Use of nardosinone in prevention or treatment of myocardial toxicity induced by anthracycline Anti-tumor drugs

Abstract

The present invention relates to pharmaceutical use of nardosinone in the prevention and treatment of cardiotoxicity, and in particular, to use of nardosinone in the prevention or treatment of myocardial toxicity induced by anthracycline anti-tumor drugs. Experiments have proved that nardosinone has a significant therapeutic effect on myocardial injury in mice induced by doxorubicin, and can significantly increase the decreased left ventricular ejection fraction and fractional shortening of the heart of mice induced by DOX, reduce the release of lactate dehydrogenase and creatine kinase in the plasma of mice induced by DOX, decrease myocardial fibrosis in mice induced by DOX, and alleviate cardiomyocyte apoptosis and endoplasmic reticulum stress injury induced by DOX. Nardosinone features low raw material costs and easy availability, and can be used as an active ingredient or a pharmaceutical formulation component for myocardial protection against anthracycline chemotherapeutic drugs, holding promising pharmaceutical application prospects.

Description

Application of naringinone in the prevention or treatment of myocardial toxicity induced by anthracycline antitumor drugs Technical Field

[0001] This invention belongs to the field of drug toxicity prevention and control technology, specifically relating to a new use of a substance, particularly the application of naringinone in the prevention or treatment of myocardial toxicity induced by anthracycline antitumor drugs. Background Technology

[0002] Cancer has become the second leading cause of disease incidence and mortality after cardiovascular disease. In my country, cancer-related deaths are nearly twice the global average, and the rate of increase is rapid. Currently, the main clinical treatments for cancer include surgery, radiotherapy, and chemotherapy. Chemotherapy, as one of the routine methods of anti-cancer treatment, is used at all stages of cancer development. Anthracyclines (such as doxorubicin, daunorubicin, aclarubicin, epirubicin, pirarubicin, idarubicin, and mitoxantrone) are commonly used antitumor chemotherapy drugs in clinical practice, and are used in various cancers with high incidence and mortality rates, including leukemia, breast cancer, and lymphoma. However, these drugs have a high affinity for myocardial tissue, producing cumulative, dose-dependent cardiotoxicity, which can further develop into irreversible myocardial damage, ultimately leading to congestive heart failure. The cardiotoxicity can occur shortly after drug administration, during treatment, after treatment, and even years or decades after drug withdrawal, thus severely limiting their clinical application.

[0003] In response to the toxic side effects of anthracyclines in clinical treatment, although liposomal doxorubicin and dextromethorphan are available to antagonize the cardiotoxicity caused by doxorubicin by changing the dosage form and chelating iron ions of anthracyclines, their use is strictly controlled in clinical practice due to their high price or the side effect of aggravating bone marrow suppression.

[0004] Currently, there are no effective and affordable preventative or treatment measures for the cardiotoxic side effects caused by anthracyclines. Therefore, research into novel drugs for the prevention or treatment of cardiotoxicity caused by anthracycline antitumor drugs, and the development of effective and cost-efficient drugs to combat cardiotoxic side effects, are of significant practical importance.

[0005] Nardostachysone is a sesquiterpene compound derived from the traditional Chinese medicine Nardostachys jatamansi. Its chemical structural formula is as follows:

[0006] .

[0007] Nardostachysone possesses a wide range of well-defined pharmacological activities, including sedation, antiepileptic activity, antidepressant activity, nerve growth promotion, and blood pressure reduction. Literature reports that naringenone can restore cardiac function and effectively alleviate aconitine-induced rapid heart rate arrhythmias in rats by inhibiting cAMP and PKA levels. However, to date, there are no research reports on the therapeutic value of naringenone in treating chemotherapy-induced myocardial injury. Technical issues

[0008] This invention aims to prevent or treat cardiotoxicity induced by anthracycline antitumor drugs. Research has revealed that naringinone can act as a cardioprotective active substance against anthracycline chemotherapy drugs. It reduces the lethality caused by doxorubicin cardiotoxicity by improving endoplasmic reticulum stress damage and alleviating cardiomyocyte apoptosis. Therefore, the application of naringinone in the prevention or treatment of cardiotoxicity induced by anthracycline antitumor drugs is proposed.

