A computer-aided method, system, or device based on Astragalus membranaceus and CAMK2B

Abstract

This invention discloses a computer-aided method, system, or device based on Astragalus membranaceus and CAMK2B. It also provides a drug screening method to screen drugs that enhance the expression of CAMK2B by Astragalus membranaceus, a method for regulating CAMK2B expression in in vitro cells, a computer-aided drug screening method based on Astragalus membranaceus and CAMK2B, a computer-aided drug screening system based on Astragalus membranaceus and CAMK2B, a computer-aided drug screening device based on Astragalus membranaceus and CAMK2B, and corresponding computer-readable storage media. Furthermore, it provides applications related to Astragalus membranaceus and CAMK2B.

Description

Technical Field

[0001] This invention belongs to the field of computer-aided design and relates to a computer-aided method, system or device based on Astragalus membranaceus and CAMK2B. Background Technology

[0002] The impact of high-altitude environments on sleep: Low air pressure and low oxygen content at high altitudes can easily lead to hypoxia in the human body. Hypoxia can damage brain cells and cerebral cortex cells, disrupt neural regulation, and consequently trigger a series of sleep problems. Furthermore, reports indicate that the longer the stay at high altitude and the higher the altitude, the worse the sleep quality. The most direct evidence is that oxygen therapy can significantly improve sleep quality and sleep structure.

[0003] Calcium / calmodulin-dependent protein kinase type II (CaMKII) is known to bind with some calcium... 2+ The interaction-dependent hyperpolarization pathway (CaMKII) is also a well-known protein family that can bind to NMDARs or L-type Ca2+ in response to Ca2+. 2+ (Channel influx). CaMKII consists of 12 catalytically active subunits, which are composed of four different subunits (CaMKIIα[Camk2a], CaMKIIβ[Camk2b], CaMKIIγ[Camk2g], and CaMKIIδ[Camk2d]), and their kinase activity is Ca 2+ rely on. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solution:

[0005] This invention proposes a drug screening method for detecting drugs that enhance the expression of CAMK2B by Astragalus membranaceus, the drug screening method comprising:

[0006] The test compound was added to cells treated with Astragalus membranaceus, and the expression level of CAMK2B in the cells was detected. The detected expression level was compared with the CAMK2B expression level obtained without the test compound. The test compound was determined to be either a drug that promotes the enhancement of CAMK2B expression by Astragalus membranaceus or a drug that inhibits the enhancement of CAMK2B expression by Astragalus membranaceus.

[0007] Furthermore, the method for determining whether the test compound is a drug that enhances the expression of CAMK2B by Astragalus membranaceus is that the expression level of CAMK2B after adding the test compound is higher than the expression level of CAMK2B when the test compound is not present.

[0008] Furthermore, the method for determining whether the test compound is a drug that inhibits the enhancing effect of Astragalus on CAMK2B expression is that the CAMK2B expression level detected after adding the test compound is lower than the CAMK2B expression level when the test compound is not present.

[0009] Furthermore, the Astragalus membranaceus includes Astragalus membranaceus extract and Astragalus membranaceus active ingredients;

[0010] Furthermore, the compounds include small molecule compounds and macromolecule compounds;

[0011] Furthermore, the compounds include protein analogs, antibodies, DNA, and RNA;

[0012] Furthermore, the DNA includes single-stranded DNA, closed circular DNA, and linker DNA;

[0013] Furthermore, the RNA includes mRNA, tRNA, rRNA, snRNA, hRNA, antisense RNA, tCRNA, dsRNA, SCRNA, catalytically active RNA, and various viral RNAs;

[0014] Furthermore, the DNA includes DNA in various conformations;

[0015] Furthermore, the antibody includes dAb, Fab, Fab', scFv, Fv, disulfide-bonded Fv, or contains a single immunoglobulin variable domain;

[0016] Furthermore, the protein analogs include proteins artificially synthesized through protein engineering.

