Method for improving endoplasmic reticulum stress reaction and regulating endoplasmic reticulum / sarcoplasmic reticulum Ca < 2 + >-ATP enzyme activity by lobetyolin and application of lobetyolin

Abstract

The invention provides a method for improving endoplasmic reticulum stress reaction and regulating endoplasmic reticulum / sarcoplasmic reticulum Ca < 2 + >-ATP enzyme activity through lobetyolin and application thereof, and relates to the technical field of biology, the method is used for improving adriamycin-induced heart failure, it is found through in-vitro experiments that lobetyolin serving as the active ingredient of codonopsis pilosula can improve endoplasmic reticulum stress in a glucose deprivation model, and the adriamycin-induced heart failure can be used for improving adriamycin-induced heart failure. The activity of Ca < 2 + >-ATP enzyme in myocardial cells is enhanced; on the basis, lobetyolin is used as a ligand, Ca < 2 + >-ATP enzyme is used as a receptor, and lobetyolin small molecules can be better combined in a Ca < 2 + >-ATP enzyme protein active cavity after the lobetyolin is butted with the Ca < 2 + >-ATP enzyme; in-vivo experiments find that the lobetyolin can improve the cardiac ejection fraction, the left ventricular short-axis shortening rate, the left ventricular end-of-systole volume and the heart stroke output under the condition of heart failure, the protection mechanism of the lobetyolin to myocardium is disclosed, and a basis is provided for clinical application of codonopsis pilosula to treatment of heart failure.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, specifically to codonopsis pilosula glycosides for improving endoplasmic reticulum stress response and regulating endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method and its application. Background Art

[0003] At present, the traditional "golden triangle" of drug treatment for heart disease in foreign clinical practice is mostly used, including angiotensin-converting enzyme inhibitors, aldosterone receptor antagonists, beta-blockers, etc., but the treatment effect is poor, and the heart function and quality of life of most patients still fail to reach the ideal state, or even deteriorate further. Non-drug treatment methods include mechanical circulation devices, cardiac resynchronization therapy, stem cell therapy, etc.; most mechanical circulation devices are expensive, require high technical operation, and have many complications; the main role of cardiac resynchronization therapy (CRT) in treatment is to maintain heart function, keep it in its current state, and delay the progression of heart failure, but it cannot reverse heart failure or restore heart function to normal; some stem cells in stem cell therapy are restricted by various ethical regulations, which determines that it cannot become a treatment method for clinical application. At the same time, the disadvantages of stem cell therapy, such as cumbersome extraction process and high price, also inhibit its large-scale promotion in clinical practice.

[0004] Heart disease is a type of disease caused by abnormal cardiac function and myocardial damage caused by factors such as cardiomyopathy, inflammation, and high hemodynamic load, which leads to weakened ejection capacity and ventricular filling capacity, and ultimately damages the heart's pumping function. The pathogenesis of heart disease is mostly due to the disorder of the heart's contractile function and (or) diastolic function, which cannot fully discharge the venous return blood from the heart, resulting in blood congestion in the venous system and insufficient blood perfusion in the arterial system. The relaxation and contraction of the myocardium depends on the excitation-contraction coupling process, which is controlled by Ca in the cytoplasm. 2+ Concentration ([Ca 2+ ]i), when [Ca 2+ When ]i increases, Ca 2+ Combined with troponin C, myocardial contraction; when [Ca 2+ ]i decreases, Ca 2+ Separated from troponin C, myocardial relaxation. SERCA2a is also known as endoplasmic reticulum Ca 2+ -ATPase, responsible for converting cytosolic Ca into 2+ Uptake into the endoplasmic reticulum, cardiac ischemia and hypoxia induce endoplasmic reticulum stress response, reduce the activity and expression of SERCA2a, and lead to endoplasmic reticulum Ca 2+ The intake of vitamins is reduced, resulting in impaired cardiac diastolic function and ultimately heart failure. Summary of the invention

[0005] In view of this, the present application provides a method for improving the endoplasmic reticulum stress response and regulating the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase method and its application solve the problem in the prior art that cardiac ischemia and hypoxia induce endoplasmic reticulum stress response, reduce endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity and expression, resulting in impaired cardiac diastolic function, leading to technical problems of heart failure.

[0006] According to the first aspect of the present application, the present application provides a method for improving the endoplasmic reticulum stress response and regulating the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method, which uses codonopsis pilosula glycoside molecules as ligands, Ca 2+ -ATPase acts as a receptor to bind codonopsis glycoside molecules to Ca 2+ -ATPase molecular docking, dangshen glycoside molecules can bind to Ca 2+ -ATPase protein active cavity, dangshen glycoside molecules can enhance Ca 2+ -ATPase activity, thereby regulating the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity.

[0007] In one embodiment, the docking steps include: step (1), preparation of the codonopsis glycoside ligand, by downloading the chemical formula of the codonopsis glycoside corresponding to the component from the database, drawing the structural formula using drawing software, and then converting it into a 3D structure using a 3D module, and then entering the compound into the force field to calculate the charge; step (2), Ca 2+ - Preparation of ATPase receptor proteins, select Ca in the protein data 2+ -ATPase and Ca 2+ The co-crystal structure of the ion is used as the receptor protein, the receptor protein is imported into the software, and then the force field is calculated as the CHARMm force field, water is removed, and the protein is saved to prepare for docking; step (3), docking operation, after adding charges to the protein, the Covalent Docking module in the software is used to perform molecular docking between the two.

[0008] In one embodiment, in step (1), the PubChem database is logged in, and the sdf format file of the chemical formula corresponding to the component is downloaded from the database using codonopsis pilosula as a keyword. The structural formula is drawn using Chemdraw software, and then converted into a 3D structure using the ChemBio3D module and saved in sdf format. The prepared compound is entered into the CHARMm force field, and the structure is minimized using an intelligent minimization algorithm.

[0009] In one embodiment, in step (2), Ca is selected from the PDB protein database. 2+ -ATPase and Ca 2+The co-crystal structure of the ion is used as the receptor protein. The receptor protein is imported into the Discovery Studio 2017R2 client software, and the protein is defined as a receptor molecule for docking. After the receptor is prepared, the central coordinates of the original ligand are defined as the active site and the receptor's own ligand is deleted. The protein is saved to prepare for docking.

