Application of forsythiaside A in preparation of medicine for treating or preventing heart injury caused by adriamycin

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of forsythiaside A in preparation of a medicine for treating or preventing heart injury caused by adriamycin. The invention discloses the application of forsythiaside A in preparing the medicine for treating or preventing heart injury caused by adriamycin for the first time, and in-vivo and in-vitro experiments prove that forsythiaside A can perform reverse inhibition on an adriamycin cardiotoxicity mechanism and does not interfere with the killing effect of adriamycin on tumor cells under an effective dose; the problem of lack of adriamycin cardiotoxicity prevention and treatment means at present is solved, and a brand new solution and a new, safe and effective candidate pharmaceutical composition are provided for clinical prevention and treatment of adriamycin cardiotoxicity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of forsythoside A in preparation of a drug for treating or preventing adriamycin-induced cardiac injury. BACKGROUND

[0002] Adriamycin is a highly efficient and broad-spectrum anthracycline antitumor antibiotic, which is widely used in the treatment of leukemia, lymphoma and various solid tumors. However, the clinical application of adriamycin is greatly limited due to its serious dose-dependent cardiotoxicity. The cardiotoxicity of adriamycin is manifested in clinical practice as acute and chronic myocardial injury, which can eventually develop into irreversible dilated cardiomyopathy and congestive heart failure, with a very high mortality rate.

[0003] Through patent retrieval, there are very limited drugs for preventing and treating adriamycin cardiotoxicity in clinical practice. Dexrazoxane is the only chemical protective agent approved by FDA for this indication, but it has bone marrow suppression, may interfere with the antitumor efficacy of adriamycin, and has the risk of causing secondary malignant tumors. Therefore, developing a new type of high-efficiency, low-toxicity and non-interference with the antitumor activity of adriamycin cardioprotective drug is a key problem to be solved in the current tumor treatment field.

[0004] Forsythoside A is a phenylethanoid glycoside compound extracted from traditional Chinese medicine Forsythia suspensa. Modern pharmacological studies have shown that it has significant antioxidant, anti-inflammatory, anti-apoptotic and neuroprotective activities. So far, no literature or patent has disclosed that forsythoside A has a therapeutic or preventive effect on adriamycin-induced cardiac injury. The present application first discovers the new use of forsythoside A in this specific field, and provides a new solution for the clinical prevention and treatment of adriamycin cardiotoxicity. SUMMARY

[0005] Through relevant patent technology and research literature retrieval, the inventors found that the mechanism of adriamycin cardiotoxicity is relatively complex, which mainly includes the following aspects: (11) Oxidative stress: adriamycin can metabolize a large amount of reactive oxygen species (ROS) in myocardial cells, and the antioxidant enzyme system of myocardial cells is relatively weak. A large amount of ROS causes oxidative stress in myocardial cells, leading to injury.

[0006] (12) Topoisomerase II beta inhibition: adriamycin can inhibit topoisomerase II beta in myocardial cells, causing DNA double-strand breaks and transcription abnormalities in cells, thereby leading to cardiac injury.

[0007] (13) Iron ion-mediated injury: adriamycin can form a complex with iron ions to catalyze the production of highly toxic hydroxyl radicals to initiate peroxidation of myocardial cell membrane lipids.

[0008] (14) Mitochondrial dysfunction: Doxorubicin can destroy the mitochondrial membrane potential, induce the opening of mitochondrial permeability transition pore (mPTP), and initiate the apoptosis program of cardiomyocytes, leading to cardiomyocyte apoptosis and necrosis.

[0009] (15) Calcium overload and inflammatory response: Doxorubicin can cause imbalance of intracellular calcium homeostasis and activate NLRP3 inflammasome and other pathways, leading to chronic inflammation of myocardial tissue.

[0010] In order to solve the problem of lack of prevention and treatment means for doxorubicin cardiotoxicity, the application first discloses the application of forsythoside A in the preparation of a drug for treating or preventing doxorubicin-induced cardiac injury, and briefly introduces the effect thereof. The specific technical scheme is as follows:

[0011] According to one aspect of the present application, the application of forsythoside A in the preparation of a drug for treating or preventing doxorubicin-induced cardiac injury is first disclosed.

