Method for screening medicine for treating chronic obstructive pulmonary disease based on rat lung organ model and application thereof

Abstract

The invention belongs to the technical field of organoid models, and particularly relates to a method for screening drugs for treating chronic obstructive pulmonary disease based on a rat lung organoid model and application of the method. Culturing the rat lung tissue subjected to enzymolysis into an organoid, identifying cell markers such as type I alveolar epithelial cells, type II alveolar epithelial cells, macrophages and the like in the lung organoid through an immunofluorescence and / or immunohistochemical method, and confirming the structural and functional integrity of the organoid; establishing a rat lung organ COPD model by using the cigarette smoke extract; then, a drug to be screened acts on the model, the drug sensitivity is evaluated by detecting cell activity, the optimal compatibility of effective components is screened in combination with an orthogonal test, then the apoptosis condition of organoid cells after the drug acts is detected by adopting flow cytometry, and finally, the drug with treatment potential is determined. The method can efficiently and accurately realize integration of drug sensitivity detection and active ingredient compatibility screening, and provides a reliable in-vitro experiment system for research and development of COPD treatment drugs.

Description

TECHNICAL FIELD The present application belongs to the technical field of organoid model, and particularly relates to a method for screening drugs for treating chronic obstructive pulmonary disease based on a rat lung organoid model and application thereof. BACKGROUND

[0001] As a three-dimensional in vitro model, lung organoids have become an important tool for studying lung development, homeostasis maintenance and pathological mechanisms by simulating the complex spatial structure and cell interaction network of lung tissue. Traditional two-dimensional (2D) cell culture systems cannot truly reflect the alveolar epithelial barrier function and mesenchymal-epithelial synergistic regulation mechanism due to the lack of cell polarity and three-dimensional microenvironment signals. Although animal models can partially restore the physiological characteristics of organs, they have limitations such as significant species differences, long experimental periods, and other core issues.

[0002] Current lung organoid construction is mainly based on embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC), which need to activate related signaling pathways in a time sequence to simulate the lung development process. However, this method has problems such as complicated culture process, low differentiation efficiency, low organoid formation rate, and significant heterogeneity in structure and function. In addition, although lung organoids derived from adult lung stem cells can express alveolar epithelial cell markers and contain some lung mesenchymal cells, their culture system is highly dependent on extracellular matrix, specific growth factors and mechanical microenvironment support, which limits the activity of the organoids and lacks functional stability. Existing literature reports that most lung organoid models focus on human cells, while rats are a classic model animal for respiratory system disease research, and there is still a gap in the standardized construction technology of rat lung organoids, especially how to balance stem cell stemness maintenance and directional differentiation efficiency, and how to simulate alveolar-capillary unit function. However, traditional lung tissue in vitro models have systematic deficiencies in simulating lung parenchymal structure and function. First, two-dimensional monolayer culture systems lack three-dimensional microenvironment, leading to disordered polarity of alveolar epithelial cells and defects in the expression of key basement membrane components, affecting cell directional differentiation and functional homeostasis. Second, static culture mode cannot reproduce respiratory-related dynamic mechanical stress, causing abnormal cell mechanical signal transduction, weakening epithelial barrier integrity and hindering injury repair mechanisms. Third, single cell type culture models lack the spatial interaction network of multiple cell populations in lung tissue, causing disruption of paracrine signaling and distortion of drug response evaluation. Fourth, disease modeling methods based on live animals have ethical controversies, involving experimental animal welfare, cross-species research reliability, and complexity of ethical review processes. SUMMARY

[0003] In view of the above problems, in a first aspect, the present application provides a method for screening drugs for treating chronic obstructive pulmonary disease based on a rat lung organoid model, comprising the following steps: culturing the rat lung tissue after enzymolysis to obtain an organoid; identifying cell markers of the lung organoid by immunofluorescence and / or immunohistochemistry; establishing a rat lung organoid model based on cigarette smoke extract using the organoid; applying the drug to be screened to the rat lung organoid model, and detecting cell viability to evaluate drug sensitivity; based on the results of the drug sensitivity experiment, using orthogonal experimental design to screen the best compatibility of the effective components; detecting the apoptosis of the organoid after drug treatment by flow cytometry; determining the drug to be screened according to the apoptosis of the organoid.

[0004] Further, the culturing of the rat lung tissue after enzymolysis to obtain an organoid comprises the following steps: mixing the sheared rat lung tissue fragments with PRS-TDE-2 digestive enzyme, oscillating and digesting for 20-30 min, then stopping the reaction and centrifuging to obtain a precipitate;

[0005] resuspending the precipitate, then sequentially performing two-stage filtration, combining all cell suspensions after filtration, adding cell lysis solution, treating under ice bath conditions, and centrifuging to obtain pure cells; mixing the pure cells into a cell suspension with Matrigel, taking the mixed solution to spot glue in a culture container, covering the organoid culture medium after solidification, replacing the organoid culture medium every 2-3 days, until an organoid with a diameter greater than 50 μm is formed;

[0006] when the diameter of the organoid reaches 50-100 μm or adhesion occurs, discard the organoid culture medium, centrifuge to collect the precipitate after blowing away the glue, dissociate the precipitate to 75-85% of the precipitate forming 2-5 cell clusters, then stop the reaction, wash after centrifugation until the glue is completely removed, and then mix with Matrigel to obtain the organoid.

