Application of acid glycoprotein AAG in promotion of glycolysis and aerobic respiratory function

Abstract

The invention belongs to the technical field of medical application, and discloses application of acid glycoprotein AAG in promotion of glycolysis and aerobic respiration. Experiments show that AAG increases the proportion of ATP (adenosine triphosphate) from glycolysis. AAG increases energy supply of glycolysis and aerobic respiratory metabolism, and glycolysis is mainly used. As the AAG is an endogenous protein of a body, the safety of the AAG as a medicine is very high, and the AAG is expected to be used for treating and / or relieving diseases caused by cell glycolysis weakening.

Description

Technical Field

[0001] The present invention relates to the technical field of medical applications, and more specifically, to the application of acidic glycoprotein AAG in promoting glycolysis and aerobic respiration. Background Art

[0002] Glycolysis is a conservative and strict biological process that breaks down glucose into pyruvate. It is a common pathway for all organisms to decompose energy. Glycolysis can be divided into two stages, which occur under anaerobic and aerobic conditions. Under aerobic conditions, pyruvate is converted to acetyl-CoA and further enters the tricarboxylic acid cycle; under anaerobic conditions, pyruvate is reduced to lactic acid. Since the enzymes that catalyze glycolysis are present in the cytoplasm, glycolysis occurs entirely in this cell compartment. Glycolysis provides part of the energy for life activities, especially for anaerobic organisms, it is the main way to obtain energy. The glycolysis pathway can also provide intermediates (provide carbon skeletons) for other metabolic pathways, and is also the main source of energy for other cells. In addition, glycolysis is also the preparatory stage for the aerobic decomposition of sugars. Glycolysis is the first choice in cells with high metabolic demands. Aerobic glycolysis can meet the expensive energy requirements of the metabolic process. It is a faster biological energy pathway than OXPHOS. Aerobic respiration is the main form of respiration for higher organisms. It is carried out in the mitochondrial matrix and inner membrane. Mitochondria are the main place for cells to carry out aerobic respiration. Aerobic respiration releases energy by completely oxidizing and decomposing organic matter.

[0003] When an organism is doing strenuous or prolonged exercise, the energy demand increases and glycolysis accelerates. At this time, breathing and circulation speed up to increase the supply of oxygen. People cannot meet the demand, and muscles are in a relatively hypoxic state. Glycolysis must be used to provide the much-needed energy. Glycolysis is an effective way for some tissues to obtain energy in the presence of oxygen. Glycolysis is the only way for mature red blood cells to obtain energy and is also an effective way for nerves, white blood cells, bone marrow and other tissue cells to obtain some energy in the presence of oxygen.

[0004] Alpha-1acid glycoprotein (AAG) is an acute phase response protein mainly synthesized by the liver. It has multiple biological activities, which prompted us to explore whether it can promote glycolysis and aerobic respiration (mainly glycolysis) for energy supply.

[0005] Currently, there is no report on AAG promoting glycolysis and aerobic respiration (mainly glycolysis) for energy supply. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned problems existing in the prior art and provide the application of acidic glycoprotein AAG in promoting cell glycolysis.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The application of acidic glycoprotein AAG in promoting cell glycolysis. The present invention shows through experiments that AAG increases the proportion of ATP derived from glycolysis. AAG increases the energy supply of glycolysis and aerobic respiration metabolism, with glycolysis as the main source.

[0009] Preferably, the acidic glycoprotein AAG and cells form an incubation system (volume is 0.08 ml), the final concentration of the acidic glycoprotein AAG in the incubation system is 0.1-10 ug / ml, and the number of the cells is 375,000 / ml.

[0010] More preferably, the promoting cell glycolysis refers to:

[0011] (1) Increased production rates of glycolytic ATP and mitochondrial ATP, and / or,

[0012] (2) Increased glycoPER / mitoOCR energy rate ratio, and / or,

[0013] (3) Improved cellular oxidative phosphorylation.

[0014] Glycolysis provides part of the energy for life activities, especially for anaerobic organisms, it is the main way to obtain energy. The glycolysis pathway can also provide intermediates (provide carbon skeletons) for other metabolic pathways, and is also the main energy source for other cells. In addition, glycolysis is also the preparatory stage for aerobic decomposition of sugars. Glycolysis is the first choice in cells with high metabolic demands. Aerobic glycolysis can meet the expensive energy needs in the metabolic process. It is a faster biological energy pathway than OXPHOS. When an organism is engaged in strenuous exercise or long-term exercise, the energy demand increases and glycolysis accelerates. At this time, even if breathing and circulation are accelerated to increase the supply of oxygen, it still cannot meet the demand. Muscles are in a relatively hypoxic state and must provide much-needed energy through glycolysis. Glycolysis is an effective way for some tissues to obtain energy in the presence of oxygen. Glycolysis is the only way for mature red blood cells to obtain energy and is also an effective way for nerves, white blood cells, bone marrow and other tissue cells to obtain part of the energy in the presence of oxygen.

