Application of nardosinone in prevention or treatment of cardiotoxicity caused by anthracycline antitumor drugs

Abstract

The invention relates to a pharmaceutical application of nardosinone in prevention and treatment of cardiotoxicity, in particular to an application of nardosinone in prevention or treatment of cardiotoxicity caused by anthracycline antitumor drugs. Experiments prove that nardosinone has a remarkable curative effect on mouse myocardial injury caused by doxorubicin, and can be used for remarkably increasing the left ventricular ejection fraction and the short-axis shortening rate of the mouse heart caused by DOX (doxorubicin); the release amount of lactic dehydrogenase and creatine kinase in mouse plasma caused by DOX is reduced, the mouse myocardial fibrosis caused by DOX is reduced, and myocardial cell apoptosis and endoplasmic reticulum stress injury caused by DOX are relieved. The nardosinone is low in raw material cost and easy to obtain, can be used as an active component or a medicine composition component for myocardial protection of anthracycline chemotherapy medicines, and has a good medication prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of drug toxicity prevention and treatment, and specifically relates to a new use of a substance, and in particular to the use of naringenone in preventing or treating myocardial toxicity caused by anthracycline anti-tumor drugs. Background Art

[0002] Cancer has become the second largest disease after cardiovascular disease in terms of morbidity and mortality. In my country, the death rate caused by cancer is nearly twice the global average, and the growth rate is relatively fast. At present, the main clinical cancer treatment methods are surgery, radiotherapy, and chemotherapy. Among them, chemotherapy, as one of the conventional means of anticancer treatment, is used in all stages of cancer lesions. Anthracyclines (doxorubicin, daunorubicin, aclarubicin, epirubicin, pirarubicin, idarubicin and mitoxantrone, etc.) are commonly used clinical anti-tumor chemotherapy drugs, and are used in a variety of cancers such as leukemia, breast cancer, and lymphoma with high morbidity and mortality. However, this type of drug has a high affinity for myocardial tissue, which will produce cumulative, dose-dependent myocardial toxicity, and further develop into irreversible myocardial damage, eventually leading to congestive heart failure. The cardiotoxicity caused by it can appear shortly after medication, during medication, after treatment, and even several years or decades after drug withdrawal, thus severely limiting its clinical application.

[0003] In response to the toxic side effects of anthracyclines in clinical treatment, although there are currently liposomal doxorubicin, dexrazoxane, etc. that antagonize the cardiotoxicity caused by doxorubicin by changing the dosage form and chelating the iron ions of anthracyclines, they are relatively strict in clinical control due to their high prices or the aggravation of side effects such as bone marrow suppression.

[0004] At present, there is no effective and affordable preventive or therapeutic measure for the cardiotoxic side effects caused by anthracyclines. Therefore, it is of great practical significance to conduct research on new drugs for the prevention or treatment of anthracycline anti-tumor drug myocardial toxicity and develop effective and affordable drugs against cardiotoxic side effects.

[0005] Nardone is a sesquiterpenoid compound derived from the Chinese medicinal material Nardone, and its chemical structure is as follows:

[0006]

[0007] Nardone has extensive and definite pharmacological activities, such as sedation, anti-epileptic, anti-depressant, nerve growth promotion, and blood pressure reduction. Literature reports that Nardone can restore cardiac function by inhibiting cAMP and PKA levels, and effectively relieve aconitine-induced tachyarrhythmia in rats. However, there has been no research and development report on the therapeutic value of Nardone in myocardial injury caused by chemotherapy drugs. Summary of the invention

[0008] The present invention is to prevent or treat myocardial toxicity caused by anthracycline anti-tumor drugs. Through research, it is found that galanthin can be used as an active substance for myocardial protection of anthracycline chemotherapy drugs, which can improve endoplasmic reticulum stress damage and alleviate myocardial cell apoptosis, thereby reducing the lethality caused by doxorubicin cardiotoxicity. Therefore, the application of galanthin in preventing or treating myocardial toxicity caused by anthracycline anti-tumor drugs is proposed.

