Computer-aided method, system or equipment based on astragalus membranaceus and CAMK2B

Abstract

The invention discloses a computer-aided method, system or equipment based on astragalus membranaceus and CAMK2B, provides a method for screening drugs influencing the enhancement effect of astragalus membranaceus on CAMK2B expression, and provides a method for regulating CAMK2B expression in in-vitro cells. The invention further provides a computer-aided drug screening method based on the astragalus membranaceus and the CAMK2B, a computer-aided drug screening system based on the astragalus membranaceus and the CAMK2B, computer-aided drug screening equipment based on the astragalus membranaceus and the CAMK2B and a corresponding computer readable storage medium. The invention further provides related application of the astragalus membranaceus and the CAMK2B.

Description

Technical Field

[0001] The invention belongs to the field of computer assistance and relates to a computer-assisted method, system or device based on astragalus and CAMK2B. Background Art

[0002] The impact of plateau environment on sleep; the air pressure in high altitude areas is low, and the oxygen content in the air is low, which can easily lead to hypoxia in the human body. Hypoxia in the body can damage brain cells and cerebral cortex cells, disrupt nerve regulation, and then cause a series of sleep problems. In addition, there are reports that the longer the time of moving to the plateau and the higher the altitude, the worse the sleep quality. The most direct evidence is that sleep quality and sleep structure can be significantly improved after oxygen therapy.

[0003] Calcium / calmodulin-dependent protein kinase type II (CaMKII), which is known to bind to some Ca 2+ The interaction-dependent hyperpolarization pathway (i.e., CaMKII) is also a well-known family of proteins that can bind to NMDARs or L-type Ca2+ in response to Ca 2+ CaMKII is composed of 12 catalytically active subunits, which are composed of four different subunits (CaMKIIα [Camk2a], CaMKIIβ [Camk2b], CaMKIIγ [Camk2g], and CaMKIIδ [Camk2d]), whose 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 solutions:

[0005] The present invention provides a method for screening drugs that affect the enhancing effect of Astragalus on CAMK2B expression, and the drug screening method comprises:

[0006] A test compound is added to cells treated with Astragalus, and the expression level of CAMK2B in the cells is detected. The detected expression level result is compared with the CAMK2B expression level obtained in the absence of the test compound, and the test compound is obtained as a drug that promotes the enhancing effect of Astragalus on CAMK2B expression, or a drug that inhibits the enhancing effect of Astragalus on CAMK2B expression.

[0007] Further, the method for determining that the test compound is a drug that promotes the enhancing effect of Astragalus on CAMK2B expression is that the CAMK2B expression level detected after adding the test compound is higher than the CAMK2B expression level in the absence of the test compound;

[0008] Further, the method for determining that 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 in the absence of the test compound;

[0009] Furthermore, the astragalus includes astragalus extract and astragalus effective ingredients;

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

[0011] Further, the compound includes protein analogs, antibodies, DNA, RNA;

[0012] Furthermore, the DNA includes single-stranded DNA, closed-circle DNA, and linked DNA;

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

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

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

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

[0017] The present invention provides a method for regulating CAMK2B expression in in vitro cells, the method comprising adding astragalus to the in vitro cells;

[0018] Furthermore, the cells include brain cells.

[0019] The present invention proposes a computer-assisted drug screening method based on Astragalus and CAMK2B, the method specifically comprising:

[0020] Obtain data on active ingredients in Astragalus and CAMK2B gene data;

[0021] The spatial structure of the active ingredients of Astragalus and the spatial structure of the CAMK2B gene were selected to determine the binding sites of the two as the binding sites of targeted drugs;

[0022] A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

[0023] Further, the method process of computer-assisted drug screening is:

[0024] Obtain the binding site of the active ingredients of Astragalus and the CAMK2B gene;

[0025] Based on the spatial structure of the binding site between the active ingredients of Astragalus and the CAMK2B gene, compounds with similar structures were screened in the molecular database;

