Molecular targeted drug screening model for stem cell mode verification and construction method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a molecular targeted drug screening model based on stem cell mode verification and a construction method and application of the molecular targeted drug screening model based on stem cell differentiation system verification. The model can be used for efficiently and quickly screening drugs according to key pathological characteristics of neurological diseases, cardiovascular diseases, liver injury, lung diseases and immune system diseases. By cloning promoters of key genes such as nerve growth factors, vascular regeneration and repair related factors, pneumonia related factors and the like and connecting quantitative reporter genes, accurate recognition and efficacy evaluation of drug targets are realized. The model is not only suitable for drug screening of chemical monomers, but also can realize dynamic analysis of an aging relationship and a dose-effect relationship aiming at the complex characteristics of traditional Chinese medicines and compound components, and effectively reveals the pharmacodynamic material basis of the traditional Chinese medicines and the compound components.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a molecular targeted drug screening model for verifying stem cell differentiation and secretion patterns, and a construction method and application thereof. Background Art

[0002] Neurological diseases, cardiovascular diseases, liver damage, lung diseases, and immune disorders remain major threats to human health. How to intuitively, efficiently, and rapidly screen for therapeutic drugs is a key scientific challenge urgently needed in the biomedical field. However, current drug screening models for these diseases have numerous limitations and cannot meet the needs for efficient and accurate drug screening.

[0003] Specifically, traditional drug screening models often struggle to effectively reveal the underlying pharmacological mechanisms of traditional Chinese medicines and compound formulas, given their complex composition. Furthermore, the screening process is time-consuming, labor-intensive, and inefficient. Furthermore, existing technologies lack a model capable of simultaneously and accurately screening and validating pharmacologically active ingredients across multiple disease pathologies.

[0004] Traditional Chinese Medicine (TCM) boasts a mature theoretical framework and extensive clinical experience. However, the complex composition and diverse ingredients of TCM formulas, the lack of clarity regarding their active ingredients, the unclear mechanisms of action, and the controversial safety of treatments have become bottlenecks hindering the modernization of TCM. Therefore, both clinical treatment and research and development urgently require a feasible drug screening model to identify the active ingredients of TCM herbs / TCM formulas and elucidate their mechanisms of efficacy.

[0005] In recent years, stem cells have demonstrated tremendous potential in drug screening due to their exceptional plasticity, self-renewal, and multidirectional differentiation. Stem cells can differentiate into specific cell types in vitro under drug induction, providing new insights and approaches for drug screening. Using stem cells as drug screening tools can fully leverage their self-renewal and multidirectional differentiation properties to screen for target components that can interfere with stem cell differentiation or secretion, thereby providing a preliminary assessment of drug efficacy.

[0006] While some stem cell-based drug screening models have been proposed, they often suffer from low screening efficiency, insufficient sensitivity, and limited applicability. This invention aims to overcome these limitations by constructing a molecularly targeted drug screening model validated using stem cell models, enabling more efficient and accurate drug screening and providing strong support for new drug development. Summary of the Invention

[0007] The purpose of the present invention is to provide a molecular targeted drug screening model for stem cell differentiation and secretion pattern verification, as well as its construction method and application. This model not only has the universal characteristics of compound efficacy prediction, but also can realize dynamic analysis of time-effect relationship and dose-effect relationship based on the complex characteristics of traditional Chinese medicine and compound ingredients, effectively revealing its material basis of efficacy, and providing important technical support for the quality control of subsequent clinical drug use.

[0008] The object of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for constructing a molecular targeted drug screening model for stem cell pattern verification, comprising the following steps:

[0010] (1) Cloning the key gene promoter related to the disease treated by the drug to be screened;

[0011] (2) Connecting the cloned gene promoter to the quantitative reporter gene to construct a recombinant plasmid;

[0012] (3) Transfecting the recombinant plasmid into a cell line, and obtaining a cell line stably expressing the reporter gene through resistance screening, thus obtaining a molecular targeted drug screening model for primary screening;

[0013] (4) Based on the preliminary test results, the test monomer is co-incubated with stem cells for 3-14 days to detect protein and gene expression to verify the efficacy of step (3).

[0014] (5) Different monomer compounds have the same dosage but different expressions, which can distinguish the strength of the monomer's predicted efficacy.

[0015] Furthermore, in step (1), the disease includes neurological disease, cardiovascular disease, liver damage, lung disease and immune disease.

[0016] Furthermore, in step (2), the quantitative reporter gene includes a luciferase gene or a red fluorescent protein gene.

[0017] Furthermore, in step (3), the cell line includes human embryonic kidney cells HEK-293T.

[0018] The present invention also provides a stem cell model-verified molecular targeted drug screening model constructed using the construction method. The stem cell model-verified molecular targeted drug screening model can perform high-throughput screening and verification of active ingredients for different targets and marker proteins.

[0019] The present invention also provides a method for drug screening, which includes the steps of screening candidate drugs using the molecular targeted drug screening model verified by the stem cell model, and evaluating the efficacy of the drug by detecting the expression of the reporter gene.

