Application of eicosapentaenoic acid in preparation of medicine for preventing and treating myocardial cell death and senescence induced by adriamycin

Abstract

The invention belongs to the technical field of medicine research, and particularly relates to application of eicosapentaenoic acid in preparation of a medicine for preventing and treating myocardial cell death and senescence induced by adriamycin. Experiments prove that a low dose (15 [mu] M) of eicosapentaenoic acid (EPA) has no obvious toxicity to H9C2 myocardial cells, and the low dose (15 [mu] M) of EPA can improve the death phenomenon of the H9C2 myocardial cells caused by adriamycin; the EPA can be used for reducing the increase of the content of beta-galactosidase in adriamycin-induced H9C2 cells. P53 and P21 related to aging are highly expressed after being treated by 0.5 uM DOX of H9C2 myocardial cells, and EPA can obviously reduce the expression of the P53 and P21. After DOX treatment, the expression of core proteins G3BP1G3BP1 and UBAP2L of stress particles in the H9C2 myocardial cells is increased, and the phenomenon can be improved by EPA (eicosapentaenoic acid). The experiments prove that eicosapentaenoic acid can improve death and aging of adriamycin-induced H9C2 myocardial cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical research, and particularly relates to application of eicosapentaenoic acid in preparation of a drug for preventing and treating doxorubicin-induced myocardial cell death and aging. BACKGROUND

[0002] Myocardial cell death and aging are the core links of heart failure and senile heart disease. Doxorubicin, as a commonly used clinical chemotherapeutic drug, has antibiotic properties and is a cornerstone for treating various chemotherapy regimens. Its main adverse reactions are dose-dependent, long-term and possibly fatal cardiotoxicity (heart failure and senile heart disease). Although the use of doxorubicin can cause cardiotoxicity, doxorubicin is still a commonly used clinical treatment drug due to the lack of suitable alternative drugs.

[0003] Currently, there are several methods for preventing and treating doxorubicin-induced cardiotoxicity: changing the dose and administration mode of doxorubicin, such as liposomal doxorubicin; doxorubicin combined with dexrazoxane for synergistic treatment; and combination with drugs for treating heart failure, such as beta blockers, ACEI / ARB drugs, and sodium glucose co-transporter 2 inhibitors. These methods have certain limitations: the use of liposomal doxorubicin can cause new adverse reactions; changing the dose of doxorubicin can reduce its adverse reactions to a certain extent but affect the treatment effect; when the cumulative dose of doxorubicin exceeds 250mg / m 2 , the benefits of dexrazoxane may outweigh the risks of subsequent tumor treatment. Therefore, the existing drugs for preventing and treating doxorubicin-induced cardiotoxicity have the defects of unclear effect and new adverse reactions. Therefore, it is necessary to develop a new strategy for preventing and treating doxorubicin-induced myocardial cell death and aging. SUMMARY

[0004] In order to develop a new strategy for preventing and treating doxorubicin-induced myocardial cell death and aging, and solve the defects of unclear effect and new adverse reactions of the existing drugs for preventing and treating doxorubicin-induced cardiotoxicity, the application provides application of eicosapentaenoic acid in preparation of a drug for preventing and treating doxorubicin-induced myocardial cell death and aging.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows.

[0006] Doxorubicin (DOX) is the most commonly used anthracycline anticancer drug, but its cardiotoxicity and other harmful side effects limit its clinical application. A large number of studies have shown that programmed cell death and aging play an important role in DOX-induced cardiotoxicity. Recent studies have mainly focused on optimizing the therapeutic index of doxorubicin by developing cardioprotective strategies (such as dexrazoxane) and using non-invasive monitoring techniques to reduce the risk of heart.

[0007] Despite extensive efforts, drugs to alleviate doxorubicin-induced cardiotoxicity, such as mitochondrial antioxidants, have not been clinically approved.

[0008] Eicosapentaenoic acid (EPA), as a kind of essential omega-3 unsaturated fatty acid, cannot be synthesized in the human body and must be supplemented by food intake. Studies have shown that eicosapentaenoic acid can reduce cardiovascular risk. Eicosapentaenoic acid promotes colon epithelial regeneration mediated by intestinal stem cells by activating the LSD 1-WNT signaling pathway. Eicosapentaenoic acid (EPA) has a protective effect on atherosclerosis. EPA / IPE (eicosapentaenoic acid ethyl ester) has multiple effects, including anti-thrombosis, anti-platelet, anti-inflammatory and dissolution.

