and the ice-pre-cooled matrix gel are mixed at a volume ratio of 1:1.

[0011] As a preferred embodiment of the method for constructing an organoid model of myocardial fibrosis according to the present invention: the mixed cell suspension is inoculated into a low-attachment 96-well plate at an inoculation rate of 25 μL / well.

[0012] As a preferred embodiment of the method for constructing an organoid model of myocardial fibrosis according to the present invention, the natural toxin is any one of azadirachtin, podophyllotoxin, or aristolochic acid.

[0013] As a preferred embodiment of the method for constructing an organoid model of myocardial fibrosis according to the present invention, the concentration of azadirachtin is 0.1-10 μM.

[0014] As a preferred embodiment of the method for constructing an organoid model of myocardial fibrosis according to the present invention, the concentration of podophyllotoxin is 0.01-0.1 μM.

[0015] As a preferred embodiment of the method for constructing an organoid model of myocardial fibrosis according to the present invention, the concentration of aristolochic acid is 1-10 μM.

[0016] As another aspect of the present invention, the present invention provides an application of a natural toxin-induced myocardial fibrosis organoid model in cardiovascular disease-related research.

[0017] As another aspect of the present invention, the present invention provides an application of a natural toxin-induced myocardial fibrosis organoid model in screening candidate drugs that can prevent, alleviate or reverse myocardial fibrosis.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] First, this model accurately reproduces the core pathological mechanism of cardiomyocyte injury driving fibrosis. Experimental results clearly show that under the induction of natural toxins such as 5 μM aristolochic acid, the structure of the cardiomyocyte marker cTNT is severely disrupted, and the expression of fibrosis genes (COL1A1, FN1, etc.) is significantly upregulated. This proves that fibrosis is a cascade reaction initiated by cardiomyocyte injury and mediated by paracrine signals, rather than the direct stimulation of fibroblasts by TGF-β in traditional models.

[0020] Second, this model simulates the process of chronic fibrosis. Low-concentration toxins induced a progressive and stable fibrotic phenotype over 28 days, characterized by large areas of abnormal collagen deposition visible on Masson staining and dense FN1 network structures shown on immunofluorescence, which is more consistent with chronic pathological processes such as metabolic diseases in clinical practice.

[0021] Third, the three-dimensional microenvironment of this model enhances the pathological complexity. The spatial relationship of the cTNT disordered region being tightly wrapped by strong FN1 signal, and the specific response of GFP reporter cells to damage, both demonstrate the dynamic processes of intercellular interactions and matrix remodeling. This more realistic and complex in vitro model is particularly suitable for screening natural products that regulate microenvironmental homeostasis through multiple targets, solving the problem of easy omissions in traditional simplified models. Attached Figure Description

[0022] Figure 1 This is a statistical graph showing the expression levels of fibrosis-related genes in the myocardial fibrosis organoid model induced by this invention.

[0023] Figure 2 The results of Masson staining (A) and statistical analysis of collagen volume fraction (B) of the myocardial fibrosis organoid model induced by this invention are shown.

[0024] Figure 3 This is an image showing the immunofluorescence staining results of the myocardial fibrosis organoid model induced by this invention. Scale bar: 100 μM.

[0025] Figure 4 This is a comparison diagram of drug screening results between the myocardial fibrosis organoid model (B) of the present invention and the traditional model (A). Detailed Implementation

[0026] The technical solutions described in this invention will now be clearly and completely described with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only a part of the feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0027] Example 1: A method for constructing an organoid model of myocardial fibrosis induced by natural toxins

[0028] Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and cardiac fibroblasts (hCFs) were mixed at a cell ratio of 7:3, with a cell density of 5 × 10⁻⁶ cells / cm². 6Cells / mL: The cell suspension was mixed with ice-cold Matrigel at a volume ratio of 1:1. The mixed cell suspension was seeded into low-attachment 96-well plates at a seeding rate of 25 μL / well and incubated at 37°C for 30 min to allow gel polymerization. Subsequently, 200 μL of cardiac organoid culture medium (containing 10% FBS and 1% penicillin-streptomycin) was added to each culture well for pre-culture for 5 days to establish stable cell-cell interactions and spontaneous beating function. From day 6, a chronic induction phase was initiated, with the culture medium containing natural toxins replaced every 3 days for 28 days. The negative control group used culture medium containing 0.1% DMSO, and the positive control group used culture medium containing 10 ng / mL transforming growth factor β1 (TGF-β1). The natural toxin was azadirachtin at a concentration of 0.1 μM.