[0010] In one embodiment, before step (1), an experiment is also included on the effect of codonopsis pilosula on alleviating the endoplasmic reticulum stress state of cells caused by glucose deficiency, wherein cardiomyocytes are inoculated in a culture medium containing 1% double antibody and 10% FBS, and after culture and subculture, the cells are grouped and processed, and then the expression of endoplasmic reticulum stress indicator proteins in cardiomyocytes is detected by Western Blot.

[0011] In one embodiment, before step (1), an experiment is also included to investigate the effect of codonopsis pilosula on alleviating the stress state of the endoplasmic reticulum of H9c2 cells after modeling. The cardiomyocytes are inoculated in a culture medium containing 1% double antibody and 10% FBS. After culture and subculture, the cells are grouped and treated, and then the ThT immunofluorescence method is used to detect the stress state of the endoplasmic reticulum in the cardiomyocytes.

[0012] In one embodiment, the cardiomyocytes are rat cardiomyocytes H9c2; the groups include: a blank group, wherein the cells are cultured in normal DMEM medium; a model group, wherein the cells are cultured in sugar-free DMEM medium; a drug administration group, wherein the codonopsis pilosula is dissolved in sugar-free DMEM medium, and the solutions are respectively prepared to contain 10, 20, and 40 μM codonopsis pilosula solution and the cells are cultured; and a positive drug administration group, wherein the cells are cultured in a solution containing 5 μM 4-PBA and 4-PBA is dissolved in sugar-free DMEM medium.

[0013] In one embodiment, before step (1), the method further comprises: reducing Ca in myocardial cells by dangshen glycosides. 2+ In the experiment of concentration, cardiomyocytes were inoculated in a culture medium containing 1% double antibody and 10% FBS. After culture and subculture, the cells were divided into groups and treated. Fluo-4 AM fluorescent probe was then used to detect intracellular Ca 2+concentration; the grouping includes a blank group, wherein the cells are cultured in normal DMEM medium; an ATP activation group, wherein the ATP activation group is prepared by adding 10 μM ATP standard solution to the DMEM medium; a SERCA inhibitor group, wherein the SERCA inhibitor group is prepared by adding 5 μM Thapsigargin to the DMEM medium; a drug administration group, wherein the drug administration group is prepared by adding 20 μM codonopsis glycoside to the DMEM medium; and a drug administration + inhibitor group, wherein the drug administration + inhibitor group is prepared by adding 20 μM codonopsis glycoside and 5 μM Thapsigargin to the DMEM medium.

[0014] In one embodiment, before step (1), the method further comprises: regulating myocardial cell Ca 2+ -ATPase activity experiment, the cardiomyocytes are rat cardiomyocytes H9c2, the cardiomyocytes are inoculated in a culture medium containing 1% double antibody and 10% FBS, cultured and subcultured, the cells are grouped, and then the total protein of the cardiomyocytes in each group is extracted, and the Ca concentration is measured according to the total protein concentration of the cardiomyocytes. 2+ -ATPase activity;

[0015] The grouping includes a basal culture medium, wherein the basal culture medium is a DMEM culture medium for culturing cells; a drug administration group, wherein 20 μM codonopsis ginsenoside, 20 μM codonopsis ginsenoside I, 20 μM notoginseng saponin R1, 20 μM notoginseng saponin R2, 20 μM notoginseng saponin Fc and 20 μM phthalide extract are dissolved in a high-glucose DMEM culture medium for culturing cells; and a positive drug administration group, wherein 10 μM ATP is dissolved in a high-glucose DMEM culture medium for culturing cells.

[0016] According to the second aspect of the present application, the present application provides a method for improving the endoplasmic reticulum stress response and regulating Ca according to the above-mentioned dangshen glycosides. 2+ -Application of ATPase activity in improving doxorubicin-induced heart failure.

[0017] The dangshen glycoside provided in the present application improves the endoplasmic reticulum stress response and regulates the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method and its application, using codonopsis pilosula glycoside molecules as ligands, Ca 2+ -ATPase acts as a receptor to bind codonopsis glycoside molecules to Ca 2+ -ATPase molecular docking, dangshen glycoside molecules can bind to Ca 2+ -ATPase protein active cavity, the codonopsis glycoside molecule can enhance the Ca 2+ -ATPase activity, codonopsis pilosula and Ca 2+After docking with the -ATPase, it has hydrogen bonding and van der Waals forces, and has good Alkyl, Pi-Alkyl and metal receptor effects. The oxygen atoms in the protein and small molecule structure have strong electron-withdrawing and hydrogen bonding forces, indicating that the dangshen glycoside small molecule can be well bound to Ca 2+ -ATPase protein active cavity, thereby exerting its effect. In vitro experiments have found that dangshen glycoside, an active ingredient of dangshen, can improve endoplasmic reticulum stress in the glucose deprivation model and enhance Ca2+ in cardiomyocytes. 2+ -ATPase activity. In vivo experiments found that codonopsis pilosula can improve cardiac ejection fraction, left ventricular short-axis shortening rate, left ventricular end-systolic volume and cardiac output in heart failure, and significantly improved doxorubicin-induced heart failure, revealing the protective mechanism of codonopsis pilosula on the myocardium, providing a basis for the clinical application of codonopsis pilosula to treat heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram showing the effect of codonopsis pilosula on the expression of endoplasmic reticulum stress protein in rat cardiomyocyte H9c2 induced by glucose deficiency provided in Example 1 of the present application;

[0019] Figure 2 Shown is a ThT fluorescence detection diagram of rat cardiomyocytes H9c2 provided in Example 2 of the present application after administration of codonopsis pilosula glycosides in a glucose deficiency model;

[0020] Figure 3 Shown is a graph of ThT fluorescence intensity of rat cardiomyocytes H9c2 provided in Example 2 of the present application after administration of codonopsis pilosula glycosides in a glucose deficiency model;

[0021] Figure 4 Shown is the Fluo4-AM fluorescence image of rat myocardial cells H9c2 in each group after adding CaCl2 provided in Example 3 of the present application;

[0022] Figure 5 The results are shown in Example 3 of the present application, which show the Ca content of rat myocardial cells H9c2 in each group after adding CaCl2. 2+ Ion content change curve;

[0023] Figure 6 The Ca2+ of rat myocardial cells H9c2 provided in Example 4 of the present application is shown. 2+ -Comparison chart of ATPase activity;

[0024] Figure 7 The results are shown in Example 5 of the present application for the combination of dangshen glycoside and Ca 2+ -ATPase protein (5ZTF) molecular docking binding diagram;

[0025] Figure 8Shown is a schematic diagram of the mouse ultrasound cardiography detection results provided in Example 6 of the present application. DETAILED DESCRIPTION

[0026] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It will be understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0027] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0028] Example 1: Detection of the expression of endoplasmic reticulum stress-related factors in rat myocardial cells by codonopsis pilosula

[0029] 1. Cell culture

[0030] Rat cardiomyocytes H9c2 (purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd., catalog number BFN60804388) were inoculated in DMEM complete medium (containing 1% double antibody and 10% FBS), and the inoculated DMEM medium was placed in a constant temperature incubator at 37°C and 5% CO2 for culture. When the cell fusion degree in the inoculated DMEM medium reached more than 70%, the cells were subcultured, and the cells were subcultured by direct bottling.