[0009] The first object of the present application is to provide the use of eicosapentaenoic acid (EPA) in the preparation of a drug for preventing and treating doxorubicin-induced myocardial cell death and aging, and the structure of the eicosapentaenoic acid is as follows: .

[0010] The eicosapentaenoic acid provided by the present application has the effect of significantly improving the survival rate of myocardial cells caused by doxorubicin and inhibiting cell aging caused by doxorubicin, and does not have new adverse reactions, overcoming the defects of existing drugs for preventing and treating doxorubicin-induced cardiotoxicity, such as unclear effect and new adverse reactions.

[0011] As a preferred embodiment of the present application, the eicosapentaenoic acid is used for preparing a drug for improving the survival rate of myocardial cells.

[0012] Further, the eicosapentaenoic acid is used for preparing a drug for improving the aging of myocardial cells.

[0013] Further, the drug takes the eicosapentaenoic acid as an active ingredient, and is supplemented with a pharmaceutically acceptable excipient or carrier.

[0014] Further, the drug is an oral preparation or an injection preparation.

[0015] Further, the drug includes granules, capsules or tablets.

[0016] It can be understood that the drug of the present application can be added with different pharmaceutically acceptable excipients to prepare suitable clinical dosage forms, which include but are not limited to the following dosage forms: tablets, capsules, granules, powders, syrup or other oral liquid preparations, pills, injections, preferably granules, capsules or tablets.

[0017] Further, the pharmaceutically acceptable excipients include, but are not limited to, any one or more of diluents, wetting agents, binding agents, disintegrants, lubricants, adjusting agents, solubilizers, co-solvents, emulsifiers, antioxidants, preservatives, pH adjusting agents, isotonic or isotonizing adjusting agents.

[0018] The diluents are selected from starch, sucrose, cellulose, inorganic salts, etc.; the wetting agents are selected from water, ethanol, etc.; the binding agents are selected from starch paste, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, etc.; the disintegrants are selected from starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, cross-linked povidone, surfactants, runteng disintegrants, etc.; the lubricants are selected from talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, micro-powder silica gel, polyethylene glycol, etc.; the adjusting agents are selected from pigments, fragrances, sweeteners, gum paste, odor correctors, etc.; the solubilizers are selected from Tween, polyoxyethylene fatty alcohol ether, soap, sulfate, sulfonate, etc.; the co-solvents are selected from organic acids and their salts, amide and amine compounds, inorganic salts, polyethylene glycol, glycerol, etc.; the emulsifiers are selected from Span, Tween, lecithin, benzyl zein, glycerol fatty acid ester, higher fatty acid salt, sulfate, sulfonate, gum arabic, tragacanth gum, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silicon dioxide, bentonite, etc.; the antioxidants are selected from sulfite, pyrosulfite, bisulfite, ascorbic acid, gallic acid and its esters, etc.; the preservatives are selected from nipagin, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils, etc.; the pH adjusting agents are selected from hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, citric acid, citrate, etc.; the isotonic or isotonizing adjusting agents are selected from glucose, sodium chloride, sodium citrate, sorbitol, and xylitol, etc.

[0019] It can be understood that the drugs involved in the embodiments of the present application are based on different excipients and prepared into different dosage forms, and accordingly, the administration modes can also be various.

[0020] Further, the carriers include any one or more of microcrystalline cellulose, magnesium stearate, and polyethylene glycol.

[0021] A second object of the present application is to provide a drug for improving the death and aging of myocardial cells induced by doxorubicin, which takes the eicosapentaenoic acid as the only effective active ingredient.

[0022] A third object of the present application is to provide a drug for improving the survival rate of myocardial cells, which takes the eicosapentaenoic acid as the only effective active ingredient.

[0023] The fourth object of the present application is to provide a drug for improving myocardial cell death, which is based on eicosapentaenoic acid as the only effective active ingredient.

[0024] Compared with the prior art, the present application has the following beneficial effects: 1. The present application provides the use of eicosapentaenoic acid in the preparation of a drug for preventing and treating adriamycin-induced myocardial cell death and aging. The eicosapentaenoic acid has the effect of significantly improving the survival rate of myocardial cells caused by adriamycin and inhibiting cell aging caused by adriamycin, and does not cause new adverse reactions. The eicosapentaenoic acid in the present application simultaneously meets the two conditions of "effective-mechanism clear" and "safe-no new toxicity" which the existing drugs cannot achieve at the same time. Through the double evidence chain of "quantifiable significant cell protection + system verified no new toxicity", the two gaps in the predictability of efficacy and safety of the existing prevention and treatment drugs are filled, and the defects of the existing drugs for preventing and treating adriamycin-induced cardiotoxicity, such as unclear effect and new adverse reactions, are overcome. Compared with the existing drugs for preventing and treating myocardial cell death and aging, the eicosapentaenoic acid provided by the present application further reduces the production of stress granules by up-regulating the expression of ADAR1 to reduce adriamycin-induced cardiotoxicity, which has made significant progress compared with the prior art.