[0015] Therefore, the present invention also protects the use of acidic glycoprotein AAG in the preparation of functional products that promote cell glycolysis, in order to use AAG to treat and / or alleviate diseases caused by weakened cell glycolysis.

[0016] Preferably, the acidic glycoprotein AAG and cells form an incubation system (volume is 0.08 ml), the final concentration of the acidic glycoprotein AAG in the incubation system is 0.1-10 ug / ml, and the number of the cells is 375,000 / ml.

[0017] More preferably, the functional product has at least the following functions:

[0018] (1) Increased production rates of glycolytic ATP and mitochondrial ATP, and / or,

[0019] (2) Increased glycoPER / mitoOCR energy rate ratio, and / or,

[0020] (3) Improved cellular oxidative phosphorylation.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention shows through experiments that AAG increases the proportion of ATP derived from glycolysis. AAG increases glycolysis and aerobic respiration metabolic energy supply, with glycolysis being the main source. Since AAG is an endogenous protein of the body, it is highly safe as a drug, and it is expected that AAG can be used to treat and / or alleviate diseases caused by weakened cellular glycolysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of Seahorse XF96 cell culture plate;

[0024] Figure 2 This is a graph of the extracellular acidification rate (ECAR) for the glycolysis rate experiment;

[0025] Figure 3 A quantitative diagram of basal and compensatory glycolysis;

[0026] Figure 4 This is a graph of the rate of mitochondrial ATP production;

[0027] Figure 5 Energy maps for oxidative phosphorylation and glycolysis. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0029] The present invention uses an extracellular flux analyzer to monitor iPSC-CM (human induced pluripotent stem cell-derived cardiomyocytes) culture in real time. Human iPSC-CM was purchased from Cosmos Biotechnology Co., Ltd. (Nanjing, China).

[0030] Example 1

[0031] Seahorse Experiment

[0032] 1. Preparation

[0033] 1. Start the detection system preheating in advance

[0034] (1) Press the switch on the lower left side of the back of the Seahorse XFe96 instrument to turn on the instrument.

[0035] (2) Press the switch on the lower right corner of the Seahorse XFe96 Controller (control computer) to turn on the control system;

[0036] (3) Open the Wave software, wait for the control system to connect successfully to the instrument host, and preheat overnight.

[0037] 2. Inoculate cells to adhere to the wall (the inoculation area of ​​each well of the XFe96 cell culture plate is 0.106cm 2 , which is 40% of the inoculation area of ​​each well of a common 96-well cell culture plate)

[0038] (1) Open the package in a clean bench and take out the XFe96 cell culture plate;

[0039] (2) Add 80 μL of cell growth medium to the background correction well of the XFe96 cell culture plate without adding cells;

[0040] (3) Collect the cultured cells (blow the cells apart as much as possible without damaging them), calculate the cell density and prepare the cell suspension, and calculate the density based on the inoculation volume of 80 μL (e.g., if 10K cells / well are inoculated, the cell suspension density is 10K cells / 80 μL / well = 12.5K cells / mL);

[0041] (4) Inoculate 80 μL of cell suspension in each well of the XFe96 cell culture plate except for the background correction well;

[0042] (5) Place the cell culture plate in a clean bench for 1 hour to allow the cells to settle naturally, which helps to evenly distribute the cells and reduce the edge effect of certain cell types (please note that this step is very important. Do not move the cell culture plate after cell inoculation. Cover it with the lid and let it sit directly).

[0043] (6) Place the cell culture plate in a 37°C CO2 cell culture incubator to allow the cells to adhere to the wall and culture overnight. The cell confluence must reach 50-90% before loading (it is recommended that the cell confluence reach about 80-90% as the best).