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

[0010] The technical solution adopted by the present invention is:

[0011] The present invention provides the use of narostane as a preventive and therapeutic drug for myocardial toxicity caused by anthracycline anti-tumor drugs, which can be used to prevent and treat myocardial toxicity caused by anthracycline anti-tumor drugs, and can be used to prepare drugs, drug mixtures and drug compositions for inhibiting myocardial damage caused by anthracycline anti-tumor drugs.

[0012] At the same time, the present invention also provides narostane for preventing, treating or inhibiting myocardial damage caused by anthracycline anti-tumor drugs and its application in such drugs.

[0013] At the same time, galanthin and its drug application can be used to alleviate myocardial cell apoptosis, endoplasmic reticulum stress injury, myocardial cell lactate dehydrogenase abnormality, cardiac function decline or myocardial fibrosis caused by anthracycline chemotherapy drugs.

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

[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 anthracycline chemotherapy-induced myocardial toxicity;

[0017] 2) Alleviate myocardial cell apoptosis caused by anthracycline chemotherapy drugs;

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

[0019] 4) Alleviate the abnormality of lactate dehydrogenase in myocardial cells caused by anthracycline chemotherapy drugs;

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

[0021] 6) Alleviate myocardial fibrosis caused by anthracycline chemotherapy drugs.

[0022] Beneficial effects of the invention:

[0023] 1. The present invention provides a new method for preventing or treating myocardial toxicity caused by anthracycline anti-tumor drugs. Nardostachysinone is the main component of Nardostachysinensis, which can be extracted from natural plants, and the extraction method is mature. The raw material cost is low and easy to obtain. As an active ingredient or drug formulation component for myocardial protection of anthracycline chemotherapy drugs, it has good prospects for use. In addition, Nardostachysinone can be administered orally, which is easy to accept and conducive to clinical promotion and application.

[0024] 2. Gansongxin can be used as an alternative drug for improving myocardial injury caused by anthracycline drugs, and is used to prevent and reduce myocardial toxicity caused by anthracycline anti-tumor drugs. The present invention interferes with the mouse myocardial injury model induced by anthracycline drugs (taking doxorubicin as an example) by using Gansongxin. The results of animal experiments show that Gansongxin can effectively improve the cardiac function damage of mice caused by anthracycline anti-tumor drugs by reducing myocardial cell apoptosis, reducing myocardial fibrosis, etc., reducing myocardial cell apoptosis and activation of endoplasmic reticulum stress signaling pathways, thereby reducing the cardiac toxicity caused by doxorubicin. Experiments have shown that Gansongxin has a significant therapeutic effect on myocardial injury caused by doxorubicin (DOX) in mice. It can significantly increase the left ventricular ejection fraction (LVEF) and short-axis shortening rate (FS) of the mouse heart caused by DOX, reduce the release of lactate dehydrogenase (LDH) and creatine kinase (CK) in mouse plasma caused by DOX, reduce myocardial fibrosis caused by DOX in mice, and reduce myocardial cell apoptosis and endoplasmic reticulum stress damage caused by DOX.

[0025] Therefore, it can be used to prevent and alleviate myocardial toxicity caused by anthracycline anti-tumor drugs and has clinical practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shown are the effects of naringenone on cardiac function in mice with DOX-induced myocardial injury (n=8-11);

[0027] Figure 1Table 1 is a comparison of the effects of galanthin on cardiac function of mice with DOX myocardial injury in Experiment 1, where A: representative echocardiograms of mice in each group; BE: bar chart statistical analysis results of LVEF%, FS%, LVIDd and LVIDs in each group. Compared with Control, ** 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%: fractional shortening; Control: blank control group; DOX: doxorubicin model group; DOX+L-Nar: low-dose narostachysin group; DOX+M-Nar: medium-dose narostachysin group; DOX+H-Nar: high-dose narostachysin group; Nar: narostachysin; DOX: doxorubicin.