[0025] 2、The application shows that low-dose (15uM) EPA itself has no obvious toxicity to H9C2 myocardial cells, and low-dose (15uM) EPA can improve the death of H9C2 myocardial cells caused by doxorubicin through CCK8 cytotoxicity experiment, beta-galactosidase staining, q-PCR, Western Blot, detection of endogenous dsRNA and Z-RNA immunofluorescence experiment, active oxygen staining experiment, detection of myocardial cell membrane potential, stress granules and other experiments; EPA can reduce the increase of beta-galactosidase content in H9C2 cells induced by doxorubicin; q-PCR results show that P53 and P21 related to aging are highly expressed after 0.5uM DOX treatment of H9C2 myocardial cells, and EPA can significantly reduce the expression; Western Blot results show that DOX activates the high expression of P53 and P21, aging markers in H9C2 myocardial cells, and EPA significantly improves this phenomenon; through the application of laser confocal immunofluorescence experiment, DOX can activate the expression of endogenous dsRNA and Z-RNA, and EPA improves this phenomenon; immunofluorescence shows that after DOX treatment, the expression of stress granule core protein G3BP1 in H9C2 myocardial cells is obviously increased and distributed in a circular shape around the nucleus, and EPA improves this phenomenon in quantity and size; q-PCR and Western Blot experiments both show that after DOX treatment, the expression of stress granule core protein G3BP1 and UBAP2L in H9C2 myocardial cells is increased, and EPA can improve this phenomenon. The above experiments prove that eicosapentaenoic acid (EPA) can improve the death and aging of H9C2 myocardial cells induced by doxorubicin. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 1 is a cell optimal drug concentration screening diagram of doxorubicin (DOX) and eicosapentaenoic acid (EPA) in the application, wherein CON is a blank group. P <0.05, P <0.01, P <0.001, P <0.0001; Figure 1 A in the figure is DOX concentration screening; Figure 1 B in the figure is eicosapentaenoic acid (EPA) + DOX concentration screening.

[0027] Figure 2Figure 1 is a schematic diagram of the expression of P53, P21, ADAR1, ZBP1, IRF3, IFNa, RUG-1, OAS1, and IL-1β after treatment of cells with a blank group (CON), doxorubicin (DOX), and eicosapentaenoic acid (EPA) + DOX, wherein Figure 2 A in the formula is P53; Figure 2 B in the formula is P21; Figure 2 C in the formula is ADAR1; Figure 2 D in the formula is ZBP1; Figure 2 E in the formula is IRF3; Figure 2 F in the formula is IFNa; Figure 2 G in the formula is RUG-1; Figure 2 H in the formula is OAS1; Figure 2 I in the formula is IL-1β; P <0.05, P <0.01, P <0.001, P <0.0001.

[0028] Figure 3 Figure 2 is a schematic diagram of the expression of IL-6, IL-1β, TNF-a, UBAP2L, and G3BP1 after treatment of cells with a blank group (CON), doxorubicin (DOX), and eicosapentaenoic acid (EPA) + DOX, wherein Figure 3 A in the formula is IL-6; Figure 3 B in the formula is IL-1β; Figure 3 C in the formula is TNF-a; Figure 3 D in the formula is UBAP2L; Figure 3 E in the formula is G3BP1; P <0.05, P <0.01, P <0.001, P <0.0001.

[0029] Figure 4 Figure 3 is a schematic diagram of the results of β-galactosidase staining after treatment of H9C2 cells with a blank group (CON), doxorubicin (DOX), and eicosapentaenoic acid (EPA) + DOX.

[0030] Figure 5Figure and image J image analysis schematic diagram of the Western Blot results of the blank group (CON), doxorubicin (DOX) and eicosapentaenoic acid (EPA) + DOX treated H9C2 cells in the present application; wherein, Figure 5 A in the figure is the result of the aging-related proteins P53 and P21 WB, and the internal reference is β-actin, wherein the samples from left to right are: CON1, CON2, CON3, DOX1, DOX2, DOX3, EPA1, EPA2, EPA3; Figure 5 B in the figure is the image analysis schematic diagram of the WB image of P53 using image J; Figure 5 C in the figure is the image analysis schematic diagram of the WB image of P21 using image J; P <0.05, P <0.01.

