Use of dihydroquercetin for prevention and treatment of cardiac toxicity induced by arsenic trioxide

Abstract

The present application relates to a new use of Dihydroquercetin for preventing and treating cardiac toxicity induced by arsenic trioxide, and belongs to the technical field of medicine. In order to solve the problem that the prior art lacks effective treatment means for cardiac toxicity induced by ATO, the present application provides a new use of Dihydroquercetin for preventing and treating cardiac toxicity induced by arsenic trioxide, that is, the application of Dihydroquercetin in the preparation of a medicine for preventing and treating cardiac toxicity induced by arsenic trioxide. The present application proves through experiments that Dihydroquercetin can alleviate cardiac toxicity induced by ATO in multiple ways, including alleviating ATO-induced apoptosis, ferroptosis, oxidative stress, mitochondrial dysfunction and DNA damage. The present application finds that Dihydroquercetin has an excellent effect of preventing and treating cardiac toxicity induced by arsenic trioxide, provides a new strategy for improving the safety of arsenic trioxide, provides an embedding point for preparing a new medicine for preventing and treating cardiac toxicity induced by arsenic trioxide, and has an important clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a new use of taxifolin for preventing and treating cardiac toxicity induced by arsenic trioxide. BACKGROUND

[0002] Arsenic trioxide (ATO) can prolong the survival time of patients with acute promyelocytic leukemia (APL), but severe cardiac toxicity limits its clinical application. Research data shows that the incidence of cardiac discomfort in APL patients receiving ATO treatment is about 51.6%, the incidence of abnormal heart rate is about 47.4%, the incidence of abnormal electrocardiogram is about 61.1%, and the incidence of abnormal myocardial enzyme is about 2.1%. The incidence of treatment-related cardiac events is the highest in the 46-60 year-old patient group, reaching 84.2%. ATO can even cause serious arrhythmia, endangering the safety of patients.

[0003] ATO can act on various cells of the heart to cause cardiac toxicity, including affecting heart development, heart shape, heart contraction and dilation, and other cardiovascular functions. ATO causes changes in mitochondrial integrity, ROS production, calcium overload and apoptosis of myocardial cells in a dose-dependent and time-dependent manner, thereby producing cardiac toxicity. In addition, ATO can also cause severe endothelial dysfunction.

[0004] Currently, there is a lack of effective treatment for ATO-induced cardiac toxicity in clinical practice, and there is an urgent need in the art for a drug that can prevent and treat arsenic trioxide-induced cardiac toxicity to improve the safety of arsenic trioxide medication. SUMMARY

[0005] To solve the problem of the lack of effective treatment for ATO-induced cardiac toxicity in the prior art, the present application provides a new use of taxifolin (TAX) for preventing and treating arsenic trioxide-induced cardiac toxicity.

[0006] Technical scheme of the present application:

[0007] Application of taxifolin in the preparation of a drug for preventing and treating arsenic trioxide-induced cardiac toxicity.

[0008] Further, the drug for preventing and treating arsenic trioxide-induced cardiac toxicity contains taxifolin as the only active ingredient or one of the active ingredients.

[0009] Further, the content of taxifolin in the drug for preventing and treating arsenic trioxide-induced cardiac toxicity is 0.1wt% to 99wt%.

[0010] Further, the drug for preventing and treating arsenic trioxide-induced cardiotoxicity also comprises a pharmaceutically acceptable adjuvant and / or carrier.

[0011] Further, the drug for preventing and treating arsenic trioxide-induced cardiotoxicity is a gastrointestinal administration dosage form.

[0012] Further, the gastrointestinal administration dosage form comprises tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent preparations, emulsions, suspensions, syrups, drops or chewable tablets.

[0013] Further, the drug for preventing and treating arsenic trioxide-induced cardiotoxicity can alleviate the decrease in cell viability of myocardial cells and endothelial cells caused by arsenic trioxide and improve the cell morphology of myocardial cells and endothelial cells.

[0014] Further, the drug for preventing and treating arsenic trioxide-induced cardiotoxicity can alleviate the decrease in cell viability of myocardial cells and endothelial cells caused by arsenic trioxide and improve the cell morphology of myocardial cells and endothelial cells.

[0015] Further, the drug for preventing and treating arsenic trioxide-induced cardiotoxicity can alleviate the decrease in cell viability of myocardial cells and endothelial cells caused by arsenic trioxide and improve the cell morphology of myocardial cells and endothelial cells.

