Use of hydroxyjasmolin in preparation of drugs for preventing and treating cardiotoxicity induced by anti-tumor drugs

Abstract

The application discloses application of hydroxyl genkwa flower ketone (HGK) in preparation of a medicine for preventing and treating cardiotoxicity induced by an anti-tumor medicine. The process is as follows: a doxorubicin (DOX) is used to induce and establish a myocardial cell H9C2 injury model, and a minimum concentration of the myocardial cell injury induced by the DOX is determined. After 20 muM of the HGK is added to a treatment group, DOX-induced H9C2 cell apoptosis can be significantly improved. The DOX is used to induce and establish a mouse myocardial injury model, compared with a doxorubicin group, the left ventricular systolic fraction (FS%) and the ejection fraction (EF%) of mice in the doxorubicin+hydroxyl genkwa flower ketone group are significantly increased, the myocardial fiber structure is improved, the cytoplasm vacuolization is reduced, the serum cTnT, CK-MB and BNP levels are reduced, and the heart tissue ROS level is reduced. Therefore, the hydroxyl genkwa flower ketone can significantly reduce the myocardial toxicity induced by the DOX.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the application of hydroxygenistein in the preparation of drugs for preventing and treating cardiotoxicity induced by antitumor drugs. Background Technology

[0002] Doxorubicin (DOX) is an anthracycline antibiotic with broad-spectrum antitumor activity, primarily used to treat various malignant tumors, such as lymphoma, breast cancer, and leukemia. DOX's mechanism of action mainly involves embedding itself into DNA molecules, interfering with DNA replication and transcription, thereby inhibiting cancer cell proliferation and inducing cell death. While DOX plays a crucial role in inhibiting tumor growth and preventing metastasis, its treatment also causes various side effects, the most common and serious being DOX-induced cardiotoxicity (DIC). Approximately 25% of DOX users experience DIC, including decreased left ventricular ejection fraction, arrhythmias, and heart failure, which can even lead to death, severely limiting DOX use. In conclusion, DOX-induced cardiotoxicity is a significant factor limiting its clinical use; researching new methods to alleviate DIC will broaden the therapeutic window of DOX and effectively prolong the survival of DOX users.

[0003] Hydroxygenkwanin (HGK) is a natural flavonoid compound with anti-inflammatory, antioxidant, antitumor, and immunomodulatory effects (C. Kerneur, CE Cano, D. Olive, Major pathways involved in macrophage polarization in cancer, Front. Immunol. 2022, 13:111038.). Plants of the genus *Daphne* are widely cultivated worldwide. As a bioactive food ingredient, HGK's medicinal properties have been recognized from traditional uses to modern research. HGK exerts its antitumor effect by regulating growth arrest and inhibiting migration (Y.-L, et al. Hydroxygenkwanin suppresses non-small cell lung cancer progression by enhancing EGFR degradation, Molecules, 2020, 25: 941.). Previous studies have shown that HGK exerts its antitumor effect on human non-small cell lung cancer by activating p21, inhibiting tumor colony formation and migration, and inducing tumor cell cycle arrest. Furthermore, HGK inhibits the activation of the NF-κB pathway and the expression of inflammatory mediators such as NO, iNOS, TNF-α, and IL-6 in lymphocytes, thereby suppressing rheumatoid arthritis (Y. Sun, Y. Bao, H. Yu, Q. Chen, F. Lu, S. Zhai, C. Zhang, F. Li, C. Wang, C. Yuan, Anti-rheumatoid arthritis effects of flavonoids from Daphne genkwa, Int. Immunopharm. 2020, 83:106384.). In cardiovascular diseases, studies have shown that HGK has anti-inflammatory activity and can act as a tissue factor (TF) inhibitor to prevent thrombosis. Therefore, we used VSMCs and a mouse femoral artery filament injury model to detect the inhibitory effect of HGK on neointimal hyperplasia and to explore its regulatory mechanism in detail. In this study, HGK exerted anti-proliferative, anti-migration, and anti-inflammatory effects, accelerated reendothelialization, and reduced neointimal hyperplasia. However, whether hydroxygenistein can improve doxorubicin-induced cardiotoxicity and the related mechanisms are currently unclear. Therefore, this application investigates whether hydroxygenistein can improve doxorubicin-induced cardiotoxicity by establishing a mouse model of doxorubicin-induced cardiotoxicity. Summary of the Invention

[0004] The purpose of this invention is to provide the application of hydroxygenistein in the preparation of a drug for preventing and treating cardiotoxicity induced by antitumor drugs.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of hydroxygenistein in the preparation of drugs for preventing and inhibiting cardiotoxicity caused by antitumor chemotherapy drugs.

[0007] The antitumor chemotherapy drug is one of doxorubicin, daunorubicin, aclarubicin, epirubicin, pirarubicin, idarubicin, or mitoxantrone.