[0007] Further, the rat lung tissue fragments are mixed with PRS-TDE-2 digestive enzyme at a volume ratio of 1:(25-50).

[0008] Further, the two-stage filtration is sequentially performed through 180-220 μm and 80-120 μm filter screens.

[0009] Further, the concentration of ammonium chloride in the cell lysate is 1.55 mol / L, the concentration of potassium bicarbonate is 0.1 mol / L, the concentration of disodium EDTA is 1 mmol / L, the concentration of N-acetyl cysteine is 20 mmol / L, and the concentration of heparin is 2.5 IU / mL; the solvent is an aqueous solution.

[0010] Further, the density of cells in the Matrigel after mixing the cell suspension and the Matrigel is 2-5 x 10 5 cells / mL. The Matrigel is composed of Matrigel, epidermal growth factor EGF, fibroblast growth factor 1 FGF1, and granulocyte-macrophage colony-stimulating factor GM-CSF in a volume ratio of 92-96:1-2:2-4:1-2.

[0011] Further, the organoid culture medium is composed of Precedo medium, B27 supplement, N2 supplement, penicillin-streptomycin, Noggin, R-spondin 1, EGF, FGF, and Y-27632 in a volume ratio of 47.78 mL:0.5-1.5 mL:0.5-1 mL:0.5-1 mL:25-75 μL:25-75 μL:25-50 μL:25-50 μL:25-50 μL; for example, Precedo medium, B27, N2, penicillin-streptomycin, Noggin, R-spondin 1, EGF, FGF, and Y-27632 in a volume ratio of 47.78 mL:1 mL:0.5 mL:0.5 mL:50 μL:50 μL:25 μL:25 μL:25 μL.

[0012] Further, the lung organoid cell markers include type I alveolar epithelial cell markers, type II alveolar epithelial cell markers, macrophage markers, smooth muscle cell markers, endothelial cell markers, and neutrophil markers.

[0013] Further, the type I alveolar epithelial cell marker is Podoplanin, the type II alveolar epithelial cell marker is SFTPC, the macrophage marker is F4 / 80, the smooth muscle cell marker is α-SMA, the endothelial cell marker is CD31, and the neutrophil marker is MPO.

[0014] In a second aspect, the application provides use of the method for screening drugs for treating chronic obstructive pulmonary disease based on a rat lung organoid model in constructing a platform for screening drugs for treating chronic obstructive pulmonary disease.

[0015] Advantages of the present application: In the process of constructing rat lung organoids, the present application retains the key cell components (such as alveolar epithelial cells, etc.), spatial structure and part of the function of the primary tissue under three-dimensional culture conditions. Compared with traditional two-dimensional cell culture, this model is closer to the real lung tissue state in rats in terms of genetic background, cell interaction and microenvironment. This makes the results of using this organoid to simulate the pathological process of CSE (cigarette smoke extract) induced chronic obstructive pulmonary disease (COPD) (such as inflammation, oxidative stress, mucus hypersecretion, epithelial damage, and possible airway remodeling) and subsequent drug screening more accurately reflect the potential effects of drugs in rats and even other mammals, significantly improving the success rate of prediction from in vitro screening to in vivo animal experiments and even clinical translation. The development of COPD involves complex interactions between multiple lung cell types (epithelial cells, immune cells, interstitial cells). The rat lung organoid model can to some extent reproduce the co-culture and signal exchange between these cells (such as epithelial-mesenchymal interaction), which cannot be matched by single cell type culture. Using this model to model CSE can more comprehensively simulate the key pathological features of COPD (such as epithelial barrier damage, inflammatory factor release, protease / anti-protease imbalance, oxidative stress response, etc.), and evaluate the effects of drugs on these complex interaction networks, more accurately identify effective compounds that can intervene in multiple targets and multiple pathways.

[0016] The drug screening platform based on the rat lung organoid model of COPD constructed by the present application can highly simulate the physiological and pathological environment of lung tissue in vivo, providing a more accurate and effective tool for COPD research and drug screening. Through a series of steps such as culture, identification and modeling of rat lung organoids, a stable and reliable in vitro model is established, which helps to further study the pathogenesis of COPD and evaluate the efficacy and safety of drugs. The constructed drug screening platform can effectively screen a large number of compounds for effectiveness and preliminary safety (such as cytotoxicity) in the early stage, and only a small number of potential candidate molecules are pushed into animal in vivo experiments. This method can significantly reduce the number of experimental animals used in the preliminary screening stage, reduce the pain caused by animal experiments, and also directly reduce the costs related to the feeding, management and experimental operation of a large number of animals, in line with the ethical and sustainable development trends of modern drug research and development.

