Use of masp-2 peptide in the preparation of a drug for preventing or treating adriamycin-induced cardiac injury

Abstract

The application provides application of masitinib in preparation of a drug for preventing or treating cardiac injury caused by doxorubicin. The application belongs to the technical field of biological medicine, and specifically provides application of masitinib or a derivative thereof in preparation of a drug for preventing or treating cardiac injury caused by doxorubicin. Through a plurality of experiments, it is proved that masitinib has good prevention and treatment effects on cardiomyopathy caused by doxorubicin and its complications.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of mastoside in the preparation of medicaments for the prevention or treatment of doxorubicin-induced cardiac injury. Background Technology

[0002] Doxorubicin (DOX), an anthracycline broad-spectrum antitumor drug, is widely used to treat various malignant tumors such as leukemia, breast cancer, lung cancer, and lymphoma, with significant efficacy. However, its clinical application is severely limited by dose-dependent cardiotoxicity. Doxorubicin-induced cardiac injury is a complex pathological process involving multiple mechanisms, including but not limited to: inhibiting nucleic acid and protein synthesis through DNA embedding, inducing mitochondrial dysfunction, leading to excessive production of reactive oxygen species (ROS), triggering cardiomyocyte apoptosis and ferroptosis, promoting myocardial interstitial fibrosis, disrupting intracellular calcium ion homeostasis, and activating a persistent inflammatory response. These pathological changes can ultimately lead to myocardial damage, decreased cardiac function, and may progress to heart failure, arrhythmias, and even dilated cardiomyopathy.

[0003] Currently, strategies for preventing and treating doxorubicin-induced cardiotoxicity are very limited. Clinically, antioxidants such as vitamin E, N-acetylcysteine, and resveratrol are often used to mitigate oxidative stress damage. However, the protective effects of these drugs are not ideal and fall far short of meeting the urgent clinical need for highly effective and specific cardioprotective agents. Therefore, exploring new drugs and targets that can effectively prevent or treat doxorubicin-induced cardiac injury has significant clinical implications and application value.

[0004] Mazdutide is a novel glucagon (GCG) / glucagon-like peptide-1 (GLP-1) dual receptor agonist. Its primary known use is as a hypoglycemic and weight-loss drug, and clinical studies have shown its potential to improve glycemic control, reduce weight, and provide multiple metabolic benefits (such as improved blood lipids, lower blood pressure, and reduced liver fat content). Based on existing research on GLP-1 receptor agonists, these drugs have been shown to potentially have beneficial effects on the cardiovascular system by increasing myocardial glucose uptake and oxidation utilization to optimize energy metabolism, reducing cardiac inflammation, inhibiting myocardial fibrosis, and reducing cardiomyocyte apoptosis. However, to date, no research has reported or suggested any role for mazdutide in the prevention or treatment of cardiomyopathy induced by chemotherapy drugs such as doxorubicin. Whether mazdutide can be used for the prevention and treatment of doxorubicin-induced cardiomyopathy remains entirely unknown. Summary of the Invention

[0005] The first objective of this invention is to provide the use of mascara and its pharmaceutically acceptable derivatives in the preparation of medicaments for the prevention or treatment of doxorubicin-induced cardiac injury.

[0006] In this invention, the masto peptide and its pharmaceutically acceptable derivatives are in the form of pharmaceutically acceptable salts.

[0007] In this invention, the mastotrieptide and its pharmaceutically acceptable derivatives are in the form of pharmaceutically acceptable acid addition salts.

[0008] This invention utilizes a pharmaceutically acceptable salt form (such as an acid addition salt) to significantly improve the physicochemical properties of mastoside, including stability, solubility, and bioavailability, thereby ensuring its effectiveness during preparation and storage and facilitating its absorption in vivo to exert a cardioprotective effect. This limitation, based on mature pharmaceutical salt chemistry technology, clarifies the specific implementation path for industrialization, enhancing the invention's practicality. Simultaneously, it reasonably expands the scope of protection, covering various common stable forms of the active ingredient, making the core inventive concept less susceptible to circumvention by simple salt substitution and improving the stability of patent rights.