[0026] Perform molecular docking between the screened compounds and the CAMK2B gene to calculate the binding energy scores of the targeted compounds, and rank them according to the scores to obtain candidate drugs;

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

[0028] Furthermore, the computer-assisted drug screening screens protein analogs / antibodies / RNA / DNA drugs based on the spatial structure of the binding site between the effective ingredients of Astragalus and the CAMK2B gene; or screens protein / antibody / RNA / DNA drugs similar to the effective ingredients of Astragalus to obtain candidate drugs;

[0029] Furthermore, computer-assisted drug screening was performed to screen small molecule enhancers based on the spatial structure or target of the binding site between the active ingredients of Astragalus and the CAMK2B gene to obtain candidate drugs.

[0030] The present invention provides a computer-assisted drug screening system based on Astragalus and CAMK2B, comprising:

[0031] Data acquisition unit: obtain the data of effective ingredients in Astragalus and CAMK2B gene data;

[0032] Site determination unit: select the spatial structure of the binding site between the effective ingredients of Astragalus and the CAMK2B gene, and determine the binding site as the binding site of the targeted drug;

[0033] Drug screening unit: using computer-assisted drug screening to obtain candidate drugs targeting the binding site.

[0034] The present invention provides a computer-assisted drug screening device based on Astragalus and CAMK2B, comprising:

[0035] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the aforementioned drug screening method, the aforementioned method for regulating CAMK2B expression in in vitro cells, or the aforementioned computer-assisted drug screening method is implemented.

[0036] The present invention provides a computer-readable storage medium having a computer program thereon, comprising:

[0037] When the computer program is executed by a processor, the aforementioned drug screening method, the aforementioned method for regulating CAMK2B expression in in vitro cells, or the aforementioned computer-assisted drug screening method is implemented.

[0038] The present invention proposes the following application of Astragalus 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 for regulating or assisting in regulating the expression level of CAMK2B;

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

[0043] Furthermore, the drug exists in the form of a pharmaceutically acceptable salt;

[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 ingestion, ointments, creams or lotions suitable for topical administration, delivery forms suitable for use as eye drops, aerosol forms suitable for administration by inhalation, 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 ingestion (such as capsules, tablets, caplets, elixirs), ointments, creams or lotions suitable for topical administration, delivery forms suitable for use as eye drops, aerosol forms suitable for administration by inhalation (such as through nasal inhalation or oral inhalation), and dosage forms suitable for parenteral administration, i.e., subcutaneous, intramuscular or intravenous injection.

[0047] The present invention provides a drug obtained based on the above-mentioned drug screening method, the above-mentioned method for regulating CAMK2B expression in in vitro cells, or the above-mentioned computer-assisted drug screening method.

[0048] Advantages of the present invention:

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

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

[0051] 3. Huangqi Qing Gao plays an important role in regulating sleep disorders caused by hypoxia by promoting the expression of CAMK2B. The spatial structure and target sites formed based on the relationship between Huangqi Qing Gao and CAMK2B or the candidate drugs obtained through screening are of great clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the 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 work.

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

[0056] Figure 4 The figure shows the WB detection result of hypoxia exposure-induced CAMK2B specific expression in the brain;

[0057] Figure 5 This is a statistical result diagram showing the specific expression of CAMK2B in the brain induced by hypoxia exposure;

[0058] Figure 6 This is the WB result of the specific expression of CAMK2B in the brain induced by hypoxia exposure after the application of Huangqi Qing Gao;

[0059] Figure 7 This is the WB result of the expression of CAMP pathway in the brain induced by hypoxia exposure after the application of Huangqi Qing Gao;

[0060] Figure 8 This is a statistical chart showing the results of PKA expression in the brain induced by hypoxia exposure after administration of Huangqi Qing Gao;

[0061] Fig. 9This is a statistical chart showing the results of P-CREB expression in the brain induced by hypoxia exposure after administration of Huangqi Qing Gao;

[0062] Fig.10 This is a statistical chart showing the results of CREB expression in the brain induced by hypoxia exposure after administration of Huangqi Qing Gao. DETAILED DESCRIPTION

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

[0064] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish between different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to be different types.