[0012] Further, the performance symptoms of the cardiac injury include cardiomyocyte apoptosis, oxidative stress injury of myocardial tissue, inflammation of myocardial tissue, myocardial fibrosis, left ventricular dysfunction, dilated cardiomyopathy or heart failure.

[0013] Further, the drug comprises a therapeutically effective amount of forsythoside A and one or more pharmaceutically acceptable carriers and excipients.

[0014] Further, the administration route of the drug includes oral administration, intravenous injection or intraperitoneal injection.

[0015] Further, the dosage form of the drug includes but is not limited to tablets, capsules, oral liquids, injections or lyophilized powders.

[0016] Further, the administration dose of forsythoside A in the drug is 10-50 mg / kg / day. It should be noted that the preferred administration dose of forsythoside A in the drug is 10-30 mg / kg / day.

[0017] According to another aspect of the present application, the present application also provides a pharmaceutical composition for treating or preventing doxorubicin-induced cardiac injury, which comprises forsythoside A as an active ingredient and a pharmaceutically acceptable carrier or excipient.

[0018] Further, the composition is prepared into an oral preparation or an injection preparation.

[0019] Further, the weight percentage of forsythoside A in the composition is 0.1-99.9%.

[0020] The application discloses application of forsythoside A in preparation of a drug for treating or preventing adriamycin-induced cardiac injury, and solves the problem of lack of prevention and treatment means for adriamycin-induced cardiac injury by reverse inhibition of the mechanism of adriamycin-induced cardiac toxicity through forsythoside A, thereby providing a brand-new solution for clinical prevention and treatment of adriamycin-induced cardiac toxicity.

[0021] Compared with the prior art, the application has at least the following beneficial effects: 16. New use of old drugs and innovative discovery: the application first discovers the specific protection of forsythoside A on adriamycin-induced cardiac toxicity, and develops a new use of forsythoside A in the specific field of prevention and treatment of adriamycin-induced cardiac toxicity, which belongs to the development of new use of known drugs.

[0022] 17. Multi-target synergistic protection: experiments prove that forsythoside A can significantly improve cardiac dysfunction and pathological injury caused by adriamycin through multiple mechanisms such as anti-oxidation (reducing ROS), anti-apoptosis (regulating Bcl-2 / Bax / Caspase-3 pathway) and anti-fibrosis.

[0023] Three, no influence on the efficacy of chemotherapy: in vitro cell experiments prove that forsythoside A does not interfere with the proliferation inhibition activity of adriamycin on tumor cells (such as MCF-7 breast cancer cells) at an effective concentration for exerting cardiac protection, which is a key advantage of forsythoside A as an adjuvant drug for chemotherapy.

[0024] Four, high safety: forsythoside A is a natural component derived from traditional Chinese medicine forsythia, which is not chemically synthesized, has low toxicity, small side effects and high safety, and has a good clinical transformation prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this application. The embodiments of the present application illustrated in the drawings and their descriptions are used to explain the present application and are not intended to limit the present application. In the drawings:

[0026] Figure 1 Figure 1 is a result graph of the influence of forsythoside A on the viability of H9c2 myocardial cells caused by adriamycin; Figure 2 Figure 2 is a line graph of the in vitro proliferation activity of MCF-7 breast cancer cells caused by adriamycin; Figure 3 Figure 3 is a graph of the observation of cardiac function and myocardial tissue section pathology of mice; Figure 4 Figure 4 is a graph of the Western Blotting detection result of the influence of forsythoside A on the core signal pathway of adriamycin-induced cardiac toxicity. DETAILED DESCRIPTION

[0027] To enable those skilled in the art to better understand the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0028] It should be noted that the terms "comprising" and "having" and any variations thereof in the specification, claims and accompanying drawings of this invention are intended to cover non-exclusive inclusion, such as a process, method, system, product or device that includes a series of steps or units, which is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products or devices.

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0030] Example 1: In vitro protective effect of forsythoside A against doxorubicin-induced H9c2 cardiomyocyte injury 1. Cell resuscitation and culture: H9c2 cardiomyocytes were rapidly thawed in a 37°C water bath. The cell suspension was transferred to a 15 mL centrifuge tube, and 4-6 mL of complete culture medium was slowly added and mixed well. The tube was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were then seeded into culture flasks containing 6-8 mL of complete culture medium and incubated overnight. The incubator parameters were set as follows: temperature 37°C, 5% CO2, humidity 70-80%.