[0031] 2. Cell Grouping and Processing

[0032] Blank group (Contol): cells cultured in normal DMEM medium;

[0033] Model group: cells cultured in sugar-free DMEM medium;

[0034] Drug administration group: Codonopsis pilosula (purchased from Yuanye Biotechnology, product number B20283) was dissolved in sugar-free DMEM medium, and prepared into solutions containing 10, 20, and 40 μM codonopsis pilosula, and cells were cultured;

[0035] Positive administration group (4-PBA): 4-PBA (4-Phenylbutyric acid, an endoplasmic reticulum stress inhibitor) was dissolved in a sugar-free DMEM medium to prepare a solution containing 5 μM 4-PBA, and the cells were cultured using the solution.

[0036] The subcultured rat cardiomyocytes H9c2 were inoculated into the culture medium of the blank group, model group, drug group and positive drug group, respectively, and the culture dishes inoculated with the subcultured rat cardiomyocytes H9c2 were placed in a 5% CO2, 37°C incubator for further culture for 12 hours.

[0037] 3. Detection of the expression of endoplasmic reticulum stress indicator proteins in rat cardiomyocytes by Western Blot

[0038] (1) Extraction of total protein from rat cardiomyocytes: Discard the cell culture medium in each culture dish, add 2 mL of PBS to each culture dish to wash rat cardiomyocytes H9c2, wash twice, add 150 μL of protein lysis buffer to rat cardiomyocytes H9c2 after washing, the protein lysis buffer is prepared by mixing RIPA lysis buffer (purchased from Beijing Solebow Technology Co., Ltd.) and phenylmethylsulfonyl fluoride (PMSF, purchased from Merk) at a ratio of 100:1, lyse the cells on ice for 30 min after adding the protein lysis buffer, scrape the cells with a cell scraper and place them in an EP tube, place the cells in the EP tube after lysis in a pre-cooled centrifuge, centrifuge at 12000 rpm for 10 min at 4°C, and collect the protein supernatant. Take 60 μL of protein supernatant from each group, add 15 μL of loading buffer (addition amount = sample volume / 5), and boil the protein in a 100°C metal bath for 3 min. The denatured rat cardiomyocyte total protein was stored in a -20°C refrigerator.

[0039] (2) Preparation of SDS-PAGE gel: Prepare a 12% separation gel mixture. Carefully mix the mixture in a 50 mL centrifuge tube, then pour the gel into the gel preparation tank. The surface of the gel is about 2 cm from the top. Add a layer of isopropanol on the surface of the separation gel to make the surface of the gel flat. After about 30 minutes, the separation gel solidifies. After the gel solidifies, pour off the liquid on the surface, and then use filter paper to absorb the isopropanol on the surface of the gel.

[0040] (3) Prepare 5% concentrated gel mixture: Add 5% concentrated gel mixture onto the separation gel and insert an electrophoresis comb of corresponding thickness. After the concentrated gel solidifies, leave it at room temperature.

[0041] (4) Western-blot detection: Take out the protein treated in step (1), add 5 μL of protein solution to each gel well, and after the sample is added, run the gel at a constant voltage. The voltage is set to 80V in the concentrated gel. When the bromophenol blue tracer dye enters the separation gel, adjust the voltage to 120V. Turn off the power until the protein reaches the bottom of the separation gel. After that, transfer the membrane using the semi-dry method. The PVDF membrane is first placed in methanol for 30 seconds for activation; it is placed in the order of positive electrode-filter paper-PVDF membrane-gel-filter paper-negative electrode, and an appropriate amount of 1× transfer buffer is added. After transferring the membrane for 17 minutes using a standard semi-dry transfer instrument (Bio-Rad, USA) at a constant voltage of 17V, the PVDF membrane is blocked with a pre-prepared 5% skim milk blocking solution for 1 hour.

[0042] (6) Primary antibody incubation: The target primary antibody used is an endoplasmic reticulum stress indicator, the antibody is derived from rabbit, and can react specifically with rat antigens. The primary antibody solution is prepared with 5% skim milk at a concentration ratio of 1:1000; the internal reference primary antibody is β-actin antibody, the antibody is derived from rabbit, and can react specifically with rat antigens. It is prepared with 5% skim milk at a concentration ratio of 1:50000. Transfer the blocked PVDF membrane in step (5) to the prepared antibody and incubate overnight at 4°C. The next day, wash the PVDF membrane three times with 1×TBST buffer, 10 minutes each time.

[0043] (7) Secondary antibody incubation: The secondary antibody used was HRP Goat anti-Rabbit IgG (Abcam, ab205718), which can react specifically with the primary antibody from rabbit; the secondary antibody solution was prepared with 5% skim milk, and the concentration ratio was secondary antigen solution: 5% skim milk = 1:10000. The washed PVDF membrane was transferred to the secondary antibody, incubated on a shaker at room temperature for 50 min, and then washed 3 times with 1×TBST buffer on a shaker for 10 min each time.

[0044] (8) Chemiluminescence development: Millipore chemiluminescence reagent (Merk Company) was mixed at a ratio of solution A: solution B = 1:1, and chemiluminescence detection was performed using the iBright multifunctional imaging analysis system (Thermofisher scientific company).

