Screening method and application of traditional Chinese medicine active ingredients of targeted neuron mitochondria

Abstract

The invention relates to a screening method and application of traditional Chinese medicine active ingredients of targeted neuron mitochondria, and the screening method comprises the following steps: (1) determining an MICU1 protein structure, carrying out information processing on the protein structure, and then carrying out energy optimization to obtain a protein binding conformation; (2) collecting a 2D structure of the traditional Chinese medicine monomeric compound database, and outputting a 3D structure through software to obtain an analysis combination conformation; and (3) importing a 3D structure of a traditional Chinese medicine monomeric compound into software, and carrying out molecular docking with the protein binding conformation, i.e., carrying out mutual docking between receptor and ligand molecules through geometric matching and energy matching, and screening out the traditional Chinese medicine active component with targeted neuron mitochondria. The method has the characteristics of high efficiency, accuracy, simplicity and low cost; meanwhile, by combining with experiments, screening results and drug effects are further optimized and verified, and the compound has the significance of accurately targeting neuron mitochondria and improving damage.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a method for screening active ingredients of traditional Chinese medicine targeting neuron mitochondria and an application thereof. Background Art

[0002] Stroke is characterized by high mortality, high disability, and high morbidity, and is one of the major global public health issues. Neurons are the most basic structural and functional units of the brain, which can receive, integrate, transmit, encode, and maintain normal brain function. However, neurons are extremely sensitive to ischemia and hypoxia, and cannot regenerate after stroke injury. Mitochondria, as the key "energy supply center" of neurons, may have serious structural and functional abnormalities in many human diseases (such as ischemic stroke, neurodegenerative diseases, dementia, etc.). Ischemic stroke can induce mitochondrial oxidative stress and energy metabolism disorders, aggravating disease progression. Stroke reduces the expression of antioxidant substances in neuronal cells such as superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH), and reduces the ability to scavenge reactive oxygen species (ROS), leading to mitochondrial oxidative stress. Mitochondrial oxidative stress in turn causes imbalance in the mitochondrial oxidative respiratory chain and calcium ion homeostasis, aggravating cellular energy metabolism disorders and leading to cell death. Therefore, targeting mitochondria to improve cellular anergy is a way to fundamentally reduce neuronal damage in ischemic stroke.

[0003] Mitochondrial calcium uptake 1 (MICU1) is an important regulator of mitochondrial calcium homeostasis located in the inner membrane of mitochondria. MICU1 regulates the function of the mitochondrial calcium uniporter (MCU) complex, the opening of the mitochondrial permeability transition pore, and mitochondrial calcium homeostasis by setting the threshold of mitochondrial calcium uptake, thereby maintaining the stability of the intracellular environment and regulating physiological and pathological functions such as cell energy metabolism, oxidative stress, ferroptosis, and apoptosis. MICU1 structural and functional disorders are closely related to the development of various diseases such as cardiovascular disease, stroke, and tumors. In particular, with the analysis and application of molecular genetics, proteomics, and artificial intelligence big data, the potential molecular mechanism of MICU1 structural and functional disorders leading to neurological diseases has gradually been recognized. The research and development needs of potential clinical application methods such as precise regulation of molecules such as MICU1 and targeting MICU1 to improve mitochondrial function are becoming increasingly urgent.

[0004] Traditional Chinese medicine has a long history of treating ischemic stroke. Traditional Chinese medicine believes that stroke is a disease caused by the imbalance of yin and yang of the internal organs due to fatigue, internal injuries, worry, anger, etc., which leads to the disorder of qi and blood, and the direct attack of the brain, resulting in the blockage of brain vessels or blood overflow outside the brain vessels. A variety of Chinese medicine prescriptions or Chinese herbal ingredients can be used to treat stroke: "Golden Chamber Synopsis" records the use of chrysanthemum, scutellaria, rhubarb and other detoxifying drugs to treat stroke; "Yi Lin Gaicuo" records that Buyang Huanwu Decoction is mainly used to treat stroke qi deficiency and blood stasis. In addition, Academician Wang Yongyan of the China Academy of Chinese Medical Sciences advocates studying "toxic evil" as an important pathogenic factor of stroke. In recent years, with the country's vigorous promotion of traditional Chinese medicine, more and more studies have been conducted on the use of traditional Chinese medicine to treat ischemic stroke. Traditional Chinese medicine compound prescriptions are complex and diverse, and traditional Chinese medicine compounds that target the regulation of mitochondrial dysfunction are increasingly valued. Similar Chinese herbal medicines often have similar medicinal effects, and the therapeutic effects they exert are often the common chemical components of plants in the same family. Therefore, studying the effective active monomer components of traditional Chinese medicine for targeted treatment of ischemic stroke is of great value in improving drug efficacy and reducing adverse reactions. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for screening active ingredients of traditional Chinese medicine targeting neuronal mitochondria and its application, which has the characteristics of high efficiency, accuracy, simplicity and low cost.