[0031] It should be noted that the complete culture medium is a 10% complete culture medium, which is prepared as follows: fetal bovine serum accounts for 10% of the culture medium, and the antibiotic solution accounts for 1%. After calculating based on the total volume, the serum and antibiotic solution are added to DMEM (1.5 g / L NaHCO3) in sequence, and the mixture is thoroughly mixed by pipetting. It is then stored at 4°C.

[0032] 2. Cell passage: When the adherent cells in the culture flask have grown to about 80%, aspirate the old culture medium, retaining 2-3 mL of culture medium, and gently pipette the cells. Collect the pipetteed cells and centrifuge at 1000 rpm for 5 min, discarding the supernatant; resuspend the cells and passage them at a ratio of 1:2 to 1:4, then seed them into culture flasks containing fresh culture medium.

[0033] 3. Experimental grouping: Cells in the logarithmic growth phase (2-3 days after passage) were divided into groups of 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of cells / well in 96-well cell culture plates and cultured for 24 h. Afterward, cells were randomly assigned to experimental groups, with 6 replicates per group. Control group: Added an equal volume of complete culture medium; Model group (Dox): treated with complete culture medium containing 1 μM doxorubicin (MCE, HY-15142R) for 72 h; Low-dose forsythoside A group (DOX+FA 50): First, add complete culture medium containing 10 μM forsythoside A for 2 h for pretreatment, discard the pretreatment solution, add complete culture medium containing 1 μM doxorubicin and 50 μM forsythoside A, and incubate for a total of 96 h. Low-dose group of forsythoside A (DOX+FA 100): The administration method was the same as above, using a complete culture medium containing 100 μM forsythoside A. Forsythoside A medium-dose group (DOX+FA 150): Administration method was the same as above, using complete culture medium containing 150 μM forsythoside A. High-dose group of forsythoside A (DOX+FA 200): The administration method was the same as above, using a complete culture medium containing 200 μM forsythoside A.

[0034] 4. Cardiac cell viability assay (CCK-8 assay): After drug treatment, add 10 μL of CCK-8 solution (Beyotime, C0038) to each well and incubate for another 2 h in an incubator; measure the OD of each well at 450 nm using a microplate reader. 450 The values ​​were placed under a microscope for observation and recording.

[0035] 5. Analysis of Experimental Results: The experimental results are as follows... Figure 1 As shown, where Figure 1 (A) is an electron micrograph of H9c2 cardiomyocytes in good condition. Figure 1 (B) represents the OD of H9c2 cardiomyocytes. 450 The bar chart shows that compared with the control group, p < 0.01; compared with the model group, p < 0.05 or p < 0.01. Figure 1 (A) It is evident that the addition of forsythoside A significantly increased cell density, indicating that forsythoside A effectively alleviated the damage to cardiomyocytes caused by doxorubicin and reduced cell death. Figure 1(B) It can be seen that compared with the control group, doxorubicin significantly reduced the viability of H9c2 cardiomyocytes. The addition of a small amount (50 μM) of FA had little effect on alleviating doxorubicin toxicity. The addition of medium and low doses (100 μM) of FA increased cell activity, showing a significant difference compared with the model group. It can be seen that forsythoside A can effectively counteract the cardiomyocyte toxicity caused by doxorubicin.

[0036] Example 2: Forsythoside A does not affect the antitumor activity of doxorubicin. 18. Cell resuscitation and culture: Take MCF-7 human breast cancer cells and thaw them rapidly in a 37°C water bath. Transfer the cell suspension to a 15 mL centrifuge tube, slowly add 4-6 mL of complete culture medium and mix well. Centrifuge at 1000 rpm for 5 min and discard the supernatant. Seed the cells into culture flasks containing 6-8 mL of complete culture medium and incubate overnight at 37°C in a 5% CO2 incubator.