[0009] In this invention, the cardiac damage caused by doxorubicin is selected from one or more of myocardial injury, heart failure, arrhythmia, and dilated cardiomyopathy.

[0010] In this invention, the drug for preventing or treating doxorubicin-induced cardiac damage is a pharmaceutical composition, which further includes a pharmaceutically acceptable carrier.

[0011] In this invention, the drug for preventing or treating doxorubicin-induced cardiac injury has a pharmaceutically acceptable dosage form.

[0012] In this invention, the dosage form is at least one of oral preparations, injectable preparations, and aerosols.

[0013] The drug for preventing or treating doxorubicin-induced cardiac injury also contains other active ingredients for preventing and / or treating one or more of the following: doxorubicin-induced myocardial injury, doxorubicin-induced heart failure, doxorubicin-induced arrhythmia, and doxorubicin-induced dilated cardiomyopathy.

[0014] A kit containing reagents for detecting substances comprising mastoside, said kit being used for use with one or more of the following:

[0015] (1) Diagnosis, risk assessment and / or evaluation of treatment efficacy and prognosis of doxorubicin-induced myocardial injury;

[0016] (2) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of doxorubicin-induced heart failure;

[0017] (3) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of arrhythmias caused by doxorubicin;

[0018] (4) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of dilated cardiomyopathy caused by doxorubicin.

[0019] The use of reagents for detecting substances containing mastolide in the preparation of a kit, said kit being used for one or more of the following:

[0020] (1) Diagnosis, risk assessment and / or evaluation of treatment efficacy and prognosis of doxorubicin-induced myocardial injury;

[0021] (2) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of doxorubicin-induced heart failure;

[0022] (3) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of arrhythmias caused by doxorubicin;

[0023] (4) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of dilated cardiomyopathy caused by doxorubicin.

[0024] This invention, through extensive experiments, demonstrates that mastipotetamide has an ameliorative effect on a mouse model of doxorubicin-induced cardiomyopathy. Cardiac function was assessed using echocardiography and hemodynamic monitoring, and ferroptosis and inflammation-related markers in mouse and primary cardiomyocytes were detected. Molecular biological analysis was used to detect the levels of inflammation-related proteins and mRNAs in mouse and primary cardiomyocytes, revealing that mastipotetamide can improve doxorubicin-induced cardiomyopathy. In conclusion, mastipotetamide is found to have potential applications in the prevention and treatment of doxorubicin-induced cardiomyopathy and its complications. Attached Figure Description

[0025] Figure 1 Based on in vivo experimental data from Example 1 of the present invention, the effects of mastodextrin on myocardial injury and deterioration of cardiac function in a mouse model of doxorubicin cardiomyopathy are demonstrated.

[0026] Figure 2 Based on in vitro experimental data from Example 2 of the present invention, the effects of mastodextrin on doxorubicin-induced cardiomyocyte damage are demonstrated.

[0027] Figure 3 Based on in vivo experimental data from Example 3 of the present invention, the effect of mastodextrin on ferroptosis in myocardial tissue of a mouse model of doxorubicin cardiomyopathy is demonstrated.

[0028] Figure 4 Based on in vitro experimental data from Example 4 of the present invention, the effect of mastolide on doxorubicin-induced ferroptosis in cardiomyocytes is demonstrated.

[0029] Figure 5 Based on in vivo experimental data from Example 5 of the present invention, the effect of mastodextrin on improving cardiac inflammatory response in a mouse model of doxorubicin cardiomyopathy is demonstrated.

[0030] Figure 6 Based on in vitro experimental data from Example 6 of the present invention, the effect of mastodextrin on improving the inflammatory response of cardiomyocytes induced by doxorubicin is demonstrated. Detailed Implementation

[0031] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the present invention.

[0032] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0033] To prevent and treat doxorubicin-induced cardiotoxicity, specific embodiments of the present invention provide a novel drug for preventing or treating doxorubicin-induced cardiac injury, specifically comprising:

[0034] The use of mastolide and its pharmaceutically acceptable derivatives in the preparation of drugs for the prevention or treatment of doxorubicin-induced cardiac injury.