[0017] Other features and advantages of the present application will be set forth in the following specification, and in part will be apparent from the specification, or can be learned by practice of the present application. The objects and other advantages of the present application will be realized and attained by the structure particularly pointed out in the specification, claims and drawings. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0019] Figure 1 The immunofluorescence detection results of organoid markers (Podoplanin, lung surfactant-associated protein C SFTPC, transmembrane glycoprotein F4 / 80, alpha-smooth muscle actin alpha-SMA, and myeloperoxidase MPO mark type I / II alveolar epithelial cells, macrophages, smooth muscle cells, and neutrophils, respectively, x20 (using 20 times magnification)) in the embodiments of the present application are shown. Figure 2 The cell-specific expression results of organoid markers Podoplanin, SFTPC, F4 / 80, alpha-SMA, and MPO characterized by immunohistochemical method in the embodiments of the present application are shown. Figure 3 The cell survival rates in lung organoids under different CSE concentrations in the embodiments of the present application are shown. Figure 4 The microscopic images of lung organoids under different CSE concentrations under 24h conditions in the embodiments of the present application are shown. Figure 5 The microscopic images of lung organoids under different CSE concentrations under 48h conditions in the embodiments of the present application are shown. Figure 6 The microscopic images of lung organoids under different CSE concentrations under 72h conditions in the embodiments of the present application are shown. Figure 7 The effects of ginsenoside Rb1 and ginsenoside Rg1, two kinds of traditional Chinese medicine monomers, on the activity (cell survival rate in lung organoids) of CSE-induced lung organoids in the embodiments of the present application are shown. Figure 8 The effects of astragaloside A and schisandrin A, two kinds of traditional Chinese medicine monomers, on the activity of CSE-induced lung organoids in the embodiments of the present application are shown. Figure 9 The effects of sinigrin and quercetin, two kinds of traditional Chinese medicine monomers, on the activity of CSE-induced lung organoids in the embodiments of the present application are shown. Figure 10 The effects of ligustrazine and ferulic acid, two kinds of traditional Chinese medicine monomers, on the activity of CSE-induced lung organoids in the embodiments of the present application are shown. Figure 11EC50 values of two traditional Chinese medicine monomers, ginsenoside Rb1 and ginsenoside Rg1, detected by CSE-induced lung organoid viability in the embodiment of the present application are shown. Figure 12 EC50 values of two traditional Chinese medicine monomers, sinigrin and astragaloside IV, detected by CSE-induced lung organoid viability in the embodiment of the present application are shown. Figure 13 EC50 values of two traditional Chinese medicine monomers, ligustrazine and ferulic acid, detected by CSE-induced lung organoid viability in the embodiment of the present application are shown. Figure 14 Cell viability of CSE-induced lung organoids under drug treatment of different experimental groups in the embodiment of the present application is shown. Figure 15 Expression level of IL-1β (interleukin-1β) in CSE-induced lung organoids under drug treatment of different experimental groups in the embodiment of the present application is shown. Figure 16 Expression level of TNF-α (tumor necrosis factor-α) in CSE-induced lung organoids under drug treatment of different experimental groups in the embodiment of the present application is shown. Figure 17 Expression level of VEGF (vascular endothelial growth factor) in CSE-induced lung organoids under drug treatment of different experimental groups in the embodiment of the present application is shown. Figure 18 Expression level of TGF-β (transforming growth factor-β) in CSE-induced lung organoids under drug treatment of different experimental groups in the embodiment of the present application is shown. Figure 19 Correlation analysis results of expression levels of multiple cytokines (TGF-β, IL-1β, VEGF, TNF-α) in different traditional Chinese medicine monomer treatment groups in the embodiment of the present application are shown. Figure 20 Apoptosis loss results of the CON group in the embodiment of the present application are shown. Figure 21 Apoptosis loss results of the MOD group in the embodiment of the present application are shown. Figure 22 Apoptosis loss results of the experimental group 2 in the embodiment of the present application are shown. Figure 23 Quantitative analysis results of apoptosis loss of the CON group, the MOD group and the experimental group 2 in the embodiment of the present application are shown. DETAILED DESCRIPTION

[0020] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0021] Some materials and reagents used in the embodiments of the present application are as follows: Cell viability detection reagent: Cell Counting-Lite 2.0 (Vazyme, item number: DD1101-02; batch number: 0919 / 1101e709); Matrigel (ABW, item number: 082703); Precedo medium (Zhongke Puresyn Biomedicals Technology Co., Ltd., item number: PRS-LCM-3D), tissue digestion enzyme (Zhongke Puresyn Biomedicals Technology Co., Ltd., item number: PRS-TDE-2); 24-well cover glass provided by Jingan Biotechnology (item number: J24001), B27 supplement (Gibco, item number: 17504044), N2 supplement (Gibco, item number: 17502048), penicillin-streptomycin double antibody (Gibco, item number: 15140122), Noggin (MCE, item number: HY-P70558), R-spondin 1 (MCE, item number: HY-P700005), EGF (sino biological, item number: 10605-H01H) and Y-27632 (MCE, item number: HY-10071).