[0037] It should be noted that the complete culture medium is a 10% complete culture medium, which is prepared as follows: fetal bovine serum accounts for 10% of the culture medium, and the antibiotic solution accounts for 1%. After calculating based on the total volume, the serum and antibiotic solution are added to DMEM (1.5 g / L NaHCO3) in sequence, and the mixture is thoroughly mixed by pipetting. It is then stored at 4°C.

[0038] 2. Cell passage: When the adherent cells in the culture flask grow to about 80-85%, aspirate the old culture medium, wash twice with sterile PBS, add 1 mL of 0.25% trypsin-EDTA digestion solution, and incubate for 2-3 minutes. Observe under a microscope. When the intercellular spaces significantly increase, the cells shrink and become rounded and begin to detach in sheets, immediately add complete culture medium to stop the digestion. Centrifuge the cell suspension at 1000 rpm for 5 minutes and discard the supernatant. After resuspending, passage at a ratio of 1:3 to 1:4 and inoculate into culture flasks containing fresh culture medium.

[0039] 3. Experimental grouping: MCF-7 cells in the logarithmic growth phase (2-3 days after passage) were digested and prepared into single-cell suspensions, and then cultured at 1×10⁻⁶ cells per cell. 4 The cells were seeded at a density of 1000 cells / well in 96-well plates and cultured for 24 h. Then, the cells were randomly assigned to experimental groups with 6 replicates per group. Doxorubicin monotherapy group (DOX): Doxorubicin concentration gradients were set (0, 0.25, 0.5, 1, 2, 4 μM); Group A of combined drug therapy (FA 50 μM): Based on the doxorubicin gradient concentration, a fixed concentration of forsythoside A 50 μM was added simultaneously; Group B of combined medication (FA 100μM): Based on the doxorubicin gradient concentration, a fixed concentration of forsythoside A 100 μM was added simultaneously.

[0040] 4. Tumor cell viability assay (MTT method): The MTT cell proliferation and cytotoxicity assay kit (Beyotime, C009M) was used to detect cytotoxicity in this experiment.

[0041] (1) Solution preparation: Dissolve 25 mg MTT in 5 mL of MTT solvent to prepare a 5 mg / mL MTT solution, and store it in the dark.

[0042] (2) Add 10 μL MTT solution to each well and continue incubation in a cell culture incubator for 4 h.

[0043] (3) Add 100 μL of Formazan dissolving solution to each well, mix well, and continue incubation in a cell culture incubator until the Formazan is completely dissolved under a regular optical microscope, and then measure the absorbance at 570 nm.

[0044] 5. Analysis of Activity Assay Results: Experimental results are as follows Figure 2 As shown, cell viability in the DOX group decreased significantly with increasing doxorubicin concentration, indicating that doxorubicin has a significant killing effect on MCF-7 cells. The curve trends observed in the FA 50μM and FA 100μM groups highly overlapped with those in the DOX group (p>0.05), meaning that when forsythoside A was used in combination, the inhibition of MCF-7 cell viability was almost entirely due to doxorubicin itself, indicating that forsythoside A does not interfere with doxorubicin's entry into tumor cells or its anti-tumor effect.

[0045] Example 3: In vivo protective effect of forsythoside A against doxorubicin-induced cardiac injury in mice. 19. Model Construction: Thirty 8-week-old C57BL / 6J mice were purchased from the Experimental Animal Center of Hubei University of Medicine. All animal experiments were conducted in accordance with the regulations of the institution's Animal Care and Use Committee. The mice were acclimatized in an SPF-grade animal room for one week (12 / 12 h light / dark cycle, free access to water and food) before being divided into groups.

[0046] 20. Model grouping and drug administration regimen: Mice were randomly divided into 3 groups (n=10): Control group: Daily intraperitoneal injection of 10 μL / g of 0.9% normal saline; Model group (DOX): On day 3, acute cardiotoxicity was induced by intraperitoneal injection of doxorubicin 20 mg / kg (doxorubicin was diluted with 0.9% saline to 2 mg / mL); Forsythoside A group (FA+DOX): 30 mg / kg forsythoside A was injected intraperitoneally daily from day 1 to day 6 (i.e., pre-administration started 3 days before modeling and continued for 3 days after modeling).