[0017] This invention proposes a method for regulating CAMK2B expression in in vitro cells, the method comprising adding Astragalus membranaceus into in vitro cells;

[0018] Furthermore, the cells include brain cells.

[0019] This invention proposes a computer-aided drug screening method based on Astragalus membranaceus and CAMK2B, the method specifically comprising:

[0020] Obtain data on the effective components and CAMK2B gene of Astragalus membranaceus;

[0021] The spatial structures of the effective components of Astragalus membranaceus and the CAMK2B gene were selected to determine their binding sites as the binding sites for targeted drugs.

[0022] Candidate drugs targeting the binding site were obtained using a computer-aided drug screening method.

[0023] Furthermore, the process of the computer-aided drug screening method is as follows:

[0024] To obtain the binding site between the effective components of Astragalus membranaceus and the CAMK2B gene;

[0025] Based on the spatial structure of the binding site between the active ingredient of Astragalus membranaceus and the CAMK2B gene, compounds with similar structures were screened from molecular databases.

[0026] The selected compounds were molecularly docked with the CAMK2B gene to calculate the binding energy score of the target compounds, and the candidate drugs were obtained by sorting them according to the score.

[0027] Furthermore, the gene includes DNA or RNA;

[0028] Furthermore, the computer-aided drug screening is based on the spatial structure of the binding site between the active ingredient of Astragalus membranaceus and the CAMK2B gene to screen for protein analogs / antibodies / RNA / DNA drugs; or to screen for protein / antibody / RNA / DNA drugs similar to the active ingredient of Astragalus membranaceus to obtain candidate drugs;

[0029] Furthermore, computer-aided drug screening uses the spatial structure or target of the binding site between the active ingredient of Astragalus membranaceus and the CAMK2B gene to screen small molecule enhancers and obtain candidate drugs.

[0030] This invention proposes a computer-aided drug screening system based on Astragalus membranaceus and CAMK2B, comprising:

[0031] Data acquisition unit: Acquire data on effective components and CAMK2B gene from Astragalus membranaceus;

[0032] Site determination unit: Select the spatial structure of the binding site between the effective components of Astragalus membranaceus and the CAMK2B gene, and determine the binding site as the binding site for targeted drugs;

[0033] Drug screening unit: Uses computer-aided drug screening methods to obtain candidate drugs that target the binding site.

[0034] This invention proposes a computer-aided drug screening device based on Astragalus membranaceus and CAMK2B, comprising:

[0035] The system includes a memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, which, when executed, implement the aforementioned drug screening method, the aforementioned method for regulating CAMK2B expression in in vitro cells, or the aforementioned computer-aided drug screening method.

[0036] This invention proposes a computer-readable storage medium having a computer program thereon, comprising:

[0037] When the computer program is executed by the processor, it implements the drug screening method described above, the method for regulating CAMK2B expression in in vitro cells described above, or the computer-aided drug screening method described above.

[0038] This invention proposes the following applications of Astragalus membranaceus and CAMK2B:

[0039] 1) Application in the preparation of drugs for regulating sleep disorders;

[0040] 2) Application in screening candidate drugs for regulating sleep disorders;

[0041] 3) Application in the preparation of drugs that regulate or assist in regulating the expression level of CAMK2B;

[0042] Furthermore, the drug includes pharmaceutically acceptable excipients;

[0043] Furthermore, the drug may exist in a pharmaceutically acceptable salt form;

[0044] Furthermore, the sleep disorder is a sleep disorder caused by hypoxia;

[0045] Furthermore, the formulation types of the drug include dosage forms suitable for injection, dosage forms suitable for oral administration, ointments, creams or lotions suitable for topical administration, delivery dosage forms suitable for use as eye drops, aerosol forms suitable for inhalation administration, and dosage forms suitable for parenteral administration.

[0046] In some embodiments, the formulation types of the drug include dosage forms suitable for injection, dosage forms suitable for oral administration (such as capsules, tablets, pouches, elixirs), ointments, creams or lotions suitable for topical administration, delivery dosage forms suitable for use as eye drops, aerosol forms suitable for inhalation (such as intranasal or oral inhalation), and dosage forms suitable for parenteral administration, i.e., subcutaneous, intramuscular or intravenous injection.