[0035] In a specific embodiment of the present invention, Mastodide specifically refers to a glucagon (GCG) / glucagon-like peptide-1 (GLP-1) dual receptor agonist, whose trade name includes "Sinmei". Mastodide has been approved for use in the preparation of weight loss drugs.

[0036] In the above embodiments, the masto peptide and its pharmaceutically acceptable derivatives are in the form of pharmaceutically acceptable salts.

[0037] In the above embodiments, the mastotrieptide and its pharmaceutically acceptable derivatives are in the form of pharmaceutically acceptable acid addition salts.

[0038] In the above embodiments, the cardiac damage caused by doxorubicin is selected from one or more of myocardial injury, heart failure, arrhythmia, and dilated cardiomyopathy.

[0039] In the above embodiments, the drug for preventing or treating doxorubicin-induced cardiac damage is a pharmaceutical composition, which further includes a pharmaceutically acceptable carrier.

[0040] In the above embodiments, the drug for preventing or treating doxorubicin-induced cardiac injury has a pharmaceutically acceptable dosage form.

[0041] In the above embodiments, the dosage form is at least one of oral preparations, injectable preparations, and aerosols.

[0042] Secondly, specific embodiments of the present invention also provide that the medicament for preventing or treating cardiac injury caused by doxorubicin further comprises other active ingredients, said other active ingredients being used to prevent and / or treat one or more of doxorubicin-induced myocardial injury, doxorubicin-induced heart failure, doxorubicin-induced arrhythmia, and doxorubicin-induced dilated cardiomyopathy.

[0043] Thirdly, specific embodiments of the present invention also provide a kit containing reagents for detecting substances comprising mastoside components, said kit being used for one or more of the following:

[0044] (1) Diagnosis, risk assessment and / or evaluation of treatment efficacy and prognosis of doxorubicin-induced myocardial injury;

[0045] (2) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of doxorubicin-induced heart failure;

[0046] (3) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of arrhythmias caused by doxorubicin;

[0047] (4) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of dilated cardiomyopathy caused by doxorubicin.

[0048] Fourthly, specific embodiments of the present invention also provide the use of reagents for detecting substances containing mastoside components in the preparation of a kit, said kit being used for one or more of the following:

[0049] (1) Diagnosis, risk assessment and / or evaluation of treatment efficacy and prognosis of doxorubicin-induced myocardial injury;

[0050] (2) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of doxorubicin-induced heart failure;

[0051] (3) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of arrhythmias caused by doxorubicin;

[0052] (4) Diagnosis, risk assessment and / or evaluation of treatment and prognosis of dilated cardiomyopathy caused by doxorubicin.

[0053] This invention is the first to demonstrate that in myocardial tissue damaged by doxorubicin, left ventricular diastolic and systolic functions are significantly reduced, while markers of myocardial toxicity are significantly increased. Both in vivo and in vitro, injection of mastolide can significantly reduce doxorubicin-induced myocardial toxicity.

[0054] The technical solutions of the present invention are further described below through specific embodiments. Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. In some cases, terms with a conventional understanding are limited herein for clarification or ease of reference, and such limitations should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and have been adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0055] It is worth mentioning that, in this invention, the term "doxorubicin myocardial injury" has a meaning known in the art, referring to left ventricular dysfunction that occurs after the use of doxorubicin.

[0056] In this invention, the term "prevention and / or treatment of doxorubicin myocardial injury" refers to inhibiting or slowing the occurrence of doxorubicin myocardial injury and related diseases.

[0057] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] Unless otherwise specified, the following embodiments are all conventional methods.

[0059] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0060] All experimental data in this invention are percentages. Chi-square tests were used to compare two samples, and Graphpad 8.0 software was used for data analysis. P < 0.05 was considered statistically significant.

[0061] In the following figures, # represents P < 0.05, indicating a significant difference between the two groups.

[0062] Example 1

[0063] Mastolide prevents myocardial damage and improves cardiac function in doxorubicin cardiomyopathy.