[0047] 21. Cardiac Function Testing: On day 6 of the experiment, mice were anesthetized using an isoflurane inhalation anesthesia system. The induction concentration was 3%, and the maintenance concentration was 1.5-2%. After the mice lost their positive reflexes, they were fixed supine on a constant-temperature heating plate (maintaining body temperature at 37°C). The fur on the anterior chest area of ​​the mice was removed to fully expose the skin. After applying ultrasound coupling gel, a small animal ultrasound imaging system (VisualSonics Vevo 2100) equipped with a 30 MHz high-frequency probe was used. The probe was placed at the left sternal border to obtain a parasternal short-axis view at the level of the left ventricular papillary muscles. The M-mode ultrasound was activated, and the left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs) were measured over 3-5 consecutive cardiac cycles. The system automatically calculated the left ventricular ejection fraction (LVEF%) and left ventricular short-axis shortening rate (LVFS%) according to the formula. Three measurements were taken for each mouse, and the average value was used. The ultrasound images are shown below. Figure 3 As shown in (A), the parameter evaluation is as follows: Figure 3 As shown in (B).

[0048] 22. Tissue Sampling: After ultrasound examination, mice were immediately euthanized by cervical dislocation. The thoracic cavity was opened, and the heart was quickly excised and placed in pre-cooled PBS buffer (4°C). The heart was gently squeezed to wash away any residual blood in the heart chambers. The heart tissue was cut along the coronal plane, and the portion containing the left ventricle was taken and immersed in 4% paraformaldehyde fixative, fixed at room temperature (25°C) for 24-48 h. The other part of the myocardial tissue was placed in cryovials and flash-frozen in liquid nitrogen, and then transferred to a freezer at -80°C for storage.

[0049] 23. Histopathological examination: (24) Embedding and sectioning: The fixed tissue was routinely dehydrated and embedded, then subjected to 70% ethanol solution for 30 min → 80% ethanol solution for 30 min → 90% ethanol solution for 30 min → 95% ethanol solution for 30 min → anhydrous ethanol for 60 min → anhydrous ethanol for 60 min → xylene for 60 min → xylene for 60 min → paraffin for 60 min → paraffin for 60 min → paraffin for 60 min. After being wrapped into a wax block, it was sectioned and placed in a slide oven. When the slides stood upright and no longer dripped water, they were placed in a 95℃ oven for 1 hour. Then, they were subjected to xylene for 5 min → xylene for 5 min → 70% ethanol solution for 2 min → 80% ethanol solution for 2 min → 90% ethanol solution for 2 min → 95% ethanol solution for 2 min → anhydrous ethanol for 2 min to dehydrate and clear the tissue.

[0050] (25) Hematoxylin-eosin (H&E) staining: This staining process was performed using the Masson trichrome staining kit (Solepro, G1120). The sample sections were gently immersed in hematoxylin staining solution for 4-5 min, then rinsed with tap water for 10 min; then differentiated with hydrochloric acid and ethanol for 2-5 s, rinsed with tap water for 10 min; and stained with eosin solution for 25 s to 1 min. The sample sections were then dehydrated sequentially with 70%, 80%, 95%, and 100% ethanol for approximately 10 s each; subsequently, the sample sections were cleared with xylene for 5 min, replaced with fresh xylene, cleared again for 5 min, and then mounted with neutral resin and observed under a microscope. The staining results are as follows: Figure 3 As shown in (C).

[0051] (26) Masson staining: This staining process was performed using the Masson trichrome staining kit (Solepro, G1340). Ponceau S and fuchsin staining solution were added to the sample slides for 5-10 min. A weak acid working solution was prepared at a ratio of distilled water to weak acid solution of 2:1, and the slides were washed for 30 s. Excess liquid was discarded, and phosphomolybdic acid solution was added for 1-2 min. The slides were washed for 30 s with the weak acid working solution; excess liquid was discarded, and aniline blue staining solution was added for 1-2 min. The slides were washed for 30 s with the weak acid working solution; rapid dehydration was performed with 95% ethanol for 2-3 s, followed by dehydration twice with anhydrous ethanol for 5-10 s each time; clearing was performed twice with xylene for 1-2 min each time; after mounting with neutral resin, the slides were observed under a microscope. The staining results are as follows: Figure 3 As shown in (C).