[0020] Furthermore, the method comprises the following steps:

[0021] S1 uses a cytotoxicity experiment to determine the optimal concentration of the candidate drug, and stimulates the molecular targeted drug screening model verified by the stem cell model;

[0022] S2 preliminarily screens the active ingredients by detecting fluorescence intensity or luciferase activity;

[0023] S3 After the initial screening, the selected components are verified by stem cell-directed induction to examine whether there is expression and secretion of relevant nutritional factors;

[0024] S4 Based on the above results, an animal model was constructed to verify the efficacy of the screened ingredients.

[0025] The present invention also provides an application of the molecular targeted drug screening model verified by the stem cell pattern in screening drugs for treating specific diseases.

[0026] Furthermore, the molecular targeted drug screening model verified by the stem cell model contains key genes related to specific diseases, and the drugs for treating specific diseases can affect the key genes.

[0027] Furthermore, the specific diseases include neurological diseases, cardiovascular diseases, liver damage, lung diseases and immune diseases.

[0028] Beneficial effects:

[0029] The molecular targeted drug screening model based on stem cell model verification proposed in the present invention has significant technological innovation and practical application value in the field of biomedicine. By cloning the key gene promoters related to the disease and connecting the reporter gene for quantification, the model realizes efficient drug screening for neurological diseases, cardiovascular diseases and lung diseases. The drug screening model constructed by the present invention can intuitively, efficiently and quickly screen out drugs with therapeutic effects, significantly improving the efficiency and accuracy of drug screening. The model has the universal characteristics of compound efficacy prediction, is suitable for drug screening for a variety of diseases, and has a wide range of applicability. In view of the complex characteristics of traditional Chinese medicine and compound ingredients, the present invention can realize the dynamic analysis of time-effect relationship and dose-effect relationship, and effectively reveal the material basis of its efficacy. Through stem cell directed induction verification, the active ingredients of the drug and its mechanism of action can be further confirmed, providing a scientific basis for the modernization and internationalization of traditional Chinese medicine.

[0030] The present invention relies on the fluorescent protein reporter gene system and the luciferase reporter gene system, adopts common, simple and fast detection technology, and reduces the cost of drug screening. The model has the ability to predict the material basis of drug efficacy, and can screen out potential drug candidates at an early stage, reducing the use of subsequent experimental animals and the cost of clinical trials. The present invention uses stem cells as a screening tool, making full use of the characteristics of stem cell self-renewal and multidirectional differentiation, and improving the sensitivity and reliability of screening. The preliminary screening results of the cell model are further verified by co-incubation of stem cells, making the screening results more reliable.

[0031] The present invention provides a powerful tool for searching for new drugs and their lead compounds for various diseases, helping to accelerate the process of new drug research and development. By revealing the mechanism of action and the material basis of the drug's efficacy, it provides a scientific basis for the clinical application of the drug, helping to promote the development of the biopharmaceutical industry. The drug screening model constructed by the present invention is not only suitable for drug screening for neurological diseases, cardiovascular diseases, and lung diseases, but can also be expanded to applications in other disease areas. With the continuous development and improvement of technology, this model is expected to become an important tool in the field of drug screening, injecting new vitality into the development of the biopharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 This is a diagram of the establishment and verification of trace ingredient pharmacological screening, where A is a cell line that stably expresses the target protein based on a stem cell model; B is a functional verification description of the screening model in neurological diseases; C is a functional verification description of the screening model in cardiovascular diseases; and D is a functional verification description of the screening model in lung diseases.

[0034] Figure 2 Figure 1 shows the efficacy validation and application results of the drug screening model in neurological diseases. A is a schematic diagram of plasmid construction; B is a fluorescence image of the hypoxia-inducible factor gene promoter, vascular endothelial growth factor gene promoter, and nerve growth factor gene plasmid observed under a fluorescence microscope at a magnification of 200 times. 1 CoCl2,f-300μmol·L- 1 Cobalt chloride, g-1μg·ml - LPS, h-100 μmol·L- 1CLE; C is the LUC result of the cell line quantified by fluorescence intensity detection using different concentrations of positive drugs, n = 3, x ± s. The significance was determined by two-sided t test, P < 0.05 compared with the control group;

[0035] Figure 3 Figure 1 shows the application and functional validation of molecular targeted screening models in neurological diseases. A: The effects of isoliquiritigenin (ISL), propiolactone (Ala), crocin, benzoylneaconitine (Benzoy), gallic acid (GA), and protocatechuic acid (PA) on vascular endothelial growth factor (VEGF) gene expression were compared with the control group by fluorescence intensity detection; BC: The effects of 17 compounds on hypoxia-inducible factor (HIF) and VEGF gene expression were detected by fluorescence intensity detection; DE: The effects of ellagic acid (EA) on nerve growth factor (NGF) and VEGF gene expression were detected by fluorescence intensity detection; FH: The effects of different concentrations of nookatone, chrysin, and tectochrysin on NGF gene expression were detected by fluorescence intensity detection; IJ: Fluorescence was observed under a fluorescence microscope at 400x magnification. The expressions of nestin and GalC (a glial cell marker protein) were observed by immunofluorescence staining. RA (all-trans retinoic acid, a positive control) was also observed. n = 3, x ± s. The significance was analyzed by two-tailed t test, *P<0.001, ***P<0.0001 vs. control;

