Application of hydroxygenkwanin in preparation of medicine for preventing and treating cardiotoxicity induced by antitumor medicine

Abstract

The invention discloses an application of hydroxygenkwanin (HGK) in preparation of a drug for preventing and treating cardiotoxicity induced by antitumor drugs. The method comprises the following steps: inducing doxorubicin (DOX), establishing a myocardial cell H9C2 injury model, and determining the DOX-induced myocardial cell injury minimum concentration; after 20 [mu] M of HGK is added into a treatment group, DOX-induced H9C2 cell apoptosis can be significantly improved. A mouse myocardial injury model is induced and established by DOX, and compared with a doxorubicin group, the left ventricular systolic fraction (FS%) and ejection fraction (EF%) of mice in the doxorubicin and hydroxygenkwanin group are remarkably increased, the myocardial fiber structure is improved, the cytoplasm vacuolation is reduced, the levels of serum cTnT, CK-MB and BNP are reduced, and the ROS level of heart tissue is reduced. Therefore, the hydroxygenkwanin can obviously reduce the cardiotoxicity induced by the DOX.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically to the use of hydroxygenkandrin in the preparation of drugs for preventing and treating cardiotoxicity induced by anti-tumor drugs. Background Art

[0002] Doxorubicin (DOX) is an anthracycline antibiotic with broad antitumor activity, mainly used to treat a variety of malignant tumors, such as lymphoma, breast cancer, leukemia, etc. The mechanism of action of DOX is mainly to embed into DNA molecules, interfere with DNA replication and transcription, thereby inhibiting the proliferation of cancer cells and inducing their death. Although DOX plays a key role in inhibiting tumor growth and preventing metastasis, DOX treatment can also cause a variety of side effects, the most common and serious of which is DOX-induced cardiotoxicity (DIC). About 25% of DOX users will experience DIC such as reduced left ventricular ejection fraction, arrhythmia and heart failure, and even lead to death of patients, which seriously limits the use of DOX. In summary, DOX-induced cardiotoxicity is an important factor limiting its clinical use; studying 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.). Genkwanin plants are widely cultivated in the world. As a bioactive food ingredient, HGK has been recognized for its medicinal properties from traditional uses to modern research. HGK exerts antitumor effects 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 antitumor effects on human non-small cell lung cancer by activating p21, inhibiting tumor colony formation and migration, and inducing tumor cell cycle arrest. In addition, HGK inhibits the activation of the NF-kB pathway and the expression of inflammatory mediators such as NO, iNOS, TNF-a, and IL-6 in lymphocytes, thereby inhibiting rheumatoid arthritis (Y. Sun, Y. Bao, H. Yu, Q. Chen, F. Lu, S. Zhai, C. Zhang, F. Li, C. Wang, C. Yuan, Anti-rheumatoidarthritiseffects 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 mouse femoral artery filament injury models to detect the inhibitory effect of HGK on neointimal hyperplasia and explore its regulatory mechanism in detail. In this study, HGK exerted anti-proliferative, anti-migratory, and anti-inflammatory effects, accelerated re-endothelialization, and reduced neointimal hyperplasia. However, it is still unclear whether hydroxygenkwain can improve doxorubicin-induced cardiotoxicity and its related mechanism. Therefore, this application establishes a mouse model of doxorubicin-induced cardiotoxicity to explore whether hydroxygenkwain can improve doxorubicin-induced cardiotoxicity. Summary of the invention

[0004] The object of the present invention is to provide an application of hydroxygenkandrin in the preparation of a drug for preventing and treating cardiotoxicity induced by anti-tumor drugs.

[0005] Based on the above purpose, the present invention adopts the following technical solutions: Application of hydroxygenkwain in the preparation of drugs for preventing and inhibiting cardiotoxicity caused by anti-tumor chemotherapy drugs.

[0006] The anti-tumor chemotherapy drug is one of doxorubicin, daunorubicin, aclarubicin, epirubicin, pirarubicin, idarubicin or mitoxantrone.

[0007] Furthermore, the structural formula of hydroxygenkwain is as follows: .

[0008] Furthermore, the effective content of hydroxygenkwain in the drug is 0.5-60 μM.

[0009] Furthermore, the drug is a drug mixture or a drug composition.

[0010] Furthermore, the drug mixture or drug composition includes hydroxygenkandrin and doxorubicin.

[0011] A drug mixture or a drug composition, the active ingredient of which includes hydroxygenkwain, and the drug mixture or the drug composition has at least one of the following functions 1) to 4): 1) To prevent and / or treat doxorubicin-induced cardiotoxicity; 2) Alleviate myocardial tissue inflammation caused by doxorubicin; 3) Reduce the increase of reactive oxygen free radicals caused by doxorubicin; 4) Alleviate myocardial cell apoptosis induced by doxorubicin.