[0008] Furthermore, the structural formula of the hydroxygenistein is as follows: .

[0009] Furthermore, the effective content of hydroxygenistein in the drug is 0.5~60μM.

[0010] Furthermore, the drug is a drug mixture or a drug composition.

[0011] Furthermore, the drug mixture or drug composition includes hydroxygenistein and doxorubicin.

[0012] A pharmaceutical mixture or composition having hydroxygenistein as its active ingredient, said pharmaceutical mixture or composition having at least one of the following functions (1) to (4):

[0013] 1) Prevention and / or treatment of doxorubicin cardiotoxicity;

[0014] 2) Relieves myocardial tissue inflammation caused by doxorubicin;

[0015] 3) Reduces the increase in reactive oxygen species induced by doxorubicin;

[0016] 4) Relieves cardiomyocyte apoptosis induced by doxorubicin.

[0017] Furthermore, the drug mixture or drug composition is any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions, and solutions.

[0018] Doxorubicin has cardiotoxicity, and hydroxygenistein may improve the cardiotoxicity caused by doxorubicin. This may provide a new approach for using hydroxygenistein to prevent and treat doxorubicin-induced cardiotoxicity, providing a theoretical basis and new therapeutic targets for the subsequent development of drugs to alleviate DIC, promoting the development of new DIC treatments, and reducing the side effects of doxorubicin, which helps to expand the therapeutic window of doxorubicin, reduce the economic burden on patients and the healthcare system caused by DIC, and has certain economic and social benefits. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the chemical structure of HGK.

[0020] Figure 2 Figure 1 shows the effects of the control group, HGK group, DOX group, and DOX+HGK group on H9C2 cardiomyocytes: (A) MTT assay analysis of the effect of HGK on cardiomyocytes. (B) MTT assay analysis of the effect of HGK on DOX-treated cardiomyocytes. (C) HGK reduces DOX-induced ROS levels, from left to right: DMSO, 20 μM HGK, 1 μM HGK, and 1 μM DOX+20 μM HGK. (D) HGK reduces DOX-induced apoptosis levels. (E) is the quantitative result of (D). HGK: hydroxygenkwanin. *, **, ***, and **** represent p<0.05, p<0.01, p<0.001, and p<0.0001, respectively.

[0021] Figure 3 The following diagram illustrates how hydroxygenistein alleviated DOX-induced cardiotoxicity in C57BL / 6 mice: A. Schematic diagram of animal experiments. B. Representative echocardiogram of C57BL / 6 mice. C. Quantitative results of heart weight / tibia length. DE. Statistical graph of left ventricular FS% and EF% in C57BL / 6 mice. FH. Serum levels of cTnT, CK-MB, and BNP in mice were detected using an ELISA kit. *, **, ***, and **** represent p<0.05, p<0.01, p<0.001, and p<0.0001, respectively.

[0022] Figure 4 To characterize the levels of cardiac inflammation, fibrosis, cardiomyocyte apoptosis, and fibrosis in C57BL / 6J mice under four different treatments, the myocardial tissues were stained with (A) H&E, (B) MASSON, (C) TUNEL, and (D) WGA. (E) is a quantitative result of (D). HGK: hydroxygenkwanin. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention. The parameters, proportions, etc. of the embodiments can be selected according to local conditions without having a substantial impact on the results.

[0024] Materials and Methods:

[0025] 1.1 Cell Culture: H9C2 cells were cultured in DMEM medium (containing 10% FBS and penicillin-streptomycin) at 37°C and 5% CO2.

[0026] 1.2 MTT Assay for Cytotoxicity: Logarithmic growth phase cells were collected, and the cell suspension concentration was adjusted. 100 μL of cell suspension was added to each well, and the cells were seeded to a density of 6000 H9C2 cardiomyocytes / well. Cells were cultured at 37°C and 5% CO2. After adhesion, HGK was added for treatment, with final HGK concentrations in the cell suspension successively set to 0 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM. After approximately 24 hours, culture medium containing MTT was added, and the cells were incubated at 37°C for 2–4 hours. The MTT solution was then discarded, and 100 μL of DMSO was added to each well. The cells were then gently shaken on a shaker for 10 minutes to fully dissolve any crystals. The absorbance (OD) of each well was measured using a microplate reader. 490 nm, the analysis results are as follows Figure 2 As shown in Figure A.

[0027] 1.3 MTT assay for cell proliferation: Cells in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted. 100 μL of cell suspension was added to each well, and the cells were seeded to achieve a density of 6000 H9C2 cardiomyocytes / well. Cells were cultured at 37℃ and 5% CO2. After cell adhesion, drugs were added in the following groups: DMSO, 20 μM HGK, 40 μM HGK, 1 μM DOX, 1 μM DOX + 20 μM HGK, and 1 μM DOX + 40 μM HGK. After 24 h of drug treatment, MTT assay was performed, and the absorbance was measured at 490 nm.