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

[0066] Figure 1 A schematic flow chart of a computer-assisted drug screening method based on Astragalus and CAMK2B provided in an embodiment of the present invention specifically includes:

[0067] S101: Obtain data on active ingredients in Astragalus and CAMK2B gene data;

[0068] In some embodiments, the astragalus is the dried root of the leguminous plant Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. It is excavated in spring and autumn, the fibrous roots and root heads are removed, and the roots are dried in the sun. This product is cylindrical, some with branches, thicker at the upper end, 30 to 90 cm long, and 1 to 3.5 cm in diameter. The surface is light brown or light brown, with irregular longitudinal wrinkles or longitudinal grooves. It is hard and tough, not easy to break, with strong fibrous and powdery cross-section, yellowish-white cortex, light yellow wood, radial texture and cracks, and the center of the old root is occasionally withered, dark brown or hollow. The smell is slight, the taste is slightly sweet, and there is a slight bean smell when chewed. It enters the lung and spleen meridians. It is sweet and warm in nature. Functions and indications: replenishing qi and strengthening the exterior, promoting diuresis and expelling toxins, draining pus, healing sores and promoting tissue regeneration. Used for qi deficiency and fatigue, poor appetite and loose stools, sinking of qi in the middle, chronic diarrhea and prolapse of the anus, bloody stool and metrorrhagia, spontaneous sweating due to exterior deficiency, edema due to qi deficiency, carbuncle that is difficult to heal, long-term ulcers that do not heal, blood deficiency and jaundice, internal heat and thirst; chronic nephritis and proteinuria, diabetes.

[0069] In some embodiments, the effective ingredients of Astragalus are Astragalus polysaccharides, saponins and flavonoids, such as Astragalus polysaccharides, Astragalosides and Astragaloside IV.

[0070] In some embodiments, CAMK2B is a calcium / calmodulin-dependent protein kinase II beta that acts autonomously after Ca(2+) / calmodulin binding and autophosphorylation and is involved in the formation of dendritic spines and synapses, neuronal plasticity, and regulation of skeletal muscle sarcoplasmic reticulum Ca(2+) transport. In neurons, it plays an important structural role in the reorganization of the actin cytoskeleton during plasticity by binding and bundling actin filaments in a kinase-independent manner. This structural function is required for the proper targeting of CaMK2A, which acts downstream of NMDARs to promote the formation of dendritic spines and synapses and maintain synaptic plasticity, thereby achieving long-term potentiation (LTP) and hippocampus-dependent learning. In developing hippocampal neurons, it promotes the arborization of the dendritic tree, 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 a role for Ca 2+The role of CaMKIIβ / calmodulin-dependent protein kinase II (CaMKII)α / CAMKIIβ as a sleep-promoting kinase. Importantly, activation or inhibition of CaMKIIβ can increase or decrease sleep time by nearly two-fold, supporting a role for CaMKIIβ as a core sleep regulator in mammals. This sleep regulation depends on the kinase activity of CaMKIIβ in excitatory neurons. Furthermore, CaMKIIβ mutants that mimic different phosphorylation states can modulate various sleep steps, including sleep induction, sleep maintenance, and sleep abolition.

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

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

[0074] First, based on the known spatial structure of the drug and the target site, the binding mode of the drug and the target site is studied to determine the 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, a large number of small molecule compounds are virtually screened using computer-aided drug design methods. In this process, the binding ability of small molecule compounds to target sites will be predicted and evaluated. Generally, small molecules with low binding energy and good shape complementarity will be selected as potential drug candidates.

[0076] Finally, the screened candidate drugs are further experimentally verified to confirm their interaction with the target site and biological activity.

[0077] In some embodiments, in virtual screening, a binding site refers to an area on a target site that binds to a small molecule compound. In order to perform a receptor-based virtual screening, it is first necessary to determine the binding site of the target site. This is usually achieved by analyzing and studying the structure of the target site.