[0029] Example 2: A method for constructing a natural hormone-induced organoid model of myocardial fibrosis

[0030] The difference between this embodiment and Example 1 is that the concentration of azadirachtin is 1 μM.

[0031] Example 3: A method for constructing an organoid model of myocardial fibrosis induced by natural toxins.

[0032] The difference between this embodiment and Example 1 is that the concentration of azadirachtin is 10 μM.

[0033] Example 4: A method for constructing an organoid model of myocardial fibrosis induced by natural toxins

[0034] The difference between this embodiment and Embodiment 1 is that the natural toxin is podophyllotoxin, and its concentration is 0.01 μM.

[0035] Example 5: A method for constructing an organoid model of myocardial fibrosis induced by natural toxins.

[0036] The difference between this embodiment and Embodiment 1 is that the natural toxin is podophyllotoxin, and its concentration is 0.05 μM.

[0037] Example 6: A method for constructing an organoid model of myocardial fibrosis induced by natural toxins

[0038] The difference between this embodiment and Embodiment 1 is that the natural toxin is podophyllotoxin, and its concentration is 0.1 μM.

[0039] Example 7: A method for constructing an organoid model of myocardial fibrosis induced by natural toxins

[0040] The difference between this embodiment and Example 1 is that the natural toxin is aristolochic acid, and its concentration is 1 μM.

[0041] Example 8: A method for constructing an organoid model of myocardial fibrosis induced by natural toxins

[0042] The difference between this embodiment and Example 1 is that the natural toxin is aristolochic acid, and its concentration is 5 μM.

[0043] Example 9: A method for constructing an organoid model of myocardial fibrosis induced by natural toxins.

[0044] The difference between this embodiment and Example 1 is that the natural toxin is aristolochic acid, and its concentration is 10 μM.

[0045] Experimental Example 1: Detection of expression levels of fibrosis-related genes (qRT-PCR)

[0046] Organoids prepared in Examples 1-9, as well as those prepared in the negative and positive control groups, were collected. Cells were lysed by rapid freezing in liquid nitrogen using TRIzol reagent and then homogenized thoroughly to extract total RNA. Subsequently, 1 μg of total RNA was synthesized into cDNA using a reverse transcription kit. Using the cDNA as a template, qRT-PCR was performed on a quantitative PCR instrument using SYBR Green premixed reagent. Specific primers targeted the core markers of fibrosis: COL1A1 (type I collagen α1), COL3A1 (type III collagen α1), FN1 (fibronectin 1), and ACTA2 (actin α2), with GAPDH used as an internal control gene. Two... -ΔΔCt The relative expression levels of each gene were calculated, and the final data were expressed as mean ± standard deviation. One-way ANOVA was used to compare statistical differences between groups.

[0047] The results of COL1A1 mRNA expression detection are as follows: Figure 1As shown in Figure A, the expression level in the negative control group was 1.2-fold, representing the baseline state of the model. The expression in the positive control group (TGF-β1) was upregulated to 5.8-fold, confirming the effective activation of the fibrosis-inducing pathway. In the treatment with the three natural toxins, azadirachtin showed a dose-dependent effect: Example 1 (0.1 μM) showed a weak 1.9-fold increase in expression; Example 2 (1 μM) significantly upregulated to 5.6-fold, approaching the level of the positive control; Example 3 (10 μM) showed a slight decrease to 5.0-fold, suggesting that excessively high concentrations may have inhibitory effects or toxic interference. Therefore, 1 μM is the optimal concentration of azadirachtin for inducing COL1A1 expression. The effect of podophyllotoxin exhibited an inverted U-shaped pattern: Example 4 (0.01 μM) showed a 1.1-fold increase, almost ineffective; Example 5 (0.05 μM) showed a 4.7-fold increase, with significant induction; Example 6 (0.1 μM) decreased to 3.3-fold, indicating that this concentration may have exceeded the optimal effect window, and toxicity or compensatory inhibition began to appear, suggesting that 0.05 μM is the optimal induction concentration for podophyllotoxin. Aristolochic acid, on the other hand, showed a strong concentration effect: Example 7 (1 μM) showed a 2.0-fold increase, with slight induction; Example 8 (5 μM) showed a high 7.2-fold increase, with induction even stronger than the classic positive control; Example 9 (10 μM) decreased to 3.5-fold, indicating that high concentrations may lead to impaired expression due to cytotoxicity, suggesting that 5 μM is the optimal induction concentration for aristolochic acid.