[0009] Experiments have shown that naringinone has a good preventive or therapeutic effect on myocardial toxicity caused by anthracyclines. Technical solutions

[0010] The technical solution adopted in this invention is as follows:

[0011] This invention provides the application of naringinone as a preventive and therapeutic drug for myocardial toxicity caused by anthracycline antitumor drugs. It can be used to prevent and treat myocardial toxicity caused by anthracycline antitumor drugs, and can be used to prepare drugs, drug mixtures and drug compositions that inhibit myocardial damage caused by anthracycline antitumor drugs.

[0012] Meanwhile, the present invention also provides the use of naringinone for the prevention, treatment or inhibition of myocardial damage caused by anthracycline antitumor drugs and its application in such drugs.

[0013] Meanwhile, naringinone and its drug applications can be used to alleviate cardiomyocyte apoptosis, endoplasmic reticulum stress damage, cardiomyocyte lactate dehydrogenase abnormalities, decreased cardiac function, or myocardial fibrosis caused by anthracycline chemotherapy drugs.

[0014] The anthracycline antitumor drugs include at least one of doxorubicin, daunorubicin, aclarubicin, idarubicin, epirubicin, or mitoxantrone. Nardostachysone can be used alone, or as a drug, drug mixture, or drug composition thereof, the active ingredient of which includes ninaflavin. The drug, drug mixture, or drug composition is any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions, and solutions, or as an oral liquid, capsule, or injection. The daily dose of ninaflavin for myocardial protection in adults (60 kg) is 150-1000 mg. It is used in the preparation of drug mixtures or drug compositions, the active ingredient of which includes ninaflavin.

[0015] The drug, drug mixture, or drug composition has at least one of the following functions: 1)-6):

[0016] 1) Prevention and / or treatment of cardiotoxicity caused by anthracycline chemotherapy drugs;

[0017] 2) Alleviates cardiomyocyte apoptosis induced by anthracycline chemotherapy drugs;

[0018] 3) Alleviate endoplasmic reticulum stress damage caused by anthracycline chemotherapy drugs;

[0019] 4) Alleviates the abnormality of myocardial cell lactate dehydrogenase caused by anthracycline chemotherapy drugs;

[0020] 5) Alleviates the decline in cardiac function caused by anthracycline chemotherapy drugs;

[0021] 6) Relieves myocardial fibrosis caused by anthracycline chemotherapy drugs. Beneficial effects

[0022] 1. This invention provides a novel method for preventing or treating cardiotoxicity induced by anthracycline antitumor drugs. Nardostachysone is the main component of Nardostachys jatamansi, which can be extracted from natural plants using well-established extraction methods. The raw material is inexpensive and readily available, making it a promising active ingredient or formulation component for cardioprotection against anthracycline chemotherapy drugs. Furthermore, ninaflavinsone can be administered orally, making it readily accepted and facilitating clinical promotion and application.

[0023] 2. Nardostachysone can be considered as an alternative drug for improving anthracycline-induced myocardial injury and for preventing and mitigating myocardial toxicity caused by anthracycline antitumor drugs. This invention utilizes a mouse model of anthracycline-induced myocardial injury induced by anthracycline drugs (using doxorubicin as an example) with the interference of ninaflavin. Animal experimental results show that ninaflavin can effectively improve cardiac function damage induced by anthracycline antitumor drugs in mice by reducing cardiomyocyte apoptosis and alleviating myocardial fibrosis, thereby reducing cardiomyocyte apoptosis and activation of endoplasmic reticulum stress signaling pathways, thus reducing doxorubicin-induced cardiotoxicity. Experiments have demonstrated that ninaflavin has a significant therapeutic effect on doxorubicin (DOX)-induced myocardial injury in mice. It can significantly increase the decrease in left ventricular ejection fraction (LVEF) and fractional shortening (FS) induced by DOX in mice, reduce the release of lactate dehydrogenase (LDH) and creatine kinase (CK) in mouse plasma induced by DOX, reduce DOX-induced myocardial fibrosis in mice, and alleviate DOX-induced cardiomyocyte apoptosis and endoplasmic reticulum stress damage.