[0078] Once the binding site is determined, the computer-aided drug design method can be used to dock the compounds in the small molecule compound library with the target site. During the docking process, the computer simulates the interaction between the compound and the binding site and evaluates their binding ability. Based on the docking results, small molecule compounds with strong binding ability to the target site can be screened as potential drug candidates. The determination of virtual screening binding sites is crucial to the success of drug design. Therefore, when determining the binding site, it is necessary to consider a variety of factors, such as the structural characteristics of the target site, the binding mode of known ligands, etc. At the same time, in the virtual screening process, the binding site also needs to be reasonably processed and optimized to improve the accuracy and efficiency of the screening.

[0079] S103: Using a computer-assisted drug screening method to obtain candidate drugs targeting the binding site.

[0080] In some embodiments, computer-aided drug screening is a technology that uses computer-aided drug design methods to screen drugs. It can help researchers quickly screen candidate drugs with strong binding ability to target sites and potential efficacy from a large number of small molecule compounds.

[0081] In some embodiments, molecular docking is a method for drug design based on the characteristics of receptors and the interaction between receptors and drug molecules. It mainly studies the interaction between molecules (such as ligands and receptors) and predicts their binding mode and affinity. This method is widely used in the early stages of drug development and can help researchers quickly screen out compounds with potential efficacy.

[0082] Molecular docking methods mainly 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 the calculation of geometric matching, grid calculation, fragment growth and other methods are usually used, while energy calculation uses simulated annealing, genetic algorithm and other methods.

[0083] According to the degree and method of simplification, molecular docking methods can be divided into rigid docking, semi-flexible docking and flexible docking. In the rigid docking method, the molecular conformations involved in the docking do not change during the calculation process, only the spatial position and posture of the molecules are changed. Semi-flexible docking allows some conformations to change during the calculation. Flexible docking allows more conformational changes.

[0084] In some embodiments, the molecular libraries used in virtual screening are mainly the following: ZINC: contains more than 250 million commercially available compounds for small molecule virtual screening. PubChem: contains biologically active 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, there are some commercial databases such as ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M and Interbioscreen, which are also often used for virtual screening.

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

[0086] In order to evaluate affinity, some scoring systems or scoring methods are usually used to quantify the interaction between molecules and receptors. These scoring methods are based on different algorithms and physical models, which can reflect the binding energy, interaction type and affinity between molecules and receptors.

[0087] In some embodiments, protein analogs refer to substances that have similar structures and functions to certain proteins in the body. They can mimic the functions of proteins and thus play an important role in the treatment of diseases.

[0088] Antibodies are immunoglobulins produced by plasma cells differentiated from B lymphocytes under antigen stimulation, which can specifically bind to the corresponding antigen. The structure of antibodies is mainly divided into two parts: constant region and variable region. In a given species, the constant regions of different antibody molecules have the same or almost the same amino acid sequence. The variable region is located at the ends of the two arms of the "Y".

[0089] RNA drugs are a class of drugs designed based on the properties of RNA and its mechanism of action in cells. RNA can act as messenger RNA (mRNA) to guide protein synthesis in cells, or as microRNA (miRNA) to regulate gene expression. Therefore, rationally designed RNA drugs can be used to regulate gene expression in cells to achieve the purpose 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 performing an inhibitory activity test on a compound are as follows:

[0091] Select appropriate target sites / genes / proteins: Select appropriate target sites / genes / proteins as experimental subjects based on the research objectives and disease targets. Ensure that the target sites / genes / proteins have potential interactions with the compounds being studied.

[0092] Prepare compounds: Synthesize or purchase the desired compounds and ensure their purity and structural accuracy. If necessary, the compounds can be modified or engineered to optimize their inhibitory activity.

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

[0094] Experimental operation: Compounds of different concentrations are mixed with the target site / gene / protein, incubated for a period of time under appropriate conditions, and then measured. Depending on the experimental design, control groups and experimental groups can be set up to compare the inhibitory activity of different compounds.