[0028] 1.4 Apoptosis Level Assay: H9C2 cells were pre-seeded in 6-well plates at a density of 20 cells / well. Approximately 24 hours later, cells were treated with DMSO, 1 μM DOX, 20 μM HGK, or 1 μM DOX + 20 μM HGK. Samples were collected 24 hours after treatment, and the assays were performed according to the Anexin V-FITC / PI kit instructions.

[0029] 1.5 Establishment of a DOX-induced mouse model of myocardial injury: Eight-week-old C57BL / 6 mice were randomly divided into a control group (Control group), a model group (doxorubicin group), a hydroxygenistein group, and a hydroxygenistein + doxorubicin group. Except for the control group, which received intraperitoneal injection of saline, mice in the model group (doxorubicin group) received a single intraperitoneal injection of doxorubicin at a dose of 18 mg / kg. In the single-drug group (HGK), HGK was administered via gavage at a dose of 1.5 mg / kg, twice daily for two weeks. Mice in the hydroxygenistein + doxorubicin group first received a single intraperitoneal injection of doxorubicin at a dose of 18 mg / kg, followed by gavage administration of HGK at a dose of 1.5 mg / kg, twice daily for two weeks.

[0030] 1.6 HE staining: Paraffin sections of embedded mouse hearts were baked (2 h, 60 ℃), dewaxed with xylene, washed with ethanol and water of different proportions, stained with hematoxylin for 2 min, washed with water, differentiated with differentiation solution (3-5 s), rinsed with tap water for 10 min, stained with eosin for 1 min, rinsed with tap water for 10 min, dehydrated with 95% EtOH (2 min) and 100% EtOH (2 min) in sequence, mounted, and photographed to record the results.

[0031] 1.7 Masson staining: For dewaxed mouse heart sections, stain with Weigert iron hematoxylin using the Masson trichrome staining kit (Solarbio, GI1340) for 5-10 min. Wash away excess staining solution with distilled water, add acidic ethanol differentiation solution, wash with water, re-blue with Masson blue solution for 3-5 min, wash with distilled water for 30 s, stain with Ponceau S and fuchsin for 5-10 min, wash with weak acid working solution for 30 s, discard excess liquid, treat with phosphomolybdic acid solution for 1-2 min, discard excess liquid again, stain with aniline blue solution for 1-2 min, wash with weak acid working solution for 30 s, dehydrate with 95% ethanol and anhydrous ethanol sequentially, clear with xylene twice, 1-2 dishes each time, mount with neutral glue, and then photograph and record.

[0032] 1.8 WGA staining: H9C2 cells were fixed with 4% formaldehyde, followed by the addition of 100 µL iFluor 647-WGA working solution. Cells were incubated with WGA working solution at room temperature for 30 minutes, then washed twice with HHBS buffer. Cells were then photographed and recorded using a fluorescence microscope with a Cy5 filter set.

[0033] 1.9 Reactive Oxygen Species (ROS) Detection: H9C2 cells were cultured under healthy cell conditions. Cells were collected and DCFH-DA was diluted 1:1000 with serum-free culture medium. After collection, the cells were resuspended in the diluted DCFH-DA working solution to achieve a cell density of 1.0 × 10⁶ cells / year. 7 The cells were incubated at 37°C in the dark for 20 minutes at a concentration of / mL, with the probe inverted every 5 minutes to ensure full contact between the probe and the cells. The cells were washed three times with serum-free culture medium, suspended in serum-free culture medium, divided into several aliquots, and then stimulated with the drug. The aliquots were incubated at 37°C in the dark for 20 minutes, and the cells were observed and recorded directly using a laser confocal microscope.

[0034] 2.0 TUNEL Apoptosis Detection: At room temperature, paraffin sections of mouse heart tissue samples were thoroughly dewaxed by immersing them in xylene. They were then washed once each in a gradient of ethanol (90%, 80%, 70%) for 3 minutes each time, gradually increasing the water content. The sections were gently rinsed with PBS, and excess liquid around the samples was carefully blotted with filter paper. Proteinase K (2 mg / mL) was diluted 1:100 with PBS. 100 µL of Proteinase K (20 µg / mL) was added to each sample, ensuring the solution covered the entire sample area, and incubated at room temperature for 20 minutes. The samples were rinsed three times with PBS, and excess liquid was gently blotted. The treated samples were kept moist in a humidified chamber. 100 µL of 1×DNase I Buffer was added to the permeabilized samples, and incubated at room temperature for 5 minutes. 1 µL of DNase I was added to the remaining 100 µL of 1×DNase I Buffer. Gently tap or aspirate the liquid, add 100 μL of buffer containing 5 µg / mL DNase I, incubate at room temperature for 10 min, and wash three times with PBS. Prepare an appropriate amount of TUNEL detection solution according to the table below, add the TUNEL detection solution to the sample, incubate at 37°C in the dark for 60 min, and wash three times with PBS. After mounting with mounting medium, observe under a fluorescence microscope.