[0016] Further, the drug for preventing and treating arsenic trioxide-induced cardiotoxicity can alleviate the decrease in cell viability of myocardial cells and endothelial cells caused by arsenic trioxide and improve the cell morphology of myocardial cells and endothelial cells.

[0017] Advantages of the present application:

[0018] The present application proves by experiments that dihydroquercetin can alleviate arsenic trioxide-induced cardiotoxicity in multiple ways, including alleviating the decrease in cell viability of myocardial cells (AC16) and endothelial cells (HUVECs) caused by arsenic trioxide and significantly improving the cell morphology of myocardial cells and endothelial cells; dihydroquercetin alleviates the apoptosis of myocardial cells and endothelial cells induced by arsenic trioxide by inhibiting the activation of Bax / Bcl-2 and inhibits the ferroptosis of myocardial cells and endothelial cells by up-regulating GPX4 / xCT; dihydroquercetin significantly reduces oxidative stress damage by reducing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in myocardial cells and endothelial cells while increasing the activities of antioxidant enzymes glutathione (GSH) and superoxide dismutase (SOD), and also reverses the DNA damage and mitochondrial membrane potential decrease induced by arsenic trioxide.

[0019] This invention creatively discovers that dihydroquercetin has excellent effects in preventing and treating arsenic trioxide-induced cardiotoxicity, providing a new strategy for improving the safety of arsenic trioxide medication and a new entry point for preparing new drugs to prevent or treat arsenic trioxide-induced cardiotoxicity, which has very important clinical application prospects. Attached Figure Description

[0020] Figure 1 The image shows a comparison of the viability of AC16 cells in different treatment groups in Example 1. A represents the control group and the ATO treatment group, B represents the control group and the ATO+TAX treatment group, and C represents the control group and the TAX treatment group.

[0021] Figure 2 The image shows a comparison of HUVECs cell viability in different treatment groups in Example 1. A represents the control group and the ATO treatment group, B represents the control group and the ATO+TAX treatment group, and C represents the control group and the TAX treatment group.

[0022] Figure 3 This is a comparison of the morphology of AC16 cells in different treatment groups in Example 2. Scale bar = 100 μm.

[0023] Figure 4 This is a comparison of the cell morphology of HUVECs in different treatment groups in Example 2. Scale bar = 100 μm.

[0024] Figure 5 Representative fluorescence images of AC16 cells stained with TUNEL in different treatment groups in Example 3, scale bar = 50 μm;

[0025] Figure 6 Representative fluorescence images of HUVECs cells from different treatment groups in Example 3, stained with TUNEL. Scale bar = 50 μm.

[0026] Figure 7 This is a comparison of the number of TUNEL-positive cells in AC16 cells from different treatment groups in Example 3, based on quantitative analysis.

[0027] Figure 8 This is a comparison of the number of TUNEL-positive cells in HUVECs from different treatment groups in Example 3, based on quantitative analysis.

[0028] Figure 9 This is a comparison of the protein expression levels of Caspase-3, Bcl-2, and Bax in AC16 cells from different treatment groups in Example 4.

[0029] Figure 10 This is a comparison of the protein expression levels of Caspase-3, Bcl-2, and Bax in HUVECs cells from different treatment groups in Example 4.

[0030] Figure 11 Figure 6: Comparison of Caspase3 / β-actin, Bcl-2 / β-actin and Bax / β-actin immunoblot quantitative analysis in different treatment groups of HUVECs cells in Example 4;

[0031] Figure 12 Figure 6: Comparison of Caspase3 / β-actin, Bcl-2 / β-actin and Bax / β-actin immunoblot quantitative analysis in different treatment groups of HUVECs cells in Example 4;

[0032] Figure 13 Figure 7: Comparison of xCT and GPX4 protein expression levels in different treatment groups of AC16 cells in Example 5;

[0033] Figure 14 Figure 7: Comparison of xCT and GPX4 protein expression levels in different treatment groups of AC16 cells in Example 5;

[0034] Figure 15 Figure 7: Comparison of xCT and GPX4 protein expression levels in different treatment groups of AC16 cells in Example 5;

[0035] Figure 16 Figure 7: Comparison of xCT and GPX4 protein expression levels in different treatment groups of AC16 cells in Example 5;

[0036] Figure 17 Figure 8: Comparison of MDA, GSH and SOD levels in different treatment groups of AC16 cells in Example 6;