[0036] Figure 4 Figure 1 shows the application and functional validation of molecular targeted screening models in cardiovascular disease. Figures AC: Effects of the complex on the expression of C-Jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK), and nuclear factor kappa-B (NFkB) compared with the control group, as measured by fluorescence intensity. CG: Calycosin-7-O-β-D-glucoside. D: Effects of various compounds at different concentrations on VEGF gene expression, as measured by fluorescence intensity. EG: mRNA expression of α-smooth muscle actin (a-SMA), VEGF, and platelet endothelial cell adhesion molecule-1 (CD31) in BMSCs after drug induction for 2 and 4 days. HM: Fluorescence was observed under a fluorescence microscope at 800x magnification. Expression of α-SMA, VEGF, and CD31 was observed by immunofluorescence staining (HJ). Protein expression was analyzed using ImageJ. n = 3, x ± s. Significance was determined using a two-tailed t-test. *P < 0.05, **P < 0.01 vs. control group. △ P < 0.05 vs simvastatin;

[0037] Figure 5Figures show the application and functional validation of molecular targeted screening models in lung diseases. A: Comparison of the insert fragment and gene sequencing results of the transforming growth factor-β (TGF-β) promoter LUC in pLenti; B: Fluorescence intensity analysis of the effects of different components of Shagan Qingfei Jiedu Granule on TGF-β gene expression; C-D: Concentration optimization for the LPS-induced alveolar epithelial cell injury model determined by assessing cell viability and quantifying IL-1β levels using an enzyme-linked immunosorbent assay (ELISA); E: Western blot analysis of the effects of different concentrations of isothiocyanate on the TGF-β / SMAD signaling pathway. n = 6, x ± s; significance was determined using a two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the control group. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Example 1

[0044] 1. Experimental Materials and Instruments

[0045] 1.1 The experimental reagents and materials involved in the present invention include: LATaq polymerase (639141), restriction endonuclease (R6161), T4 DNA ligase (2011A) (TaKaRa, Japan); Pfu DNA polymerase (M7741, Fermentas, Canada); Taq polymerase (1096608), mammalian cell transfection reagent (Lipofectamine 2000, 11668-027) (Invitrogen, USA); EZNA TM Plasmid miniprep kit (D6943, Omega, USA); luciferase assay kit (E1500, Promega, USA); protein concentration assay kit (23225, Thermo Fisher Scientific, USA); aminoglycoside antibiotic G418 (E859-5G, Amresco, USA); dimethyl sulfoxide (D2650), CoCl2 (409332), lipopolysaccharide (SMB00704), clenbuterol (C-081) (Sigma, USA); DMEM high-glucose culture medium (SH30022.01B), penicillin and streptomycin (SV30010), trypsin (SH30042), ampicillin (SR0136), L-DMEM (SH30525.01) (Hyclone, USA); FBS (0500, Sciencell, USA); PBS buffer (G4202-500 ML, Sevier Company, China); Trizol (15596018CN, Invitrogen, USA); nestin antibody (ab105389), glial cell marker protein antibody (ab240638) (Abcam, UK); immunofluorescence secondary antibody (C3278, Beyotime, China); 4% paraformaldehyde (BL539A, Lanjieke Technology Co., Ltd., Beijing, China); nuclear factor κB plasmid (pNFκB-Luc), extracellular regulated kinase plasmid (pERK-Luc), c-Jun N-terminal kinase plasmid (pJNK-Luc) (kindly provided by Harvard Medical School); endotoxin-free plasmid extraction kit (CW0504, Kangwei Century Biotechnology Co., Ltd., Beijing, China); other chemical reagents were domestically produced and of analytical grade unless otherwise specified.

[0046] 1.2 The experimental instruments involved in this invention include: S1000 TMThermal Cycler PCR instrument, ChemiDoc XRS + Gel imaging analysis system (Bio-Rad Biomedical Products, Inc., USA); DYY-III electrophoresis instrument (Beijing Liuyi Instrument Factory); Nanodrop 2000 ultra-micro spectrophotometer, Flash microplate reader (Thermo Fisher Scientific, USA); DMIL microscope, DMI 4000B inverted phase-contrast fluorescence microscope (Leica Instruments, Germany); MACO-15ACCO2 incubator (Sanyo Electric Co., Ltd., Japan); SW-CJ-2N clean bench (Harbin Donglian Company).