[0031] Figure 6 Figure of dsRNA immunofluorescence of the blank group (CON), doxorubicin (DOX) and eicosapentaenoic acid (EPA) + DOX treated H9C2 cells in the present application.

[0032] Figure 7 Figure of Z-RNA immunofluorescence of the blank group (CON), doxorubicin (DOX) and eicosapentaenoic acid (EPA) + DOX treated H9C2 cells in the present application.

[0033] Figure 8 Figure and image J image analysis schematic diagram of the G3BP1 immunofluorescence of the blank group (CON), doxorubicin (DOX) and eicosapentaenoic acid (EPA) + DOX treated H9C2 cells in the present application; wherein, Figure 8 A in the figure is the G3BP1 immunofluorescence image; Figure 8 B in the figure is the image analysis schematic diagram using image J; P <0.05, P <0.01, P <0.001. DETAILED DESCRIPTION

[0034] The present application will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but should not be understood as limiting the present application. If not specially stated, the technical means used in the following examples are the conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specially stated, can be obtained from commercial channels.

[0035] Doxorubicin is a broad-spectrum anti-tumor drug with dose-dependent cardiotoxicity, including various types of arrhythmia, ventricular dilation, and eventually developing into congestive heart failure, which greatly limits the clinical use of DOX.

[0036] Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid mainly found in deep-sea fish and some seaweed. It has multiple benefits for human health, including reducing the risk of cardiovascular disease, anti-inflammatory effects, improving lipid levels, and protecting the nervous system. However, more uses of Eicosapentaenoic acid (EPA) remain to be explored.

[0037] The present application can improve the death and aging of H9C2 myocardial cells induced by doxorubicin through CCK8 cytotoxicity experiment, β-galactosidase staining, q-PCR, Western Blot, detection of endogenous dsRNA and Z-RNA immunofluorescence experiment, reactive oxygen species staining experiment, detection of myocardial cell membrane potential, stress granules and other experiments.

[0038] The eicosapentaenoic acid involved in the following examples has the following structure: .

[0039] Eicosapentaenoic acid is purchased from Huazhong Haiwei, 98% (mass ratio) high purity.

[0040] Example 1 I. Method: 1. Cell proliferation experiment (1) H9C2 cell suspension (100 μL / well) was inoculated in a 96-well plate and pre-cultured in a humidified incubator for 24 h (at 37℃, 5% CO2).

[0041] Among them, the preparation method of H9C2 cell suspension is to blow and centrifuge after 0.25% trypsin digestion of iron wall cells for 1 min, and then discard the supernatant and add complete medium for resuspension.

[0042] H9C2 cells are purchased from Pnuo Sai.

[0043] (2) Dissolve eicosapentaenoic acid (EPA) and doxorubicin (DOX) in 1% (v / v) serum medium to a final concentration of 0.5 μΜ DOX and 15 μΜ EPA, discard the original culture medium of the 96-well plate, and then divide into three groups: a blank group is added with 1% (v / v) serum medium 100 μΐ,, a doxorubicin group is added with 0.5 μΜ DOX (1% (v / v) serum medium 100 μΐ, is added in the first 24 h, and 0.5 μΜ DOX 100 μΐ, is added in the last 24 h), and eicosapentaenoic acid is added with 15 μΜ EPA and 0.5 μΜ DOX (15 μΜ EPA 100 μΐ, is added in the first 24 h, and 0.5 μΜ DOX 100 μΐ, is added in the last 24 h), and incubate in a humidified incubator for 48 h (at 37 °C, 5% CO2).

[0044] wherein the doxorubicin is purchased from Macklin (purity 98%, mass ratio).

[0045] (3) Remove the drug solution in each well, add 100 μΐ, serum-free medium and 10 μΐ, CCK-8 solution to each well of the plate, and incubate the plate in an incubator for 4 h.

[0046] (4) Measure the absorbance at 450 nm using a microplate reader, and analyze the data.

[0047] wherein the cell survival rate is calculated according to the following formula: Cell survival rate = [ (A s - A b ) / (A0- A b )] x 100%; in the formula, the unit of the cell survival rate is %; A s is the absorbance of the experimental well (the absorbance of the well containing cells, medium, CCK-8 and the test compound); A b is the absorbance of the blank well (the absorbance of the well containing medium and CCK-8); and A0is the absorbance of the control well (the absorbance of the well containing cells, medium and CCK-8).