[0064] (1) Establishment of a mouse model of myocardial injury caused by doxorubicin

[0065] Using a randomized table method, C57BL / 6J mice were first injected subcutaneously with Mazdutide, and then simultaneously injected intraperitoneally with DOX into the following four groups: vehicle+NS group, vehicle+DOX group, Mazdutide+NS group, and Mazdutide+DOX group, with 10 mice in each group. Figure 1 A represents the survival analysis curves for each group of mice.

[0066] (2) Weighing the heart of small animals and measuring the tibia length of mice

[0067] The results show: See Figure 1 B, Figure 1 B indicates the ratio of heart weight to tibia length in each group of mice; compared with the vehicle+DOX group, the Mazdutide+DOX group showed increased body weight, heart weight, and HW / TL, suggesting that Mazdutide alleviates myocardial damage and cardiac function deterioration in doxorubicin cardiomyopathy.

[0068] (3) Small animal ultrasound evaluation of mouse cardiac diastolic and systolic function and hemodynamic assessment of mouse cardiac function

[0069] Mice were anesthetized with isoflurane, and echocardiography was performed using a MyLab 30CV system. Left ventricular (LV) morphology was measured over five consecutive cardiac cycles, including left ventricular end-systolic diameter (LVESd), left ventricular end-diastolic diameter (LVEDd), left ventricular fractional shortening (LVFS), and left ventricular ejection fraction (LVEF). Hemodynamics were measured using cardiac catheterization, as previously described. Hemodynamic parameters such as cardiac output (CO), maximum pressure incidence (dP / dtmax), and minimum pressure decay rate (dP / dtmin) were examined.

[0070] The results are as follows Figure 1 C- Figure 1 The I-display shows: Figure 1 C- Figure 1 F represents the LVEF, LVFS, LVEDd, and LVESd values ​​of mice in each group, assessed by ultrasound. Figure 1 G- Figure 1 I represents hemodynamic parameters for each group, including cardiac output (CO), rate of maximum pressure (dP / dt max), and rate of minimum pressure decay (dP / dt min).

[0071] Compared to the vehicle+DOX group mice, the Mazdutide+DOX group mice had higher LVEF, LVFS, CO, (dP / dtmax), and (dP / dtmin) values, and lower LVEDd and LVESd values. Mazdutide improved myocardial damage and cardiac function deterioration in doxorubicin cardiomyopathy.

[0072] (4) Expression of serum myocardial injury markers CK-MB, LDH and cTNT

[0073] The expression of myocardial injury markers CK-MB, LDH, and cTNT in the serum of mice in the Mazdutide +DOX group and the vehicle +DOX group was detected by ELISA to determine the degree of myocardial injury.

[0074] The results are as follows Figure 1 J- Figure 1 The L display: Figure 1 J- Figure 1 L represents the ELISA assay for myocardial injury markers CK-MB, CTNT, and LDH in mice across different groups. Compared to the vehicle+DOX group, the Mazdutide+DOX group showed significantly reduced expression of myocardial injury markers CK-MB, LDH, and cTNT, demonstrating that Mazdutide can improve myocardial injury and cardiac function deterioration in doxorubicin cardiomyopathy.

[0075] Example 2

[0076] Mastopeptide alleviates doxorubicin-induced myocardial cell damage

[0077] (1) Detection of NRVM cell viability by CCK-8 assay

[0078] The experimental method involved adding 100 μL of cell suspension to a 96-well plate and culturing for 24 h. Each group was divided into 6 replicates. After DOX treatment for 24 h in the PBS group and Mazdutide group, 10 μL of CCK-8 solution was added to each well. The culture plate was incubated in an incubator for 4 h, and the absorbance of each group of cells at 450 nm was measured using a microplate reader.

[0079] The results show: See Figure 2 A, Figure 2 A is used to evaluate the cell viability level of each group, from Figure 2 As can be seen from A, compared with the vehicle+DOX group, the Mazdutide+DOX group had higher cell activity, indicating that Mazdutide alleviated the cardiomyocyte damage caused by doxorubicin.

[0080] (2) LDH release experiment

[0081] Procedure: LDH release into the supernatant is considered an indicator of cytotoxicity. Following the specified treatment, culture supernatant was collected using a commercially available kit to detect LDH release, following the manufacturer's instructions. The absorbance of the samples was measured at 490 nm and 600 nm using a spectrophotometer.