[0024] Therefore, it can be used to prevent and reduce myocardial toxicity caused by anthracycline antitumor drugs, and has clinical applicability. Attached Figure Description

[0025] Figure 1 shows the effect of nardostachyne on cardiac function in mice with DOX-induced myocardial injury (n=8-11).

[0026] Figure 1 and Table 1 show the comparative effects of naringin on cardiac function in DOX-injured mice in Experiment 1. A: Echocardiograms of representative mice in each group; BE: Statistical analysis results of bar charts for LVEF%, FS%, LVIDd, and LVIDs in each group. Compared with the Control group, ** p<0.01, *** p<0.001; compared with the DOX model group, # p<0.05, ## p<0.01, ### p<0.001. LVIDd: Left ventricular diastolic diameter; LVIDs: Left ventricular systolic diameter; LVEF%: Left ventricular ejection fraction; FS%: Short axis shortening rate; Control: Blank control group; DOX: Doxorubicin model group; DOX+L-Nar: Low-dose naringinone group; DOX+M-Nar: Medium-dose naringinone group; DOX+H-Nar: High-dose naringinone group; Nar: Narcissus; DOX: Doxorubicin.

[0027] Figure 2 shows the effects of nardostachyl on heart weight and cardiac index in DOX myocardial injury model mice (n=8-11).

[0028] Figure 2 and Table 2 show the effects of naringin on heart weight and cardiac index in DOX-injured mice in Experiment 2. A: Statistical analysis of heart-to-body ratio in each group; B: Statistical analysis of left ventricular weight in each group. Compared with the Control group, ** p<0.01; compared with the DOX model group, # p<0.05, ## p<0.01. HWI: heart weight ratio; LVWI: left ventricular index; Control: blank control group; DOX: doxorubicin model group; DOX+L-Nar: low-dose naringinone group; DOX+M-Nar: medium-dose naringinone group; DOX+H-Nar: high-dose naringinone group; Nar: naringinone; DOX: doxorubicin.

[0029] Figure 3 shows the effect of nardostachyne on plasma LDH and CK levels in DOX-injured mice (n=8-11).

[0030] Figure 3 and Table 3 show the effects of nardostachysone on LDH and CK release in the plasma of DOX-injured mice in Experiment 3. A: Bar chart of LDH activity levels in plasma of mice in each group; B: Bar chart of CK activity levels in plasma of mice in each group. Compared with the Control group, * p<0.05; compared with the DOX model group, # p<0.05. LDH: lactate dehydrogenase; CK: creatine kinase; Control: blank control group; DOX: doxorubicin model group; DOX+L-Nar: low-dose naringinone group; DOX+M-Nar: medium-dose naringinone group; DOX+H-Nar: high-dose naringinone group; Nar: naringinone; DOX: doxorubicin.

[0031] Figure 4 shows the effect of naringinone on myocardial structure in mice with DOX-induced myocardial injury (HE staining, n=3).

[0032] Figure 4 shows the comparative effects of naringinone on myocardial structure in mice with DOX-induced myocardial injury in Experiment 4. In the figure, Control: blank control group; DOX: doxorubicin model group; DOX+L-Nar: low-dose naringinone group; DOX+M-Nar: medium-dose naringinone group; DOX+H-Nar: high-dose naringinone group; Nar: naringinone; DOX: doxorubicin.

[0033] Figure 5 shows the effect of naringinone on myocardial fibrosis in DOX-induced myocardial injury mice (Masson staining, n=3).

[0034] Figure 5 shows a comparison of the effects of naringin on myocardial fibrosis in DOX-injured mice in Experiment 5. A: Representative Masson staining images (200X) from each group; B: Statistical analysis of myocardial collagen area in each group (bar chart). Compared with the Control group, *** p<0.001; compared with the DOX model group, # p<0.05, ## p<0.01. Control: Blank control group; DOX: Doxorubicin model group; DOX+L-Nar: Low-dose naringin group; DOX+M-Nar: Medium-dose naringin group; DOX+H-Nar: High-dose naringin group; Nar: naringin; DOX: Doxorubicin.