[0044] XF assays are performed in XF96 cell culture plates and XFe96 test plates. The cell culture plates are designed as a typical 96-well plate, such as Figure 1 The inoculation area of ​​each well is 0.106 cm 2, which is smaller than a typical 96-well cell culture plate (only 40% of the normal 96-well bottom area), but larger than a 384-well plate. The following is the recommended method for seeding adherent cells:

[0045] a. Collect and resuspend cells in 80 μL growth medium to the desired concentration. The optimal cell seeding density ranges widely, generally 5,000 to 40,000 cells / well, and the cell number needs to be determined based on experience;

[0046] b. Inoculate 80 μL of cell suspension in each well; do not inoculate cells in the background correction wells (A1, A12, H1, H12, i.e., the four corners), and ensure that only culture medium (no cells) is added to the background correction wells;

[0047] c. Leave the cell plate on a clean bench at room temperature for 1 hour. Do not move or shake the plate during this period. This will promote uniform distribution of cells and reduce edge effects. Observe the cell adhesion under a microscope;

[0048] d. Place the cells in a cell culture incubator for overnight culture and observe the growth and status of the cells under a microscope.

[0049] 3. Hydration probe plate

[0050] (1) Add at least 20 mL of XF Calibrant into a 50 mL centrifuge tube and incubate overnight in a 37°C CO2-free cell culture incubator (CO2-free means no additional CO2 supplementation).

[0051] (2) Open the Seahorse XFe96 FluxAssay Kit and remove the probe plate assembly, including the bottom tool plate, the green probe plate, and the cover;

[0052] (3) Remove the cover and probe board from the tool board and place them upside down on the laboratory bench (to protect the sensor on the probe board from damage);

[0053] (4) Add 200 μL of sterile water to each well of the tool plate;

[0054] (5) Take the cover and the probe plate and align them with the holes on the tool plate. Put them back on the tool plate to restore the entire device so that the sensor is immersed in sterile water.

[0055] (6) Check again whether the sterile water level is higher than all sensors to ensure that all sensors are immersed in sterile water;

[0056] (7) Place the entire probe card assembly in a 37°C CO2-free cell culture incubator and incubate overnight.

[0057] 2. On the day of the experiment - on-machine testing

[0058] 1. Continue to hydrate the probe plate

[0059] (1) Remove the centrifuge tube and probe card assembly incubated with XF calibrant from the 37°C CO2-free cell culture incubator;

[0060] (2) Remove the cover and probe card from the tool plate and place them upside down on the laboratory bench;

[0061] (3) Discard the sterile water in the tool plate;

[0062] (4) Add 200 ml of XF calibrant to each well of the tool plate;

[0063] (5) Align the cover and the probe plate with the holes of the tool plate and replace them on the tool plate to restore the entire device so that the sensor is immersed in the XF calibration solution;

[0064] (6) Place the entire probe card assembly in a 37°C CO2-free cell culture incubator to hydrate for 45 to 60 minutes and wait for drug preparation.

[0065] 2. Prepare Seahorse test solution

[0066] Prepare: 103575-100 Seahorse XF DMEM Medium, pH 7.4 or 103576-100 Seahorse XF RPMI Medium, pH 7.4;

[0067] 103577-100Seahorse XF 1.0M Glucose Solution;

[0068] 103578-100Seahorse XF 100mM Pyruvate Solution;

[0069] 103579-100Seahorse XF 200mM Glutamine Solution (Note: Store at -20°C).

[0070] (1) Aliquot 97 mL of 103575-100 or 103576-100 for later use;

[0071] (2) Add 1 mL of glucose, 1 mL of pyruvate, and 1 mL of glutamine to 97 mL of culture medium and mix well. The test solution is ready (the volume of added substrate can be adjusted according to the concentration required for the experiment);

[0072] (3) Place the prepared test solution in a 37°C CO2-free cell culture incubator for incubation until ready for use (or use directly after incubation in a 37°C water bath).

[0073] 3. Washing cells

[0074] (1) Take out the incubated test solution and prepare to replace the cell solution;

[0075] (2) Take out the cultured adherent cells from the CO2 cell culture incubator and observe the cell status under a microscope;

[0076] (3) Aspirate 60 μL of the growth medium from all wells of the cell culture plate, but leave 20 μL;

[0077] (4) Wash cells: add 200 μL of detection solution to all wells, and then aspirate and discard 200 μL;

[0078] (5) Repeat the previous step until 20 μL of detection solution remains in the well;

[0079] (6) Add 160 μL of detection solution to all wells to make the final volume 180 μL;

[0080] (7) Observe under a microscope to ensure that no cells are washed away or scratched during the fluid change process, causing uneven distribution;

[0081] (8) Place the cell culture plate in a 37°C CO2-free cell culture incubator for 60 minutes and wait for testing.