[0082] The results show: See Figure 2 B, Figure 2 B was used to characterize LDH release assays to evaluate LDH levels in each group of cells, from Figure 2 As can be seen from B, the Mazdutide+DOX group had a lower LDH ratio compared to the vehicle+DOX group, indicating that Mazdutide alleviated cardiomyocyte damage caused by doxorubicin.

[0083] Example 3

[0084] Mastolide alleviates doxorubicin cardiomyopathy by preventing ferroptosis.

[0085] (1) Expression levels of MDA / non-hene iron in serum and cardiac homogenate

[0086] For operating instructions, please refer to the MDA / non-hene iron handling kit.

[0087] The results are as follows Figure 3 A- Figure 3 The D display shows: Figure 3 A- Figure 3 B was used to assess the expression levels of MDA in mouse serum and heart tissue homogenates. Figure 3 C- Figure 3 The D-value was used to assess the expression levels of non-heme iron in mouse serum and heart tissue homogenates. Compared with the vehicle+DOX group, the expression of MDA / non-heniron in serum and heart homogenates was significantly decreased in the Mazdutide+DOX group, and Mazdutide alleviated doxorubicin cardiomyopathy by preventing ferroptosis.

[0088] (2) Expression levels of GSH and NAPDH in cardiac homogenate

[0089] For operating instructions, please refer to the GSH and NAPDH kit instructions.

[0090] The results are as follows Figure 3 E- Figure 3 The F display shows that Figure 3 E was used to assess the expression level of NAPDH in mouse heart tissue homogenate. Figure 3The F-value was used to assess the expression level of GSH in mouse heart tissue homogenate. Compared with the vehicle+DOX group, the expression level of NAPDH in heart homogenate of the Mazdutide+DOX group was significantly decreased, and the expression level of GSH was significantly upregulated. Mazdutide alleviated doxorubicin cardiomyopathy by preventing ferroptosis.

[0091] Example 4

[0092] Mastodextrin improves doxorubicin cardiomyopathy by preventing ferroptosis.

[0093] (1) Expression levels of GSH, NAPDH and MDA in primary cardiomyocyte homogenate proteins.

[0094] For operating instructions, please refer to the GSH, NAPDH, and MDA kits.

[0095] The results are as follows Figure 4 A- Figure 4 The C-value shows: Figure 4 A was used to assess the expression level of MDA in primary cardiomyocytes of each group. Figure 4 B was used to assess the expression level of GSH in primary cardiomyocytes of each group. Figure 4 C was used to assess the expression level of NAPDH in primary cardiomyocytes of each group. Compared with the DOX group, the expression levels of MDA and NAPDH were significantly reduced and the expression level of GSH was significantly upregulated in the Mazdutide + DOX group.

[0096] Example 5

[0097] Mastodextrin improves inflammation in doxorubicin cardiomyopathy

[0098] (1) Detection of mRNA expression of TNF-α, IL-6 and IL-1β in cardiac tissue by quantitative real-time PCR.

[0099] The procedure is as follows: Add 1 mL of Trizol to the left ventricle (LV) tissue, let stand for 10 min, extract with chloroform, add chloroform at a ratio of 200 μL / mL of Trizol, let stand at room temperature for 5 min, centrifuge at 12000 rpm at 4°C for 30 min, the supernatant after centrifugation is RNA, transfer to a new EP tube, add 500 μL of isopropanol, let stand at room temperature for 10 min, centrifuge at 12000 rpm at 4°C for 10 min, discard the supernatant, wash with 75% ethanol, centrifuge, dry at room temperature for 10 min, dissolve in DEPC water, and determine the RNA concentration and purity using a spectrophotometer, and store in a -80°C freezer.

[0100] The results show: See Figure 5 A, Figure 5The A-value was used to evaluate the expression levels of TNF-α, IL-1β, and IL-6 mRNA in mice by quantitative real-time PCR. Compared with the vehicle+DOX group, the expression levels of TNF-α, IL-6, and IL-1β mRNA in the Mazdutide+DOX group were significantly reduced, indicating that Mazdutide can improve inflammation in doxorubicin cardiomyopathy.