[0028] Figure 2 Shown are the effects of naringenone on the heart weight and cardiac index of mice with DOX myocardial injury model (n=8-11);

[0029] Figure 2 Table 2 is a comparison of the effects of galanthin on the heart weight and cardiac index of mice with DOX myocardial injury in Experiment 2, where A: a bar chart of the heart body ratio of each group of mice; B: a bar chart of the left ventricular weight of each group of mice. Compared with Control, ** p<0.01; compared with the DOX model group, # p<0.05, ## p<0.01. HWI: heart-to-weight ratio; LVWI: left ventricular index; Control: blank control group; DOX: doxorubicin model group; DOX+L-Nar: low-dose narostane group; DOX+M-Nar: medium-dose narostane group; DOX+H-Nar: high-dose narostane group; Nar: narostane; DOX: doxorubicin.

[0030] Figure 3 Shown are the effects of naringenone on the plasma LDH and CK levels in mice with DOX myocardial injury (n=8-11);

[0031] Figure 3 Table 3 is a comparison of the effects of galanthin on the release of LDH and CK in the plasma of mice with DOX myocardial injury in Experiment 3, where A: a bar chart of statistical analysis of the activity level of LDH in the plasma of mice in each group; B: a bar chart of statistical analysis of the activity level of CK in the plasma of mice in each group. Compared with Control, *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 naringenin group; DOX+M-Nar: medium-dose naringenin group; DOX+H-Nar: high-dose naringenin group; Nar: naringenin; DOX: doxorubicin.

[0032] Figure 4 Shown is the effect of naringenin on the myocardial structure of mice with DOX-induced myocardial injury (HE staining, n=3);

[0033] Figure 4 This is a comparison chart of the effects of narostane on the myocardial structure of mice with DOX myocardial injury in Experiment 4, where Control: blank control group; DOX: doxorubicin model group; DOX+L-Nar: low-dose narostane group; DOX+M-Nar: medium-dose narostane group; DOX+H-Nar: high-dose narostane group; Nar: narostane; DOX: doxorubicin.

[0034] Figure 5 Shown is the effect of naringenin on myocardial fibrosis in mice with DOX-induced myocardial injury (Masson staining, n=3);

[0035] Figure 5 This is a comparison of the effects of galanthin on myocardial fibrosis in mice with DOX myocardial injury in Experiment 5, where A: representative Masson staining images of each group (200X); B: statistical analysis of the myocardial collagen area of ​​mice in each group. Compared with Control, *** 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 naringenin group; DOX+M-Nar: medium-dose naringenin group; DOX+H-Nar: high-dose naringenin group; Nar: naringenin; DOX: doxorubicin.

[0036] Figure 6 Shown is the effect of naringenin on cardiomyocyte apoptosis in mice with DOX-induced myocardial injury (Tunnel staining, n=3);

[0037] Figure 6This is a comparison of the effects of galanthin on myocardial cell apoptosis in mice with DOX myocardial injury in Experiment 6, where A: Representative images of myocardial self-tunnel staining in each group of mice (200X); B: Statistical analysis of the number of apoptotic myocardial cells in each group of mice. Compared with Control, *** 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 naringenin group; DOX+M-Nar: medium-dose naringenin group; DOX+H-Nar: high-dose naringenin group; Nar: naringenin; DOX: doxorubicin.

[0038] Figure 7 Shown are the effects of naringenin on the expression of apoptotic proteins in myocardial tissue and the expression of proteins in the endoplasmic reticulum stress signaling pathway in mice with adriamycin-induced myocardial injury (n=3);

[0039] Figure 7 This is a comparison chart of the effect of galanthin on the expression of proteins in the endoplasmic reticulum stress signaling pathway in the myocardial tissue of mice with DOX myocardial injury in Experiment 7, where A: The effect of galanthin 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 the bar graph of galanthin 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 Control, ** 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 naringenin group; DOX+M-Nar: medium-dose naringenin group; DOX+H-Nar: high-dose naringenin group; Nar: naringenin; DOX: doxorubicin. DETAILED DESCRIPTION

[0040] In order to make the technical concept and advantages of the present invention more clearly understood, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings. It should be understood that the following embodiments are only used to explain and illustrate the preferred implementation methods of the present invention, and should not be regarded as and do not constitute a limitation on the scope of patent protection claimed by the present invention.

[0041] 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.

[0042] Nardosinone used in the following examples: Molecular formula C 15 H 22 O3 was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., catalog number: 32476-67-8.