[0047] 2. Experimental Procedure

[0048] 2.1. Construction of recombinant plasmids for drug screening models

[0049] Using rat genomic DNA as a template and HIF, VEGF, NGF, and TGF-β as examples, primers were designed to introduce MuI and AfIII restriction sites upstream and downstream of the rHIF1a, rVEGF, rNGF, and rTGF-β promoter sequences, respectively. PCR amplification yielded the following promoter fragments: rHIFlaprol (Hp1) (-583 to +23 bp), rHIF1apro2 (Hp2) (+59 to +588 bp), rVEGFpro (Vp) (-1106 to +113 bp), rNGFpro (Np) (-615 to +50 bp), and rTGF-βpro (Tp) (835 bp) (+1 represents the gene transcription start site). The promoter fragments were identified by 1% agarose gel electrophoresis and subsequently recovered by ethanol precipitation. After double enzyme digestion of the vector and each promoter fragment, agarose gel electrophoresis was performed for verification, the target fragment was recovered, the vector and promoter fragment were connected, Escherichia coli was transformed, and ampicillin plate resistance screening was performed. Positive transformants were selected and rapid lysis identification was performed. After the positive ones were expanded and cultured, the plasmids were extracted, enzyme digestion and PCR identification were performed, and the successfully identified plasmids were sequenced to finally obtain the target gene recombinant plasmid.

[0050] 2.2. Plasmid amplification

[0051] Add 0.5 μL of plasmid solution to 50 μL of DH5α competent cell suspension, mix well, place on ice for 30 minutes, then heat shock the suspension at 42°C for 90 seconds, and then quickly place it on ice for 5 minutes. After the ice bath, shake and culture on a constant temperature shaker in an air bath for 1 hour. After the growth state is restored, spread the bacterial solution on a 100 μg ml -1 Then, the transformed strains were selected and inoculated in LB medium containing 100 μg ml -1Amp was cultured in LB liquid medium with shaking overnight, and finally extracted using an endotoxin-free plasmid extraction kit.

[0052] 2.3. Establishment of stable cell lines

[0053] The target gene recombinant plasmids for different diseases, such as pHp1-E2, pHp2-E2, pVp-E2, pNp-E2, pHpl-Luc, pHp2-Luc, pVp-Luc, pNp-Luc, pNFkB-Luc, pERK-Luc, pJNK-Luc, and pTp-Luc, were transfected into human embryonic kidney cells (HEK-293T) respectively. When the confluence reached 70%-90%, plasmid DNA-liposome complexes prepared using Lipofectamine 2000 reagent were added. Subsequently, the cells were trypsinized 36-48 hours after transfection, and according to the transfection efficiency, an appropriate amount was taken and dispersed in culture dishes for culture. When the cells attached to the wall, 800ng·μL was added. -1 Screening begins with G418. After 2-3 weeks, the cells form separate, widely spaced colonies in the culture dish. Select the appropriate cell colony under an inverted microscope and mark the location of the cell colony on the bottom of the culture dish with a marker. Using a strictly aseptic technique, carefully remove all cells around the marked point with a beveled pipette tip. Rinse and remove the scraped cells with culture medium, retain the marked cells, and then add fresh culture medium for culturing. As the selected cell colony gradually expands, detect the presence of foreign cells in real time and remove them promptly. When the cell colony grows to an appropriate size, digest it with an enzyme and dilute it in a gradient into a 96-well plate. After the cells adhere to the wall, select the wells containing single cells under a fluorescence microscope, mark them, and monitor and culture them closely. When the cells grow into clones of appropriate size in the 96-well plate, expand the culture and freeze them promptly.

[0054] 2.4. Cell line culture and functional identification

[0055] All stably transfected cell lines were cultured using 293T culture conditions in a 37°C, 5% CO2 incubator. Cell number and morphology were observed daily. When cells reached 80% confluence, they were trypsinized and passaged or plated in 24-well or 96-well plates as needed. Cells in the logarithmic growth phase were selected for experiments. Blank control and experimental groups were established. Monomers known to be upregulated, such as LPS and CLE, were established as experimental groups and diluted with culture medium to the corresponding concentrations. Different concentrations of the experimental group were added to the experimental group, while the blank control group was added with the corresponding volume of culture medium.

[0056] 2.5. Cellular luciferase activity detection

[0057] Place the cell line to be tested at 4°C and centrifuge at 12,000 × g for 3 minutes to obtain the supernatant. Accurately pipette the supernatant into a well plate, then add the luciferase working solution and mix thoroughly. Incubate at room temperature for 5 minutes to allow the luminescence signal to stabilize. Luciferase activity is then measured using a microplate reader. Protein concentration is then determined using a protein concentration assay kit. A standard curve is plotted to calculate the protein content of the sample. The final luciferase activity is calculated as follows: Relative luciferase activity units = luciferase activity / protein content.

[0058] 2.6. Isolation and Culture of BMSCs

[0059] BMSCs were isolated from 4-6 week old SD male rats. The legs were disinfected with alcohol and the tibia with flesh was removed. The muscle was removed and the epiphyseal segments were cut to expose the epiphyseal cavity. The bone marrow cavity was then repeatedly flushed with L-DMEM until it turned white. The flushing solution was evenly pipetted and cultured. The medium was changed according to the cell adhesion. When the cell confluence reached 85%, the cells were passaged and purified. Specific markers on the surface of BMSCs were detected by flow cytometry. Common positive markers CD29 and CD90 and negative marker CD45 on the surface of BMSCs were selected for measurement to determine whether the cells isolated and extracted from living animals could meet the experimental requirements.