[0045] Figure 1 The figure shows the effect of codonopsis ginsenoside on the expression of endoplasmic reticulum stress proteins in H9c2 rat cardiomyocytes induced by glucose deficiency provided in Example 1 of the present application. The expression of P-IRE1α (phosphorylated inositol-requiring kinase 1α), ATF6, GRP78 (glucose-regulated protein) and β-action (actin) was detected by WB method. β-action (actin) was used as an internal reference protein, which was not affected by the expression of endoplasmic reticulum stress proteins in cells. Figure 1 As shown in a, compared with the blank group (Contol), the expression of P-IRE1α and GRP78 proteins in rat cardiomyocytes in the model group (Model) was significantly upregulated; Figure 1 b. Figure 1 c. Figure 1 As shown in Figure d, compared with the model group (Model), after adding different concentrations of codonopsis pilosula (Q10, Q20, Q40), the expression of P-IRE1α, ATF6, and GRP78 proteins were downregulated. The results showed that different doses of codonopsis pilosula can alleviate the endoplasmic reticulum stress caused by glucose deficiency.

[0046] Example 2: Detection of ThT in rat myocardial cells by codonopsis pilosula

[0047] 1. Cell culture

[0048] Rat cardiomyocytes H9c2 (purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd., catalog number BFN60804388) were inoculated in DMEM complete medium (containing 1% double antibody and 10% FBS), and the inoculated DMEM medium was placed in a constant temperature incubator at 37°C and 5% CO2 for culture. When the cell fusion degree in the inoculated DMEM medium reached more than 70%, the cells were subcultured, and the cells were subcultured by direct bottling.

[0049] 2. Cell Grouping and Processing

[0050] Blank group (Contol): cells cultured in normal DMEM medium;

[0051] Model group: cells cultured in sugar-free DMEM medium;

[0052] Drug-treated group: Codonopsis pilosula was dissolved in sugar-free DMEM medium to prepare solutions containing 10, 20, and 40 μM codonopsis pilosula, and cells were cultured using solutions containing 10, 20, and 40 μM codonopsis pilosula;

[0053] Positive administration group (4-PBA): 4-PBA (4-Phenylbutyric acid, an endoplasmic reticulum stress inhibitor) was dissolved in a sugar-free DMEM medium to prepare a solution containing 5 μM 4-PBA, and the cells were cultured using the solution.

[0054] The subcultured rat cardiomyocytes H9c2 were inoculated into the culture medium of the blank group, model group, drug group and positive drug group, respectively, and the confocal dishes inoculated with the subcultured rat cardiomyocytes H9c2 were placed in a 5% CO2, 37°C incubator for further culture for 12 hours.

[0055] 3ThT (Thioflavin T) immunofluorescence detection

[0056] Endoplasmic reticulum stress is a pathological state of endoplasmic reticulum dysfunction in which cells respond to conditions such as misfolded and unfolded protein aggregation and calcium ion balance disorders in the endoplasmic reticulum lumen. Thioflavin T (ThT) is a benzothiazole dye that shows enhanced fluorescence after binding to amyloid fibrils. ThT can bind to structural proteins containing β-folds, such as a-synuclein fibrils. Once bound, the emission spectrum of Thioflavin T will red-shift to produce green fluorescence, and the fluorescence intensity will be significantly enhanced. This is a good method for detecting the formation of fibrils and unfolded proteins, and is usually used to diagnose unfolded proteins and amyloid fibrils.

[0057] (1) Fixation: 4% paraformaldehyde was added to confocal microplates (purchased from Wuxi Nice Life Science Co., Ltd.) of rat cardiomyocytes H9c2 in the blank group, model group, drug group, and positive drug group after culture in the incubator for 12 h, and the rat cardiomyocytes H9C2 were fixed for 20 min at room temperature and washed three times with PBS, each time for 5 min.

[0058] (2) Membrane disruption: 0.1% Triton was added to the confocal microplates of each group, and the microplates were allowed to stand at room temperature for 15 min. The microplates were then washed with PBS for 3 times, each time for 5 min.

[0059] (3) Blocking: Add 5% goat serum blocking solution to the confocal microplates of each group and infiltrate the microplates with goat serum blocking solution at room temperature for 2 h.

[0060] (4) ThT fluorescence incubation: Add diluted Thioflavin T (ThT) fluorescence reagent (AbMole) to each confocal dish, incubate at 37°C for 30 min, and then wash three times with PBS, each time for 5 min.

[0061] (5) Nuclear staining: Add 1× DAPI staining solution to the confocal microplates of each group, treat at room temperature for 5 min, and wash three times with PBS, each time for 5 min.

[0062] (6) Sealing: The slides were sealed and stored with anti-fluorescence quencher (Southern Biotech).

[0063] (7) Photography: Photography was performed using a STELLARIS5Cryo confocal microscope (Leica Microsystems, Germany).

[0064] Figure 2Shown is a ThT fluorescence detection diagram of H9c2 cells provided in Example 2 of the present application after administration of codonopsis pilosula glycosides in a glucose deficiency model; Figure 3 Shown is a ThT fluorescence intensity diagram of H9c2 cells provided in Example 2 of the present application after administration of codonopsis pilosula glycosides in a glucose-deficient model; Figure 2 As shown in the figure, ThT, as a fluorescent indicator of endoplasmic reticulum stress, can fully reflect the situation of endoplasmic reticulum stress in cells according to the fluorescence intensity. In rat cardiomyocytes H9c2, compared with the Control group, the Model group increased the green fluorescence intensity of ThT, indicating that the model group caused endoplasmic reticulum stress; Figure 3 As shown, administration of drugs such as codonopsis pilosula (10 μM, 20 μM, 40 μM) and 4-PBA (5 μM) can alleviate the endoplasmic reticulum stress state of H9c2 cells after modeling, and the fluorescence is weakened.

[0065] Example 3. Ca2+ in rat myocardial cells 2+ Fluorescence assay (Fluo4-AM)

[0066] 1. Cell culture

[0067] Rat cardiomyocytes H9c2 (Qingqi (Shanghai) Biotechnology Development Co., Ltd., catalog number BFN60804388) were inoculated in DMEM complete medium (containing 1% double antibody and 10% FBS), and the inoculated DMEM medium was placed in a constant temperature incubator at 37°C and 5% CO2 for culture. When the cell confluence in the inoculated DMEM medium reached more than 70%, the cells were subcultured by direct flask division.