[0035] Table 1. TUNEL Detection Solution Components

[0036]

[0037] 2. Experimental Results:

[0038] (1) Hydroxygenin can alleviate DOX-induced cardiotoxicity in cardiomyocytes.

[0039] This application first performed an MTT assay to determine the cytotoxic effect of hydroxygenistein in H9C2 cardiomyocytes. HGK (structural formula shown) was then used in H9C2 cardiomyocytes. Figure 1As shown in the figure, the treatment with HGK resulted in the growth of H9C2 cells being unaffected by increasing HGK concentration, indicating that HGK has no or low cytotoxicity. Figure 2 A). Subsequently, this application performed an MTT assay to determine the combined cytotoxic effects of DOX and hydroxygenistein in H9C2 cardiomyocytes. The results showed that DOX reduced cell viability (…). Figure 2 B), and the addition of HGK significantly reversed DOX-induced cell death, indicating that HGK has a protective effect against DOX-induced cardiomyocyte damage. This application also examined ROS levels and apoptosis levels in H9C2 cells, and the results showed that DOX treatment increased cellular ROS levels (B). Figure 2 C) and the proportion of apoptosis ( Figure 2 D), and the addition of HGK significantly reversed these adverse effects of DOX on cells ( Figure 2 These results demonstrate that HGK can alleviate DOX-induced cardiotoxicity in cardiomyocytes.

[0040] (2) Hydroxygenin can reduce DOX-induced cardiotoxicity in mice.

[0041] This application investigated the effects of hydroxygenistein on disseminated intravascular coagulation (DIC). To this end, a DIC model was constructed using male C57BL / 6 mice, as follows: Figure 3 As shown in Figure A, as expected, the ratio of ventricular weight to tibial length was significantly decreased in the DOX model group, while it was significantly increased in the hydroxygenistein combined with other drugs group, indicating that hydroxygenistein alleviated DOX-induced cardiotoxicity. Figure 3 C). Echocardiography showed ( Figure 3 B), when mice received DOX intervention, the left ventricular systolic fraction (FS%) and ejection fraction (EF%) were significantly reduced. Figure 3 DE (destructive reaction) indicated a significant decrease in cardiac function in mice; while the left ventricular systolic fraction (FS%) and ejection fraction (EF%) were significantly increased in the hydroxygenistein combined with other drugs group. Furthermore, compared to the DOX group, the levels of myocardial injury markers (i.e., cTnT, CK-MB, and BNP) in myocardial tissue homogenates from C57BL / 6 mice treated with hydroxygenistein combined with other drugs were also significantly reduced. Figure 3 (FH). In summary, hydroxygenistein alleviated DOX-induced cardiotoxicity in mice.

[0042] (3) Histological analysis showed that hydroxygenistein alleviated DOX-induced cardiotoxicity in mice.

[0043] This application also found through histological analysis that H&E staining results showed that DOX enhanced inflammatory infiltration of cardiomyocytes, while the addition of HGK alleviated this phenomenon. Figure 4A); MASSON staining results showed that DOX-treated myocardial tissue inflammation levels were significantly upregulated, while HGK significantly downregulated DOX-induced myocardial tissue inflammation levels. Figure 4 B); DOX induces an increase in cardiomyocyte apoptosis, while HGK reduces this increased level of apoptosis (B). Figure 4 C); Cardiac cardiomyocytes in DIC mice shrank, while HGK administration significantly restored the size of cardiomyocytes (C). Figure 4 DE). The above results indicate that hydroxygenistein can alleviate DOX-induced cardiotoxicity in mice.

Claims

1. The application of hydroxygenistein in the preparation of drugs for preventing and inhibiting cardiotoxicity caused by antitumor chemotherapy drugs, characterized in that, The hydroxygenistein has at least one of the following functions: 1) to 4): 1) Prevention and / or treatment of doxorubicin cardiotoxicity; 2) Relieves myocardial tissue inflammation caused by doxorubicin; 3) Reduces the increase in reactive oxygen species induced by doxorubicin; 4) Alleviates doxorubicin-induced cardiomyocyte apoptosis; The antitumor chemotherapy drug is doxorubicin; the structural formula of the hydroxygentodin is as follows: .

2. The application according to claim 1, characterized in that, The effective content of hydroxygenistein in the drug is 0.5~60μM.

3. The application according to claim 1, characterized in that, The drug is a pharmaceutical composition.

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