[0022] Ginsenoside Rg1 (batch number: 231129), ginsenoside Rb1 (batch number: 240109), astragaloside IV (batch number: 240120), schisandrin A (batch number: 230108), sinigrin potassium salt (batch number: 231203), quercetin (batch number: 231030), ferulic acid (batch number: 240509) and tetramethylprazine (batch number: 240105) are all from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd. The mass fraction (HPLC) of the above components is all greater than or equal to 98%.

[0023] Example 1 Culture and subculture of organoids: After the SD rats were ethically sacrificed, they were fixed in a supine position on a sterile operating table. After the skin of the chest and abdomen was disinfected with alcohol, the lung tissue of the rat was aseptically separated and washed three times in pre-cooled PBS. The whole process of lung tissue processing should be completed within 4 hours.

[0024] Primary culture of organoids: 1) Tissue digestion: 1 mm3 rat lung tissue pieces were mixed with 25-50 times volume of PRS-TDE-2 digestion enzyme and shaken at 37°C for 25 min to obtain a flocculent tissue. The reaction was terminated by adding DMEM / F12 containing 10% FBS and centrifuged at 1500 x g for 5 min to obtain the precipitate.

[0025] The shaking digestion time was 20-30 min. When the digestion time was less than 20 min, the rat lung tissue pieces were not fully dissociated, and it was difficult to pass through the filter screen, resulting in a significant decrease in the yield of effective cell clusters and a decrease in the efficiency of organoid formation. When the digestion time was more than 30 min, the cell-cell junctions and microenvironment structure were destroyed, causing immune cells and epithelial cells to die or lose function, and finally only epithelial ball structures with a single cell type could be formed, which could not achieve the goal of immune-epithelial co-culture. Through repeated experimental verification, the digestion window of 20-30 min using a special enzyme preparation (PRS-TDE-2) under shaking conditions can efficiently generate cell clusters with appropriate size and complete structure, and maximally preserve fragile cell types and their original connection relationships.

[0026] 2) Cell separation: The precipitate was resuspended in PBS and passed through a two-stage filter system of 200 μm + 100 μm, and the lung tissue that did not pass through the repeated digestion was repeated. After the cell suspensions were combined, a cell lysis solution (prepared from 155 mM (0.83%) ammonium chloride, 10 mM (0.10%) potassium bicarbonate, 0.1 mM (0.00372%) disodium EDTA, 2.0 mM (0.03%) N-acetyl cysteine, 2.5 IU / mL (0.00147%) heparin, and 100 mL distilled water) was used for 5 min (ice bath), and the pure cells were obtained by centrifugation at 1500 x g.

[0027] The first-stage 200 μm filter screen removed the large tissue pieces that were not fully digested, preventing them from interfering with subsequent culture; the second-stage 100 μm filter screen precisely intercepted cell clusters with a diameter greater than 100 μm and excluded single cells and small clusters, efficiently preserving the immune-epithelial complex structure with physiological significance. This systematic filtration design effectively avoids the serious problems caused by conventional 40 μm filter screens. Since most macrophages and neutrophils form a complex with epithelial cells (rather than existing as single cells), using a small-pore filter screen will irreversibly lose these key immune components, resulting in a lack of immune microenvironment in the organoids. On the other hand, through previous experiments, it was found that the conventional method of using only a large-pore filter screen (such as 200 μm or 500 μm) would introduce large clusters, leading to central necrosis, asynchronous and heterogeneous organoid formation, and other problems during culture, which is not conducive to subsequent quantitative analysis.

[0028] N-acetyl cysteine in the cell lysate can be used as an antioxidant to neutralize the excess active oxygen generated during the lysis process, effectively prevent the oxidative stress and excessive activation of immune cells such as neutrophils, and thus maintain their subsequent functional response capacity in 3D culture; heparin can be used as a chemokine stabilizer to protect the integrity of cell surface receptors and temperature-sensitive signaling molecules in the solution, and also to retain key molecular basis for normal communication and interaction between cells in the subsequent organoid co-culture system.

[0029] 3) Matrigel embedding: the cell suspension is mixed with Matrigel at a volume ratio of 1:1 (final concentration 5-8 x 10 5 cells / mL), 50 μL of the mixed solution is spotted on a preheated 24-well plate, and 700 μL of organoid culture medium is covered after 37°C solidification for 30 minutes. The organoid culture medium is replaced every 2-3 days (preparation of organoid culture medium: 47.78 mL of Precedo as the basic medium, and 1 mL of B27 supplement with a volume concentration of 2%, 0.5 mL of N2 supplement with a volume concentration of 1%, 0.5 mL of penicillin-streptomycin with a volume concentration of 1%, 50 μL of Noggin with a concentration of 100 ng / mL, 50 μL of R-spondin 1 with a concentration of 100 ng / mL, 25 μL of epidermal growth factor EGF with a concentration of 50 ng / mL, 25 μL of fibroblast growth factor FGF with a concentration of 50 ng / mL, and 25 μL of Y-27632 with a concentration of 5 μM). The standard for organoid formation is a diameter >= 50 μm.