[0101] (2) Expression of TNF-α, IL-6 and IL-1β in serum and cardiac homogenate

[0102] The operation is described in Example 1.

[0103] The results are as follows Figure 5 B- Figure 5 The D display: Figure 5 B- Figure 5 D represents the expression levels of TNF-α, IL-1β, and IL-6 in mouse serum as assessed by ELISA. Compared to the vehicle+DOX group, the Mazdutide+DOX group showed significantly lower protein expression levels of TNF-α, IL-6, and IL-1β in serum, indicating that Mazdutide can improve inflammation in doxorubicin-induced cardiomyopathy.

[0104] (3) Western blot was used to detect the expression levels of TNF-α, IL-6 and IL-1β proteins in cardiac tissue.

[0105] The procedure involves using the BCA protein assay kit to assess proteins extracted from cardiac tissue. Left ventricular (LV) tissue was added at a ratio of 10 mg / 100 μL to protein lysis buffer (1 mL of protein lysis buffer contained 10 μL of 1 mmol / L benzyl sulfonyl fluoride, 50 μL of 1 mmol / L sodium chloride, 10 μL of 1 mmol / L sodium vanadate, 100 μL of Roche phosphatase inhibitor, 100 μL of complete lysate, and 730 μL of RIFA strong lysis buffer). Cells from each group were collected and placed in 1.5 mL EP tubes. LV tissue was then lysed 15-20 times using an ultrasonic lysis device (5 kHz) and centrifuged at 12000 rpm for 30 min at 4 °C. Protein concentration was quantified using the BCA method. After protein denaturation at 72 °C, the cells were stored at -80 °C. Protein electrophoresis was performed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to PVDF membranes and blocked for 2 hours, then primary antibody was added and the membrane was incubated overnight at 4°C with shaking. The next day, the membranes were washed with TBST buffer, incubated with secondary antibody for 1.5 hours, and then developed using a chemical scanning membrane analyzer. Protein bands were analyzed using ImageLab software, with GADPH as an internal control.

[0106] The results are as follows Figure 5 E- Figure 5H display: Figure 5 E- Figure 5 H represents the comparison of protein expression levels of TNF-α, IL-1β, and IL-6 in different mouse groups using Western blot. Compared with the vehicle+DOX group, the protein expression levels of TNF-α, IL-6, and IL-1β were significantly reduced in the Mazdutide+DOX group, indicating that Mazdutide can improve inflammation in doxorubicin-induced cardiomyopathy.

[0107] Example 6

[0108] Mastodextrin improves inflammation in doxorubicin cardiomyopathy

[0109] (1) Detection of mRNA expression of TNF-α, IL-6 and IL-1β in primary cardiomyocytes by quantitative real-time PCR.

[0110] Experimental procedures are as follows: ① RNA extraction: Add 1 mL of Trizol to a six-well plate inoculated with cells, let stand for 10 min, extract with chloroform, add chloroform at a ratio of 200 μL / mL Trizol, let stand at room temperature for 5 min, centrifuge at 12000 rpm at 4℃ for 15 min, the supernatant is RNA, transfer to a new EP tube, add 500 μL of isopropanol, let stand at room temperature for 10 min, centrifuge at 12000 rpm at 4℃ for 10 min, discard the supernatant, wash with 75% ethanol, centrifuge, dry at room temperature for 10 min, dissolve in DEPC water, and determine the RNA concentration and purity using a spectrophotometer, store at -80℃. ② RT-PCR detection: Detect the mRNA expression levels of apoptosis and oxidative stress-related factors according to the LightCycler 480 SYBR Green I Master instructions, using GADPH as an internal control.

[0111] The results are as follows Figure 6 Display A: Figure 6 The A-value was used to evaluate the expression levels of TNF-α, IL-1β, and IL-6 mRNA in mice by quantitative real-time PCR. Compared with the vehicle+DOX group, the expression levels of TNF-α, IL-6, and IL-1β mRNA in the Mazdutide+DOX group were significantly reduced, indicating that Mazdutide can improve inflammation in doxorubicin cardiomyopathy.