[0082] 4. Dispense the medicine and load it into the probe port

[0083] 1. Dispensing medicine

[0084] (1) Take out a pack of aluminum foil bagged medicine and a lid opener from the test kit;

[0085] (2) Open the aluminum foil bag, take out three drug tubes containing oligomycin (blue cap), FCCP (yellow cap) and Rot / AA (red cap), and place the three drug tubes on an appropriate tube rack (Table 1 is the configuration system);

[0086] (3) Use the cap opener to open the medicine tube;

[0087] (4) Take out the prepared 37°C test solution and add it to the corresponding drug tubes according to the volume shown in Table 1 below. Use a pipette to gently blow and mix (blow and mix about 10 times or vortex to mix) to allow the drug to fully dissolve.

[0088] Table 1

[0089] Compound Volumeofassaymedium Stockoncentration Capcolor Oligomycin 630μL 100μM Blue FCCP 720μL 100μM Yellow Rot / AA 540μL 50μM Red

[0090] (5) Use the detection solution to dilute the resuspended drug into the drug working solution of the required concentration. Prepare about 2 to 3 mL of each drug working solution (the drug working solutions are all 10x concentrations and will be loaded into the probe plate. Please ensure that the pH of the drug working solution is 7.4±0.1, 37°C). You can dilute according to Table 2 below. After the drug working solution is prepared, wait for the addition of drugs (the dosing system is shown in Table 2).

[0091] Table 2

[0092]

[0093] 2. Load the drug into the probe card drug loading port

[0094] (1) Take out the hydrated probe card from the 37°C CO2-free cell culture incubator, remove the cover, and ensure that the probe card is placed on the tool plate;

[0095] (2) Take the corresponding dosing auxiliary plate and place it on the green probe plate;

[0096] (3) Take out the prepared drug working solution (37°C), and add the corresponding drugs in the corresponding volumes into the corresponding drug adding chambers of the probe card in sequence (the volume added to each series of drug adding chambers is: 20 μL for A-drug adding chamber, 22 μL for B-drug adding chamber, 25 μL for C-drug adding chamber, and 27 μL for D-drug adding chamber).

[0097] 5. Run the XF test on the machine

[0098] 1. Select the template of the corresponding test kit in the Wave software template interface to open it (double-click the template or click OpenFile);

[0099] 2. After opening the template, complete the group definition settings according to the experimental design, generate groups that meet the design, and then complete the group layout in the plate layout interface, enter the program setting interface to check the program, enter the run experiment interface and click Start Run, then a dialog box will pop up to select the save location of the experimental data, and then the instrument will pop up the plate tray;

[0100] 3. After the carrier tray pops up, place the probe card and tool board combination (the cover of the probe card must be removed) on the tray according to the software prompts, pay attention to the placement direction of the probe card (the AH row logo or S / N barcode of the probe card is on the left), then click I'mready, the tray enters the instrument, and calibration begins (about 20 minutes);

[0101] 4. After the calibration is completed, the software pops up. At this time, take out the cell culture plate from the 37℃ CO2-free cell culture incubator (observe the cell status under a microscope), click Open Tray, the tray pops up, remove the tool plate and place the cell culture plate on the tray, and click Load Cell Plate;

[0102] 5. After the cell plate enters the instrument, the instrument starts the cell energy metabolism measurement phase. After the measurement is completed, click Eject, the tray pops out to remove the probe plate and cell culture plate, click OK, and the tray enters the instrument;

[0103] 6. The entire energy metabolism measurement process is completed. Click Assay Result to view the results or click Wave Home to return to the main interface;

[0104] 7. After the experiment is over, take out the cell culture plate and probe plate, check whether all the drugs in the probe plate drug addition chamber are injected into the cell wells, whether there are any injection failures, and record them for subsequent data analysis;

[0105] 8. Quantify the samples in each well of the cell culture plate and then standardize the data.

[0106] ATP rate assay

[0107] The day before the experiment: Turn on the Seahorse XF96 Analyzer and allow the temperature to stabilize. For adherent cells, seed the Seahorse XF microplates at a predetermined density using appropriate cell growth medium. Rehydrate the probe plate overnight in a 37°C non-CO2 incubator. Design an experiment in Wave or use the ATP production rate experiment template.

[0108] On the day of the test

[0109] 1. Prepare the assay solution: Aseptically add 10 mmol / L XF glucose, 1 mmol / L XF sodium pyruvate, and 2 mmol / L XF glutamine to 100 mL Seahorse XF DMEM medium, pH 7.4; warm the assay solution to 37°C; incubate at 37°C until ready to use.