[0006] The present invention provides a method for screening active ingredients of traditional Chinese medicine targeting neuron mitochondria, comprising the following steps:

[0007] (1) Clarify the MICU1 protein structure, process the protein structure information, and then perform energy optimization to obtain the protein binding conformation;

[0008] (2) Collect the 2D structure of the Chinese medicine monomer compound database, output the 3D structure through the software, and obtain the analytical binding conformation;

[0009] (3) The 3D structure of the monomer compound of traditional Chinese medicine is imported into the software for molecular docking with the above-mentioned protein binding conformation, that is, the receptor and ligand molecules are docked with each other through geometric matching and energy matching, so as to screen out the active ingredients of traditional Chinese medicine that target neuronal mitochondria.

[0010] Preferably, the information processing in step (1) adopts The Protein Preparation Wizard module in the software hydrogenates and removes water from the MICU1 protein and deletes ion information.

[0011] Preferably, the Chinese medicine monomer compound database in step (2) includes Traditional Chinese Medicine Active Compound Library and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.

[0012] Preferably, the software in step (2) is LigPrep Module in the software.

[0013] Preferably, the molecular docking in step (3) is carried out using The Glide module in the software is used.

[0014] Preferably, in the step (3), a Virtual Screening Workflow module is used to perform virtual screening based on molecular docking: when the database is Traditional Chinese Medicine Active Compound Library, the small molecule compounds prepared in the database are screened using the standard mode in the Glide module, and the small molecule compounds with the top 20% of the scores are selected and a second round of screening is performed using the high-precision mode to obtain the ranking of the small molecule compounds; when the database is Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, the small molecule compounds prepared in the database are screened using the high-throughput screening mode in the Glide module, and the small molecule compounds with the top 20% of the scores are selected and a second round of screening is performed using the standard mode; then the small molecule compounds with the top 20% of the scores are selected and a third round of screening is performed using the high-precision mode to obtain the ranking of the small molecule compounds.

[0015] Preferably, the active ingredients of traditional Chinese medicine screened in step (3) are manually reviewed or visually reviewed.

[0016] Furthermore, the active ingredients of traditional Chinese medicine screened in step (3) include echinacoside from Cistanche tubulosa and schizonepetaside from Callicarpa genus of Verbenaceae.

[0017] Furthermore, the echinacoside and scutellaria baicalensis glycoside are used in the preparation of drugs for ischemic stroke.

[0018] Furthermore, the echinacoside and scutellarin are used in the preparation of drugs for chronic cerebral ischemic diseases.

[0019] Beneficial Effects

[0020] The present invention realizes the molecular docking of monomeric active ingredients of traditional Chinese medicine and target proteins through the interaction of geometric matching and energy matching, and has the characteristics of high efficiency, precision, simplicity and low cost. At the same time, combined with experiments, the screening results and drug effects are further optimized and verified, which has the significance of accurately targeting neuronal mitochondria and improving damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the screening method of the present invention.

[0022] Figure 2 The top 36 monomer compounds ranked by docking scores in the virtual screening of the Traditional Chinese Medicine Active Compound Library database.

[0023] Figure 3 The top 36 monomer compounds ranked by docking scores were virtually screened in the TCMSP database.

[0024] Figure 4 This is the docking diagram for the virtual screening of the traditional Chinese medicine monomer rugosin E; A is the 2D and 3D docking mode diagram; B is the local enlarged binding site.

[0025] Figure 5 A shows the status of HT22 hippocampal neurons in different groups under a two-light microscope; B shows the results of CCK8 detection. * represents p<0.05, ** represents p<0.001.