[0035] Figure 6 shows the effect of naringin on cardiomyocyte apoptosis in DOX-induced myocardial injury mice (Tunnel staining, n=3).

[0036] Figure 6 shows a comparison of the effects of naringin on cardiomyocyte apoptosis in DOX-injured mice in Experiment 6. A: Representative images of the myocardium from each group after Tunnel staining (200X); B: Statistical analysis of the number of apoptotic cardiomyocytes in each group (bar chart). Compared with the Control group, *** p<0.001; compared with the DOX model group, ## p<0.01, ### p<0.001. Control: Blank control group; DOX: Doxorubicin model group; DOX+L-Nar: Low-dose naringinone group; DOX+M-Nar: Medium-dose naringinone group; DOX+H-Nar: High-dose naringinone group; Nar: naringinone; DOX: Doxorubicin.

[0037] Figure 7 shows the effects of naringinone on the expression of apoptosis proteins and endoplasmic reticulum stress signaling pathway proteins in myocardial tissue of mice with doxorubicin-induced myocardial injury (n=3).

[0038] Figure 7 shows a comparison of the effects of nardostachysone on the expression of endoplasmic reticulum stress signaling pathway proteins in the myocardial tissue of DOX-injured mice in Experiment 7. A: Effects of nardostachysone on the expression of Bax, Bcl2, p-PERK, PERK, GRP78, p-eIF-2α, eIF-2α, and CHOP proteins in the myocardial tissue of each group of mice; BF: Statistical analysis of bar charts showing the effects of nardostachysone on the expression of Bcl2 / Bax, p-PERK / PERK, GRP78 / β-Actin, p-eIF-2α / eIF-2α, and CHOP / β-Actin proteins in the myocardial tissue of each group of mice. Compared with the Control group, ** p<0.01; compared with the DOX model group, # p<0.05, ## p<0.01. Control: Blank control group; DOX: Doxorubicin model group; DOX+L-Nar: Low-dose naringin group; DOX+M-Nar: Medium-dose naringin group; DOX+H-Nar: High-dose naringin group; Nar: naringin; DOX: Doxorubicin. Embodiments of the present invention

[0039] To make the technical concept and advantages of this invention clearer, the technical solution of this invention will be further described in detail below with reference to the accompanying drawings. It should be understood that the following embodiments are merely preferred embodiments for explaining and illustrating this invention, and should not be considered as, nor constitute a limitation on, the scope of patent protection claimed by this invention.

[0040] Unless otherwise specified, the instruments and equipment involved in the following examples or experiments are all conventional instruments and equipment; the reagents or raw materials involved are all commercially available conventional products; the detection methods involved, unless otherwise specified, are all conventional methods.

[0041] The nardosinone used in the following examples: molecular formula C 15 H 22 O3, purchased from Sichuan Victy Biotech Co., Ltd., product number: 32476 - 67 - 8. Example

[0042] Nardosinone was formulated into a gastric lavage solution of 5 mg / mL using 0.5% sodium carboxymethylcellulose solution, and doxorubicin (DOX) was used as an example for the following corresponding experiments.

[0043] 1. The materials used are as follows:

[0044] Experimental animals: 70 SPF - grade male Kunming mice were purchased from Beijing Huafukang Biotech Co., Ltd., license number SCXK Jing 2013 - 0007, with a body weight of 25 - 28 g.

[0045] LDH detection kit: purchased from Changchun Huili Biotechnology Co., Ltd., product number: C058 - e.

[0046] CK detection kit: purchased from Changchun Huili Biotechnology Co., Ltd., product number: C059 - e.

[0047] HE staining kit: purchased from Wuhan Sevier Biotechnology Co., Ltd., product number: G1005 - 100 mL.

[0048] Masson staining kit: purchased from Wuhan Sevier Biotechnology Co., Ltd., product number: G1006 - 100 mL.

[0049] Tunel apoptosis detection kit: purchased from Wuhan Sevier Biotechnology Co., Ltd., product number: G1507 - 50 T.