[0095] Data analysis: The IC50 value of the compound (i.e., the concentration of the compound required to inhibit 50% of the target) is determined by statistical analysis of the experimental data. The smaller the IC50 value, the stronger the inhibitory activity of the compound. In addition, a dose-effect curve can be drawn to intuitively display the inhibitory activity of the compound.

[0096] Interpretation and discussion of results: Based on the experimental results, analyze the inhibitory activity of the compound on the target site / gene / protein and discuss it in combination with its structure and properties. Explore the possible binding mode, mechanism of action and comparison with known inhibitors. In addition, the effect of the compound on cells or organisms can be further studied to evaluate its potential drug candidate.

[0097] Figure 2 A schematic diagram of a computer-aided drug screening system based on Astragalus and CAMK2B provided in an embodiment of the present invention specifically includes:

[0098] Data acquisition unit: obtain the data of effective ingredients in Astragalus and CAMK2B gene data;

[0099] Site determination unit: select the spatial structure of the binding site between the effective ingredients of Astragalus and the CAMK2B gene, and determine the binding site as the binding site of the targeted drug;

[0100] Drug screening unit: using computer-assisted drug screening to obtain candidate drugs targeting the binding site.

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

[0102] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the aforementioned drug screening method, the aforementioned method for regulating CAMK2B expression in in vitro cells, or the aforementioned computer-assisted drug screening method is implemented.

[0103] Example

[0104] 1. Experimental Materials

[0105] 1.1 Animals, cells, and drugs

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

[0107] Astragalus Paste: 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 buffer and other reagents for the experiment

[0114] For 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 the Molecular Cloning Experiment Guide for details.

[0115] 1.4 Experimental instruments and equipment

[0116] The information of 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 given a 12-h light and 12-h dark cycle, with lights on at 7:00 am and off at 7:00 pm every day. The ambient temperature was maintained at 22-24°C. Normal diet and drinking water were given every day. All animal husbandry and experiments followed the principles of animal ethics and welfare.

[0123] 2.2 Western Blot (WB)

[0124] a. Quantify BSA in the lysed tissue samples. First, dilute the standard sample 10 times (2μl sample + 18μl PBS). According to the sample volume, prepare a mixture of BCA reagent solution and B solution (50:1), and shake it evenly at room temperature. Add 200uL of the mixture to each sample tube and standard tube, incubate at 37℃ for 30min, and then use an enzyme reader to detect the OD value (570nm) and draw a calibration curve (R 2 >0.99), calibrate the sample protein concentration and prepare the sample.

[0125] b. Prepare 8%-10% gel, load 30 μL of protein, 100 μg of protein, select constant voltage 60V (10-15min), after the sample enters the separation gel, adjust to 120-160V, and terminate the electrophoresis when bromophenol blue reaches the bottom edge of the gel (the entire electrophoresis time is about 1.5-2 hours).

[0126] c. Transfer the protein after electrophoresis to PVDF membrane (60V constant voltage transfer for 3 hours). After transfer, take out the membrane, add it to TBST and shake it for 1-2 minutes to remove the formaldehyde in the transfer solution. Use TBST to prepare 5% skim milk or 5% BSA as blocking solution, and then put the membrane into the blocking solution and block it at room temperature for 1-2 hours.

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

[0128] e. After overnight incubation, remove the primary antibody and wash the membrane three times with 1×TBST, 10 min each time, add the corresponding secondary antibody (rabbit antibody or mouse antibody) diluted in proportion, and incubate at room temperature for 1-2 h.

[0129] f. After the secondary antibody incubation, wash the membrane 5 times with 1×TBST, 8 min each time, then add ECL developer and place it in a developer, and save the developed image.

[0130] 2.3 Hypoxic stress paradigm

[0131] Altitude 6000 meters. Mice were subjected to hypoxic stress for 72 hours at an altitude of 6000 meters, and given a 12-hour light and 12-hour dark cycle, with lights on at 7:00 am and off at 7:00 pm every day. The ambient temperature was maintained at 22-24°C. Normal diet and drinking water were given every day.