[0052] (27) Analysis of experimental results: The results of left ventricular ejection fraction (LVEF%) and left ventricular fractional shortening (LVFS%) are as follows: Figure 3 As shown in (B), the addition of doxorubicin significantly decreased LVEF (p<0.05) and LVFS (p<0.05) in the model group, indicating successful establishment of the heart failure model. The addition of FA significantly increased both values, bringing them close to those in the control group, demonstrating that forsythoside A effectively improved cardiac contractile function and alleviated heart failure induced by doxorubicin cardiotoxicity. H&E staining results. Figure 3 (C) shows (p<0.05) that the model group exhibited myocardial fiber rupture, disordered arrangement, and significant inflammatory cell infiltration, while the forsythoside A group showed a more intact myocardial structure and a significantly reduced inflammatory response. Masson staining results are as follows. Figure 3 (C) shows that blue collagen deposition was observed in the model group slices, while the blue collagen deposition was significantly reduced in the forsythoside A group, indicating that forsythoside A effectively inhibited doxorubicin-induced myocardial fibrosis and curbed the cardiotoxicity of doxorubicin.

[0053] 28. Mechanism Research: New Functions (29) Protein extraction: Take the frozen myocardial tissue, add RIPA lysis buffer containing PMSF, homogenize and lyse on ice for 30 min, then place it in a refrigerated centrifuge at 12000 rpm for 15 min (4℃), and take the supernatant.

[0054] (30) Gel preparation: Select a 1.0 mm glass plate, carefully clean the glass plate with a test tube brush and detergent powder, and rinse it with clean water. Align the glass plates with the shorter side facing inward, insert them into both sides of the gel casting machine, place them on the base, and tighten the knobs on both sides at the same time to fix the glass plates. To check if the equipment is leaking, fill the glass plate with distilled water and observe for a few minutes. If the liquid level does not drop, the seal is good. At this time, pour out the water and invert the plate to dry. Select the appropriate concentration of the gel casting solution according to the molecular weight of the protein. Take a small beaker and add various reagents in sequence to prepare the separating gel. After adding TEMED and shaking well, pour the gel immediately. When pouring the gel, use a pipette to draw up the gel solution and slowly add it along the side of the glass plate to avoid generating air bubbles. Add 95% ethanol to the top layer of the gel solution for liquid sealing. Let it stand at room temperature on a horizontal surface for about 30 minutes. When a horizontal fold line is observed between the water and the gel, it indicates that the separating gel has solidified. At this time, pour out the upper layer of ethanol. The remaining liquid can be absorbed with filter paper. Take a small beaker and add the required reagents to prepare a 5% stacking gel. Fill the remaining space in the glass plate with the stacking gel in the same way. Insert a dry, clean comb horizontally into the stacking gel and let it stand at room temperature until it solidifies. After solidification, hold both ends of the comb with your hands and gently pull it upwards. Transfer the glass plate into the electrophoresis tank and add electrophoresis buffer to submerge it.

[0055] (31) Sample loading and electrophoresis: Remove the sample and pre-stained protein marker from the refrigerator in advance, thaw them, and shake to mix. Add the marker and sample as needed, determining the loading amount based on 30-50 μg of protein per well and the desired development effect. Add the sample gently to avoid spilling it into other wells. Connect the positive and negative electrodes of the electrophoresis apparatus and turn on the power. Set the voltage and start electrophoresis. Electrophoresis is complete when the marker separation is obvious and the blue bromophenol blue band is about 1 cm from the bottom of the gel.

[0056] (32) Transfer: Prepare the transfer buffer in advance, and cut the filter paper and PVDF membrane. Immerse the PVDF membrane in methanol for about 1 minute to activate it, then transfer it to the transfer buffer and soak it together with the filter paper for later use. After electrophoresis, remove the glass plate, gently pry it open, and cut the gel according to the molecular weight of the protein to be tested, referring to the position of the marker. Keep the gel moist throughout the operation. Make a "sandwich" structure on the electrophoresis apparatus clamp, placing three layers of filter paper at the bottom, then placing the gel, PVDF membrane, and three layers of filter paper in sequence. Gently roll with a glass rod to remove air bubbles, and pour the transfer buffer into the electrophoresis tank. Cover the tank, set the current and the corresponding time. To prevent the effects of excessive heat, the electrophoresis tank should be kept in an ice bath during the transfer process.