[0026] Figure 6 A is a typical Western Blot image of the effect of Chinese medicine monomers ECH and POL on the expression of mitochondrial-related protein MICU1; B and C are statistical analysis of the relative expression levels of proteins. * represents p<0.05. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0028] Example 1

[0029] 1. Establishment of virtual screening model based on molecular docking ( Figure 1 )

[0030] (1) Protein preparation: Identify the target protein, consult protein databases (such as Protein Data Bank (PDB): https: / / www.rcsb.org / ; UniProt: https: / / www.uniprot.org / ; InterPro: https: / / www.ebi.ac.uk / interpro / , etc.), and download protein three-dimensional structure data. If there is no clear structure of the protein, use the alphafold website to predict the structure of the target protein: for example, RAT MICU1 (Calcium uptake protein 1, mitochondrial, uniport ID: Q6P6Q9) has no experimentally resolved three-dimensional structure, and its alphafold predicted structure is AF-Q6P6Q9-F1. Download the RAT MICU1 protein structure from the alphafold website.

[0031] (2) Protein structure information processing: The Protein Preparation Wizard module in the software performs hydrogenation, dehydration, and ion information processing on the protein; energy optimization is performed (OPLS2005 force field, RMSD is ). Use the Receptor Grid Generation module to create a grid file for the processed protein: For example, generate a grid file centered on the key amino acids of MICU1 protein: ALA122 / GLU115 / ARG323 / GLU326 / GLY331 / ARG316 / GLY338, and set the box size to

[0032] (3) Preparation of TCM monomer compounds: The Traditional Chinese Medicine Active Compound Library (containing 2.8K compounds, Cat. No: HY-L065, MCE, TCM monomer compound library) and the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, TCM Systems Pharmacology Database and Analysis Platform) (containing 14.2K compounds) were used in 2D format. The software LigPrep Module performs hydrogenation, energy optimization and other processing, and outputs 3D structures for virtual screening;

[0033] (4) Computer molecular docking virtual screening: Use the Virtual Screening Workflow module for virtual screening, import the prepared compounds, and use the Glide module for molecular docking, that is, the receptor and ligand molecules are docked with each other through geometric matching and energy matching:

[0034] (5) Import of active monomers from TCM monomer compound library: First, the standard (SP) mode in the Glide module is used to screen the small molecule compounds prepared in the database, and the small molecule compounds with the top 20% scores are selected for a second round of screening using the high precision (XP) mode to obtain the ranking of the small molecule compounds.

[0035] (6) Import of active monomers of traditional Chinese medicine from the TCMSP database: First, the small molecule compounds prepared in the database were screened using the high-throughput screening (HTVS) mode in the Glide module, and the small molecule compounds with the top 20% scores were selected for the second round of screening using the standard (SP) mode; then, the top 20% scores were selected for the third round of screening using the high-precision (XP) mode to obtain the ranking of the small molecule compounds.

[0036] (7) Manual review of molecular docking results: Manual review of the binding force between the target and the compound, the compound structure, etc., and selection of the top 100 compounds in the HY-L065 Traditional Chinese Medicine Active Compound Library (including the top 36 such as Figure 2 As shown in the table), the top 100 TCM active monomers ranked by TCMSP (the top 36 of which are as shown in the table Figure 3 shown).

[0037] (8) Visual virtual screening of molecular docking: Based on the molecular docking score (the higher the absolute value of the score, the stronger the binding force between the compound and the protein), the active monomers of traditional Chinese medicine are selected, and the docking pattern diagram (2D / 3D) is output using AutoDock software or Pymol software, and the key sites are marked; the higher the absolute value of the molecular docking score, the stronger the binding force between the compound and the protein. For example, the absolute value of the molecular docking score of rugosin E is 17.068, and the docking pattern diagram (2D / 3D) is as follows: Figure 4As shown in the figure, rugosin E forms 10 hydrogen bonds and 3 salt bridges with the target protein: the hydroxyl group of rugosin E can form 5 hydrogen bonds with PRO160 / ASN161 / GLU162 of the target protein; the carbonyl group of rugosin E can form 4 hydrogen bonds with GLN348 / LYS103 / ARG105 / ARG327 of the target protein; the ester group can form 1 hydrogen bond with GLN328 of the target protein; in addition, the oxygen anion of rugosin E can form 3 salt bridges with ARG105 / LYS352 / ARG327 of the target protein.

[0038] (9) Experimental verification of the efficacy of Chinese medicine monomers: Select Chinese medicine monomers of interest, such as echinacoside (ECH) from Cistanche tubulosa and poliumoside (POL) from Callicarpa genus of the Verbenaceae family, and conduct in vivo and in vitro experiments to verify the protective effect of Chinese medicine active monomers on nerve cells.