[0030] The composition of Matrigel includes Matrigel, epidermal growth factor EGF, fibroblast growth factor 1 FGF1, and granulocyte-macrophage colony-stimulating factor GM-CSF at a volume ratio of 96:1:2:1; it can more accurately simulate the biochemical properties of the basement membrane of lung tissue and enhance the biophysical and biochemical complexity of the matrix, not only improving the stability of the three-dimensional structure, but also providing a more in-vivo niche signal for different types of cells.

[0031] Subculture: when the organoid diameter reaches 50-100 μm or adhesion occurs, discard the culture medium and add 1 mL of pre-cooled DMEM / F12 to disperse the gel, and collect the precipitate by centrifugation at 2500 x g. The precipitate is dissociated by PRS-ODR (37°C, 10-20 minutes) to 80% to form 2-5 cell clusters, and the reaction is terminated by adding an equal volume of DMEM / F12 containing 10% FBS. Repeat the washing until the gel is completely removed after centrifugation at 2500 x g. Mix with Matrigel at a density of 2-5 x 10 5 cells / mL, and follow the same subsequent steps as the primary culture. The cell viability is determined by trypan blue staining method, and 3 independent fields are selected for statistics.

[0032] Example 2 Pulmonary organoid cell marker immunofluorescence identification: After the organoids in Example 1 were passaged and cultured, they were fixed with 4% paraformaldehyde, washed with PBS 3 times (3 min / time), and treated with a mixture of 0.3% Triton X-100 and 0.3% H2O2 (prepared with PBS) at room temperature for 30 min in the dark. 5% BSA (diluted with PBS) was blocked for 30 min, and specific primary antibodies were added in turn: anti-Podoplanin (type I alveolar epithelial cells), anti-SFTPC (type II alveolar epithelial cells), anti-F4 / 80 (macrophages), anti-α-SMA (smooth muscle cells), anti-CD31 (endothelial cells), and anti-MPO (neutrophils). After incubation at 37°C for 1 h, they were transferred to 4°C overnight. PBS was washed 3 times, and the corresponding species Alexa Fluor 488 / 594-labeled secondary antibodies were added and incubated in the dark for 1 h, and DAPI was used for nuclear staining for 5 min. The sections were mounted with an anti-quenching mounting medium (ProLong Gold), and images were collected under a fluorescence microscope.

[0033] The collected images are shown in Figure 1 The immunofluorescence detection results show that the Rat lung-PDO-P1 organoids highly express the type I alveolar epithelial cell marker Podoplanin and the type II alveolar epithelial cell marker SFTPC, and also detect very low levels of the macrophage marker F4 / 80 and the neutrophil marker MPO, but do not detect the expression of the smooth muscle cell marker α-SMA. The above results show that the model successfully constructs a lung organoid with typical alveolar epithelial characteristics, which can be used for subsequent experimental research.

[0034] Example 3 Pulmonary organoid cell marker immunohistochemical identification: After the lung organoid tissue in Example 1 was fixed with 4% paraformaldehyde, it was dehydrated with gradient ethanol, transparentized with xylene, embedded with paraffin, and sectioned. After dewaxing and rehydrating, antigen retrieval was performed at 95-100°C for 20-30 min, 3% H2O2 was blocked for 10-15 min, and 5% goat serum was blocked for 30 min. The primary antibody (anti-Podoplanin (type I alveolar epithelial cells), anti-SFTPC (type II alveolar epithelial cells), anti-F4 / 80 (macrophages), anti-α-SMA (smooth muscle cells), anti-CD31 (endothelial cells), and anti-MPO (neutrophils)) was incubated at 4°C overnight, the HRP secondary antibody was incubated at 37°C for 30-60 min, DAB was developed for 5-15 min, hematoxylin was re-stained, and neutral resin was mounted. The whole process was washed with PBS to ensure the specificity of the staining.

[0035] The staining results are shown in Figure 2As shown, the immunohistochemical detection results showed that the Rat lung-PDO-P1 organoids presented high expression levels of podoplanin (PDPN), a marker of type I alveolar epithelial cells, and pro-SFTPC, a marker of type II alveolar epithelial cells. At the same time, the macrophage marker F4 / 80 and the neutrophil marker MPO were detected to be extremely lowly expressed, while the smooth muscle cell marker a-SMA and the endothelial cell marker CD31 were not detected. The above results showed that the model successfully simulated the structure of lung organoids with typical alveolar epithelial characteristics, which was suitable for subsequent research.