[0037] Figure 18 Figure 8: Comparison of MDA, GSH and SOD levels in different treatment groups of AC16 cells in Example 6;

[0038] Figure 19 Figure 9: Representative fluorescence images of ROS staining in different treatment groups of AC16 cells and HUVECs cells in Example 6;

[0039] Figure 20 Figure 10: Comparison of ROS fluorescence intensity in different treatment groups of AC16 cells in Example 6;

[0040] Figure 21 Figure 10: Comparison of ROS fluorescence intensity in different treatment groups of AC16 cells in Example 6;

[0041] Figure 22Representative immunofluorescence images of H2AX staining in AC16 cells of different treatment groups in Example 7, scale bar = 50 μm, red represents H2AX, and blue represents the nucleus;

[0042] Figure 23 Representative immunofluorescence images of H2AX staining in HUVECs cells of different treatment groups in Example 7, scale bar = 50 μm, red represents H2AX, and blue represents the nucleus;

[0043] Figure 24 Quantitative analysis comparison chart of H2AX fluorescence staining in AC16 cells of different treatment groups in Example 7;

[0044] Figure 25 Quantitative analysis comparison chart of H2AX fluorescence staining in HUVECs cells of different treatment groups in Example 7;

[0045] Figure 26 Representative fluorescence images of JC-1 staining in AC16 cells of different treatment groups in Example 8, scale bar = 50 μm;

[0046] Figure 27 Representative fluorescence images of JC-1 staining in HUVECs cells of different treatment groups in Example 8, scale bar = 50 μm;

[0047] Figure 28 Quantitative analysis comparison chart of relative fluorescence values of JC-1 staining in AC16 cells of different treatment groups in Example 8;

[0048] Figure 29 Quantitative analysis comparison chart of relative fluorescence values of JC-1 staining in HUVECs cells of different treatment groups in Example 8. DETAILED DESCRIPTION

[0049] The technical solutions of the present application are further described below in conjunction with the examples, but are not limited thereto, and any modification or equivalent replacement to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application shall be encompassed in the protection scope of the present application. The process equipment or device not specifically mentioned in the following examples is the conventional equipment or device in the art, and if not specifically indicated, the raw materials used in the examples of the present application are commercially available; if not specifically indicated, the technical means used in the examples of the present application are conventional means well known to those skilled in the art.

[0050] Sources of chemicals and reagents used in the examples of the present application:

[0051] Arsenic trioxide (ATO) was purchased from Harbin Medical Pharmaceutical Co., Ltd. of Heilongjiang Province, sodium arsenite chloride injection, concentration of 1 mg / mL.

[0052] Dihydroquercetin (TAX) (purity 99.97%) was purchased from MedChemExpress (MCE) (Monmouth Junction, NJ, USA).

[0053] MDA, GSH and SOD kits were purchased from Jiancheng Bioengineering Institute (Nanjing).

[0054] CCK-8 kit, ROS fluorescent detection kit, TUNEL detection kit and JC-1 mitochondrial membrane potential detection kit were all purchased from Biyun Tian (Shanghai).

[0055] The cardiomyocytes (AC16) and human umbilical vein endothelial cells (HUVECs) used in this example were from the myocardial ischemia laboratory of Harbin Medical University, China.

[0056] All data in this example were expressed as mean ± SD. All data were statistically analyzed and plotted using GraphPad Prism software version 9.0 (GraphPad Software, Inc., CA, USA). Comparison between two groups used unpaired t test, and comparison among multiple groups used one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant between groups.

[0057] Example 1

[0058] This example investigated the ability of dihydroquercetin TAX to improve the decrease in cell viability caused by ATO.

[0059] Both AC16 cells and HUVECs cells were cultured in high glucose DMEM with 10% fetal bovine serum, 1% penicillin / streptomycin (v / v). The incubator temperature was set at 37°C, and provided with 95% air and 5% carbon dioxide.

[0060] To determine the cytotoxicity of ATO and TAX on AC16 cells and HUVECs cells, CCK-8 assay was used to detect cell viability. According to the kit instructions, 5000 cells in each 100 μL cell suspension were seeded into a 96-well culture plate.