[0050] Antibodies: p-PERK (Thr982), purchased from Beyotime Biotechnology Co., Ltd., catalog number: AF5902, 1:1000 dilution; PERK: purchased from Jiangsu Qinke Biotechnology Research Center Co., Ltd., catalog number: AF5304, 1:1000 dilution; p-eIF-2α (Ser51): purchased from Beyotime Biotechnology Co., Ltd., catalog number: AF5803, 1:1000 dilution; eIF-2α: purchased from Beyotime Biotechnology Co., Ltd., catalog number: AF6771, 1:1000 dilution; GRP78: purchased from Beyotime Biotechnology Co., Ltd., catalog number: AF0171, 1:1000 dilution; Bcl2: purchased from Jiangsu Qinke Biotechnology Research Center. Limited Company, Product No.: BF9013, 1:1000 dilution; Bax: Purchased from Wuhan Saiweier Biotechnology Co., Ltd., Product No.: GB12960, 1:1000 dilution; CHOP: Purchased from Jiangsu Qinke Biotechnology Research Center Co., Ltd., Product No.: DF60254, 1:1000 dilution; β-Actin: Purchased from Beyotime Biotechnology Co., Ltd., Product No.: AF5003, 1:3000 dilution; BRD4: Purchased from Wuhan Saiweier Biotechnology Co., Ltd., Product No.: GB11999, 1:1000 dilution; HRP-labeled goat anti-rabbit IgG: Purchased from Wuhan Saiweier Biotechnology Co., Ltd., Product No.: GB23301, 1:4000 dilution.

[0051] 2. Experimental Procedure

[0052] Seventy SPF-grade male Kunming mice were randomly divided into four groups: a normal control group (n=10, Control group), a model group (n=12, DOX group), a low-dose naringin group (DOX+L-Nar group, 25 mg / kg / d), a medium-dose naringin group (DOX+M-Nar group, 50 mg / kg / d), a high-dose naringin group (DOX+H-Nar group, 100 mg / kg / d), and a positive control group (DOX+Captopril group, 100 mg / kg / d). Animals in the normal control and model groups were administered 0.5 mL of 0.5% sodium carboxymethyl cellulose solution by gavage; animals in the drug-treated groups were administered naringin and captopril by gavage at the above doses for 28 days. In the normal control group, mice were intraperitoneally injected with 0.2 mL of physiological saline. Mice in the model group and drug-treated groups were intraperitoneally injected with doxorubicin (Beijing Solarbio Science & Technology Co., Ltd., batch number 125316-40-9) 3 mg / kg / day (prepared with 0.2 mL of physiological saline), every other day for a total of 5 times. During the DOX modeling process, 6 mice died (3 in the DOX model group, and 1 each in the low-dose, medium-dose, and captopril positive control groups). No echocardiography was performed 28 days after the administration of DOX. The following day, after enucleation and blood collection, the mice were dissected, and heart tissue was collected for further examination and analysis.

[0053] 3. Indicator Observation and Detection Methods

[0054] (1) Echocardiography: After the experiment, mice were placed in an induction box and anesthetized with 4% isoflurane. After the mice were comatose, they were placed in a supine position on the physiological information monitoring platform, and their limbs were fixed to the monitoring platform with medical tape. At the same time, the mice inhaled 2% isoflurane for continuous anesthesia, and the precordial area of ​​the mice was shaved. Then, medical ultrasound coupling agent was applied to the shaved area, and ultrasound detection was performed using a Vevo 2100 M type echocardiography system and a 12L-RS Linear type high-frequency probe. The short-axis view was obtained, the conversion energy frequency was 13MHz, the depth was 2cm, and the frame rate per second was set to the maximum. Five cardiac cycles were continuously detected. The main indicators measured were left ventricular diameter at end-systole (LVIDs), left ventricular diameter at end-diastole (LVIDd), left ventricular ejection fraction (LVEF, %), and fractional shortening (FS, %), and statistical analysis was performed.