[0047] This invention proposes a drug obtained based on the aforementioned drug screening method, the aforementioned method for regulating CAMK2B expression in in vitro cells, or the aforementioned computer-aided drug screening method.

[0048] Advantages of this invention:

[0049] 1. Based on the relationship between Astragalus membranaceus and CAMK2B expression, computer-based virtual drug screening can help improve the screening efficiency of enhancement drugs and shorten the screening cycle.

[0050] 2. Screen small molecule compounds by spatial structure or protein analogs / antibodies / RNA drugs by protein active sites, or screen protein / antibody / RNA drugs similar to the active ingredients of Astragalus membranaceus to obtain candidate drugs. Screen useful candidate compounds in multiple ways to improve the usability of the screened compounds.

[0051] 3. Astragalus extract plays an important role in regulating hypoxia-induced sleep disorders by promoting the expression of CAMK2B. The spatial structure and target sites formed based on the relationship between Astragalus extract and CAMK2B, or the candidate drugs obtained through screening, have important clinical significance. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 A schematic diagram of a computer-aided drug screening method based on Astragalus membranaceus and CAMK2B provided in an embodiment of the present invention;

[0054] Figure 2 A schematic diagram of a computer-aided drug screening system based on Astragalus membranaceus and CAMK2B provided in an embodiment of the present invention;

[0055] Figure 3 A schematic diagram of a computer-aided drug screening device based on Astragalus membranaceus and CAMK2B provided for an embodiment of the present invention;

[0056] Figure 4 The image shows the results of Western blot analysis of CAMK2B-specific expression in the brain induced by hypoxia exposure.

[0057] Figure 5 Statistical results of CAMK2B-specific expression in the brain induced by hypoxia exposure;

[0058] Figure 6 The image shows the results of Western blotting (WB) of the brain inducing specific expression of CAMK2B in the brain after administration of Astragalus membranaceus extract followed by hypoxia.

[0059] Figure 7 The image shows the Western blot results of brain cAMP pathway expression induced by hypoxia exposure after administration of Astragalus membranaceus extract.

[0060] Figure 8 A statistical graph showing the results of PKA expression in the brain induced by hypoxia exposure after administration of Astragalus membranaceus extract;

[0061] Figure 9Statistical graph showing the results of P-CREB expression in the brain induced by hypoxia exposure after administration of Astragalus membranaceus extract;

[0062] Figure 10 A statistical graph showing the results of CREB expression in the brain induced by hypoxia exposure after administration of Astragalus membranaceus extract. Detailed Implementation

[0063] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0064] In some of the processes described in the specification, claims, and accompanying drawings of this invention, multiple operations appearing in a specific order are included. However, it should be clearly understood that these operations may not be executed in the order they appear herein, or may be executed in parallel. The operation numbers, such as 101, 102, etc., are merely used to distinguish different operations and do not represent any execution order. Furthermore, these processes may include more or fewer operations, and these operations may be executed sequentially or in parallel. It should be noted that the descriptions such as "first," "second," etc., in this document are used to distinguish different messages, devices, modules, etc., and do not represent a sequential order, nor do they limit "first" and "second" to different types.

[0065] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0066] Figure 1 This invention provides a schematic flowchart of a computer-aided drug screening method based on Astragalus membranaceus and CAMK2B, which specifically includes:

[0067] S101: Obtain data on the effective components and CAMK2B gene of Astragalus membranaceus;