[0012] Furthermore, the drug mixture or drug composition is in any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions and solutions.

[0013] Doxorubicin is cardiotoxic, and hydroxygenkwain may improve the cardiotoxicity caused by doxorubicin. This may provide a new idea for the use of hydroxygenkwain to prevent and treat cardiotoxicity caused by doxorubicin, provide a theoretical basis and new therapeutic targets for the subsequent development of drugs to relieve DIC, promote the development of new DIC treatment methods, and reduce the side effects of doxorubicin, which will help expand the treatment window of doxorubicin and reduce the economic burden on patients and medical systems caused by DIC, with certain economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the chemical structure of HGK.

[0015] Figure 2 The results of the control group, HGK group, DOX group and DOX+HGK group on cardiomyocytes H9C2: (A) MTT assay to analyze the effect of HGK on cardiomyocytes. (B) MTT assay to analyze the effect of HGK on DOX-treated cardiomyocytes. (C) HGK can reduce the level of ROS induced by DOX, from left to right: DMSO, 20μM HGK, 1μM HGK and 1μM DOX+20μM HGK. (D) HGK reduces the level of apoptosis induced by DOX, (E) is the quantitative result of (D). HGK: hydroxygenkwanin. *, **, *** and **** indicate p<0.05, p<0.01, p<0.001 and p<0.0001, respectively.

[0016] Figure 3 Hydroxygenkandrin 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 graphs of left ventricular FS% and EF% in C57BL / 6 mice. FH, ELISA kits were used to detect the levels of cTnT, CK-MB and BNP in mouse serum. *, **, *** and **** indicate p<0.05, p<0.01, p<0.001 and p<0.0001, respectively.

[0017] Figure 4 Myocardial tissues of C57BL / 6J mice treated with four different treatments were subjected to (A) H&E staining; (B) MASSON staining; (C) TUNEL staining; and (D) WGA staining to characterize the levels of cardiac inflammation, fibrosis, cardiomyocyte apoptosis, and fibrosis in mice. (E) is the quantitative result of (D). HGK: hydroxygenkwanin. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the embodiments of the drawings. It should be understood that the embodiments described in this specification are only for explaining the present invention, not for limiting the present invention, and the parameters, proportions, etc. of the embodiments can be selected according to local conditions without substantial impact on the results.

[0019] Materials and methods 1.1 Cell culture: H9C2 cells were cultured in DMEM medium (containing 10% FBS and penicillin-streptomycin) at 37°C and 5% CO2.

[0020] 1.2 MTT cytotoxicity test: Collect logarithmic phase cells, adjust the concentration of cell suspension, add 100 μL cell suspension to each well, plate the H9C2 cardiomyocytes to be tested at a density of 6000 cells / well, and culture at 37°C and 5% CO2. After the cells adhere to the wall, add HGK for drug treatment, so that the final concentration of HGK in the cell suspension is 0 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM, respectively; after about 24 hours, add culture medium containing MTT, incubate at 37°C for 2-4 hours, discard the MTT solution, add 100 μL DMSO to each well, place on a shaker for 10 minutes to fully dissolve the crystals, and use an enzyme marker to measure the absorbance value of each well, i.e., OD 490 nm, the analysis results are as follows Figure 2 As shown in A.

[0021] 1.3 MTT method for cell proliferation assay: Collect logarithmic phase cells, adjust the concentration of cell suspension, add 100 μL of cell suspension to each well, plate the H9C2 cardiomyocytes to be tested to a density of 6000 cells / well, and culture at 37°C and 5% CO2. Add drugs after cell adhesion, and divide into groups of 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 hours of drug treatment, add MTT treatment and measure the absorbance at 490 nm.

[0022] 1.4 Determination of cell apoptosis level: H9C2 cells were seeded in 6-well plates in advance with a seeding density of 20w cells / well. Drug treatment was performed about 24 hours later. The drug treatment groups were DMSO, 1μM DOX, 20μM HGK, and 1μM DOX+20μM HGK. Samples were collected 24 hours after drug treatment, and the experiment was performed according to the instructions of the AnexinV-FITC / PI kit and measured on the machine.