[0036] Example 4 Pre-experiment of CSE organoid modeling The absorbance of cigarette smoke extract (CSE) culture medium was measured at a wavelength of 320 nm by ultraviolet spectrophotometry, a standard curve was established, and it was confirmed that an OD value of 1.0 corresponded to a CSE stock solution concentration of 100%. Based on this, CSE culture media with concentration gradients of 250%, 200%, 150%, 100%, 50%, 20%, 10%, and blank control (0%) were prepared, and each concentration gradient was obtained by sterile PBS dilution of the stock solution. The matured rat lung organoids were randomly divided into groups and placed in the above different concentration CSE culture media for continuous intervention for 24 h, 48 h, and 72 h, with 3 biological replicates in each group. After the intervention, ATP quantification analysis was performed using a 3D cell viability detection kit: after removing the culture medium, an equal volume of reagent was added to each well, the organoids were shaken and lysed for 15 min, and after incubation at room temperature for 25 min in the dark, the chemiluminescence intensity was detected using a microplate reader. Blank wells and background correction wells were set throughout the experiment, and after normalization of the data, the relative cell viability value was calculated based on the blank control group. Microscopic imaging was used to observe the organoids.

[0037] The results of cell survival rate under different time and concentration CSE treatments are shown in Figure 3 As can be seen, after treating the rat lung organoids with different concentrations of CSE, the organoid viability was weakened to varying degrees, and the weakening of the organoid viability showed a time-dependent manner as the treatment time was prolonged. Under the condition of 24 h, compared with 0% CSE, 150%, 200%, and 250% CSE had statistical significance (#P<0.05); under the condition of 48 h, compared with 0% CSE, 100%, 150%, 200%, and 250% CSE had statistical significance (P<0.05); under the condition of 72 h, compared with 0% CSE, 50%, 100%, 150%, 200%, and 250% CSE had statistical significance (&P<0.05).

[0038] The microscopic images of the lung organoids are shown in Figures 4-6 ​As shown, after different concentrations of CSE treatment for 48h, the morphology of the organoids was damaged to varying degrees compared with the control group, and the damage was most obvious at 72h. According to microscopic imaging and organoid viability detection, 100% CSE treatment for 72h was preferred as the modeling condition for subsequent drug sensitivity tests.

[0039] Example 5 CSE organoid drug sensitivity experiment: Based on the results of the pre-experiment, the concentration of cigarette smoke extract (CSE) was selected to establish the model. The organoids constructed in Example 1 were randomly divided into: Blank control group: Matrigel + organoid culture medium; Normal group (CON group): Organoids + Matrigel + organoid culture medium; Model group (MOD group): Organoids + Matrigel + organoid culture medium + 100% CSE; Drug group: Organoids + Matrigel + organoid culture medium + 100% CSE + traditional Chinese medicine monomers.

[0040] Each group had 3 replicates. After grouping, the culture was continued for 72 hours. After the culture ended, an equal volume of CTG reagent was added to each well, and it was incubated in the dark for 10 minutes. Then, the chemiluminescence signal intensity was detected using a microplate reader. The cell survival rate was calculated according to the following formula: Cell survival rate (%) = [(drug group luminescence value - blank control group luminescence value) / (normal control group luminescence value - blank control group luminescence value)] x 100%.

[0041] The traditional Chinese medicine monomers included eight monomer components: ginsenoside Rb1, ginsenoside Rg1, astragaloside, schisandrin A, sinigrin, quercetin, ligustrazine, and ferulic acid. The effects of each traditional Chinese medicine monomer on the cell survival rate in CSE-induced lung organoids were as follows Figures 7-10 As shown, compared with the normal group, the MOD group had statistical significance (#P<0.05); compared with the MOD group, 40μg / mL and 80μg / mL of ginsenoside Rb1 had statistical significance (P<0.05). Compared with the MOD group, 40μg / mL and 80μg / mL of ginsenoside Rg1 had statistical significance (P<0.05). Compared with the MOD group, 200μg / mL and 400μg / mL of astragaloside had statistical significance (P<0.05). Compared with the MOD group, 8μg / mL and 16μg / mL of sinigrin had statistical significance (P<0.05). Compared with the MOD group, 100μg / mL and 200μg / mL of astragaloside had statistical significance (P<0.05). ​​​​P<0.05). Compared with the MOD group, there was no statistical significance in different concentrations of quercetin and schisantherin A. Compared with the MOD group, the cell survival rate of the 3.125 μg / mL ligustrazine treatment group was slightly increased, but there was no statistical significance, and its effect was unstable and difficult to predict the effective concentration range; and higher concentrations (6.25-50 μg / mL) of ligustrazine led to a significant decrease in cell survival rate in a dose-dependent manner. Therefore, the application selects ferulic acid for subsequent optimization research. Compared with ginsenoside Rg1, ginsenoside Rb1 has lower cost, both of which can increase cell survival rate, and there is no statistical difference. In summary, the lung qi group selects ginsenoside Rb1 and astragaloside A, the phlegm group selects sinigrin, and the blood group selects ferulic acid for the next step experiment.