[0068] In some embodiments, Astragalus is the dried root of *Astragalus membranaceus* (Fisch.) Bge. var. *mongholicus* (Bge.) Hsiao or *Astragalus membranaceus* (Fisch.) Bge., belonging to the Fabaceae family. It is harvested in spring and autumn, with fibrous roots and root heads removed, and then sun-dried. The root is cylindrical, sometimes branched, thicker at the upper end, 30–90 cm long, and 1–3.5 cm in diameter. The surface is light brownish-yellow or light brownish-brown, with irregular longitudinal wrinkles or grooves. It is hard and tough, not easily broken, with a strongly fibrous and powdery fracture surface. The cortex is yellowish-white, the xylem is light yellow, with radial striations and fissures. Older roots may occasionally have a decayed, dark brown, or hollow center. It has a faint odor, a slightly sweet taste, and a slightly beany flavor when chewed. It enters the Lung and Spleen meridians. Its properties are sweet and warm. Functions and indications: Tonifies Qi and strengthens the exterior, promotes diuresis and eliminates toxins, drains pus, and promotes wound healing and tissue regeneration. Used for Qi deficiency and fatigue, poor appetite and loose stools, sinking of middle Qi, chronic diarrhea and rectal prolapse, hematochezia and metrorrhagia, spontaneous sweating due to exterior deficiency, edema due to Qi deficiency, carbuncles that are difficult to heal, chronic ulcers that do not heal, anemia and chlorosis, internal heat and thirst; proteinuria in chronic nephritis, and diabetes.

[0069] In some embodiments, the effective components of Astragalus membranaceus are astragalus polysaccharides, saponins, and flavonoids, such as astragalus polysaccharides, astragalus saponins, and astragaloside A.

[0070] In some embodiments, CAMK2B is a calcium / calmodulin-dependent protein kinase II beta that functions autonomously after Ca(2+) / calmodulin binding and autophosphorylation, participating in the regulation of dendritic spine and synapse formation, neuronal plasticity, and Ca(2+) transport in the sarcoplasmic reticulum of skeletal muscle. In neurons, it plays a crucial structural role in the remodeling of the actin cytoskeleton during plasticity by binding and binding actin filaments in a kinase-independent manner. This structural function is essential for the proper targeting of CaMK2A, which acts downstream of NMDAR, promoting dendritic spine and synapse formation and maintaining synaptic plasticity, thereby enabling long-term enhancement (LTP) and hippocampal-dependent learning. In developing hippocampal neurons, it promotes dendritic dendrite branching, and in mature neurons, it promotes dendritic remodeling. CAMK2B also regulates the migration of developing neurons.

[0071] The reduced sleep duration observed in Camk2a and Camk2b knockout mice reveals Ca 2+The role of calmodulin-dependent protein kinase II (CaMKII)α / CAMKIIβ as a sleep-promoting kinase. Importantly, activation or inhibition of CaMKIIβ can increase or decrease sleep duration by nearly two-fold, thus supporting the role of CaMKIIβ as a core sleep regulator in mammals. This sleep regulation depends on the kinase activity of CaMKIIβ in excitatory neurons. Furthermore, CaMKIIβ mutants mimicking different phosphorylation states can regulate various sleep steps, including sleep induction, sleep maintenance, and sleep cancellation.

[0072] S102: Select the spatial structure of the effective components of Astragalus membranaceus and the spatial structure of the CAMK2B gene, and determine the binding sites of the two as the binding sites of the targeted drug.

[0073] In some embodiments, virtual screening is an important method in targeted drug design. The process is roughly as follows:

[0074] First, based on the known spatial structures of the drug and target sites, the binding modes between the drug and target sites are studied to identify key amino acid residues. This step helps to understand how the drug interacts with the target site and provides a basis for subsequent virtual screening.

[0075] Next, computer-aided drug design methods are used to virtually screen a large number of small molecule compounds. During this process, the binding affinity of small molecule compounds to target sites is predicted and evaluated. Typically, small molecules with low binding energies and good shape complementarity are selected as potential drug candidates.

[0076] Finally, the selected candidate drugs were further experimentally validated to confirm their interaction with the target site and their biological activity.

[0077] In some embodiments, in virtual screening, the binding site refers to the region on the target site that binds to a small molecule compound. To perform receptor-based virtual screening, the binding site of the target site must first be determined. This is typically achieved through analysis and study of the structure of the target site.