[0048] The mRAN expression levels of COL3A1, FN1, and ACTA2 were detected as follows: Figure 1 B. Figure 1 C and Figure 1 As shown in D, compared with the control group, the positive control group showed a significant increase, indicating that the fibrosis induction pathway was activated. In the induction models of Examples 1-9, the trend of the experimental results was similar to that of COL1A1 mRNA expression, with the expression levels of each gene first increasing and then decreasing. This indicates that the induction ability of the three natural toxin inducers, azadirachtin, podophyllotoxin and aristolochic acid, is enhanced with increasing concentration. However, at high doses, the induction effect may be reduced due to cytotoxicity.

[0049] Therefore, combining Figure 1 It was found that in the myocardial fibrosis organoid model, the optimal concentrations of the three natural toxins for inducing the expression of COL1A1, COL3A1, FN1, and ACTA2 were 1 μM azadirachtin, 0.05 μM podophyllotoxin, and 5 μM aristolochic acid, respectively. These concentrations achieved the most significant and stable pro-fibrotic effects at the molecular level, providing crucial evidence for subsequent model construction and mechanistic studies.

[0050] Experimental Example 2: Assessment of Collagen Deposition

[0051] The myocardial fibrosis organoid model collected in Example 8 was fixed overnight (approximately 12-16 hours) at 4°C with 4% paraformaldehyde. Subsequently, a standard paraffin embedding procedure was performed: dehydration was carried out sequentially with gradient ethanol (70%, 85%, 95%, 100%), followed by xylene clearing, and finally paraffin embedding to form paraffin blocks. The paraffin blocks were serially sectioned using a paraffin microtome to a thickness of 5 μm, and the sections were mounted on glass slides. For staining, dewaxing and hydration were performed first, followed by Masson's trichrome staining: Weigert iron hematoxylin was used to stain the cell nuclei, Ponceau S and acid fuchsin were used to stain the cytoplasm and muscle, and after differentiation, phosphomolybdic acid was used to treat the differentiated tissues, followed by aniline blue staining of collagen fibers. After staining, the slides were mounted with neutral resin. Finally, the images were observed and acquired under an optical microscope. Image analysis software such as Image-Pro Plus or ImageJ was used to calculate the percentage of blue collagen fiber area to the total tissue area in each field of view, i.e., collagen volume fraction (CVF), and statistical comparisons were performed between groups.

[0052] The Masson trichrome staining results of the myocardial fibrosis organoids obtained in Example 8 are as follows: Figure 2 As shown in Figure A, compared to the negative control group, which had a dense structure and contained only a small amount of blue collagen fibers, the induced myocardial organoids exhibited large-area, dense blue collagen deposition in the extracellular matrix. These collagen fibers were disordered and intertwined, forming a typical fibrotic network structure widely distributed among cardiomyocytes. Quantitative analysis is as follows: Figure 2 B showed that its collagen volume fraction was significantly higher than that of the negative control group. This result directly confirms that aristolochic acid successfully induced abnormal excessive deposition of extracellular matrix in myocardial organoids, and proves the successful establishment of the myocardial fibrosis model from the perspective of histomorphology.