[0048] 2. q-PCR (1) Divide the H9C2 cells into three groups: a blank group (CON group), a doxorubicin group (DOX group), and an eicosapentaenoic acid + DOX (EPA + DOX group) group.

[0049] H9C2 cells in the CON group were added with 5 mL of culture medium containing 10% (v / v) serum (48 h), H9C2 cells in the DOX group were added with 1% (v / v) serum culture medium 5 mL for the first 24 h and 0.5 uM DOX 5 mL for the last 24 h, H9C2 cells in the EPA+DOX group were added with 15 uM EPA 100 5 mL for the first 24 h and 0.5 uM DOX 100 mL for the last 24 h, and were incubated in an incubator for 48 h.

[0050] (2) RNA was extracted after lysis of the cells in each of the above groups.

[0051] (3) An RT system was configured to reverse transcribe the RNA into DNA.

[0052] (4) A Real Time PCR system was configured to perform Real Time PCR reaction.

[0053] The method for extracting RNA was Animal Total RNA Isolation Kit (FOREGENE).

[0054] The method for reverse transcribing the RNA into DNA was RT EasyTMII, FOREGENE.

[0055] The conditions, reaction system, reaction procedure and primers of Real Time PCR reaction are shown in Table 1.

[0056] Table 1 Primer sequence 3, β-galactosidase staining (1) H9C2 cells in a 10 mm culture dish were inoculated in a 6-well plate and divided into three groups, 1% serum medium 100 uL was added to the blank group, 0.5 uM DOX (1% serum medium 100 uL was added for the first 24 h and 0.5 uM DOX 100 uL was added for the last 24 h) was added to the doxorubicin group, 15 uM EPA and 0.5 uM DOX (15 uM EPA 100 uL was added for the first 24 h and 0.5 uM DOX 100 uL was added for the last 24 h) was added to the eicosapentaenoic acid group, and was cultured in a humidified incubator for 48 h (at 37°C, 5% CO2).

[0057] (2) The cell culture solution was aspirated, washed with PBS twice, and then 1 mL of β-galactosidase staining fixative was added, and fixed at room temperature for 15 min.

[0058] The PBS was purchased from Siboly Kang, with a concentration of 10X and a pH of 7.4.

[0059] (3) Discard the fixing solution, wash with PBS for 3 times, 2 min each time.

[0060] (4) After the positive cells are observed under the ordinary optical microscope, remove the β-galactosidase staining working solution, clean the staining solution with PBS, and finally add 1 mL of PBS to cover the cells and observe.

[0061] (5) After the positive cells are observed under the ordinary optical microscope, remove the β-galactosidase staining working solution, clean the staining solution with PBS, and finally add 1 mL of PBS to cover the cells and observe.

[0062] 4. Western Blot (1) Protein extraction: divide the cells into three groups, add 5 mL of culture medium containing 10% (v / v) serum to the H9C2 cells in the CON group (48 h), add 5 mL of culture medium containing 1% (v / v) serum to the H9C2 cells in the DOX group for the first 24 h, and then add 5 mL of 0.5 uM DOX for the last 24 h, add 100 mL of 15 uM EPA to the H9C2 cells in the EPA+DOX group for the first 24 h, and then add 100 mL of 0.5 uM DOX for the last 24 h, and incubate in an incubator for 48 h.

[0063] After treatment, centrifuge to collect the suspended cells. Discard the original culture medium in the centrifuge tube, add 1 mL of pre-cooled PBS to resuspend the cells, and then transfer to a 4°C high-speed centrifuge for centrifugation (3000 rpm, 5 min). After centrifugation, aspirate the supernatant, add an appropriate amount of pre-configured protein lysis buffer (RIPA lysis buffer: Protease Inhibitor Cocktail, volume ratio = 100:1), vortex to mix, and lyse at 4°C for 30 min. After lysis, centrifuge at 4°C (12000 rpm, 15 min), and after centrifugation, transfer the supernatant to a new EP tube, add 5x loading buffer in proportion, mix, heat in a 100°C metal bath for 10 min, and cool to room temperature after heating.

[0064] (2) Electrophoresis: configure the separation gel and the concentrated gel in proportion, fill them, insert the comb, remove the comb after the gel solidifies, aspirate the residual gel with a syringe, add the sample to the sample well, and perform constant voltage electrophoresis at 80V for the concentrated gel and 120V for the separation gel.