[0057] (33) Blocking and Antibody Incubation: Prepare defatted blocking solution in advance. After the transfer is complete, turn off the power, remove the PVDF membrane with tweezers, immerse it in the blocking solution, and wash it on a shaker at 90 rpm / min for 1.5 h. After blocking, transfer the membrane to PBST buffer and wash it on a shaker at 120 rpm / min for 10 min / 4 times. Then, place the PVDF membrane in the prepared primary antibody solution. Incubate at 4 ℃. After the time is up, remove the PVDF membrane, place it in PBST buffer, wash it on a shaker, and then transfer it to the prepared secondary antibody solution to immerse the PVDF membrane. Set the shaker to 80 rpm / min and incubate for 2 h.

[0058] (34) Development and Image Analysis: After incubation, rinse with PBST buffer for 10 min, three times in total. Take the EP tube covered with aluminum foil and prepare the luminescent solution under dark conditions. Place the rinsed PVDF membrane in the dark compartment of the gel imaging system and evenly cover the membrane surface with the luminescent solution. React in the dark for about 1 min, then expose and image the membrane. The experimental results are as follows: Figure 4 As shown.

[0059] (35) Analysis of experimental results: Figure 4 (A) The WB gel image shows that doxorubicin can induce the expression of cleaved caspase-3 and Bax. In the forsythoside A group, the bands of cleaved caspase-3 and Bax were significantly reduced, indicating that forsythoside A can effectively downregulate the expression of cleaved caspase-3 and Bax, interrupt the cell apoptosis program and reduce cardiomyocyte damage. Figure 4 (B) is a bar chart of Cleaved Caspase-3 and Bcl2 / Bax activity expression (p<0.05). It can be seen that the level of Cleaved Caspase-3 in the forsythoside A group was significantly downregulated compared with the model group; the level of Bcl2 / Bax ratio was significantly upregulated compared with the model group; the treatment of forsythoside A significantly reversed the downregulation of Bcl-2 expression induced by doxorubicin and the upregulation of Bax and Cleaved Caspase-3 expression, and restored the anti-apoptotic ability of mitochondria.

[0060] The above description represents the preferred embodiments of the present invention. It should be noted that all reagents and materials used in the embodiments of the present invention, unless otherwise specified, are standardized products that can be obtained through conventional commercial channels. However, it should be understood that those skilled in the art may choose equivalent products from other suppliers, which does not depart from the scope of protection of the present invention. For those skilled in the art, several improvements and modifications can be made without departing from the principles described in the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of forsythoside A in the preparation of drugs for treating or preventing doxorubicin-induced cardiac injury.

2. The application according to claim 1, characterized in that, The symptoms of cardiac injury include: cardiomyocyte apoptosis, myocardial tissue oxidative stress damage, myocardial tissue inflammation, myocardial fibrosis, left ventricular dysfunction, dilated cardiomyopathy, or heart failure.

3. The application according to claim 1, characterized in that, The drug comprises a therapeutically effective amount of forsythoside A and one or more pharmaceutically acceptable carriers and excipients.

4. The application according to claim 1, characterized in that, The drug can be administered orally, intravenously, or via intraperitoneally.

5. The application according to claim 1, characterized in that, The dosage forms of the drug include, but are not limited to, tablets, capsules, oral liquids, injections, or lyophilized powders.

6. The application according to claim 1, characterized in that, The dosage of forsythoside A in the drug is 10-50 mg / kg / day.

7. The application according to claim 6, characterized in that, The dosage of forsythoside A in the drug is 10-30 mg / kg / day.

8. A pharmaceutical composition for treating or preventing doxorubicin-induced cardiac injury, characterized in that, The pharmaceutical composition comprises forsythoside A as the active ingredient and a pharmaceutically acceptable carrier or excipient.

9. The pharmaceutical composition according to claim 8, characterized in that, The composition is prepared into an oral or injectable formulation.

10. The pharmaceutical composition according to claim 8, characterized in that, The composition contains forsythoside A in a weight percentage of 0.1-99.9%.

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