[0023] 1.5 Establishment of DOX-induced myocardial injury model in mice: 8-week-old C57BL / 6 mice were used in the experiment and randomly divided into control group (Control group), model group (doxorubicin group), hydroxygenkwain group, and hydroxygenkwain + doxorubicin group. Except for the control group, which was intraperitoneally injected with normal saline, the mice in the model group (doxorubicin group) were intraperitoneally injected with doxorubicin at a dose of 18 mg / kg, once; in the single-dose group (HGK), HGK was administered by gavage at a dose of 1.5 mg / kg, once every two days, for two consecutive weeks. The mice in the hydroxygenkwain + doxorubicin group were first intraperitoneally injected with doxorubicin at a dose of 18 mg / kg, and then HGK was administered by gavage at a dose of 1.5 mg / kg, once every two days, for two consecutive weeks.

[0024] 1.6 HE staining: The embedded experimental mouse heart paraffin sections were baked (2h, 60℃), dewaxed with xylene, washed with different proportions of ethanol, stained with hematoxylin for 2min, washed with water, differentiated with differentiation solution (3-5s), rinsed with tap water for 10min, stained with eosin for 1min, rinsed with tap water for 10min, dehydrated with 95% EtOH (2min) and 100% EtOH (2min) in sequence, and then sealed and photographed to record the results.

[0025] 1.7 Masson staining: The dewaxed experimental mouse heart sections were stained with Masson trichrome staining kit (Solarbio, GI1340) and Weigert iron hematoxylin for 5-10 minutes, the excess stain was washed off with distilled water, acidic ethanol differentiation solution was added, washed with water, Masson blueing solution was used to return to blue for 3-5 minutes, washed with distilled water for 30 seconds, stained with Ponceau fuchsin staining solution for 5-10 minutes, washed with weak acid working solution for 30 seconds, the excess liquid was poured off, phosphomolybdic acid solution was added for treatment for 1-2 minutes, the excess liquid was poured off, aniline blue staining solution was added for staining for 1-2 minutes, washed with weak acid working solution for 30 seconds, dehydrated with 95% ethanol and anhydrous ethanol in turn, transparentized with xylene twice, 1-2 dishes each time, sealed with neutral glue and photographed and recorded.

[0026] 1.8 WGA staining: H9C2 cells were fixed with 4% formaldehyde, and then 100 µL of iFluor 647-WGA working solution was added. The cells were incubated with WGA working solution for 30 minutes at room temperature, and then washed twice with HHBS buffer and photographed on a fluorescence microscope using a Cy5 filter set.

[0027] 1.9 Reactive oxygen species (ROS) detection: H9C2 cells were cultured under the condition of ensuring the health of cells. The cells were collected and DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000. After the cells were collected, they were resuspended in the diluted DCFH-DA working solution to a cell density of 1.0×10 7 / mL, incubate in a 37℃ cell culture incubator in the dark for 20 min, invert and mix every 5 min to make the probe and cells fully contact. Wash the cells 3 times with serum-free culture medium, suspend the cells in serum-free culture medium, divide the cells into several equal parts, and then stimulate with drugs, incubate in a 37℃ cell culture incubator in the dark for 20 min, and directly observe and record under a laser confocal microscope.

[0028] 2.0 TUNEL apoptosis assay: At room temperature, paraffin sections of mouse heart tissue samples were immersed in xylene to completely remove paraffin, and then washed in gradient ethanol (90, 80, 70%) once for 3 minutes each time, gradually increasing the water content. Gently rinse the sections with PBS, and carefully absorb the excess liquid around the samples on the slide with filter paper. Dilute Proteinase K (2mg / mL) with PBS at a ratio of 1:100. Add 100 µL of Proteinase K (20µg / mL) to each sample, so that the solution covers the entire sample area, and incubate at room temperature for 20 minutes. Rinse the samples with PBS 3 times, gently absorb the excess liquid, and keep the processed samples moist in a humidified box. Take 100µL of 1×DNase I Buffer and add it to the permeabilized samples, and incubate at room temperature for 5 minutes. Add 1μL DNase I to the remaining 100μL 1×DNase I Buffer. Gently tap or aspirate the liquid, add 100μL of buffer containing 5μg / mL DNase I, incubate at room temperature for 10 minutes, and wash 3 times with PBS. Prepare an appropriate amount of TUNEL detection solution according to the table below, add TUNEL detection solution to the sample, incubate at 37℃ in the dark for 60 minutes, and wash 3 times with PBS. After sealing with sealing agent, observe under a fluorescence microscope.