[0039] 2. CCK8 kit was used to detect the effects of echinacoside and bromocoside on the growth of neuronal cells in an in vitro stroke model - oxygen-glucose deprivation (OGD)

[0040] (1) Establishment of OGD: HT22 hippocampal neurons (GNM47, Cell Bank of the Chinese Academy of Sciences);

[0041] (2) Cell culture: DMEM high-glucose medium containing 10% fetal bovine serum (FBS) was used, and 100 U / mL penicillin and 100 μg / mL streptomycin were added, and the cells were cultured in an incubator at 37° C. and 5% CO 2 .

[0042] (3) Cell medium replacement and cell subculturing: The cell growth status was observed under a light microscope for 1-2 days, and the cells were digested with 0.25% ETDA trypsin for 1-2 min before medium replacement and subculturing.

[0043] (4) Cell grouping: The cells were divided into normal control group (Con group), model group (OGD group), OGD + echinacoside intervention group (ECH group), OGD + chrysanthemum indica intervention group (POL group), and OGD + proanthocyanidin C1 intervention group (PCC1 group);

[0044] (5) Cell counting and plating, 3-5*10 4 / well, Nunc 24-well plate (124275, Thermo Fisher)

[0045] (6) The cells in the OGD group and the drug intervention group were treated with PBS for three times, and then DMEM sugar-free culture medium was added. The cells were placed in a hypoxic incubator (Stemcell, Canada, model: 27310, with conditions set at 1% O2, 5% CO2, and 94% N2) for 4 hours.

[0046] (7) After the cell hypoxia ended, the DMEM sugar-free medium was replaced with a 10% fetal bovine serum HT22 cell-specific medium (NO.GZ20011-500M, Servicebio), and the cells were placed back into the cell culture incubator for continued culture. The reoxygenation detection time was set at 12 h, 24 h, and 48 h, respectively.

[0047] (8) Observe the changes in cell morphology using an inverted optical microscope.

[0048] (9) Enhanced CCK-8 kit (C0041, Bio-Tech, Shanghai) was used to measure cell viability: the original culture medium was discarded, and 10 μL of enhanced CCK-8 solution was added to each well; the cells were incubated at 37°C in a cell culture incubator for 1 hour;

[0049] (10) The absorbance (OD, D) value was measured at 450 nm by an enzyme-labeled instrument, and the cell survival rate was calculated by the ratio (p) of the D (450 nm) value of each group to the D (450 nm) value of the normal control group.

[0050] Figure 5 The results suggest that ECH (echinacoside) and POL (paemonicoside) promote the survival of OGD neurons and have a neuroprotective effect.

[0051] 3. Detection of the effects of echinacoside and scutellaria glycoside on the expression of mitochondrial MICU1 protein in OGD neurons

[0052] (1) Western Blot was used to detect the expression of MICU1 protein in neuronal mitochondria;

[0053] (2) Protein sample extraction: cells were washed with PBS three times, 1 min each time; M-PER was added TM Mammalian protein extraction reagent (78501, Thermo Scientific TM ) to fully dissolve the protein. In order to reduce protein degradation, a protease inhibitor cocktail (P8340, Sigma) can be added.

[0054] (3) BCA method for protein quality control: Pierce TM BCA Protein Assay Kits (A65453, ThermoScientificTM ) Protein concentration determination;

[0055] (4) Electrophoresis: Take BeyoGel TM SDS-PAGE precast gel (Tris-Gly, 10%, 12 holes) (P0052B, Biyuntian) was fixed in the electrophoresis tank. The prepared protein sample was added to the gel wells, and a protein marker was added to a separate well to indicate the molecular weight of the protein. Electrophoresis was performed at a constant voltage of 80V. After the protein sample entered the separation gel from the concentrated gel and the protein marker was significantly separated, the voltage was increased to 120-150V. The electrophoresis could be terminated when the molecular size of the target protein was significantly separated.

[0056] (5) Transfer: Cut the NC membrane into 6.5 cm × 9 cm size, pour in the pre-cooled transfer solution (1×), place the black plate of the transfer clamp at the bottom, and place the white sponge, 3 filter papers (Biyuntian), gel, NC membrane, 3 filter papers (Biyuntian), white sponge, white plate in sequence, and clamp the black and white plates to fix them. Transfer at low temperature and constant voltage of 100 V for 2 h.