[0043] Example 1

[0044] 0.5% sodium carboxymethylcellulose solution was used to prepare 5 mg / mL intragastric solution of naringenin, and the following corresponding experiments were carried out using doxorubicin (DOX) as an example.

[0045] 1. The materials used are as follows:

[0046] Experimental animals: 70 SPF male Kunming mice were purchased from Beijing Huafukang Biotechnology Co., Ltd., license number SCXK2013-0007, weighing 25-28 g.

[0047] LDH detection kit: purchased from Changchun Huili Biotechnology Co., Ltd., catalog number: C058-e.

[0048] CK detection kit: purchased from Changchun Huili Biotechnology Co., Ltd., catalog number: C059-e.

[0049] HE staining kit: purchased from Wuhan Saiweier Biotechnology Co., Ltd., product number: G1005-100 mL.

[0050] Masson staining kit: purchased from Wuhan Saiweier Biotechnology Co., Ltd., catalog number: G1006-100 mL.

[0051] Tunel apoptosis detection kit: purchased from Wuhan Saiweier Biotechnology Co., Ltd., catalog number: G1507-50 T.

[0052] Antibodies: p-PERK (Thr982), purchased from Biotech Co., Ltd., catalog number: AF5902, 1:1000 dilution; PERK: purchased from Jiangsu Qinke Biological Research Center Co., Ltd., catalog number: AF5304, 1:1000 dilution; p-eIF-2α (Ser51): purchased from Biotech Co., Ltd., catalog number: AF5803, 1:1000 dilution; eIF-2α: purchased from Biotech Co., Ltd., catalog number: AF6771, 1:1000 dilution; GRP78: purchased from Biotech Co., Ltd., catalog number: AF0171, 1:1000 dilution; Bcl2: purchased from Jiangsu Qinke Biological Research Center Co., Ltd., catalog number: BF9013, 1:1000 dilution; Bax: purchased from Wuhan Saiweier Biotechnology Co., Ltd., catalog number: GB12960, 1:1000 dilution; CHOP: purchased from Jiangsu Qinke Biological Research Center Co., Ltd., catalog number: DF60254, 1:1000 dilution; β-Actin: purchased from Biyuntian Biotechnology Co., Ltd., catalog number: AF5003, 1:3000 dilution; BRD4: purchased from Wuhan Saiweier Biotechnology Co., Ltd., catalog number: GB11999, 1:1000 dilution; HRP-labeled goat anti-rabbit IgG: purchased from Wuhan Saiweier Biotechnology Co., Ltd., catalog number: GB23301, 1:4000 dilution.

[0053] 2. Experimental process

[0054] 70 SPF male Kunming mice were divided into 10 normal control group (Control group), 12 model group (DOX group), 12 low-dose naringenone group (DOX+L-Nar group, 25 mg / kg / d), medium-dose naringenone group (DOX+M-Nar, 50 mg / kg / d), high-dose naringenone group (DOX+H-Nar group, 100 mg / kg / d), and positive drug captopril group (DOX+Captopril group, 100 mg / kg / d). The animals in the normal control group and the model group were given 0.5 mL of 0.5% sodium carboxymethyl cellulose solution by gavage; the animals in the drug administration group were given naringenone and captopril by gavage according to the above doses for 28 days. The normal control group animals were intraperitoneally injected with 0.2 mL of saline, and the model group and the drug group mice were intraperitoneally injected with doxorubicin (Beijing Solaibao Technology Co., Ltd., batch number 125316-40-9) 3 mg / kg / d (normal saline was prepared into 0.2 mL), injected every other day, for a total of 5 times. During the process of intraperitoneal injection of DOX into mice, 6 mice died (3 in the DOX model group, 1 in each of the low-dose, medium-dose, and positive drug captopril groups). No mouse heart ultrasound examination was performed 28 days after the end of oral administration. The next day, after removing the eyeballs and taking blood, the mouse heart tissue was dissected for further examination and analysis.