[0078] Once the binding site is identified, computer-aided drug design methods can be used to dock compounds from a small molecule library with the target site. During docking, the computer simulates the interaction between the compound and the binding site and evaluates their binding affinity. Based on the docking results, small molecule compounds with strong binding affinity to the target site can be screened as potential drug candidates. The determination of the binding site in virtual screening is crucial to the success of drug design. Therefore, multiple factors need to be considered when determining the binding site, such as the structural characteristics of the target site and the binding modes of known ligands. Simultaneously, the binding site needs to be appropriately processed and optimized during virtual screening to improve the accuracy and efficiency of the screening.

[0079] S103: A candidate drug targeting the binding site is obtained by using a computer-aided drug screening method.

[0080] In some embodiments, computer-aided drug screening is a technique that uses computer-aided drug design methods to screen drugs. It can help researchers quickly screen a large number of small molecule compounds for candidate drugs that have strong binding affinity to target sites and potential pharmacological effects.

[0081] In some embodiments, molecular docking is a method for drug design based on the characteristics of the receptor and the interaction between the receptor and drug molecules. It is a theoretical simulation method that primarily studies intermolecular interactions (such as ligand-receptor interactions) and predicts their binding modes and affinities. This method is widely used in the early stages of drug development to help researchers quickly screen compounds with potential pharmacological effects.

[0082] Molecular docking methods primarily focus on spatial matching and energy matching. Spatial matching refers to the geometric complementarity between the drug molecule and the receptor target, while energy matching refers to the minimization of the interaction between the drug molecule and the receptor target. For geometric matching calculations, methods such as lattice calculations and fragment growth are commonly used, while energy calculations employ methods such as simulated annealing and genetic algorithms.

[0083] Based on the degree and method of simplification, molecular docking methods can be divided into rigid docking, semi-flexible docking, and flexible docking. In rigid docking, the conformation of the docking molecules remains unchanged during the calculation; only their spatial position and orientation are altered. Semi-flexible docking allows for partial conformational changes during the calculation. Flexible docking allows for even more conformational changes.

[0084] In some embodiments, the molecular libraries used in virtual screening primarily include the following: ZINC: Contains over 250 million commercially available compounds for small molecule virtual screening. PubChem: Contains bioactive substances for small molecule virtual screening. DrugBank: Contains drugs and small molecules for drug design and discovery. ChEMBL: Contains small molecules for drug discovery and chemical genomics research. ChemDB: Contains a large number of known small molecules for chemical genomics research and drug discovery. HMDB: Contains a large number of known small molecules for chemical genomics research and drug discovery. BindingDB: Contains a large number of known small molecules for chemical genomics research and drug discovery. SMPDB: Contains a large number of known small molecules for chemical genomics research and drug discovery. In addition, some commercial databases such as ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M, and Interbioscreen are also frequently used for virtual screening.

[0085] In some embodiments, during molecular docking, affinity depends on the interactions between the molecule and the receptor, including hydrogen bonds, van der Waals forces, electrostatic interactions, etc. These interactions collectively determine the binding mode between the molecule and the receptor, thereby affecting affinity.

[0086] To assess affinity, scoring systems or methods are typically used to quantify the interaction between molecules and receptors. These scoring methods are based on different algorithms and physical models and can reflect the binding energy, interaction type, and affinity between molecules and receptors.

[0087] In some embodiments, protein analogs refer to substances that have a similar structure and function to a protein in the body. They can mimic the function of proteins, thus playing an important role in disease treatment.

[0088] Antibodies are immunoglobulins produced by plasma cells, differentiated from B lymphocytes, in response to antigen stimulation by the body's immune system. They specifically bind to the corresponding antigens. The structure of an antibody is mainly divided into two parts: a constant region and a variable region. In a given species, the constant regions of different antibody molecules have the same or nearly identical amino acid sequences. The variable regions are located at the ends of the two arms of the "Y" shape.