[0057] (6) Blocking: After transfer, remove the NC membrane and block it with 5-10% skim milk at room temperature for 1 hour. Wash it with TBST for 3 times, 5-8 minutes each time.

[0058] (7) Antibody incubation: Add the primary antibody MICU1 Monoclonal Antibody (40490, Signalway Antibody, LLC) (Western blotting: dilution 1:1000) and incubate overnight at 4°C. The next day, recover the primary antibody and wash the NC membrane with TBST three times, 5-8 min each time. Add the secondary antibody (anti-mouse, 1:5000 dilution) and incubate on a shaker at room temperature for 1 h. Discard the secondary antibody and wash the NC membrane with TBST three times, 5-8 min each time.

[0059] (8) Luminescence development: BeyoECL Plus (Ultra-sensitive ECL chemiluminescence kit, P0018S, Beyotime), and use a chemiluminescence instrument for development to obtain bands.

[0060] Figure 6 The results suggested that the Chinese medicine monomers ECH and POL could promote the expression of MICU1.

Claims

1. A method for screening active ingredients of traditional Chinese medicine targeting neuronal mitochondria, comprising the following steps: (1) Clarify the MICU1 protein structure, process the protein structure information, and then perform energy optimization to obtain the protein binding conformation; (2) Collect the 2D structure of the Chinese medicine monomer compound database, output the 3D structure through the software, and obtain the analytical binding conformation; (3) The 3D structure of the monomer compound of traditional Chinese medicine is imported into the software for molecular docking with the above-mentioned protein binding conformation, that is, the receptor and ligand molecules are docked with each other through geometric matching and energy matching, so as to screen out the active ingredients of traditional Chinese medicine that target neuronal mitochondria.

2. The method for screening active ingredients of traditional Chinese medicine targeting neuronal mitochondria according to claim 1, characterized in that: The information processing in step (1) is carried out using The Protein Preparation Wizard module in the software hydrogenates and removes water from the MICU1 protein and deletes ion information.

3. The method for screening active ingredients of traditional Chinese medicine targeting neuronal mitochondria according to claim 1, characterized in that: The Chinese medicine monomer compound database in step (2) includes Traditional Chinese Medicine Active Compound Library and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.

4. The method for screening active ingredients of traditional Chinese medicine targeting neuronal mitochondria according to claim 1, characterized in that: The software in step (2) is LigPrep Module in the software.

5. The method for screening active ingredients of traditional Chinese medicine targeting neuronal mitochondria according to claim 1, characterized in that: The molecular docking in step (3) is carried out using The Glide module in the software is used.

6. The method for screening active ingredients of traditional Chinese medicine targeting neuronal mitochondria according to claim 1, characterized in that: In the step (3), the Virtual Screening Workflow module is used to perform virtual screening based on molecular docking: when the database is Traditional Chinese Medicine Active Compound Library, the small molecule compounds prepared in the database are screened using the standard mode in the Glide module, and the small molecule compounds with the top 20% of the scores are selected and the second round of screening is performed using the high-precision mode to obtain the ranking of the small molecule compounds; when the database is Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, the small molecule compounds prepared in the database are screened using the high-throughput screening mode in the Glide module, and the small molecule compounds with the top 20% of the scores are selected and the second round of screening is performed using the standard mode; then the small molecule compounds with the top 20% of the scores are selected and the third round of screening is performed using the high-precision mode to obtain the ranking of the small molecule compounds.

7. The method for screening active ingredients of traditional Chinese medicine targeting neuronal mitochondria according to claim 1, characterized in that: The active ingredients of traditional Chinese medicine screened in step (3) are manually reviewed or visually reviewed.

8. The method for screening active ingredients of traditional Chinese medicine targeting neuronal mitochondria according to claim 1, characterized in that: The active ingredients of traditional Chinese medicine screened in step (3) include echinacoside from Cistanche tubulosa and schizonepetaside from Callicarpa spp. of the Verbenaceae family.

9. The method for screening active ingredients of traditional Chinese medicine targeting neuronal mitochondria according to claim 8, characterized in that: The application of the echinacoside and scutellaria baicalensis in the preparation of medicines for ischemic stroke.

10. The method for screening active ingredients of traditional Chinese medicine targeting neuron mitochondria according to claim 8, characterized in that: The application of echinacoside and scutellarin in preparing medicine for chronic cerebral ischemia disease.

Images