[0112] (2) Western blot was used to detect the expression levels of TNF-α, IL-6 and IL-1β proteins in primary cardiomyocytes.

[0113] Experimental Procedure: Protein Extraction and Quantification: Protein concentration was standardized before all Western blotting. Cells from each group were seeded into six-well plates and cultured for 24 h. Then, 30 mL of protein lysis buffer (1 mL of protein lysis buffer contains 10 μL of 1 mmol / L benzyl sulfonyl fluoride, 50 μL of 1 mmol / L sodium chloride, 10 μL of 1 mmol / L sodium vanadate, 100 μL of Roche phosphatase inhibitor, 100 μL of complete lysate, and 730 μL of RIFA strong lysis buffer) was added to each well. Cells from each group were collected and transferred to 1.5 mL EP tubes. Cells were then lysed 15-20 times using an ultrasonic lysate system (5 kHz) and centrifuged at 12000 rpm for 15 min at 4 °C. Protein concentration was quantified using the BCA method. After protein denaturation at 72 °C, the cells were stored at -80 °C. ② Western blotting procedure: Protein electrophoresis was performed using 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Proteins were transferred to a PVDF membrane and blocked for 2 hours, then primary antibody was added and the membrane was incubated overnight at 4°C with shaking. The next day, the membrane was washed with TBST buffer, incubated with secondary antibody for 1.5 hours, and then developed using a chemical scanning membrane analyzer. Protein bands were analyzed using ImageLab software, with GADPH as an internal control.

[0114] The results are as follows Figure 6 B- Figure 6 The E display shows: Figure 6 B- Figure 6 E represents the comparison of protein expression levels of TNF-α, IL-1β, and IL-6 in different mouse groups using Western blot. Compared with the vehicle+DOX group, the protein expression levels of TNF-α, IL-6, and IL-1β were significantly reduced in the Mazdutide+DOX group, indicating that Mazdutide can improve inflammation in doxorubicin-induced cardiomyopathy.

[0115] As can be seen from the above embodiments, the mastolide of the present invention has an ameliorative effect on the doxorubicin-induced cardiomyopathy model in mice. By detecting cardiac function through echocardiography and hemodynamics, ferroptosis and inflammation-related indicators in mouse and primary cardiomyocytes were detected. Molecular biology was used to detect the levels of inflammation-related proteins and mRNA in mouse and primary cardiomyocytes, and it was found that mastolide can improve doxorubicin-induced cardiomyopathy.

[0116] Furthermore, based on the above embodiments, this invention constructed a mouse model of doxorubicin-induced cardiomyopathy and used mascara for functional verification, confirming that mascara can improve myocardial injury and cardiac dysfunction in mice with doxorubicin-induced cardiomyopathy, and also confirming that mascara can improve primary cardiomyocyte injury in rats; mascara reduces ferroptosis, inflammation, and reactive oxygen species levels in myocardial tissue of mice with doxorubicin-induced cardiomyopathy. Mascara improves ferroptosis, inflammation, and reactive oxygen species levels in a primary cardiomyocyte injury model in rats with doxorubicin-induced cardiomyopathy. Therefore, mascara can be used to prevent or treat doxorubicin-induced myocardial injury and related diseases, providing new evidence for the prevention, diagnosis, and treatment of doxorubicin-induced myocardial injury in clinical medicine. These results indicate that mascara can be used to prevent or treat doxorubicin-induced myocardial injury and related diseases by improving ferroptosis.

[0117] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. The use of a mastoside in the preparation of a drug for the prevention or treatment of doxorubicin-induced myocardial injury.

2. The application as described in claim 1, characterized in that, The drug for preventing or treating myocardial injury caused by doxorubicin is a pharmaceutical composition, which further includes a pharmaceutically acceptable carrier.

3. The application as described in claim 1, characterized in that, The drug for preventing or treating doxorubicin-induced myocardial injury has a pharmaceutically acceptable dosage form.

4. The application as described in claim 3, characterized in that, The dosage form is at least one of oral preparations, injectable preparations, and aerosols.

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