[0068] 2. Grouping of rat cardiomyocytes H9c2

[0069] Rat cardiomyocytes H9c2 were divided into:

[0070] Blank group (Contol), cells were cultured with normal DMEM medium;

[0071] ATP activation group (ATP(A)), 10 μM ATP standard solution was added to the DMEM medium after subculture;

[0072] SERCA inhibitor group (Thapsigargin (TG)), 5 μM Thapsigargin (sarco / endoplasmic reticulum Ca 2+ -ATPase inhibitors);

[0073] In the drug-treated group (Lobetyolin (L)), 20 μM codonopsis pilosula was added to the DMEM medium after subculture, and the drug-treated group was incubated for 12 h in advance;

[0074] In the drug administration + inhibitor group (Lobetyolin (L) + Thapsigargin (TG)), 20 μM of codonopsis pilosula and 5 μM of Thapsigargin were added to the DMEM medium after subculture, and the drug administration group was incubated at 37°C for 12 h in advance.

[0075] 3. Fluo-4 AM fluorescent probe loading in H9c2 rat cardiomyocytes

[0076] Fluo-4 AM (fluorescent dye) is often used as a fluorescent probe in cell biology experiments to detect changes in intracellular calcium ion concentration. The detection principle is based on the fact that Fluo-4 AM can penetrate the cell membrane and enter the cell, and then be cleaved by esterases in the cell to form Fluo-4, which is then retained in the cell. The free ligand of Fluo-4 is almost non-fluorescent, but when it binds to intracellular calcium ions, it can produce strong fluorescence, which increases by 60 to 80 times.

[0077] Take out the rat cardiomyocytes H9c2 cultured in group in step 2, remove the culture medium, and wash the cells 3 times with HBSS solution. Add the prepared Fluo-4 AM solution to the 96-well plate for culturing cells and incubate at 37°C for 30 minutes, then aspirate the Fluo-4 AM solution. Wash the cells 3 times with HBSS solution to fully remove the residual Fluo-4 AM, and incubate in a cell culture incubator for about 20-30 minutes. During the whole process, the 96-well plate is wrapped with tin foil to avoid light.

[0078] 4. Calcium imaging in rat cardiomyocytes

[0079] Place the cells loaded with Fluo-4 AM in step 3 on the stage of a fluorescence microscope and detect the cells using a live cell workstation with an excitation wavelength of 494 nm and an emission wavelength of 516 nm. The exposure frequency is 1 Hz for 30 s, and the calculated ratio represents the relative calcium level. When perfusion of a CaCl2 solution (1.5 mM) that can induce an increase in intracellular calcium is performed, changes in fluorescence intensity can be observed. Specifically, when the intracellular calcium concentration increases, the fluorescence intensity at an excitation wavelength of 494 nm increases, while the fluorescence intensity at an excitation wavelength of 516 nm decreases, so the ratio of relative calcium levels increases.

[0080] Fluorescence intensity of Fluo-4 reflects intracytoplasmic Ca in rat cardiomyocytes 2+ Before the addition of CaCl2 solution, the cells emitted weak green fluorescence under 488nm excitation light. Figure 4 The figure shows the fluorescence images of Fluo4,AM in H9c2 cells in each group after adding CaCl2 provided in Example 3 of the present application; Figure 4As shown in the figure, after adding 1.5mM CaCl2 solution, under the action of incubation with 5μM Thapsigargin (TG, SERCA2a inhibitor), the Fluo-4 fluorescence intensity of H9c2 cells was significantly stronger than that of the blank group (Contol) and the drug group (Lobetyolin (L)), which indicates that the addition of CaCl2 to H9c2 cells can inhibit the activity of SERCA2a and increase the intracytoplasmic Ca 2+ In cells treated with 20 μM codonopsis glycoside or 10 μM ATP (SERCA2a agonist), the Fluo-4 fluorescence intensity of H9c2 cells was significantly weakened compared with the normal group, indicating that 20 μM codonopsis glycoside and ATP have SERCA2a agonist effects. After the cells were treated with 20 μM codonopsis glycoside + SERCA2a inhibitor 5 μM Thapsigargin, the Fluo-4 fluorescence intensity increased compared with the 20 μM codonopsis glycoside group, and the intracellular Ca 2+ The increase in concentration indicated that Thapsigargin could inhibit SERCA2a and block the stimulatory effect of codonopsis pilosula on SERCA2a. At the end of the observation, the Fluo-4 fluorescence intensity of each group increased by more than 2 times.

[0081] Figure 5 The figure shows the intracellular Ca concentration of H9c2 cells in each group after adding CaCl2 provided in one embodiment of the present application. 2+ Ion content change curve; Figure 5 As shown in the figure, it can be seen that the Fluo-4 fluorescence intensity of H9c2 cells in the drug group (Lobetyolin (L)) and ATP activation group (ATP (A)) was significantly weakened compared with the normal group, which indicates that the addition of 20 μM codonopsis glycoside to H9c2 cells has a SERCA2a stimulating effect similar to ATP, and can reduce cytoplasmic Ca by enhancing SERCA2a activity. 2+ The concentration of 5-6-dose dapoxetine was 44.3%, while the Fluo-4 fluorescence intensity of H9c2 cells in the blank group (Contol), SERCA inhibitor group (Thapsigargin (TG)), and drug administration + inhibitor group (Lobetyolin (L) + Thapsigargin (TG)) was significantly enhanced compared with other groups. Because Thapsigargin can inhibit SERCA2a and block the stimulatory effect of codonopsis pilosula on SERCA2a, the activity of SERCA2a is decreased, and the cytoplasmic Ca 2+ Therefore, dangshen glycosides can enhance the activity of SERCA2a and reduce cytoplasmic Ca 2+ concentration.

[0082] Example 4: Codonopsis pilosula regulates Ca2+ in rat myocardial cells 2+-ATPase activity detection

[0083] 1. Cell culture

[0084] Rat cardiomyocytes H9c2 (purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd., catalog number BFN60804388) were inoculated in DMEM complete medium (containing 1% double antibody and 10% FBS), and the inoculated DMEM medium was placed in a constant temperature incubator at 37°C and 5% CO2 for culture. When the cell fusion degree in the inoculated DMEM medium reached more than 70%, the cells were subcultured, and the cells were subcultured by direct bottling.

[0085] 2. Cell Grouping and Processing

[0086] Basic culture medium: normal DMEM medium to culture cells;

[0087] Drug administration group: Codonopsis pilosula (20 μM), Codonopsis pilosula I (20 μM), notoginsenoside R1 (20 μM), notoginsenoside R2 (20 μM), notoginsenoside Fc (20 μM) and phthalide extract (20 μM) were dissolved in high-glucose DMEM medium;

[0088] Positive drug administration group: 10 μM ATP was dissolved in high-glucose DMEM medium to prepare a solution containing 10 μM ATP, and the cells were cultured using this solution.