[0065] (3) Membrane transfer: use fast membrane transfer solution, transfer at a constant current of 350 mA for 40 min.

[0066] (4) Antibody incubation: The transferred membrane was washed with TBST twice (70 rpm, 5 min), and the rapid blocking solution was added for blocking at room temperature (60 rpm, 10 min). After blocking, the blocking solution was removed, and the membrane was washed with TBST three times (70 rpm, 5 min). The diluted primary antibody was added, and the membrane was incubated on a shaker at 4°C overnight (60 rpm). The primary antibody was recovered, and the membrane was washed with TBST three times (70 rpm, 5 min). The diluted secondary antibody was added, and the membrane was incubated at room temperature for 60 min (60 rpm). After incubation, the secondary antibody was recovered, and the membrane was washed with TBST three times (60 rpm).

[0067] (5) Chemiluminescence detection: The membrane was taken out, and the TBST was absorbed with filter paper. Freshly prepared ECL mixed solution was added to the protein side of the membrane in the developing box, and the membrane was exposed in the darkroom.

[0068] 5. dsRNA detection The H9C2 cells were divided into a blank group (CON), an experimental group (DOX), and a treatment group (EPA+DOX). The H9C2 cells in each group were inoculated in a confocal culture dish. In the CON group, 5 mL of culture medium containing 10% (v / v) serum was added to the H9C2 cells (48 h). In the DOX group, 1% v (v / v) serum culture medium was added to the H9C2 cells for the first 24 h, and 0.5 uM DOX was added for the last 24 h. In the EPA+DOX group, 15 uM EPA was added to the H9C2 cells for the first 24 h, and 0.5 uM DOX was added for the last 24 h. The cells were incubated in an incubator for 48 h.

[0069] After treatment, the culture medium was discarded, and the cells were washed with PBS twice. 4% (v / v) formaldehyde fixing solution (without methanol) was added, and the cells were incubated on a shaker at room temperature for 15 min. The formaldehyde was removed, and the cells were washed with PBS three times, each for 3 min. The cells were incubated with 0.5% (v / v) permeation buffer on a shaker at room temperature for 45 min, and then washed with PBS three times, each for 3 min. The cells were incubated with 3% (v / v) blocking buffer on a shaker at room temperature for 45 min, and then washed with PBS three times, each for 3 min. Then, the diluted primary antibody was added, and the cells were incubated at 4°C overnight. The primary antibody was discarded, and the cells were washed with PBS three times, each for 3 min. The cells were incubated with fluorescent secondary antibody in the dark at room temperature for 1 hour. After incubation, the secondary antibody was discarded, and the cells were washed with PBS three times, each for 3 min. Then, the cells were incubated with DAPI at room temperature for 15 min, and then washed with PBS three times, each for 3 min. Finally, 1.5 mL of PBS was added to cover the cells, and the cells were observed under a confocal microscope.

[0070] 6. Z-RNA detection H9C2 cells were divided into blank group (CON), experimental group (DOX) and treatment group (EPA+DOX), and H9C2 cells of each group were inoculated in confocal culture dishes. 5 mL of culture medium containing 10% (v / v) serum was added to the H9C2 cells in the CON group (48 h), 5 mL of 1% (v / v) serum culture medium was added to the H9C2 cells in the DOX group for the first 24 h, and 0.5 uM DOX 5 mL was added for the last 24 h. 100 mL of 15 uM EPA was added to the H9C2 cells in the EPA+DOX group for the first 24 h, and 0.5 uM DOX 100 mL was added for the last 24 h, and incubated in an incubator for 48 h. After the treatment was completed, the culture medium was removed and washed with PBS for 2 times, fixed with 4% (v / v) formaldehyde for 15 min, and washed with PBS for 3 times, each for 3 min. After incubation with 0.1% (v / v) Triton X-100 for 10 min, wash with PBS for 2 times. 0.008 U / mL of Proteinase K was treated at 37°C for 10 min, washed with PBS once, and incubated with 2% (v / v) BSA for 1 h, washed with PBS for 3 times, each for 3 min, and incubated with the first antibody at 4°C overnight. Then wash the cells with PBS for 3 times, each for 3 min, incubate with the fluorescent second antibody at room temperature for 1 hour, and wash with PBS for 3 times, each for 3 min. DAPI was incubated at room temperature for 15 min, washed with PBS for 3 times, each for 3 min. Finally, add appropriate amount of PBS to cover the sample and observe.