[0053] Experimental Example 3: Immunofluorescence Staining

[0054] To systematically evaluate the pathological characteristics of the myocardial fibrosis organoid model, this invention performed a multiplex immunofluorescence staining comparative analysis on the negative control group and the sample from Example 8 (induced by 5 μM aristolochic acid). The experiment strictly followed standard procedures: First, the two groups of organoids cultured to the predetermined endpoint were fixed in 4% paraformaldehyde, embedded in paraffin, and then prepared into 5 μm thick serial sections. After dewaxing, hydration, and antigen heat retrieval with sodium citrate buffer, the sections were blocked with 10% goat serum at room temperature for 1 hour. Subsequently, the sections were co-incubated overnight at 4°C with the following primary antibody mixture: anti-fibronectin 1 (FN1) mouse monoclonal antibody (1:200 dilution), anti-green fluorescent protein (GFP) rabbit polyclonal antibody (1:500 dilution), and anti-cardiac troponin T (cTNT) mouse monoclonal antibody (1:100 dilution). The following day, after thorough washing with PBST, the slides were incubated at room temperature for 2 hours in the dark with the corresponding fluorescent secondary antibodies: Alexa Fluor 647-labeled goat anti-mouse IgG (recognizing FN1 and cTNT, 1:400 dilution) and Alexa Fluor 488-labeled goat anti-rabbit IgG (recognizing GFP, 1:500 dilution). Finally, slides were mounted with DAPI-containing anti-quenching mounting medium, and multi-channel images were acquired using the same imaging parameters under a laser scanning confocal microscope.

[0055] Multiplex immunofluorescence staining results as follows Figure 3 As shown, in the negative control group, FN1 red fluorescence was weak; the GFP green signal, as a marker of specific cell types, showed a uniform distribution pattern; the cardiomyocyte marker cTNT (gray) showed a clear and regular striation structure, indicating that the cardiomyocytes were morphologically intact and functionally sound; the signals of each channel were clearly layered in the merged image. In contrast, Example 8 showed typical fibrotic pathological features: FN1 expression was strongly activated, forming a dense red fibrous network structure widely distributed in the tissue; at the same time, the GFP signal pattern changed, suggesting that a specific cell population responded to the injury; the cTNT signal intensity was significantly weakened and the structure was completely disordered; the cardiomyocyte striation feature disappeared, indicating that aristolochic acid caused severe cardiomyocyte structural damage. In the merged image, it can be seen that the damaged cardiomyocytes (cTNT disordered areas) were tightly wrapped by an over-deposited fibronectin network (strong FN1 signal), and this spatial distribution relationship indicates that cardiomyocyte injury triggered extracellular matrix remodeling. This result confirms at the protein localization level that aristolochic acid successfully triggered a secondary fibrotic response by inducing cardiomyocyte injury, providing direct evidence for the validation of the model mechanism.

[0056] Comparative Example 1: Drug screening comparison between natural toxin-induced model and traditional TGF-β direct stimulation model

[0057] 1. Model Construction and Drug Processing

[0058] Traditional model (control): Purified human cardiac fibroblasts (hCFs) were cultured in a two-dimensional monolayer. Twenty-four hours after cell seeding, the medium was replaced with either 10 ng / mL TGF-β1 and various concentrations of SB-431542 (1, 5, 10 μM) or 10 ng / mL TGF-β1 and various concentrations of resveratrol (10, 25, 50 μM), and treated for a total of 48 hours.

[0059] The experimental model of this invention: Three-dimensional myocardial organoids (hPSC-CMs co-cultured with hCFs) were constructed according to the method in Example 8. Intervention was initiated on day 25 of chronic induction with 5 μM aristolochic acid. The medium was replaced with fresh medium containing either 5 μM aristolochic acid and various concentrations of SB-431542 (1, 5, 10 μM) or 5 μM aristolochic acid and various concentrations of resveratrol (10, 25, 50 μM). The medium was changed every 24 hours for a total of 72 hours. Both models had corresponding solvent control groups (containing an equal volume of DMSO, final concentration ≤0.1%).