[0132] 2.4 Oral gavage of mice

[0133] After the animals were divided into groups, the treatment group was given 200ul of Huangqi Qing Gao every day. The control group was given an equal amount of normal saline for two weeks. After the treatment, hypoxic stress was performed to detect changes in related indicators.

[0134] 2.5 Data processing and analysis

[0135] The differences between the two groups were compared using the independent sample t test, and the differences between the three groups or more were compared using the one-way ANOVA test. Grayscale analysis and fluorescence quantification were analyzed using ImageJ. Data were statistically analyzed using SPSS20.0 and graphed using Graph-padPrism 6. p<0.05 indicated statistical significance.

[0136] 3. Experimental results

[0137] 3.1 Hypoxia exposure induces a specific decrease in CAMK2B expression in the brain

[0138] The results are as follows Figure 4 , Figure 5 As shown, the results showed that after hypoxic stress, behavioral studies confirmed that sleep was significantly affected after hypoxic stress, and it was found that the Camk2B molecule in the brain tissue was significantly downregulated. There are also literature reports that the Camk2B molecule is significantly correlated with sleep effects.

[0139] 3.2 Huangqi Qing Gao can significantly alleviate the decrease in CAMK2B expression in the brain induced by hypoxia exposure

[0140] The results are as follows Figure 6As shown, the results showed that after hypoxic stress, behavioral studies confirmed that sleep was significantly affected after hypoxic stress, and it was found that the Camk2B molecule in the brain tissue was significantly downregulated. After gavage of Astragalus Paste to mice, it was found that the Camk2B molecule was significantly upregulated, and behavioral tests showed that the hypoxic stress sleep of mice was significantly restored after administration.

[0141] 3.3 Huangqi Qing Gao alleviates hypoxia exposure-induced reduction in brain CAMK2B expression, not through the CAMP pathway

[0142] The results are as follows Figure 7 , Figure 8 , Fig. 9 , Fig.10 As shown, the results showed that after hypoxic stress, behavioral studies confirmed that sleep was significantly affected after hypoxic stress, and it was found that Camk2B molecules in brain tissue were significantly downregulated. After mice were treated with Astragalus Paste by gavage, it was found that Camk2B molecules were significantly upregulated, and behavioral tests showed that the hypoxic stress sleep of mice was significantly restored after administration. It has been reported in the literature that Astragalus can exert its effect through the CAMP pathway, and the CAMP pathway mainly exerts its effect through PKA and P-CREBS133 molecules. Therefore, whether the CAMP pathway has changed is confirmed by detecting whether the two molecules have changed. The experimental results confirmed that the recovery of Camk2B after Astragalus Paste was treated in mice was not achieved through the CAMP pathway.

[0143] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for screening drugs that affect the enhancing effect of Astragalus on CAMK2B expression, the drug screening method comprising: A test compound is added to cells treated with Astragalus, and the expression level of CAMK2B in the cells is detected. The detected expression level result is compared with the CAMK2B expression level obtained in the absence of the test compound, and the test compound is obtained as a drug that promotes the enhancing effect of Astragalus on CAMK2B expression, or a drug that inhibits the enhancing effect of Astragalus on CAMK2B expression.

2. The drug screening method according to claim 1, wherein the method for determining that the test compound is a drug that promotes the enhancing effect of Astragalus on CAMK2B expression is that the CAMK2B expression level detected after adding the test compound is higher than the CAMK2B expression level in the absence of the test compound; Optionally, the method for determining that 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 in the absence of the test compound; Optionally, the astragalus includes astragalus extract and astragalus effective ingredients; Optionally, the compound includes a small molecule compound and a macromolecular compound; Optionally, the compound includes protein analogs, antibodies, DNA, RNA; Optionally, the DNA includes single-stranded DNA, closed-circle DNA, or linked DNA; Optionally, the RNA includes mRNA, tRNA, rRNA, snRNA, hRNA, antisense RNA, tCRNA, dsRNA, SCRNA, catalytically active RNA, and various viral RNAs; Optionally, the DNA includes DNA in various conformations; Optionally, the antibody comprises a dAb, a Fab, a Fab', a scFv, a Fv, a disulfide-bonded Fv, or comprises a single immunoglobulin variable domain; Optionally, the protein analogs include proteins artificially synthesized by protein engineering.