[0060] 2.7. Verification of BMSCs Induction

[0061] When BMSCs reached P3, cells with good morphology and growth were selected and treated with the same drug concentration to create a screening drug group, an upregulation drug control group, and a blank control group. Cells were photographed based on their growth patterns. Subsequently, cells were harvested using Trizol reagent to extract total RNA, and the expression levels of stem cell-specific marker proteins were measured using immunofluorescence techniques.

[0062] Protein immunofluorescence detection

[0063] Cells were fixed with 4% paraformaldehyde, permeabilized with Triton solution, blocked with 5% BSA blocking solution at room temperature for 1 hour, and then incubated with primary antibody overnight. The next day, secondary antibody was incubated at room temperature in the dark for 2 hours, followed by rinsing with PBS solution.

[0064] Statistical analysis

[0065] Graphpad Prism 9.0, SPSS 25.0, and ImageJ-Fiji 20240425 were used to analyze the experimental data. The results are expressed as mean ± standard deviation (x ± s). The significance was determined by a two-sided t-test. P < 0.05 indicated a significant difference between the two groups of data and was statistically significant.

[0066] 3. Experimental Conclusion

[0067] 3.1 Establishment and functional identification of molecular targeted drug screening models validated by stem cell models

[0068] The inventors have successfully constructed drug screening models targeting brain neurotrophic factors, vascular endothelial growth factors, and lung inflammatory factors. These models can accurately screen and verify the efficacy of different disease characteristics and pathological features, and simultaneously measure the target and efficacy during the screening process ( Figure 1 The present invention constructs two reporter gene systems, wherein the reporter gene E2 is a red fluorescent protein, which is used for rapid qualitative observation and judgment; luciferase (LUC) is used for quantitative determination ( Figure 2 A). After the constructed recombinant plasmid was confirmed to be functional by transient transfection, G418 resistance screening was performed to obtain stably transfected monoclonal cell lines 293T-pHp1-E2, 293T-pHp2-E2, 293T-pVp-E2, and 293T-pNp-E2. CoCl2 upregulates hypoxia-inducible factor (HIF) by simulating hypoxia signals, inhibiting hypoxia-inducible factor, and activating the PI3K / Akt signaling pathway; lipopolysaccharide (LPS) upregulates vascular endothelial growth factor (VEGF) by binding to Toll-like receptors (TLR4), initiating mitogen-activated protein kinase (MAPK) and other signaling pathways, inducing cytokine release, and triggering oxidative stress response; Clenbuterol (CLE) upregulates nerve growth factor (NGF) by activating the β-adrenergic receptor signaling pathway, regulating intracellular calcium ion concentration, and inducing differentiation and proliferation of nerve cells. When the corresponding positive stimuli were added, the target gene was upregulated and the expression of red fluorescent protein was significantly increased ( Figure 2 B); The expression of luciferase in LUC cell lines increases with the increase of positive stimulus concentration ( Figure 2 C).

[0069] Example 2: Validation and application of drug efficacy screening model in neurological diseases

[0070] Although some progress has been made in the development of drugs to treat neurological diseases, existing drugs still have many side effects, such as nausea, vomiting, and palpitations after treatment. Ischemic cerebrovascular disease (cerebral ischemia) is a leading cause of death and long-term disability worldwide. Currently, effective treatments for cerebral ischemia primarily involve the body's adaptation to hypoxia / ischemia, angiogenesis, and neuronal protection and regeneration. Numerous studies have shown that VEGF, HIF, and NGF are functional genes associated with cerebral ischemia.

[0071] Traditional Chinese medicine has a long history of treating ischemic cerebrovascular disease. A variety of compound preparations have significant therapeutic effects in preventing ischemic cerebrovascular disease. Based on the construction of a cell screening model targeting VEGF and HIF promoters, the inventors screened out six monomeric compounds derived from different traditional Chinese medicine active ingredients that activate VEGF gene transcription ( Figure 3 A), namely isoliquiritigenin (165%), calendula lactone (156%), crocin (163%), aconitine (217%), gallic acid (154%), and protocatechuic acid (184%), but the above monomers had no obvious upregulation effect on HIF gene transcription.