[0089] The subcultured rat cardiomyocytes H9c2 were inoculated into the culture medium of the blank group, model group, drug group and positive drug group, respectively, and the culture dishes inoculated with the subcultured rat cardiomyocytes H9c2 were placed in a 5% CO2, 37°C incubator for further culture for 12 hours.

[0090] 3. Extraction of total protein from rat cardiomyocytes: discard the cell culture medium in each culture dish, add 2 mL of PBS to each culture dish to wash rat cardiomyocytes H9c2, wash twice, add 150 μL of protein lysis buffer to rat cardiomyocytes H9c2 after washing, the protein lysis buffer is prepared by RIPA lysis buffer (purchased from Beijing Solebow Technology Co., Ltd.) and phenylmethylsulfonyl fluoride (PMSF, purchased from Merk) in a ratio of 100:1, add protein lysis buffer, lyse cells on ice for 30 minutes, scrape cells with a cell scraper, place in EP tube, place cells in EP tube after lysis in a pre-cooled centrifuge, centrifuge at 12000 rpm for 10 minutes at 4 ° C, and collect protein supernatant. Take 60 μL of protein supernatant from each group, add 15 μL of loading buffer (addition amount = sample volume / 5), and boil protein in 100 ° C metal bath for 3 minutes. Store denatured rat cardiomyocyte total protein in a -20 ° C refrigerator.

[0091] 4. Ca 2+ -Measurement of ATPase activity

[0092] In this experiment, ultra-trace Ca 2+ -ATPase detection kit (Jiangsu Addison Biotechnology Co., Ltd.) was used to analyze the enzyme activity. The microplate reader was preheated for more than 30 minutes, the wavelength was adjusted to 700 nm, all reagents were thawed to room temperature (25°C), and ultra-trace Ca 2+ -In the ATPase detection kit, add 100μL of reagent 1, 100μL of reagent 2, and 100μL of the sample to be tested, incubate at 37℃ for 20min, then add 40μL of reagent 3, mix well, centrifuge at 12000rpm, and 4℃ for 5min, and the supernatant is to be tested: add 100μL of extract and 100μL of reagent 2 to the EP control tube in sequence, incubate at 37℃ for 20min, then add 100100μL of the sample to be tested, mix well, centrifuge at 12000rpm, and 4℃ for 5min, and the supernatant is to be tested.

[0093] Color reaction: Take 50 μL of the supernatant from each group and add 200 μL of ultra-trace Ca 2+ -Reagent 4 in the ATPase assay kit, then mix well, let stand at room temperature for 3 minutes, read the absorbance of each group at a wavelength of 700nm, and determine ΔA=A-A control (each sample is made into a self-control).

[0094] According to Ca 2+ The calculation formula given by the -ATPase kit was used to calculate the Ca2+ between each group of cardiomyocytes (blank control group and codonopsis ginsenoside, codonopsis ginsenoside I, notoginseng saponin R1, notoginseng saponin R2, notoginseng saponin Fc and phthalide extract). 2+-ATPase activity, the specific experimental results are shown in Table 1.

[0095] Table 1 Ca2+ in rat myocardial cells H9c2 2+ -ATPase activity assay table

[0096]

[0097] 5. Statistical analysis

[0098] GraphPad5 software was used to record and organize the absorbance data of each group. The experimental results were analyzed using SPSS (Social Science Standards Institute).

[0099] Statistical analysis was performed using the Statistical Software Package 21.0, and the mean value plus or minus the standard deviation was used. All experimental results are shown in Table 1. One-way ANOVA was used to analyze the differences among the groups. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001, indicating that the differences among the groups are statistically significant.

[0100] Figure 6 The Ca2+ of rat myocardial cells H9c2 provided in Example 4 of the present application is shown. 2+ -Comparison chart of ATPase activity, such as Figure 6 As shown in the figure, compared with the blank control group, dangshen glycoside, dangshen glycoside I, notoginsenoside R1, notoginsenoside R2 and notoginsenoside Fc all had the effect of increasing myocardial cell Ca 2+ -ATPase activity (P < 0.05).

[0101] Example 5, Codonopsis pilosula and Ca 2+ -ATPase Molecular Docking

[0102] 1. Preparation of Codonopsis pilosula component (ligand)

[0103] Log in to PubChem database (URL: https: / / pubchem.ncbi.nlm.nih.gov / ), use the name of Lobetyolin as the keyword to download the chemical formula sdf format file of the component, draw the structural formula using Chemdraw software, convert the 3D structure through ChemBio3D module, and save it in sdf format. Type the prepared main compounds into the CHARMm force field, and the partial charges are calculated by the Momany-Rone (Momany and Rone, 1992) option. Use the Smart Minimizer algorithm to minimize the obtained structure, which performs 1000 steps of steepest descent, RMS gradient tolerance of 3, and conjugate gradient minimization.

[0104] 2. Ca2+ - Preparation of ATPase receptor protein (PDB: 5ZTF)

[0105] In the PDB protein database (http: / / www.rcsb.org / ), select Ca 2+ -ATPase and Ca 2+ The co-crystal structure of the ion (PDB ID: 5ZTF) is used as the receptor protein, and the receptor protein is imported into the Discovery Studio 2017R2 client software. In the toolbar, expand the Receptor Ligand Interactions tool panel, click DefineReceptor, and define the selected protein molecule 5ZTF as the receptor molecule for docking. Prepare the receptor protein in the Macromolecules section: Click Prepare Protein in Automatic Preparation, set some parameters to Use CHARMm Minimization, the protonation pH is 7.4, the force field used for calculation is the CHARMm force field, remove water, and keep other parameters as default settings. After the receptor is prepared, define the central coordinates of the original ligand as the active site and delete the receptor's own ligand, and save the protein to prepare for docking.

[0106] 3. Docking operation

[0107] Select the Docking Ligands (CDOCKER) semi-flexible docking mode in the Receptor-ligand Interaction section for molecular docking. Parameter setting: Expand the Hop Hits parameter, set the Top Hit to 10, and the Pose ClusterRadius, i.e., the RMSD threshold, to 0.5 to ensure that the docking conformations are as diverse as possible, and set other parameters to fixed values. After the protein is charged, the Covalent Docking module is used to perform molecular docking on the two. The software automatically calculates and stops when the calculation is optimal. The docking results sort each docking pose according to the energy value. The higher the score (the larger the negative value), the more stable the docking system and the better the ligand binds to the receptor. Table 2 shows the scoring results. Codonopsis pilosula can bind to Ca 2+ -ATPase binds well and enters the protein cavity for reasonable interaction.