[0089] RNA drugs are a class of drugs designed based on the properties of RNA and its mechanisms of action within cells. Within cells, RNA can act as messenger RNA (mRNA) to guide protein synthesis, or as microRNA (miRNA) to regulate gene expression. Therefore, well-designed RNA drugs can be used to regulate gene expression within cells, thereby achieving the goal of treating diseases. For example, many studies are exploring the use of mRNA vaccines to prevent and treat various infectious diseases.

[0090] In one embodiment, the general steps for conducting an inhibitory activity experiment on a compound are as follows:

[0091] Selecting appropriate target sites / genes / proteins: Based on research objectives and disease goals, select suitable target sites / genes / proteins as experimental subjects. Ensure that the target sites / genes / proteins have potential interactions with the compounds under study.

[0092] Compound preparation: Synthesize or purchase the desired compound, ensuring its purity and structural accuracy. The compound may be modified or altered as needed to optimize its inhibitory activity.

[0093] Assay: Design appropriate assay methods to evaluate the inhibitory activity of compounds on target sites / genes / proteins. Ensure that the assay methods are reliable, sensitive, and accurately reflect the interaction between the compound and the target site / gene / protein.

[0094] Experimental procedure: Mix different concentrations of the compound with the target site / gene / protein, incubate under appropriate conditions for a period of time, and then measure the results. Depending on the experimental design, control and experimental groups can be set up to compare the inhibitory activity of different compounds.

[0095] Data Analysis: Statistical analysis of experimental data determines the IC50 value of the compound (i.e., the concentration of the compound required to inhibit 50% of the target). The smaller the IC50 value, the stronger the inhibitory activity of the compound. Furthermore, dose-response curves can be plotted to visually demonstrate the inhibitory activity of the compound.

[0096] Results Interpretation and Discussion: Based on the experimental results, the inhibitory activity of the compounds on the target sites / genes / proteins is analyzed, and their structures and properties are discussed. Possible binding modes, mechanisms of action, and comparisons with known inhibitors are explored. Furthermore, the effects of the compounds on cells or organisms can be further investigated to assess their potential drug candidate status.

[0097] Figure 2 This invention provides a schematic diagram of a computer-aided drug screening system based on Astragalus membranaceus and CAMK2B, specifically including:

[0098] Data acquisition unit: Acquire data on effective components and CAMK2B gene from Astragalus membranaceus;

[0099] Site determination unit: Select the spatial structure of the binding site between the effective components of Astragalus membranaceus and the CAMK2B gene, and determine the binding site as the binding site for targeted drugs;

[0100] Drug screening unit: Uses computer-aided drug screening methods to obtain candidate drugs that target the binding site.

[0101] Figure 3 A schematic diagram of a computer-aided drug screening device based on Astragalus membranaceus and CAMK2B provided in this embodiment of the invention specifically includes:

[0102] The system includes a memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, which, when executed, implement the aforementioned drug screening method, the aforementioned method for regulating CAMK2B expression in in vitro cells, or the aforementioned computer-aided drug screening method.

[0103] Example

[0104] 1. Experimental materials

[0105] 1.1 Animals, cells, and drugs

[0106] Animals: C57BL / 6J mice (purchased from Spiford (Beijing) Biotechnology Co., Ltd.).

[0107] Astragalus Clearing Paste: A gift from Sichuan Huakang Youxin Biotechnology Co., Ltd.

[0108] 1.2 Experimental Reagents

[0109] The experimental reagents are shown in Table 1.

[0110] Table 1

[0111]

[0112]

[0113] 1.3 Preparation of experimental buffer solutions and other reagents

[0114] For details on the preparation of 1.5M Tris-HCl (pH 8.8), 1.0M Tris-HCl (pH 6.8), 30% acrylamide solution, 10% SDS solution, 10% APs solution, 1×PBS buffer, 1×TBS buffer, 1×TAE buffer, 1×Running Buffer, and 1×Transfer Membrane Buffer, please refer to "Molecular Cloning: A Laboratory Manual".

[0115] 1.4 Experimental Instruments and Equipment

[0116] Information on the experimental instruments and equipment is shown in Table 2.