[0055] (2) Heart weight and cardiac index: The mice were weighed the day after ultrasound examination. After dissection, the heart was removed. The heart of each mouse was perfused and cleaned with physiological saline until the saline was clear and colorless. After the surface of the heart was dried with filter paper, the mouse heart was weighed. Then, the left ventricle was separated by cutting along the short axis of the heart with a clean blade, the left ventricle was weighed and recorded, and the heart-to-body ratio and left ventricular index were calculated.

[0056] (3) Mouse plasma myocardial enzyme test: On the second day after ultrasound examination, mice in each group underwent enucleation to collect blood. The blood was stored in 1.5 ml centrifuge tubes containing heparin sodium and centrifuged at 13,000 g for 15 minutes at 4°C. The supernatant plasma was aliquoted into 1.5 ml centrifuge tubes and stored at -80°C for later use. For testing, the plasma was thawed from the freezer, and the activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in the mouse plasma of each group were measured using a Roche COBAS Integra 400 Plus / C501 fully automated biochemical analyzer. The experimental results were recorded and statistically analyzed.

[0057] (4) Observation of myocardial tissue pathology: After the experiment, the hearts of mice in each group were taken, weighed, and tissue from the apex of the left ventricle was taken along the short axis of the heart and immediately fixed in 4% paraformaldehyde solution. The tissue was embedded in paraffin, sectioned routinely, and the section thickness was 4 μm. After HE and Masson staining, the tissue was observed and analyzed under a light microscope.

[0058] (5) Observation of cardiomyocyte apoptosis: After the experiment, the hearts of mice in each group were taken, weighed, and tissue from the apex of the left ventricle was taken along the short axis of the heart and immediately fixed in 4% paraformaldehyde solution. Then, the tissue was embedded in paraffin, sectioned routinely, and the section thickness was 4 μm. The tissue was stained with TUNEL kit, and the apoptosis was observed and counted.

[0059] (6) Immunoblot detection: After the experiment, the hearts of mice in each group were taken, weighed, and left ventricular tissue was taken along the short axis of the heart. The tissue was then cut into small pieces on ice with surgical scissors and placed in a 1.5 mL EP tube. 300 μL of pre-cooled cell lysis buffer containing protease inhibitors and 3 grinding beads were added. The tube was then flash-frozen in liquid nitrogen and ground three times at 70 Hz for 30 s each time at 4℃ until no obvious heart tissue was visible. The tube was then placed in an ice box and vortexed every 10 min for a total of three times. After lysis, the tube was centrifuged at 13,000 rpm for 10 min at 4℃. The supernatant was then collected in a 1.5 mL EP tube for protein quantification and detection of relevant proteins according to the conventional immunoblotting method.

[0060] Experiment 1: Effects of naringinone on cardiac function in mice with doxorubicin-induced myocardial injury

[0061] LVIDd, LVIDs, LVEF% and FS% are the most commonly used indicators of cardiac function. Therefore, Doppler ultrasound was used to test cardiac function in each group of mice. As shown in Figure 1 and Table 1, compared with the Control group, the LVEF% and FS% of mice in the DOX model group were significantly decreased (p < 0.001), while LVIDd and LVIDs were significantly increased (p < 0.01). After gavage administration of naringinone and captopril, the LVEF% and FS% of mice in the medium- and high-dose naringinone groups and the captopril group were significantly increased (p < 0.01), while LVIDd and LVIDs were significantly decreased compared with the model group (p < 0.05), and there was no significant difference between the medium- and high-dose naringinone groups and the positive control drug captopril group (p > 0.05). These results confirm that naringinone has a strong ameliorative effect on doxorubicin-induced cardiac function damage.

[0062] Table 1: Effects of naringinone on cardiac function in DOX-injured mice (n=8~11)

[0063]

[0064] Experiment 2: Effects of naringinone on heart weight and cardiac index in mice with doxorubicin-induced myocardial injury

[0065] The effects of nardostachyl on cardiac indices in mice with doxorubicin-induced myocardial injury were observed by measuring heart weight ratio (HWI) and left ventricle / heart ratio (LVWI). As shown in Figure 2 and Table 2, compared with the control group, the cardiac indices HWI and LVWI of mice in the DOX model group were significantly increased (p < 0.01). Compared with the DOX model group, after continuous gavage administration of nardostachyl and the positive control drug captopril, the cardiac indices HWI and LVWI of mice in the medium- and high-dose nardostachyl groups and the positive control drug group were significantly decreased (p < 0.05), and there was no significant difference between the high-dose nardostachyl group and the positive control drug group (p > 0.05). These results confirm that nardostachyl can effectively alleviate doxorubicin-induced myocardial hypertrophy.