[0072] Modern medical pharmacological research has confirmed the role of Tibetan medicine in treating cerebral ischemia. Twenty-five Coral Pills, Ruyi Treasure Pills, and Twenty Agarwood Pills are all classic Tibetan medicine prescriptions. In clinical practice, a combination of these three prescriptions is often used to treat neurological diseases, with significant efficacy. The inventors extracted and isolated 17 chemical monomer components from these three classic Tibetan medicine prescriptions and screened their activity. They found that monomers numbered 2, 4, 5, 6, 9, and 12 could upregulate HIF gene expression ( Figure 3 B), monomers numbered 1, 3, 4, 7, 9, 11, and 12 can upregulate the VEGF gene ( Figure 3 C). Ellagic acid (EA) is derived from the classic Tibetan medicine compound Twenty-five Flavors Pearl Pills for treating cardiovascular and cerebrovascular diseases. Through analysis of blood components, the inventors found that EA is a monomer with a relatively high content in the compound, but 80 μmol·L -1 EA within the concentration range has no significant promoting effect on the proliferation and growth of microglia and neurons. The existing neural cell models cannot clearly define the pharmacological potential of EA and its related mechanism of action. After constructing a cell screening model with NGF and VEGF promoters as the target, the inventors found that EA can significantly upregulate the expression of NGF and VEGF ( Figure 3 This result shows that EA has the potential for neuroprotection and plays a positive auxiliary role in the clinical exploration of the mechanism of action of related diseases, and is expected to provide new directions and ideas for subsequent research and treatment.

[0073] Neural stem cells (NSCs) are adult stem cells with multidirectional differentiation potential. They not only have the ability to self-renew, but can also produce neurons and astrocytes through proliferation and differentiation, thereby playing a role in repairing neural tissue damage. Studies have shown that effectively upregulating the transcription level and protein expression of NGF can significantly promote the proliferation activity of NSCs and guide their directional differentiation into functional nerve cells. Modern pharmacological studies have found that Alpinia oxyphylla has pharmacological effects of neuroprotection, including promoting sleep, sedation and combating nerve damage. The inventors conducted a preliminary screening of six main ingredients extracted from Alpinia oxyphylla, chrysin, and jasminoides. The results showed that Alpinia oxyphylla, chrysin, and jasminoides all had an upregulating effect on NGF gene transcription ( Figure 3 FH), and based on this, the initial screening of drugs and co-incubation with BMSCs revealed that chrysin and oleanolic acid could upregulate the expression of neuronal and glial cell marker proteins, and had a certain ability to induce BMSCs to differentiate into neural cells ( Figure 3 IJ).

[0074] Example 3: Validation and application of drug screening model in cardiovascular disease

[0075] Atherosclerosis is the pathological basis for cardiovascular and cerebrovascular diseases such as coronary heart disease and myocardial infarction, and is also an important cause of death in patients. A large amount of research data shows that there are three important typical signaling pathways in the pathogenesis of atherosclerosis: nuclear factor κB (NFκB), extracellular regulated kinase (ERK), and c-Jun amino-terminal kinase (INK). Based on this, a screening model with anti-atherosclerotic disease characteristics was constructed. A large amount of research data shows that Fritillaria, nuts, mangoes and Astragalus have certain effects in preventing and treating cardiovascular diseases. Therefore, the inventors extracted ten related Chinese medicine monomer components from the above natural medicinal materials and screened out four monomer compounds, namely, ligustrazine, CB-301, formononetin and CB-202, which have significant inhibitory effects on JNK ( Figure 4 A); CB-301, fritillin B, calycosin and β-sitosterol inhibit ERK expression ( Figure 4 B); and four monomeric compounds, β-sitosterol, CB-202, CB-301 and calycosin isoflavone glycosides, which inhibit NFkB expression ( Figure 4 C).

[0076] Vascular regeneration and repair is a complex biological process involving a complex regulatory network of multiple factors and cells. α-Smooth muscle actin (a-SMA) and VEGF are specific marker genes for smooth muscle cells and vascular endothelial cells, respectively. Platelet endothelial cell adhesion molecule (CD31) is often present in tight junctions between endothelial cells. The temporal expression of these three specific marker genes, α-SMA, VEGF, and CD31, can, to a certain extent, reflect the entire process of angiogenesis. Ruyi Zhenbao Pills, Ershiwuwei Shanhu Pills, and Ershiwei Chenxiang Pills, based on the clinical characteristics of different pathological stages of ischemic stroke (acute, recovery, and sequelae), adopt a sequential combination administration regimen in the early, middle, and late stages. This approach achieves the therapeutic goal of improving brain tissue blood perfusion through a triple mechanism of action: improving microcirculatory disorders in the acute phase of blood stasis, regulating vasomotor function, and increasing cerebral blood flow. The inventors selected ten drug monomer compounds from the above three classic Tibetan medicine prescriptions and found that four compounds, namely, safflower glycoside II, costus lactone and mangiferin, could significantly enhance the transcription and expression of the reporter gene luciferase regulated by the VEGF promoter ( Figure 4 D), and qPCR revealed that the four monomeric compounds could promote BMSCs to transcribe and synthesize α-SMA, VEGF, and CD31 gene mRNA to varying degrees, among which α-SMA gene ( Figure 4 E) The relative expression level of BMSCs was the best when induced by drugs for 4 days, while VEGF ( Figure 4 F) and CD31 gene ( Figure 4 The optimal induction time for G) is 2 days. A large amount of research data shows that simvastatin promotes angiogenesis and repair by regulating endothelial progenitor cells and promoting the proliferation and migration of smooth muscle cells. Therefore, it is used as a positive control in drug screening. Immunofluorescence was used to detect the expression of α-SMA, VEGF and CD31 proteins in situ in BMSCs ( Figure 4 HJ), and the corresponding fluorescence intensity was analyzed and quantified ( Figure 4 KM). It was found that compared with the blank control group, after 4 days of induction of BMSCs with safflower glycosides I and II, costunolide, and mangiferin, all significantly promoted the expression of α-SMA protein in BMSCs. Among them, safflower glycosides II and costunolide significantly promoted the expression of α-SMA protein in BMSCs. After 2 days of drug induction of BMSCs, all of them could significantly upregulate the protein expression of VEGF and CD31.