[0055] 3. Index observation and detection methods

[0056] (1) Cardiac ultrasound examination: After the experiment, the mice were placed in an induction box and anesthetized with 4% isoflurane. After the mice fell into a coma, they were placed in a supine position on a 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 through their mouths for continuous anesthesia, and the anterior area of ​​the heart of the mice was depilated. Then medical ultrasound coupling agent was applied to the depilated area, and ultrasound detection was performed using a Vevo 2100M echocardiography system and a 12L-RS Linear high-frequency probe. The short-axis view was obtained, the conversion energy frequency was 13MHz, the depth was 2cm, and the number of frames per second was set to the maximum. Five cardiac cycles were continuously detected, and the main indicators measured were the left ventricular internal diameter at the end of systole (LVIDs), the left ventricular internal diameter at the end of diastole (LVIDd), the left ventricular ejection fraction (LVEF, %), and the short-axis shortening rate (FS, %), and statistical analysis was performed.

[0057] (2) Heart weight and cardiac index: The mice in each group were weighed the next day after ultrasound examination. After dissection, the heart was removed. Each mouse heart was perfused and cleaned with saline until the saline was clear and colorless. After absorbing the water on the surface of the heart with filter paper, the mouse heart was weighed. Then, a clean blade was used to cut along the short axis of the heart to separate the left ventricle, and the left ventricle was weighed and recorded, and the heart-to-body ratio and left ventricular index were calculated.

[0058] (3) Mouse plasma myocardial enzyme examination: On the second day after the ultrasound examination of each group of mice, the eyeballs of the mice were removed and blood was collected. The blood was stored in a 1.5 ml centrifuge tube with sodium heparin and centrifuged at 13,000 g for 15 minutes at 4°C. The upper layer of plasma was divided into 1.5 mL centrifuge tubes and stored at -80°C for later use. During the test, the plasma was taken out of the refrigerator and thawed, and the lactate dehydrogenase (LDH) and creatine kinase (CK) activities of the mouse plasma of each group were measured using the Roche COBAS Integra 400 Plus / C501 automatic biochemical analyzer. The experimental results were recorded and statistically analyzed.

[0059] (4) Observation of myocardial tissue pathology: After the experiment, the hearts of mice in each group were taken and weighed. The tissues of the left ventricular apex were taken along the short axis of the heart and immediately fixed in 4% paraformaldehyde solution. The tissues were embedded in paraffin and sliced ​​with a thickness of 4 μm. After HE and Masson staining, the tissues were observed and analyzed under a light microscope.

[0060] (5) Observation of cardiomyocyte apoptosis: After the experiment, the hearts of mice in each group were taken and weighed, and the tissues of the left ventricular apex were taken along the short axis of the heart and immediately fixed in 4% paraformaldehyde solution. Then, the tissues were embedded in paraffin, and conventional tissue sections were made with a thickness of 4 μm. The sections were stained with a Tunel kit, and the cell apoptosis was observed and counted.

[0061] (6) Western blot detection: After the experiment, the hearts of mice in each group were taken and weighed. The left ventricular tissue was taken along the short axis of the heart, and the tissue was cut as finely as possible on ice with surgical scissors and placed in a 1.5 mL EP tube. 300 μL of pre-cooled cell lysis solution containing protease inhibitors and 3 grinding beads were added. After quick freezing in liquid nitrogen, the tissue was ground three times at 4°C at a frequency of 70 Hz for 30 seconds each time until there was no obvious heart tissue. After that, the heart was placed in an ice box and vortexed once every 10 minutes for a total of three times. After the lysis was completed, the supernatant was centrifuged at 4°C and 13,000 rpm for 10 minutes, and then the supernatant was placed in a 1.5 mL EP tube for protein quantification. The relevant protein detection was performed according to the conventional Western blot detection method.

[0062] Experiment 1: Effects of naringenin on cardiac function in mice with adriamycin-induced myocardial injury

[0063] LVIDd, LVIDs, LVEF% and FS% are the most commonly used indicators of cardiac function. Therefore, cardiac function of each group of mice was tested by Doppler ultrasound. Figure 1As shown in Table 1, compared with the Control group, the LVEF% and FS% of mice in the DOX model group were significantly reduced (p<0.001), and LVIDd and LVIDs were significantly increased (p<0.01). After intragastric administration of gansongxin and captopril, the LVEF% and FS% of mice in the gansongxin medium and high dose groups and captopril group were significantly increased (p<0.01), and LVIDd and LVIDs were significantly lower than those in the model group (p<0.05), and there was no significant difference between the gansongxin medium and high dose groups and the positive drug captopril group (p>0.05). The above results confirmed that gansongxin has a strong improvement effect on the cardiac function damage caused by doxorubicin.