[0042] The EC50 values of the single components of traditional Chinese medicine for CSE-induced lung organoid viability detection are shown in Table 2. Figures 11-13 The EC50 values of ginsenoside Rb1, ginsenoside Rg1, sinigrin, astragaloside A, ligustrazine and ferulic acid for lung organoid survival rate are 26.04 μg / mL, 27.28 μg / mL, 8.02 μg / mL, 187.29 μg / mL, 3.78 μg / mL and 105.26 μg / mL, respectively; the remaining single components failed to generate a dose-effect curve.

[0043] Example 6 Screening of optimal compatibility of active components Based on the CSE organoid drug sensitivity experiment results, SPSS software was used, and the orthogonal test design principle was used (Table 1: 5-factor 5-level design) to generate an experimental scheme containing 25 candidate components (Table 2).

[0044] Table 1 L of the ratio of each group 25 (5 6 ) orthogonal table

[0045] Table 2 L of the ratio of each group 25 (5 6 ) orthogonal design

[0046] The organoids constructed in Example 1 were randomly divided into: a blank control group, a normal group, a MOD group (CSE) and a drug administration group (20% CSE+ candidate components), with 3 replicate holes in each group. After administration, the treatment was continued for 72 h. After the treatment was completed, the CTG method was used to detect the survival rate of the organoids. The results are shown in Table 2. Figure 14 Compared with the normal group, the survival rate of the MOD group was decreased (#P<0.05); compared with the MOD group, the survival rates of experiments 2, 9, 10, 16 and 21 were increased (P<0.05). P<0.05), and the survival rate was higher than 100%.

[0047] According to the survival rate results, the experimental group with better effect was screened out, and the supernatant of the organoids was collected. The concentrations of TNF-α, VEGF, IL-1β and TGF-β in the supernatant were detected by ELISA method. The ELISA experimental results are shown in Figures 15-18 P <0.05), <0.05), P <0.05). It can be seen that IL-1β: compared with the normal group, the MOD group had statistical difference (#P<0.05), compared with the MOD group, experiments 2, 9, 10, 16 and 21 had statistical difference (#P<0.05), and there was no statistical difference between experiments 2, 9, 10, 16 and 21 ( Figure 15 ). TNF-α: compared with the normal group, the MOD group had statistical difference (#P<0.05), compared with the MOD group, experiments 2, 9, 10, 16 and 21 had statistical difference (#P<0.05); compared with experiments 10 and 16, experiments 2, 9 and 21 had statistical difference, and decreased more ( Figure 16 ). VEGF: compared with the normal group, the MOD group had statistical difference (#P<0.05), compared with the MOD group, experiments 2, 9, 10, 16 and 21 had statistical difference (#P<0.05); compared with experiments 10, 16 and 21, experiments 2 and 9 had statistical difference, and decreased the most ( Figure 17 ). TGF-β: compared with the normal group, the MOD group had statistical difference (#P<0.05), compared with the MOD group, experiments 2, 9, 16 and 21 had statistical difference (#P<0.05); and there was no statistical difference between experiments 2, 9, 16 and 21 ( Figure 18 ). The correlation analysis results of the expression levels of various cytokines in different experimental groups are shown in Figure 19

[0048] Based on the CTG experimental results (drug screening results), the ratio between groups was further optimized, and orthogonal design combined with cell model activity evaluation was used to optimize the efficacy group ratio.

[0049] F(VEGF) = (M / Xn) / max(M / X1:M / Xn) x 100 F(IL-1β) = (M / Xn) / max(M / X1:M / Xn) x 100 F(TNF-α) = (M / Xn) / max(M / X1:M / Xn) x 100​​ F(TGF-β) = (M / Xn) / max(M / X1:M / Xn) x 100 Comprehensive score = [F(VEGF) + F(IL-1β) + F(TNF-α) + F(TGF-β)] / 4 M is the MOD group value, Xn is the drug intervention value, and max(M / X1:M / Xn) is the maximum value of M / X1 to M / Xn.

[0050] The cell experiment results and comprehensive score results of each MOD group are shown in Table 3, according to the statistical analysis results. Preferred experiments 2 and 9 are consistent with the comprehensive score. In combination with market price analysis, experiment 2 is the best choice.

[0051] Table 3

[0052] Example 7 Flow cytometry detection of apoptosis: First, the organoids were dissociated into a single cell suspension using a special dissociation reagent for organoids, and filtered through a 100 μm cell filter. Then, resuspend the cell pellet with pre-cooled 1x PBS, centrifuge at 1500xg for 5 minutes, and discard the supernatant. This washing step is repeated twice to thoroughly remove impurities. After discarding the supernatant, add an appropriate amount of 1x Annexin V binding buffer to resuspend the cells and adjust the concentration. Take 100 μL of the cell suspension into a 1.5 mL EP tube, add 5 μL of FITC Annexin V and 5 μL of propidium iodide (PI) staining solution, mix gently, and incubate at room temperature (20-25°C) for 15 minutes. After incubation, immediately add 400 μL of 1x Annexin V binding buffer to each tube to dilute the sample, and ensure that the flow cytometer completes the detection analysis within 1 hour after staining is complete.