[0108] Table 2 Molecular docking scoring results

[0109]

[0110] By analyzing the interaction between the ligand small molecule and the receptor macromolecule, the intermolecular affinity is analyzed. In this process, the binding of the ligand and the receptor must meet the principle of mutual matching, that is, the complementary matching of the ligand and receptor geometry, electrostatic interaction, hydrogen bond interaction, hydrophobic interaction, etc. The recognition between molecules is achieved through the interaction force between the receptor and the ligand. The interaction force between the receptor and the ligand mainly includes hydrogen bonds, van der Waals forces, hydrophobic interactions and π-π interactions.

[0111] Figure 7 The results are shown in Example 5 of the present application for the combination of dangshen glycoside and Ca 2+ -ATPase protein (5ZTF) molecular docking binding diagram, such as Figure 7 As shown, codonopsis pilosula and Ca 2+ -ATPase interaction process, the binding with the active site has hydrogen bonding and van der Waals force, and has good Alkyl effect, Pi-Alkyl effect and metal receptor effect. Figure 7 The two-dimensional diagram shows that the oxygen atoms in the protein and small molecule structure have strong electron-withdrawing and hydrogen-bonding forces, which indicates that the dangshen glycoside small molecule can bind well to Ca 2+ -ATPase protein active cavity, thereby playing a role, can be used to enhance the stimulatory effect of SERCA2a, enhance the activity of SERCA2a, reduce cytoplasmic Ca 2+ concentration.

[0112] Example 6: Use of Codonopsis pilosula to Improve Adriamycin-Induced Heart Failure in Mice

[0113] 1. Experimental Animals

[0114] 60 clean-grade healthy C57BL / 6 mice weighing 18-22g were used. All animals were kept in the SPF animal room of Tianjin University of Traditional Chinese Medicine, with a relative humidity of 40-60% and a room temperature of 22±2℃. The Animal Experiment Center provided sterilized pellet feed. Before the experiment, mice were adaptively fed, had free access to water, had no adverse reactions, and were included in the experiment with normal eating, drinking, and activities. The handling procedures of experimental animals complied with the relevant regulations on the breeding and use of experimental animals of Tianjin University of Traditional Chinese Medicine. The animal experiment ethics approval number is TCM-LAEC2021224.

[0115] 2. Animal grouping and drug treatment

[0116] C57BL / 6 mice were divided into 4 groups by random array method: blank group, model group, codonopsis pilosula treatment group, and valsartan treatment group. In this experiment, doxorubicin was used to establish a mouse heart failure model. Doxorubicin is an antitumor antibiotic that can inhibit the synthesis of RNA and DNA. It has the strongest inhibitory effect on RNA, a wide antitumor spectrum, and has effects on a variety of tumors. It is a cycle-nonspecific drug and has a killing effect on tumor cells in various growth cycles. Because doxorubicin has no selectivity and its affinity for the heart is particularly prominent, it has obvious cardiotoxicity and can induce myocardial oxidative stress damage. It is a commonly used induction drug for simulating myocardial heart failure.

[0117] Treatment method: The mice in the blank group were intraperitoneally injected with normal saline 0.9% NaCl; the mice in the model group, codonopsis pilosula treatment group, and valsartan treatment group were injected with doxorubicin (5 mg / kg) once a week for three weeks; while modeling, the model group was gavaged with deionized water, the codonopsis pilosula treatment group was gavaged with codonopsis pilosula 10 mg / kg / d, and the valsartan treatment group was gavaged with valsartan 6 mg / kg / d.

[0118] 3. Small animal ultrasound cardiology testing

[0119] Mice in the blank group, model group, codonopsis ginsenoside treatment group, and valsartan treatment group were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg) and then depilated. The ultra-high-resolution small animal ultrasound real-time imager (Vevo2100) was turned on and ready for use. The probe MS-250 was fixed, the lift table adjusted the probe height, and the probe direction was adjusted by rotation. The mouse abdomen was fixed to the operating table with tape, and the temperature was set to 37°C; a layer of ultrasound coupling agent was evenly applied on the rat's chest, the operating table was tilted 30-45° to the lower right, and the position of the operating table was fine-tuned by X and Y. Parasternal long axis, B-mode ultrasound long axis section (two-dimensional imaging), the left ventricle, aortic arch and mitral valve leaflets were visible, and images were obtained; M-mode ultrasound long axis section, M-Mode image, the sampling line was located at the maximum cavity diameter (tangent to the papillary muscle). By measuring the systolic and diastolic periods of mice, the left ventricular anterior wall (LVAW), left ventricular internal diameter (LVID), and left ventricular posterior wall (LVPW) at end-systole and end-diastole were detected and averaged. The left ventricular ejection fraction (EF), left ventricular shortening (FS), stroke volume (SV), and left ventricular end systolic volume (LVESV) were calculated by software.

[0120] Figure 8The figure shows the results of ultrasound cardiography of mice provided in Example 6 of the present application. Ultrasonic cardiac examination can directly reflect the ejection capacity, contraction and relaxation capacity of the heart, and is one of the indispensable examination items in the diagnosis of heart failure. The left ventricular ejection fraction and the left ventricular short axis shortening rate characterize the contractile function of the left ventricle of the mouse, and the left ventricular end-systolic volume and the cardiac output per stroke characterize the blood output of the mouse heart. By stimulating with doxorubicin, heart failure in mice was induced, such as Figure 8 As shown in the data, the ventricular wall contractility of mice with heart failure was significantly decreased. After being given 10 mg / kg / d of codonopsis pilosula and 6 mg / kg / d of valsartan, the ventricular wall contractility of mice was improved. According to the results of echocardiography, it was found that the cardiac ejection fraction and short-axis shortening rate of mice with heart failure were significantly reduced. Codonopsis pilosula treatment can improve the cardiac ejection fraction and short-axis shortening rate of mice; the left ventricular end-systolic volume of mice with heart failure was increased, and the stroke volume was significantly reduced. After being treated with codonopsis pilosula, the left ventricular end-systolic volume and stroke volume of mice were improved. This shows that codonopsis pilosula can be used to improve doxorubicin-induced heart failure in mice, providing an experimental basis for the clinical application of Codonopsis pilosula in the treatment of heart failure.