[0117] Table 2

[0118]

[0119]

[0120] 2. Experimental Methods

[0121] 2.1 Animals

[0122] C57BL / 6J mice were provided with a 12-hour light and 12-hour dark cycle, with lights turned on at 7:00 AM and off at 7:00 PM daily. The ambient temperature was maintained at 22–24°C. Normal food and water were provided daily. All animal husbandry and experimental procedures adhered to animal ethics and welfare principles.

[0123] 2.2 Western Blot (WB) for Protein Immunoblotting

[0124] a. Quantify the lysed tissue samples using BSA. First, serially dilute the standard and the sample 10-fold (2 μl sample + 18 μl PBS). Based on the sample volume, prepare a mixture of BCA reagent and solution B (50:1), and mix thoroughly at room temperature. Add 200 μL of the mixture to each sample tube and standard tube, incubate at 37°C for 30 min, and then measure the OD value (570 nm) using a microplate reader. Plot a standard curve (R²). 2 >0.99), calibrate the protein concentration of the sample and prepare the sample.

[0125] b. Prepare an 8%-10% gel, with a protein loading volume of 30μL (100μg protein). Select a constant voltage of 60V (10-15min). After the sample enters the separating gel, adjust the voltage to 120-160V. Electrophoresis is stopped when the bromophenol blue reaches the bottom edge of the gel (the entire electrophoresis time is approximately 1.5-2 hours).

[0126] c. Transfer the electrophoresed proteins onto a PVDF membrane (60V constant voltage transfer for 3h). After transfer, remove the membrane and add TBST and shake for 1-2min to remove formaldehyde from the transfer solution. Use TBST to prepare 5% skim milk or 5% BSA as blocking solution, and then place the membrane in the blocking solution and block at room temperature for 1-2h.

[0127] d. Select the appropriate dilution factor according to the primary antibody instructions and dilute with the corresponding antibody blocking solution, then place in a refrigerator at 4°C overnight.

[0128] e. After incubation overnight, remove the primary antibody and wash the membrane three times with 1×TBST for 10 minutes each time. Add the corresponding secondary antibody (rabbit or mouse antibody) diluted according to the ratio and incubate at room temperature for 1-2 hours.

[0129] f. After the secondary antibody incubation is completed, wash the membrane 5 times with 1×TBST for 8 minutes each time, then add ECL developing solution and place it in the developing instrument for development, and save the developed images.

[0130] 2.3 Hypoxia Stress Paradigm

[0131] Mice were kept at an altitude of 6000 meters, subjected to 72 hours of hypoxic stress, and given a 12-hour light-dark cycle. Lights were turned on at 7:00 AM and off at 7:00 PM daily. The ambient temperature was maintained between 22 and 24°C. Mice were provided with normal food and water daily.

[0132] 2.4 Gavage treatment of mice

[0133] After dividing the animals into groups, the treatment group was given 200 μL of Astragalus extract daily. The control group received an equal volume of physiological saline. This treatment continued for two weeks. After the administration was completed, the animals were subjected to hypoxic stress, and changes in relevant indicators were monitored.

[0134] 2.5 Data Processing and Analysis

[0135] The independent samples t-test was used to compare differences between two groups, and the one-way ANOVA was used for three or more groups. Data were presented as follows: Gray-scale analysis and fluorescence quantification were performed using ImageJ. Data were analyzed using SPSS 20.0 and plotted using Graph-pad Prism 6. p < 0.05 was considered statistically significant.

[0136] 3. Experimental Results

[0137] 3.1 Hypoxia exposure induces decreased CAMK2B-specific expression in the brain

[0138] The results are as follows Figure 4 , Figure 5 As shown, the results indicate that, through behavioral studies, sleep was significantly affected by hypoxia stress. The study found that Camk2B molecules were significantly downregulated in brain tissue, and there are literature reports that Camk2B molecules are significantly associated with sleep.

[0139] 3.2 Astragalus extract can significantly alleviate the decrease in CAMK2B-specific expression in the brain induced by hypoxia exposure.