[0077] Based on existing pharmacological research evidence, simvastatin regulates the function of endothelial progenitor cells through multiple pathways, synchronously activates the proliferation and migration of smooth muscle cells, and exhibits significant efficacy in promoting angiogenesis and tissue repair. Therefore, it has been established as a standard positive control drug in angiogenesis research. The present invention uses immunofluorescence labeling technology to systematically evaluate the in situ expression of α-SMA, VEGF and CD31 key proteins in BMSCs ( Figure 4 HJ), and the fluorescence intensity was quantitatively analyzed ( Figure 4 Experimental data showed that compared with the blank control group, BMSCs treated with the four active ingredients, saffron, crocin IⅡ, costunolide, and mangiferin, exhibited differentiated differentiation-promoting effects: within a 4-day induction period, all tested compounds were able to effectively induce BMSCs to highly express α-SMA protein, with saffron, crocin II, and costunolide groups showing statistically significant differences. On the other hand, just 2 days of drug intervention significantly upregulated the protein expression levels of two angiogenesis markers, VEGF and CD31. Among them, all four compounds were able to extremely significantly promote the expression of VEGF protein in BMSCs, while costunolide and crocin IⅡ were able to extremely significantly promote the expression of CD31 protein in BMSCs. Compared with the positive control simvastatin, costunolide also showed significant differences in the expression of CD31 protein in BMSCs.

[0078] Example 4: Validation and Application of Drug Screening Model for Pulmonary Diseases

[0079] Acute lung injury is a common respiratory disease in clinical practice, and it is also a common critical illness with a high mortality rate. Acute lung injury is often accompanied by symptoms such as inflammation, diffuse alveolar damage and pulmonary edema, and may even lead to acute respiratory distress syndrome (ARDS). Research data show that transforming growth factor (TGF-β) activates downstream SMAD proteins (SMAD2 / 3) by binding to cell surface receptors (TβRI / III), forms a complex and then transfers to the cell nucleus to regulate the transcription of target genes (a-SMA). The activation of ARDS can lead to pulmonary fibrosis, inflammatory cascades and alveolar epithelial damage. The inventors established a TGF-β cell screening model by constructing the TGF-β core promoter region into a luciferase reporter gene vector and transfecting 293T cells. Figure 5A). Tibetan and Mongolian medicine usually uses sea buckthorn to treat lung diseases, lung heat, cough and asthma, etc. On the basis of clinical compatibility, a new prescription, Shagan Qingfei Jiedu Granules, is formed. This prescription has the effects of clearing heat and removing phlegm, moistening the lungs and discharging pus. It has been used for many years for various lung diseases caused by lung heat with stable efficacy. The inventors systematically screened and evaluated the nine main monomer components in Shagan Qingfei Jiedu Granules (salidroside, glycyrrhizic acid, kaempferol, liquiritin, ammonium glycyrrhizate, gallic acid, chlorogenic acid, isoliquiritin, and cinnamaldehyde). The experimental results showed that the three compounds, salidroside, isoliquiritin and cinnamaldehyde, had a significant inhibitory effect on TGF-β promoter activity. Among them, the inhibitory activity of isoliquiritin was particularly prominent. After treatment, the activity of TGF-β promoter only maintained 75% of the baseline level ( Figure 5 B) The inventors noted that under the same experimental conditions, the inhibitory potency of isoliquiritin was more significant than that of other active ingredients, demonstrating its potential superior pharmacological activity in regulating the TGF-β signaling pathway.

[0080] Based on this, this application uses in vitro experiments to explore whether isoliquiritin can inhibit the expression of inflammatory factors and reduce cell apoptosis by inhibiting the activity of TGF-β in mouse alveolar epithelial cells. First, the optimal induction conditions of the LPS-induced alveolar epithelial cell injury model were explored. The experimental results showed that 10μg·ml -1 The optimal induction condition was when LPS was incubated with cells for 24 h. Figure 5 CD). Subsequently, the effect of isoliquiritin on TGF-β / SMAD pathway proteins was investigated. Compared with the control group, the levels of TGF-β and SMAD2 / 3 in the LPS-induced model group were significantly increased, while the SMAD level was significantly decreased. This indicates that the TGF-β / SMAD pathway plays an important role in the LPS-induced alveolar epithelial cell injury model ( Figure 5 E). Compared with the model group, low concentration of isoliquiritin significantly downregulated TGF-β and upregulated SMAD7, while high concentration of isoliquiritin downregulated SMAD2 / 3 levels ( Figure 4 E). The above results show that isoliquiritin can significantly reduce the level of TGF-β, and play a protective role against alveolar epithelial cell damage by downregulating SMAD2 / 3 levels and upregulating SMAD7 content.