[0053] The detection results are shown in Table 3. Figures 20-23 As can be seen, compared with the CON group, the early, middle-late, and total apoptosis levels of the MOD group are increased (##P<0.01). Compared with the MOD group, the early, middle-late, and total apoptosis levels of experiment 2 group are reduced ( P<0.01).

[0054] Although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent replacements can be made to part of the technical features; and such modifications or replacements do not cause the essence of the corresponding technical solution to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for screening drugs for treating COPD based on a rat lung organoid model, characterized in that, Includes the following steps: Organoids were obtained by culturing enzymatically hydrolyzed rat lung tissue. Cellular markers of lung organoids were identified using immunofluorescence and / or immunohistochemistry. A rat lung organoid model was established using the organoid disease based on cigarette smoke extract; The drug to be screened was applied to the rat lung organoid model, and cell viability was detected to evaluate drug sensitivity. Based on the results of drug sensitivity experiments, an orthogonal experimental design was used to screen the optimal combination of active ingredients. Flow cytometry was used to detect apoptosis in organoids after drug treatment; Drugs to be screened are determined based on the apoptosis status of organoids.

2. The method for screening drugs for treating COPD based on a rat lung organoid model according to claim 1, wherein culturing enzymatically hydrolyzed rat lung tissue to obtain organoids includes the following steps: The shredded rat lung tissue fragments were mixed with PRS-TDE-2 digestive enzyme and shaken for 20-30 minutes to digest the enzyme. The reaction was then terminated and centrifuged to obtain the precipitate. The precipitate was resuspended and then filtered in two stages. After filtration, all cell suspensions were combined, cell lysis buffer was added, and the cells were treated under ice bath conditions and then centrifuged to obtain pure cells. The purified cells were prepared into a cell suspension and mixed with matrix gel. The mixture was then dispensed into a culture container, and after solidification, it was covered with organoid culture media. When the organoid diameter reaches 50-100 μm or adhesion occurs, discard the organoid culture medium, disperse the colloid, centrifuge to collect the precipitate, dissociate the precipitate until 75-85% of the precipitate forms 2-5 cell clusters, terminate the reaction, centrifuge and wash until the colloid is completely removed, then mix with matrix gel to obtain organoids.

3. The method for screening drugs for treating COPD based on a rat lung organoid model according to claim 2, characterized in that, The rat lung tissue fragments were mixed with PRS-TDE-2 digestive enzyme at a volume ratio of 1:(25-50).

4. The method for screening drugs for treating COPD based on a rat lung organoid model according to claim 2, characterized in that, The two-stage filtration involves sequentially passing the material through an 180-220μm filter and an 80-120μm filter.

5. The method for screening drugs for treating COPD based on a rat lung organoid model according to claim 1, characterized in that, The cell lysate contained ammonium chloride at a concentration of 1.55 mol / L, potassium bicarbonate at a concentration of 0.1 mol / L, disodium EDTA at a concentration of 1 mmol / L, N-acetylcysteine ​​at a concentration of 15-25 mmol / L, and heparin at a concentration of 2-3 IU / mL; the solvent was an aqueous solution.

6. The method for screening drugs for treating COPD based on a rat lung organoid model according to claim 1, characterized in that, The cell density in the matrix gel after the cell suspension and matrix gel are mixed is 2-5 × 10⁻⁵. 5 cells / mL; The matrix gel is composed of Matrigel, epidermal growth factor, fibroblast growth factor 1, and granulocyte-macrophage colony-stimulating factor in a volume ratio of 92-96:1-2:2-4:1-2.

7. The method for screening drugs for treating COPD based on a rat lung organoid model according to claim 1, characterized in that, The organoid culture medium consisted of Precedo medium in a volume ratio of 47.78 mL: 0.5-1.5 mL: 0.5-1 mL: 0.5-1 mL: 25-75 μL: 25-75 μL: 25-50 μL: 25-50 μL: 25-50 μL, B27 supplement, N2 supplement, penicillin-streptomycin, Noggin, R-spondin 1, EGF, FGF, and Y-27632.

8. The method for screening drugs for treating COPD based on a rat lung organoid model according to claim 1, characterized in that, The lung organoid cell markers include type I alveolar epithelial cell markers, type II alveolar epithelial cell markers, macrophage markers, smooth muscle cell markers, endothelial cell markers, and neutrophil markers.

9. The method for screening drugs for treating COPD based on a rat lung organoid model according to claim 1, characterized in that, The markers for type I alveolar epithelial cells are Podoplanin, for type II alveolar epithelial cells SFTPC, for macrophages F4 / 80, for smooth muscle cells α-SMA, for endothelial cells CD31, and for neutrophils MPO.

10. The application of the method for screening drugs for treating COPD based on a rat lung organoid model according to any one of claims 1-9 in the construction of a platform for screening COPD drugs.

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