[0029] Table 1 TUNEL detection solution components 2. Experimental results: (1) Hydroxygenkandrin can alleviate DOX-induced cardiotoxicity in cardiomyocytes The present application firstly conducted an MTT experiment to determine the killing effect of hydroxygenkwain in H9C2 cardiomyocytes. HGK (structural formula: Figure 1 The results showed that the growth of H9C2 was not affected by the increase of HGK concentration, indicating that HGK had no cytotoxicity or had low toxicity ( Figure 2 A). The applicant then conducted an MTT experiment to determine the combined killing effect of DOX and hydroxygenkwain in H9C2 cardiomyocytes. The results showed that DOX reduced the cell survival rate ( Figure 2 B), and the addition of HGK significantly reversed the cell death induced by DOX, indicating that HGK has a protective effect on DOX-induced cardiomyocyte injury. This application also detected the ROS level and cell apoptosis level in H9C2 cells. The results showed that DOX treatment increased the cellular ROS level ( Figure 2 C) and apoptotic cell ratio ( Figure 2 D), and the addition of HGK significantly reversed these adverse effects of DOX on cells ( Figure 2CE). These results demonstrate that HGK can alleviate DOX-induced cardiotoxicity in cardiomyocytes.

[0030] (2) Hydroxygenkandrin can alleviate DOX-induced cardiotoxicity in mice.

[0031] The present application studies the effect of hydroxygenkwain on DIC. To this end, the present application constructed a DIC model using C57BL / 6 male mice, such as Figure 3 As shown in A. As expected, the ratio of ventricular weight to tibia length in the DOX model group decreased significantly, while the ratio of ventricular weight to tibia length in the hydroxygenkwain combined group increased significantly, indicating that hydroxygenkwain alleviated DOX-induced cardiotoxicity ( Figure 3 C). Echocardiography showed ( Figure 3 B) When treated with DOX, the left ventricular contraction fraction (FS%) and ejection fraction (EF%) of mice were significantly reduced ( Figure 3 DE), indicating that the cardiac function of mice was significantly reduced; while the left ventricular contraction fraction (FS%) and ejection fraction (EF%) of mice in the hydroxygenkwain combined treatment group increased significantly. In addition, compared with the DOX group, the levels of myocardial injury markers (i.e., cTnT, CK-MB, and BNP) in the myocardial tissue homogenate of C57BL / 6 mice treated with hydroxygenkwain were also significantly reduced ( Figure 3 FH). In summary, hydroxygenkwain alleviated DOX-induced cardiotoxicity in mice.

[0032] (3) Histological analysis showed that hydroxygenkwain alleviated DOX-induced myocardial toxicity in mice The present application also found through histological analysis that H&E staining results showed that DOX enhanced myocardial cell inflammatory infiltration, while the addition of HGK alleviated this phenomenon ( Figure 4 A); MASSON staining results showed that the level of myocardial inflammation in DOX-treated myocardial tissue was significantly upregulated, and HGK could significantly downregulate the level of DOX-induced myocardial inflammation ( Figure 4 B); DOX induced an increase in the level of cardiomyocyte apoptosis, while HGK reduced this increase in apoptosis ( Figure 4 C); The cardiomyocytes of DIC mice shrank, and the administration of HGK also significantly restored the size of cardiomyocytes ( Figure 4 DE). The above results indicate that hydroxygenkwain can alleviate DOX-induced cardiotoxicity in mice.

Claims

1. Application of hydroxygenkwain in the preparation of drugs for preventing and inhibiting cardiotoxicity caused by anti-tumor chemotherapy drugs.

2. The use according to claim 1, characterized in that: The anti-tumor chemotherapy drug is one of doxorubicin, daunorubicin, aclarubicin, epirubicin, pirarubicin, idarubicin or mitoxantrone.

3. The use according to claim 1, characterized in that: The structural formula of hydroxygenkwain is as follows: 。 4. The use according to claim 1, characterized in that: The effective content of hydroxygenkwain in the drug is 0.5-60 μM.

5. The use according to claim 1, characterized in that: The medicine is a medicine mixture or a medicine composition.

6. The use according to claim 5, characterized in that: The drug mixture or drug composition comprises hydroxygenkwain and doxorubicin.

7. A drug mixture or a drug composition, characterized in that: The active ingredient includes hydroxygenkwain, and the drug mixture or drug composition has at least one of the following functions 1) to 4): 1) To prevent and / or treat doxorubicin-induced cardiotoxicity; 2) Alleviate myocardial tissue inflammation caused by doxorubicin; 3) Reduce the increase of reactive oxygen free radicals caused by doxorubicin; 4) Alleviate myocardial cell apoptosis induced by doxorubicin.

8. The pharmaceutical mixture or pharmaceutical composition according to claim 7, characterized in that: The drug mixture or drug composition is in any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions and solutions.

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