[0140] The results are as follows Figure 6As shown, the results indicated that, through behavioral studies, sleep was significantly affected by hypoxia stress, and Camk2B molecules were found to be significantly downregulated in brain tissue. After administering Astragalus extract to mice via gavage, Camk2B molecules were found to be significantly upregulated, and behavioral studies showed that sleep was significantly restored after administration of the drug to mice.

[0141] 3.3 Astragalus extract alleviates the reduction in specific CAMK2B expression in the brain induced by hypoxia exposure, but this reduction is not achieved through the cAMP pathway.

[0142] The results are as follows Figure 7 , Figure 8 , Figure 9 , Figure 10 As shown, the results indicated that, behavioral studies confirmed that sleep was significantly affected by hypoxia stress, with a significant downregulation of Camk2B molecules in brain tissue. Furthermore, gavage treatment of mice with Astragalus membranaceus extract resulted in a significant upregulation of Camk2B molecules, and behavioral studies showed a significant recovery of hypoxia-stressed sleep in mice after administration. Previous literature has reported that Astragalus membranaceus exerts its effects through the cAMP pathway, which primarily functions through PKA and P-CREBS133 molecules. Therefore, detecting changes in these two molecules was used to confirm whether the cAMP pathway was altered. The experimental results confirmed that the recovery of Camk2B in mice treated with Astragalus membranaceus extract was not achieved through the cAMP pathway.

[0143] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A drug screening method for detecting the enhancing effect of Astragalus extract on CAMK2B expression, the drug screening method comprising: The test compound was added to cells that had been treated with Astragalus extract, and the expression level of CAMK2B in the cells was detected. The detected expression level was compared with the CAMK2B expression level obtained without the test compound. The test compound was identified as either a drug that promotes the enhancement of CAMK2B expression by Astragalus extract or a drug that inhibits the enhancement of CAMK2B expression by Astragalus extract. The method for determining whether the test compound is a drug that enhances the expression of CAMK2B by Astragalus Extract is: the CAMK2B expression level detected after adding the test compound is higher than the CAMK2B expression level when the test compound is not present. The method for determining whether the test compound is a drug that inhibits the enhancing effect of Astragalus extract on CAMK2B expression is that the CAMK2B expression level detected after adding the test compound is lower than the CAMK2B expression level when the test compound is not present.

2. The drug screening method according to claim 1, wherein the compound includes small molecule compounds and macromolecule compounds.

3. The drug screening method according to claim 1, wherein the compound comprises protein analogs, DNA, and RNA.

4. The drug screening method according to claim 1, wherein the compound comprises an antibody.

5. The drug screening method according to claim 3, wherein the DNA comprises single-stranded DNA.

6. The drug screening method according to claim 3, wherein the DNA comprises closed circular DNA.

7. The drug screening method according to claim 3, wherein the DNA comprises linker DNA.

8. The drug screening method according to claim 3, wherein the RNA includes mRNA, tRNA, rRNA, snRNA, hRNA, and SCRNA.

9. The drug screening method according to claim 3, wherein the RNA comprises tCRNA.

10. The drug screening method according to claim 3, wherein the RNA comprises antisense RNA.

11. The drug screening method according to claim 3, wherein the RNA comprises dsRNA.

12. The drug screening method according to claim 3, wherein the RNA comprises RNA with catalytic activity.

13. The drug screening method according to claim 3, wherein the RNA comprises various viral RNAs.

14. The drug screening method according to claim 3, wherein the DNA comprises DNA of various conformations.

15. The drug screening method according to claim 4, wherein the antibody comprises dAb, Fab, Fab', scFv, and Fv.

16. The drug screening method according to claim 4, wherein the antibody comprises a disulfide-bonded Fv.

17. The drug screening method according to claim 4, wherein the antibody comprises a monoimmunoglobulin variable domain.

18. The drug screening method according to claim 3, wherein the protein analogue comprises a protein artificially synthesized by protein engineering.

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