[0066] Table 2: Effects of naringinone on heart weight and cardiac index in DOX myocardial injury model mice (n=8~11)

[0067]

[0068] Experiment 3: Effects of naringinone on plasma LDH and CK release in mice with doxorubicin-induced myocardial injury

[0069] The effect of nardostachyl on DOX-induced myocardial injury in mice was investigated by evaluating the expression levels of LDH and CK myocardial enzymes in mouse plasma. As shown in Figure 3 and Table 3, the levels of LDH and CK in the plasma of mice in the DOX model group were significantly higher than those in the control group (p < 0.01). After intervention with nardostachyl and the positive control drug captopril, medium and high doses of nardostachyl and captopril significantly reduced the levels of LDH and CK in the plasma of mice with myocardial injury (p < 0.05), while there was no significant difference between the high-dose nardostachyl group and the captopril group (p > 0.05). These results confirm that nardostachyl can effectively reduce the release of LDH and CK induced by doxorubicin-induced myocardial injury.

[0070] Table 3: Effects of naringinone on plasma LDH and CK levels in DOX-injured mice (n=8~11)

[0071]

[0072] Experiment 4. Effects of naringinone on myocardial structure in mice with doxorubicin-induced myocardial injury

[0073] HE staining was used to evaluate the effect of naringin on the structural changes of cardiac tissue in mice with DOX-induced myocardial injury. As shown in Figure 4, HE staining of cardiac sections in the Control group showed no obvious lesions or fibrosis, and the cardiomyocytes were arranged in neat strips with uniform nuclei. Compared with the Control group, cardiac sections in the DOX model group showed fibrous tissue proliferation, myocardial fiber breakage, disordered arrangement of cardiomyocytes, and scattered nuclei within the myocardial fibers. Compared with the DOX model group, the cardiomyocytes in the medium- and high-dose naringin groups and the captopril positive control group were more neatly arranged, with reduced myocardial fiber breakage, and the cardiomyocyte nuclei were relatively evenly distributed in the center of the myocardial fibers. These staining results directly confirm that naringin can effectively improve the myocardial structural disorder induced by doxorubicin.

[0074] Experiment 5. Effects of naringin on myocardial fibrosis in mice with doxorubicin-induced myocardial injury

[0075] Masson staining was used to evaluate the effect of naringin on myocardial fibrosis in mice with doxorubicin-induced myocardial injury. As shown in Figure 5-A: no obvious abnormalities were observed in the heart sections of the Control group, and the myocardial fibers in the left ventricle of the mice were neatly arranged with almost no obvious blue collagen fibers between the myocardial cells; while in the heart sections of the DOX model group, the myocardial cells were more disordered, and the blue collagen fibers in the intercellular spaces were significantly increased. After intervention with naringin and captopril, the distribution of blue collagen fibers in the intercellular spaces of the myocardial cells was significantly lower than that in the DOX model group. Further quantitative analysis of the collagen fiber distribution area after Masson staining was performed. As shown in Figure 5-B: compared with the Control group, the collagen fiber area in the heart tissue of mice in the DOX model group was significantly increased (p < 0.001), while the collagen fiber area in the left ventricle of mice in the low, medium, and high dose naringin groups and the positive drug captopril group was significantly decreased (p < 0.05). The high dose naringin group showed the most significant improvement, and there was no significant difference compared with the positive drug group (p > 0.05). The above results confirm that naringinone can effectively alleviate myocardial fibrosis induced by doxorubicin.

[0076] Experiment 6. Effect of naringinone on cardiomyocyte apoptosis in mice with doxorubicin-induced myocardial injury.