[0071] 7. Stress granule detection H9C2 cells were divided into blank group (CON), experimental group (DOX) and treatment group (EPA+DOX), and H9C2 cells of each group were inoculated in confocal culture dishes. 5 mL of culture medium containing 10% (v / v) serum was added to the H9C2 cells in the CON group (48 h), 5 mL of 1% (v / v) serum culture medium was added to the H9C2 cells in the DOX group for the first 24 h, and 0.5 uM DOX 5 mL was added for the last 24 h, 100 mL of 15 uM EPA 1005 mL was added to the H9C2 cells in the EPA+DOX group for the first 24 h, and 0.5 uM DOX 100 mL was added for the last 24 h, and incubated in an incubator for 48 h. After the treatment was completed, the culture medium was removed and washed twice with PBS, and the cells were fixed in 4% (v / v) paraformaldehyde at room temperature for 15 minutes, permeabilized with 0.1% (v / v) Triton X-100 in PBS at room temperature for 15 minutes, and then blocked with 1% (v / v) BSA in 10% (v / v) negative goat serum at room temperature for 1 hour. Then the cells were incubated with 1 / 50 diluted anti-G3BP antibody (HA721314) in 1% BSA PBST solution at 4°C overnight. Goat anti- IgG H&L (iFluor488, HA1121) was used as the secondary antibody, with a dilution ratio of 1 / 500. PBS (phosphate buffered saline) instead of the first antibody as the secondary antibody was used as a control only. Nuclear DNA was labeled with DAPI as blue.

[0072] II. Results 1. CCK8 results showed that different doses of eicosapentaenoic acid given to cardiomyocytes did not produce toxicity to cardiomyocytes, as shown in A of Figure 1 . After 24H of treatment with 0.5uM doxorubicin, different doses of EPA were used for intervention, and the results showed that 1-15uM eicosapentaenoic acid could improve the phenomenon of doxorubicin-induced cardiomyocyte death and inhibition of proliferation, as shown in B of Figure 1 .

[0073] 2. q-PCR results showed that after treating cardiomyocytes with eicosapentaenoic acid, the relative expression of aging-related genes P53 and P21 was reduced compared to cells treated with doxorubicin only, indicating that eicosapentaenoic acid could improve doxorubicin-induced cardiomyocyte aging, as shown in A of Figure 2 and B of Figure 2 . Further detection of dsRNA recognizer-related genes ADAR1, ZBP1, IRF3 and dsRNA-induced interferon response-related genes IFNa, OAS1 and RIG1 found that eicosapentaenoic acid significantly improved the upregulation of these genes caused by doxorubicin, indicating that eicosapentaenoic acid could reduce the increase of dsRNA and the induced interferon response caused by doxorubicin, as shown in C of Figure 2 .Figure 2 D in Figure 2 E in Figure 2 F in Figure 2 G and Figure 2 As shown in H in the diagram.

[0074] 3. q-PCR results showed that after treatment of cardiomyocytes with eicosapentaenoic acid (EPA), the relative expression of inflammation-related genes IL-18, IL-6, TNFα, and IL-1β was significantly reduced compared to cells treated with doxorubicin alone. This indicates that EPA can alleviate doxorubicin-induced inflammation, as shown in the figure. Figure 3 I in Figure 3 A~ Figure 3 As shown in C.

[0075] 4. q-PCR results showed that after treatment of cardiomyocytes with eicosapentaenoic acid (EPA), the relative expression of genes related to stress granule formation, UBAP2L and G3BP1, was significantly reduced compared to cells treated with doxorubicin alone. This indicates that EPA can improve the doxorubicin-induced stress granule formation. Figure 3 D and Figure 4 As shown in E in the figure.

[0076] 5. β-galactosidase staining results showed a significant increase in the blue portion in the doxorubicin group, while the blue portion decreased after eicosapentaenoic acid (EPA) treatment, indicating that EPA can improve doxorubicin-induced cardiomyocyte senescence. Figure 5 As shown.

[0077] 6. Western blot analysis showed that doxorubicin increased the expression of aging-related proteins P53 and P21, while eicosapentaenoic acid (EPA) intervention reduced their expression, indicating that EPA can improve doxorubicin-induced cardiomyocyte senescence. Figure 6 As shown.