[0060] 2. Analysis of relative expression levels of FN1 mRNA

[0061] The relative expression levels of FN1 mRNA in each group were detected according to the detection method in Example 1. The experimental results after treating the traditional model with different concentrations of SB-431542 and resveratrol are as follows: Figure 4 As shown in Figure A, 10 μM SB-431542 showed the best inhibitory effect on TGF-β1-induced FN1 overexpression (reducing expression by 4.0-fold), while resveratrol showed limited inhibitory effect even at a high concentration of 50 μM (6.5-fold). The relative expression levels of FN1 mRNA after treating the model obtained in Example 8 with different concentrations of SB-431542 and resveratrol are as follows: Figure 4 As shown in Figure B, resveratrol at 50 μM significantly reduced aristolochic acid-induced FN1 expression by 4.5-fold, which was superior to 10 μM SB-431542 (3.0-fold). Therefore, traditional models are more suitable for screening direct inhibitors of the TGF-β pathway (such as SB-431542), while the model of this invention is more sensitive to natural products that act through multiple targets (such as resveratrol), and can more effectively screen for potential antifibrotic drug candidates in complex pathological environments.

[0062] In summary, this invention successfully established an organoid model of myocardial fibrosis based on natural toxin-induced and simulated cardiomyocyte damage initiation mechanisms. Through chronic stimulation of three-dimensional myocardial organoids with azadirachtin, podophyllotoxin, and aristolochic acid, the model reproduced typical fibrotic pathological features at the gene level (significant upregulation of COL1A1, FN1, etc.), tissue morphology (abnormal collagen deposition), and cellular level (disrupted cTNT structure). Key comparative examples demonstrate that, compared with the traditional TGF-β direct stimulation model, the model of this invention has significantly higher sensitivity for screening multi-target natural products (such as resveratrol), effectively identifying candidate drugs that exert their effects through upstream mechanisms such as protecting cardiomyocytes. This model provides a highly pathologically relevant and reliable platform for studying the pathological mechanisms of myocardial fibrosis and screening novel, multi-target anti-fibrotic drugs.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing an organoid model of myocardial fibrosis based on natural toxin-induced myocardial fibrosis, characterized in that, The specific steps of the model construction method are as follows: Cardiomyocytes derived from human pluripotent stem cells were mixed with cardiac fibroblasts in a specific ratio to obtain a cell suspension. The cell suspension was then mixed with pre-chilled matrix gel in a specific ratio. The mixed cell suspension was then seeded into low-attachment 96-well plates at a certain volume and incubated at 37°C for 30 min to allow gel polymerization. Subsequently, 200 μL of cardiac organoid culture medium containing 10% FBS and 1% penicillin-streptomycin was added to each culture well. The cells were pre-cultured for 5 days to establish stable intercellular interactions and spontaneous beating function. From day 6, a chronic induction phase was initiated, with the culture medium containing natural toxins replaced every 3 days for 28 days to finally obtain a cardiac fibrosis organoid model. The natural toxins were compounds that could induce endoplasmic reticulum stress in cardiomyocytes.

2. The method for constructing an organoid model of myocardial fibrosis as described in claim 1, characterized in that, The human pluripotent stem cell-derived cardiomyocytes and cardiac fibroblasts were mixed in a cell ratio of 7:

3.

3. The method for constructing an organoid model of myocardial fibrosis as described in claim 1, characterized in that, The cell density of the cell suspension was 5 × 10⁻⁶. 6 cells / mL.

4. The method for constructing an organoid model of myocardial fibrosis as described in claim 1, characterized in that, The cell suspension was mixed with ice-precooled matrix gel at a volume ratio of 1:

1.

5. The method for constructing an organoid model of myocardial fibrosis as described in claim 1, characterized in that, The mixed cell suspension was inoculated into low-attachment 96-well plates at an inoculation rate of 25 μL / well.

6. The method for constructing an organoid model of myocardial fibrosis as described in claim 1, characterized in that, The natural toxin is any one of azadirachtin, podophyllotoxin, or aristolochic acid.

7. The method for constructing an organoid model of myocardial fibrosis as described in claim 6, characterized in that, The concentration of azadirachtin is 0.1-10 μM.

8. The method for constructing an organoid model of myocardial fibrosis as described in claim 6, characterized in that, The concentration of podophyllotoxin is 0.01-0.1 μM.

9. The method for constructing an organoid model of myocardial fibrosis as described in claim 6, characterized in that, The concentration of aristolochic acid is 1-10 μM.

10. The use of a myocardial fibrosis organoid model prepared by any one of claims 1-9 in screening candidate drugs that can prevent, alleviate or reverse myocardial fibrosis.

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