[0064] Table 1 Effects of naringenone on cardiac function in mice with DOX-induced myocardial injury (n=8-11)

[0065]

[0066] Experiment 2: Effects of naringenone on heart weight and cardiac index in mice with adriamycin-induced myocardial injury

[0067] The effects of naringenin on cardiac index of mice with doxorubicin-induced myocardial injury were observed by measuring heart-to-body weight ratio (HWI) and left ventricle / heart (LVWI). Figure 2 As shown in Table 2, compared with the Control group, the heart index HWI and LVWI of mice in the DOX model group were significantly increased (p<0.01); compared with the DOX model group, after continuous intragastric administration of gansuxinone and the positive drug captopril, the heart index HWI and LVWI of mice in the gansuxinone medium and high dose groups and the positive drug group were significantly reduced (p<0.05), and there was no significant difference between the gansuxinone high dose group and the positive drug group (p>0.05). The results confirmed that gansuxinone can effectively reduce doxorubicin-induced myocardial hypertrophy.

[0068] Table 2 Effects of pyraclostrobin on heart weight and cardiac index of mice with DOX myocardial injury model (n=8-11)

[0069]

[0070] Experiment 3: Effects of naringenone on the release of LDH and CK in the plasma of mice with adriamycin-induced myocardial injury

[0071] The effects of naringenone on DOX-induced myocardial injury in mice were investigated by evaluating the expression levels of LDH and CK myocardial enzymes in mouse plasma. Figure 3As shown in 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), and after the intervention of gansongxinone and the positive drug captopril, the medium and high doses of gansongxinone and the positive drug captopril could significantly reduce 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 group of gansongxinone and the positive drug captopril group (p>0.05). The above results confirmed that gansongxinone can effectively reduce the release of LDH and CK caused by doxorubicin myocardial injury.

[0072] Table 3 Effects of pyraclostrobin on plasma LDH and CK levels in mice with DOX-induced myocardial injury (n=8-11)

[0073]

[0074] Experiment 4: Effects of naringenin on myocardial structure in mice with adriamycin-induced myocardial injury

[0075] HE staining was used to evaluate the effect of galanthin on the structural changes of cardiac tissue in mice with DOX-induced myocardial injury. Figure 4 As shown, HE staining of the heart sections of the Control group showed no obvious lesions and fibrosis, and the myocardial cell layers were arranged neatly and the nuclei were uniform in size; compared with the Control group, the heart sections of the DOX model group showed fibrous tissue hyperplasia, myocardial fiber rupture, disordered myocardial cell arrangement, and scattered nuclei in the myocardial fibers; compared with the DOX model group, the myocardial cells in the medium and high doses of galanthus quinone groups and the positive drug captopril group were arranged more neatly and the myocardial fiber rupture phenomenon was reduced, and the myocardial cell nuclei were relatively evenly distributed in the center of the myocardial fibers. The staining results directly confirmed that galanthus quinone can effectively improve the myocardial structural disorder caused by doxorubicin.

[0076] Experiment 5: Effects of naringenin on myocardial fibrosis in mice with adriamycin-induced myocardial injury

[0077] Masson staining was used to evaluate the effect of naringenin on myocardial fibrosis in mice with doxorubicin-induced myocardial injury. Figure 5 -As shown in Figure 1-A: There was no obvious abnormality in the heart sections of the Control group, the myocardial fibers of the left ventricle of the mice were arranged neatly, and there was almost no obvious blue collagen fibers between the myocardial cells; while in the heart sections of the mice in the DOX model group, the myocardial cells were arranged in a disordered manner, and the blue collagen fibers in the intercellular spaces increased significantly. After intervention with naringapine 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. The distribution area of ​​collagen fibers after Masson staining was further quantitatively analyzed. Figure 5-B shows that 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 groups of galanthus quinone and the positive drug captopril group was significantly reduced (p<0.05), among which the high dose group of galanthus quinone had the most obvious improvement effect, and there was no significant difference compared with the positive drug group (p>0.05). The above results confirm that galanthus quinone can effectively reduce myocardial fibrosis caused by doxorubicin.