[0077] The effect of nardostachysone on doxorubicin-induced apoptosis of mouse cardiomyocytes was evaluated using tunnel staining. As shown in Figure 6-A, only a few cardiomyocytes in the Control group stained pale yellow, while the number of cardiomyocytes stained yellowish-brown significantly increased in the DOX model group. After intervention with nardostachysone and captopril, the number of brown-stained cells significantly decreased. As shown in Figure 6-B, the quantitative analysis results of tunnel staining showed that the number of apoptotic cells in the left ventricle of mice in the DOX model group was significantly higher than that in the Control group (p < 0.001). The number of apoptotic cells in the heart tissue of mice in the low, medium, and high dose groups of nardostachysone, as well as the positive control group of captopril, was significantly reduced, with the high dose group of nardostachysone showing the most significant effect (p < 0.001). These experimental results confirm that nardostachysone can effectively alleviate DOX-induced cardiomyocyte apoptosis.

[0078] Experiment 7. Effects of naringinone on the expression of apoptosis proteins and endoplasmic reticulum stress signaling pathway proteins in myocardial tissue of mice with doxorubicin-induced myocardial injury.

[0079] The effects of nardostachysone on the expression of apoptosis proteins and endoplasmic reticulum stress signaling pathway proteins in the heart tissue of mice in each group were evaluated using Western blotting. The results are shown in Figure 7. Statistical analysis of protein bands using ImageJ software revealed that, compared with the Control group, the Bcl2 / Bax ratio in the heart tissue of mice in the DOX model group was significantly decreased (p < 0.01), while the p-PERK / PERK, GRP78 / β-Actin, p-eIF-2α / eIF-2α, and CHOP / β-Actin ratios were significantly increased (p < 0.01). However, after intervention with nardostachysone and captopril, the Bcl2 / Bax ratio in the heart tissue of mice in the medium- and high-dose nardostachysone groups and the captopril positive control group was significantly increased (p < 0.05), while the p-PERK / PERK, GRP78 / β-Actin, p-eIF-2α / eIF-2α, and CHOP / β-Actin ratios were significantly decreased (p < 0.05). The experimental results further confirm that naringinone can reduce cardiomyocyte apoptosis by inhibiting the activation of the endoplasmic reticulum stress PERK / eIF-2α / CHOP signaling pathway, thereby exerting a cardioprotective effect.

[0080] Animal experiments showed that naringinone can improve cardiac function damage induced by anthracycline antitumor drugs in mice, reduce cardiomyocyte apoptosis and endoplasmic reticulum stress signaling pathway activation, thereby reducing doxorubicin-induced cardiotoxicity.

[0081] Therefore, this invention can be used to prevent and reduce myocardial toxicity caused by anthracycline antitumor drugs, and has clinical applicability. Based on the experimental dosage, the converted dosage for an adult (60kg) with myocardial protection is 150-1000mg per day.

Claims

1. Application of naringinone in the prevention and treatment of myocardial toxicity caused by anthracycline antitumor drugs.

2. Application of naringinone in the preparation of drugs for the prevention and treatment of myocardial toxicity induced by anthracycline antitumor drugs.

3. Application of naringinone in the prevention, treatment or inhibition of myocardial injury induced by anthracycline antitumor drugs.

4. The use of naringinone in the preparation of drugs for the prevention, treatment or inhibition of myocardial injury caused by anthracycline antitumor drugs.

5. Application of naringinone in alleviating cardiomyocyte apoptosis, endoplasmic reticulum stress injury, cardiomyocyte lactate dehydrogenase abnormalities, decreased cardiac function, or myocardial fibrosis induced by anthracycline chemotherapy drugs.

6. Application of naringinone in the preparation of drugs to alleviate cardiomyocyte apoptosis, endoplasmic reticulum stress injury, cardiomyocyte lactate dehydrogenase abnormalities, decreased cardiac function, or myocardial fibrosis induced by anthracycline chemotherapy drugs.

7. Use according to any one of claims 1 to 6, characterized in that: The daily dose of naringin for myocardial protection in adults (60 kg) is 150-1000 mg.

8. Use according to any one of claims 1 to 6, characterized in that: Used in the preparation of pharmaceutical mixtures or compositions, wherein the active ingredient of the pharmaceutical mixture includes naringinone.

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