[0078] 7. Cell immunofluorescence confirmed that some cells in the doxorubicin group showed green fluorescence, indicating that doxorubicin induced the production of dsRNA in the cytoplasm. The proportion of green fluorescence in the eicosapentaenoic acid (EPA) group was significantly reduced, indicating that EPA can reduce the abnormal increase in dsRNA induced by doxorubicin. Figure 7 As shown.

[0079] 8. Cell immunofluorescence confirmed that some cells in the doxorubicin group showed green fluorescence, indicating that doxorubicin induced the production of z-RNA in the cytoplasm. The proportion of green fluorescence in the eicosapentaenoic acid (EPA) group was significantly reduced, indicating that EPA can reduce the abnormal increase in z-RNA induced by doxorubicin. Figure 8 As shown.

[0080] 9、Cellular immunofluorescence confirmed that part of the cells in the doxorubicin group appeared green fluorescence, indicating that doxorubicin caused stress granules in the cytoplasm, and the proportion of green fluorescence in the eicosapentaenoic acid group decreased significantly, indicating that eicosapentaenoic acid can reduce the abnormal increase of stress granules caused by doxorubicin, as shown in A of Figure 8 and B of Figure 8 . Among them, Figure 8 B in ​ is the quantitative column chart of green fluorescence in A.

[0081] The present application shows that low-dose (15uM) EPA itself has no obvious toxicity to H9C2 myocardial cells, and low-dose (15uM) EPA can improve the phenomenon of doxorubicin-induced H9C2 myocardial cell death; EPA can reduce the increase of β-galactosidase content in H9C2 cells induced by doxorubicin; q-PCR results show that P53 and P21 related to aging are highly expressed after 0.5uM DOX treatment of H9C2 myocardial cells, and EPA can significantly reduce their expression; Western Blot results show that DOX activates the high expression of P53 and P21, aging markers in H9C2 myocardial cells, and EPA significantly improves this phenomenon; through the application of laser confocal immunofluorescence experiment, DOX can activate the expression of endogenous dsRNA and Z-RNA, and EPA improves this phenomenon; immunofluorescence shows that after DOX treatment, the expression of G3BP1, the core protein of stress granules in H9C2 myocardial cells, is significantly increased and distributed in a circular shape around the nucleus, and EPA improves this phenomenon in quantity and size; q-PCR and Western Blot experiments both show that after DOX treatment, the expression of G3BP1 and UBAP2L, the core proteins of stress granules in H9C2 myocardial cells, is increased, and EPA can improve this phenomenon. The above experiments prove that eicosapentaenoic acid (EPA) can improve the death and aging of H9C2 myocardial cells induced by doxorubicin.

[0082] From the above experimental results, it can be seen that eicosapentaenoic acid can significantly improve the decrease of myocardial cell survival rate caused by doxorubicin and inhibit the cell aging caused by doxorubicin, and is an effective drug for improving the cardiotoxicity of doxorubicin.

[0083] It should be noted that when numerical ranges are involved in the present application, both endpoints of each numerical range and any number between the two endpoints can be selected, and in order to prevent repetition, the present application describes preferred embodiments.

[0084] While the preferred embodiments of the application have been described, additional variations and modifications can be made to these embodiments by those skilled in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application.

Claims

1. Application of eicosapentaenoic acid in the preparation of drugs for preventing and treating doxorubicin-induced cardiomyocyte death and aging.

2. The application according to claim 1, characterized in that, The eicosapentaenoic acid is used to prepare drugs that improve the survival rate of cardiomyocytes.

3. The application according to claim 1, characterized in that, The eicosapentaenoic acid is used to prepare drugs that improve myocardial cell aging.

4. The application according to claim 1, characterized in that, The drug uses eicosapentaenoic acid as its active ingredient, supplemented with pharmaceutically acceptable excipients or carriers.

5. The application according to claim 4, characterized in that, The drug is an oral or injectable formulation.

6. The application according to claim 5, characterized in that, The drug may be in the form of granules, capsules, or tablets.

7. The application according to claim 4, characterized in that, The excipients include any one or more of the following: diluents, wetting agents, binders, disintegrants, lubricants, regulators, solubilizers, and cosolvents.

8. The application according to claim 4, characterized in that, The carrier includes any one or more of microcrystalline cellulose, magnesium stearate, and polyethylene glycol.

9. A drug for improving doxorubicin-induced cardiomyocyte death and senescence, characterized in that, It uses eicosapentaenoic acid as described in claim 1 as its sole effective active ingredient.

10. A drug for improving the survival rate of cardiomyocytes, characterized in that, It uses eicosapentaenoic acid as described in claim 1 as its sole effective active ingredient.

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