[0078] Experiment 6: Effect of naringapolone on cardiomyocyte apoptosis in mice with doxorubicin-induced myocardial injury.

[0079] Tunnel staining was used to evaluate the effect of naringenin on doxorubicin-induced apoptosis of mouse cardiomyocytes. Figure 6 -A, only a few cardiomyocytes in the control group were stained light yellow, while the number of cardiomyocytes stained yellow-brown in the heart tissue of mice in the DOX model group increased significantly. After the intervention of naringenin and captopril, the number of brown-stained cells decreased significantly. Figure 6 -B Tunnel staining quantitative analysis results show 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), and the number of apoptotic cells in the heart tissue of mice in the low, medium, and high dose groups of galanthus quinone and the positive drug captopril group was significantly reduced, among which the high dose group of galanthus quinone had the most obvious effect (p<0.001). The above experimental results confirm that galanthus quinone can effectively reduce the apoptosis of myocardial cells caused by DOX.

[0080] Experiment 7: Effects of gadolinium on the expression of apoptosis proteins in myocardial tissue and the expression of proteins in the endoplasmic reticulum stress signaling pathway in mice with doxorubicin-induced myocardial injury.

[0081] The effect of galanthin on the expression of apoptosis proteins and endoplasmic reticulum stress signaling pathway proteins in cardiac tissue of each group of mice was evaluated by immunoblotting. Figure 7As shown in the figure, the protein bands were statistically analyzed using Image J software and it was found that compared with the Control group, the Bcl2 / Bax ratio in the heart tissue of the mice in the DOX model group was significantly decreased (p<0.01), and the p-PERK / PERK, GRP78 / β-Actin, p-eIF-2α / eIF-2α and CHOP / β-Actin ratios were significantly increased (p<0.01); after the intervention of gansuxinone and captopril, the Bcl2 / Bax ratio in the heart tissue of the mice in the gansuxinone medium and high dose groups and the positive drug captopril group was significantly increased (p<0.05), and 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 confirmed that galanthin can alleviate cardiomyocyte apoptosis by inhibiting the activation of the endoplasmic reticulum stress PERK / eIF-2α / CHOP signaling pathway, thereby exerting a cardioprotective effect.

[0082] The results of animal experiments showed that galanthin can improve the cardiac function damage in mice caused by anthracycline anti-tumor drugs, reduce myocardial cell apoptosis and activation of endoplasmic reticulum stress signaling pathway, and thus reduce the cardiotoxicity caused by doxorubicin.

[0083] Therefore, the present invention can be used to prevent and alleviate myocardial toxicity caused by anthracycline anti-tumor drugs, and has clinical practicability. According to the test dose, the dosage of myocardial protective drugs for adults (60 kg) is 150-1000 mg per day.

Claims

1. Application of galanthinone in the prevention and treatment of myocardial toxicity caused by anthracycline anti-tumor drugs.

2. The use of galanthin in the preparation of drugs for preventing and treating myocardial toxicity induced by anthracycline anti-tumor drugs.

3. Application of gadolinium in preventing, treating or inhibiting myocardial damage caused by anthracycline anti-tumor drugs.

4. The use of naringenone in the preparation of drugs for preventing, treating or inhibiting myocardial damage caused by anthracycline anti-tumor drugs.

5. Application of galanthinone in alleviating myocardial cell apoptosis, endoplasmic reticulum stress injury, myocardial cell lactate dehydrogenase abnormality, decreased cardiac function or myocardial fibrosis caused by anthracycline chemotherapy drugs.

6. Use of galanthinone in the preparation of drugs for alleviating myocardial cell apoptosis, endoplasmic reticulum stress injury, myocardial cell lactate dehydrogenase abnormality, decreased cardiac function or myocardial fibrosis caused by anthracycline chemotherapy drugs.

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

8. The use according to any one of claims 1 to 6, characterized in that: The invention is used for preparing a drug mixture or a drug composition, wherein the active ingredient of the drug comprises naringenone.

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