3. A method for regulating CAMK2B expression in in vitro cells, the method comprising adding Astragalus to the in vitro cells; Optionally, the cells comprise brain cells.

4. A computer-assisted drug screening method based on Astragalus and CAMK2B, the method specifically comprising: Obtain data on active ingredients in Astragalus and CAMK2B gene data; The spatial structure of the active ingredients of Astragalus and the spatial structure of the CAMK2B gene were selected to determine the binding sites of the two as the binding sites of targeted drugs; A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

5. The computer-assisted drug screening method according to claim 4, wherein the method process of the computer-assisted drug screening is: Obtain the binding site of the active ingredients of Astragalus and the CAMK2B gene; Based on the spatial structure of the binding site between the active ingredients of Astragalus and the CAMK2B gene, compounds with similar structures were screened in the molecular database; Perform molecular docking between the screened compounds and the CAMK2B gene to calculate the binding energy scores of the targeted compounds, and rank them according to the scores to obtain candidate drugs; Optionally, the computer-assisted drug screening screens protein analogs / antibodies / RNA / DNA drugs based on the spatial structure of the binding site between the effective ingredients of Astragalus and the CAMK2B gene; Or screen for protein / antibody / RNA / DNA drugs similar to the active ingredients of Astragalus to obtain candidate drugs; Optionally, computer-assisted drug screening is performed to screen small molecule enhancers based on the spatial structure of the binding site between the active ingredients of Astragalus and the CAMK2B gene or the target site to obtain candidate drugs.

6. A computer-aided drug screening system based on Astragalus and CAMK2B, comprising: Data acquisition unit: obtain the data of effective ingredients in Astragalus and CAMK2B gene data; Site determination unit: select the spatial structure of the binding site between the effective ingredients of Astragalus and the CAMK2B gene, and determine the binding site as the binding site of the targeted drug; Drug screening unit: using computer-assisted drug screening to obtain candidate drugs targeting the binding site.

7. A computer-aided drug screening device based on Astragalus and CAMK2B, comprising: A memory and a processor, wherein the memory is used to store program instructions; The processor is used to call program instructions, and when the program instructions are executed, the drug screening method described in claim 1 or 2, the method for regulating CAMK2B expression in in vitro cells described in claim 3, or the computer-assisted drug screening method described in claim 4 or 5 is implemented.

8. A computer-readable storage medium having a computer program thereon, comprising: When the computer program is executed by a processor, the drug screening method according to claim 1 or 2, the method for regulating CAMK2B expression in in vitro cells according to claim 3, or the computer-assisted drug screening method according to claim 4 or 5 is implemented.

9. The following applications of Astragalus and CAMK2B: 1) Application in the preparation of drugs for regulating sleep disorders; 2) Application in screening candidate drugs for regulating sleep disorders; 3) Application in the preparation of drugs for regulating or assisting in regulating the expression level of CAMK2B; Optionally, the drug includes a pharmaceutically acceptable excipient; Optionally, the drug exists in a form including a pharmaceutically acceptable salt form; Optionally, the formulation types of the drug include dosage forms suitable for injection, dosage forms suitable for oral ingestion, ointments, creams or lotions suitable for topical administration, delivery forms suitable for use as eye drops, aerosol forms suitable for administration by inhalation, and dosage forms suitable for parenteral administration.

10. A drug obtained based on the drug screening method according to claim 1 or 2, the method for regulating CAMK2B expression in in vitro cells according to claim 3, or the computer-assisted drug screening method according to claim 4 or 5.

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