[0110] 2. Prepare the Seahorse XF cell culture microplate: Remove the cell culture microplate from the 37°C CO2 incubator and examine the cells under a microscope to confirm the consistency of the plate and normal cell morphology; discard the cell growth medium in the cell culture microplate. Use a multichannel pipette to wash the cells once with preheated detection solution, and then place them in a 37°C CO2-free incubator and incubate with detection solution for 45 to 60 minutes; before starting the XF experiment, discard the detection solution again and add fresh, preheated detection solution to each well.

[0111] 3. Prepare compound stock solution: Take out an aluminum foil bag from the kit, then open the bag and take out the oligomycin (blue cap) and rotenone / antimycin A (red cap) tubes; flick the tubes to ensure that the powder is at the bottom of the tube before opening the tube; resuspend each component with the appropriate volume of detection solution according to Table 3. Vortex for about 1 minute to ensure that the compound is completely resuspended.

[0112] Table 3

[0113]

[0114] 4. Prepare the compounds to be added to the probe plate drug wells: Prepare 3 mL of each compound with the detection solution as described in Table 4. It is recommended to use 1.5 μmol / L oligomycin and 0.5 μmol / L rotenone + antimycin A (final concentration).

[0115] Table 4

[0116]

[0117] 5. Add the compound to the probe plate drug well

[0118] Standard experiment: No acute injections prior to oligomycin and rotenone / antimycin A. Compounds were added.

[0119] In the drug-adding wells of the hydrated probe plate: Drug-adding well A: oligomycin; Drug-adding well B: rotenone / antimycin A.

[0120] 6. Run the XF Real-Time Test ATP Rate

[0121] (1) Select Seahorse XF Real-Time ATP Rate Assay from the list of available templates and click Open File.

[0122] (2) Group Definitions: Confirm or modify the default experimental groups and conditions;

[0123] (3) Plate Map: confirm or modify experimental grouping;

[0124] (4) Protocol: confirm or modify the instrument operation procedure;

[0125] (5) RunAssay: Click Start Run when you are ready.

[0126] (6) When prompted, place the pre-dosed probe cartridge and calibration plate into the Seahorse XFe analyzer and click I'm Ready. Calibration takes approximately 15 to 30 minutes.

[0127] (7) After calibration is complete, the Wave Controller will display a message to load the cell tray. Click Open Tray to eject the calibration tray and load the cell tray. Make sure to remove the cell tray cover before loading.

[0128] (8) Click Load Cell Plate to run the experiment.

[0129] result: Figure 2 Plot of the Extracellular Acidification Rate (ECAR) for the Glycolytic Rate Assay. AAG treatment increased basal and maximal levels of glycolysis, as evidenced by an increase in the Extracellular Acidification Rate (ECAR) using the SeahorseXF Glycolytic Rate Assay. Figure 3 As a quantitative plot of basal and compensatory glycolysis, AAG treatment increased glycolysis at both basal and maximum levels. Figure 4 Indicating the rate of mitochondrial ATP production, AAG treatment also significantly improved OXPHOS, as manifested by an increase in the rate of mitochondrial ATP production. Figure 5 The increase in oxidative phosphorylation and glycolysis is shown in the energy diagram. AAG shifts mitoOCR / glycoPER to glycolysis and decreases the energy rate ratio of mitoOCR / glycoPER. These data suggest that AAG increases the proportion of ATP derived from glycolysis. In short, AAG increases the metabolic energy supply of glycolysis and aerobic respiration, with glycolysis being the main source.

[0130] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. Application of acidic glycoprotein AAG in promoting cellular glycolysis.

2. The use according to claim 1, characterized in that: The final concentration of the acidic glycoprotein AAG is 0.1-10ug / ml, and the number of the cells is 375,000 / ml.

3. The use according to claim 1, characterized in that: The said promoting cell glycolysis refers to: (1) Increased production rates of glycolytic ATP and mitochondrial ATP, and / or, (2) Increased glycoPER / mitoOCR energy rate ratio, and / or, (3) Improved cellular oxidative phosphorylation.

4. Application of acidic glycoprotein AAG in the preparation of functional products that promote cellular glycolysis.

5. The use according to claim 4, characterized in that: The final concentration of the acidic glycoprotein AAG is 0.1-10ug / ml, and the number of the cells is 375,000 / ml.

6. The use according to claim 1, characterized in that: The functional product has at least the following functions: (1) Increased production rates of glycolytic ATP and mitochondrial ATP, and / or, (2) Increased glycoPER / mitoOCR energy rate ratio, and / or, (3) Improved cellular oxidative phosphorylation.

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