[0121] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Codonopsis pilosula improves endoplasmic reticulum stress response and regulates endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method, characterized in that, Using codonopsis pilosula glycoside as ligand, Ca 2+ -ATPase acts as a receptor to bind codonopsis glycoside molecules to Ca 2+ -ATPase molecular docking, dangshen glycoside molecules can bind to Ca 2+ -ATPase protein active cavity, the codonopsis glycoside molecule can enhance the Ca 2 + -ATPase activity, thereby regulating the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity.

2. The dangshen glycoside according to claim 1 improves the endoplasmic reticulum stress response and regulates the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method, characterized in that, The following docking steps are included: Step (1), preparation of the codonopsis glycoside ligand, by downloading the chemical formula of the codonopsis glycoside corresponding to the component from a database, drawing the structural formula using a drawing software, and then converting it into a 3D structure using a 3D module, and then entering the compound into a force field to calculate the charge; Step (2), Ca 2+ - Preparation of ATPase receptor proteins, select Ca in the protein data 2+ -ATPase and Ca 2+ The co-crystal structure of the ion was used as the receptor protein, the receptor protein was imported into the software, the force field was calculated, and the protein was saved in preparation for docking; Step (3), docking operation, after adding charges to the protein, the two are molecularly docked using the Covalent Docking module in the software.

3. The dangshen glycoside according to claim 2 improves the endoplasmic reticulum stress response and regulates the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method, characterized in that, In the step (1), the PubChem database is logged in, and the sdf format file of the chemical formula corresponding to the component is downloaded from the database by using codonopsis pilosula as a keyword. The structural formula is drawn using Chemdraw software and then converted into a 3D structure using the ChemBio3D module and saved in sdf format. The prepared compound is entered into the CHARMm force field, and the structure is minimized using the intelligent minimization algorithm.

4. The dangshen glycoside according to claim 2 improves the endoplasmic reticulum stress response and regulates the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method, characterized in that, In the step (2), select Ca in the PDB protein database 2+ -ATPase and Ca 2+ The co-crystal structure of the ion is used as the receptor protein. The receptor protein is imported into the Discovery Studio2017R2 client software. The protein is defined as a receptor molecule for docking. After the receptor is prepared, the central coordinates of the original ligand are defined as the active site and the receptor's own ligand is deleted. The protein is saved in preparation for docking.

5. The dangshen glycoside according to claim 2 improves the endoplasmic reticulum stress response and regulates the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method, characterized in that, Prior to step (1), the method also includes an experiment on the effect of codonopsis pilosula on alleviating the endoplasmic reticulum stress state of cells caused by glucose deficiency, wherein the cardiomyocytes are inoculated in a culture medium containing 1% double antibody and 10% FBS, and after culture and subculture, the cells are grouped and processed, and then the expression of endoplasmic reticulum stress indicator proteins in the cardiomyocytes is detected by Western Blot.

6. The dangshen glycoside according to claim 2 improves the endoplasmic reticulum stress response and regulates the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method, characterized in that, Prior to step (1), the method also includes an experiment on the effect of codonopsis pilosula on alleviating the stress state of the endoplasmic reticulum of H9c2 cells after modeling, wherein the cardiomyocytes are inoculated in a culture medium containing 1% double antibody and 10% FBS, and after culture and subculture, the cells are grouped and processed, and then the stress state of the endoplasmic reticulum in the cardiomyocytes is detected by the ThT immunofluorescence method.

7. The dangshen glycoside according to claim 5 or 6 improves the endoplasmic reticulum stress response and regulates the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method, characterized in that, The cardiomyocytes are rat cardiomyocytes H9c2; the groups include: a blank group, in which cells are cultured in normal DMEM medium; a model group, in which cells are cultured in sugar-free DMEM medium; a drug administration group, in which codonopsis pilosula is dissolved in sugar-free DMEM medium, and the solutions are respectively prepared to contain 10, 20, and 40 μM codonopsis pilosula glycoside and the cells are cultured; and a positive drug administration group, in which 4-PBA is dissolved in sugar-free DMEM medium, and the solution is prepared to contain 5 μM 4-PBA and the cells are cultured.

8. The dangshen glycoside according to claim 2 improves the endoplasmic reticulum stress response and regulates the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method, characterized in that, Before the step (1), the method further comprises: 2+ In the fluorescence measurement experiment, cardiomyocytes were inoculated in a culture medium containing 1% double antibody and 10% FBS. After culture and subculture, the cells were divided into groups and treated. Fluo-4 AM fluorescent probe was then used to detect intracellular Ca 2+ concentration; The groups include a blank group, in which cells are cultured in normal DMEM medium; an ATP activation group, in which 10 μM ATP standard solution is added to the DMEM medium; a SERCA inhibitor group, in which 5 μM Thapsigargin is added to the DMEM medium; a drug administration group, in which 20 μM codonopsis glycoside is added to the DMEM medium; and a drug administration + inhibitor group, in which 20 μM codonopsis glycoside and 5 μM Thapsigargin are added to the DMEM medium.

9. The dangshen glycoside according to claim 2 improves the endoplasmic reticulum stress response and regulates the endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -ATPase activity method, characterized in that, Before the step (1), the method further comprises: regulating myocardial cell Ca 2+ -ATPase activity experiment, the cardiomyocytes are rat cardiomyocytes H9c2, the cardiomyocytes are inoculated in a culture medium containing 1% double antibody and 10% FBS, cultured and subcultured, the cardiomyocytes are grouped, and then the total protein of the cardiomyocytes in each group is extracted, and the Ca concentration is measured according to the total protein concentration of the cardiomyocytes. 2+ -ATPase activity; The grouping includes a basal culture medium, wherein the basal culture medium is a DMEM culture medium for culturing cells; a drug administration group, wherein 20 μM codonopsis ginsenoside, 20 μM codonopsis ginsenoside I, 20 μM notoginseng saponin R1, 20 μM notoginseng saponin R2, 20 μM notoginseng saponin Fc and 20 μM phthalide extract are dissolved in a high-glucose DMEM culture medium for culturing cells; and a positive drug administration group, wherein 10 μM ATP is dissolved in a high-glucose DMEM culture medium for culturing cells.

10. The dangshen glycoside according to any one of claims 1 to 9 improves endoplasmic reticulum stress response and regulates endoplasmic reticulum / sarcoplasmic reticulum Ca 2+ -Application of ATPase activity in improving doxorubicin-induced heart failure.

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