[0081] In summary, the present invention has created a drug screening model based on stem cell model verification, "molecular targeted screening + stem cell directed induction". At present, the construction of drug screening models (neurotrophic factors, vascular endothelial growth factors and lung inflammatory factors) has been completed, which is used for high-throughput screening and verification of active ingredients for different targets and marker proteins.

[0082] Given the complex composition, numerous trace elements, and unclear identification of active ingredients in traditional Chinese medicines (TCMs), serum pharmacochemistry methods offer an effective approach. This approach can track active ingredients in compound formulas that enter the bloodstream or directly identify key components within a compound. Using established stable cell lines, high-throughput screening of these key components is performed. First, cytotoxicity assays are used to determine the optimal concentration of the component, and the stably transfected cell lines are stimulated. Subsequently, fluorescence intensity or luciferase activity is measured to preliminarily identify the active ingredient. Following this initial screening, the selected components are validated through stem cell-directed induction to investigate the expression and secretion of relevant trophic factors. Finally, based on the predicted results of the model, animal models are constructed to verify the efficacy of the selected components. This completes a systematic drug screening and validation process from the cellular to the animal level, providing a scientific basis and effective method for identifying the active ingredients of TCMs and compound formulas.

[0083] The drug screening model constructed by the present invention has the ability to predict the material basis of drug efficacy, and its characteristics are significant, mainly covering the following five aspects: First, the model requires a small amount of test sample during the screening process, which is conducive to appropriately increasing biological replicates for high-throughput screening, effectively improving the precision of drug screening. Second, the model relies on the fluorescent protein reporter gene system and the luciferase reporter gene system, and can use common, simple and fast detection technologies to efficiently carry out qualitative and quantitative analysis. In this process, the dual results presented by fluorescence intensity and enzyme activity can confirm each other, thereby enhancing the accuracy of the screening results. Third, given that the plasmids involved in the model are transformable, it is possible to construct drug screening models for various disease targets, thereby evaluating drug efficacy, and showing a wide range of applicability in practical applications. Fourth, with the predictive advantage possessed by the model, the use of experimental animals and the cost required for clinical trials in the future can be effectively controlled, reducing R&D costs. Fifth, by leveraging the development-dependent biological information during the directed differentiation of stem cells, more potential drug efficacy material bases can be further excavated.

[0084] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for constructing a molecular targeted drug screening model for stem cell model verification, characterized in that: The following steps are involved: (1) Cloning the key gene promoter related to the disease treated by the drug to be screened; (2) Connecting the cloned gene promoter to the quantitative reporter gene to construct a recombinant plasmid; (3) Transfecting the recombinant plasmid into a cell line, and obtaining a cell line stably expressing the reporter gene through resistance screening, thus obtaining a molecular targeted drug screening model for primary screening; (4) Based on the preliminary test results, the test monomer is co-incubated with stem cells for 3-14 days to detect protein and gene expression to verify the efficacy of step (3). (5) Different monomer compounds have the same dosage but different expressions, which can distinguish the strength of the monomer's predicted efficacy.

2. The construction method according to claim 1, wherein In step (1), the diseases include neurological diseases, cardiovascular diseases, liver damage, lung diseases and immune diseases.

3. The construction method according to claim 1, wherein In step (2), the quantitative reporter gene includes a luciferase gene or a red fluorescent protein gene.

4. The construction method according to claim 1, wherein In step (3), the cell line includes human embryonic kidney cells HEK-293T.

5. A molecular targeted drug screening model validated by a stem cell model constructed using the construction method according to any one of claims 1 to 4, characterized in that: The molecular targeted drug screening model verified by the stem cell model can perform high-throughput screening and verification of active ingredients for different targets and marker proteins.

6. A method for drug screening, characterized in that: The method comprises the steps of screening candidate drugs using the molecular targeted drug screening model verified by the stem cell model according to claim 5, and evaluating the efficacy of the drug by detecting the expression of the reporter gene.

7. The method according to claim 6, wherein The method comprises the following steps: S1 uses a cytotoxicity experiment to determine the optimal concentration of the candidate drug, and stimulates the molecular targeted drug screening model verified by the stem cell model; S2 preliminarily screens the active ingredients by detecting fluorescence intensity or luciferase activity; S3 After the initial screening, the selected components are verified by stem cell-directed induction to examine whether there is expression and secretion of relevant nutritional factors; S4 Based on the above results, an animal model was constructed to verify the efficacy of the screened ingredients.

8. Use of the stem cell pattern-validated molecular targeted drug screening model as claimed in claim 5 in screening drugs for treating specific diseases.

9. The use according to claim 8, characterized in that The molecular targeted drug screening model verified by the stem cell pattern contains key genes related to specific diseases, and the drugs for treating specific diseases can affect the key genes.

10. The use according to claim 9, characterized in that The specific diseases include neurological diseases, cardiovascular diseases, liver damage, lung diseases and immune diseases.

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