[0061] The AC16 cells were randomly divided into different groups:

[0062] (a) Control group: treated with culture medium for 24 h;

[0063] (b) TAX group: treated with 25, 50, 100 and 200 μM TAX for 24 h, respectively;

[0064] (c) ATO group: treated with 1.58, 3.16, 6.32, 9.46, 12.64 and 15.8 μM ATO for 24 h, respectively;

[0065] (d)ATO+TAX group: 5 μM ATO was incubated for 24 h after 25, 50, 100 and 200 μM TAX were pretreated for 12 h, respectively.

[0066] The HUVECs cells were randomly divided into different groups:

[0067] (e)Control group: incubated with medium for 24 h;

[0068] (f)TAX group: incubated with 25, 50, 100 and 200 μM TAX for 24 h, respectively;

[0069] (g)ATO group: incubated with 1.58, 3.16, 6.32, 9.46, 12.64 and 15.8 μM ATO for 24 h, respectively;

[0070] (h)ATO+TAX group: 5 μM ATO was incubated for 24 h after 25, 50, 100 and 200 μM TAX were pretreated for 12 h, respectively.

[0071] After treatment, 10 μL CCK-8 solution was used to incubate the cells. Incubation was carried out at 37 °C for 1 h, and light was avoided as much as possible during the whole operation process.

[0072] Bubbles should be avoided during the operation process, otherwise the optical density value (OD) will be affected.

[0073] The absorbance value at 450 nm was measured by using a microplate reader (BioTek, Vermont, USA) to investigate the effect of ATO and TAX on the viability of AC16 and HUVECs cells, and the results are shown in Figure 1 and Figure 2 The data in the figure are the mean ± SD of each group. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3.

[0074] As shown in Figure 1 -A and Figure 2 -A, the viability of AC16 cells and HUVECs cells decreased in a dose-dependent manner after 24 h of ATO treatment, and the IC50 values were 5.19 μM and 8.26 μM, respectively. As shown in Figure 1 -B and Figure 2 -B, 50 μM concentration of TAX can significantly alleviate the decrease in the viability of HUVECs cells induced by ATO, but it has no obvious improvement on the viability of AC16 cells. However, 100 μM concentration of TAX can significantly improve the viability of AC16 cells. As shown in Figure 1 -C and Figure 2 -C, the viability of HUVECs and AC16 cells did not decrease significantly when treated with 100 μM concentration of TAX alone.

[0075] This indicates that ATO induces the decrease of AC16 cell, HUVECs cell viability in a dose-dependent manner. TAX improves the adverse effect of ATO on AC16 cell, HUVECs cell viability in a dose-dependent manner. TAX (25-200 μM) alone has no cytotoxic effect on AC16 cell, HUVECs.

[0076] Example 2

[0077] This example investigates the ability of TAX to improve the cell morphology change caused by ATO.

[0078] This example further observes the cell morphology of AC16, HUVECs cells exposed to ATO and TAX using brightfield inverted microscope.

[0079] AC16 cells were randomly divided into different groups:

[0080] (a) Control group: treated with medium for 24 h;

[0081] (b) TAX group: treated with 100 μM TAX for 24 h;

[0082] (c) ATO group: treated with 5 μM ATO for 24 h;

[0083] (d) ATO+TAX group: pretreated with 100 μM TAX for 12 h, then incubated with 5 μM ATO for 24 h.

[0084] HUVECs cells were randomly divided into different groups:

[0085] (e) Control group: treated with medium for 24 h;

[0086] (f) TAX group: treated with 100 μM TAX for 24 h;

[0087] (g) ATO group: treated with 8 μM ATO for 24 h;

[0088] (h) ATO+TAX group: pretreated with 100 μM TAX for 12 h, then incubated with 8 μM ATO for 24 h.

[0089] Results are shown in Figure 3 and Figure 4 , AC16 and HUVECs cells in TAX group alone have no change in cell morphology compared with control group; both AC16 and HUVECs cells exposed to ATO have significant morphological changes, with smaller cell volume, deformation, even shrinkage, rounding and then shedding; the cell morphology is significantly improved after the combined use of TAX. This indicates that arsenic exposure can cause cell morphology changes, and TAX can improve the changes in cell morphology.

[0090] Example 3

[0091] This embodiment investigated the ability of dihydroquercetin (TAX) to improve ATO-induced cell death.

[0092] Deoxynucleotide terminal transferase-mediated dUTP nick-end labeling (TUNEL) is a method for detecting DNA fragmentation during cell death. This example uses a TUNEL kit to label fragmented DNA and then detect cell death. After TUNEL staining, the nuclear morphology of AC16 cells and HUVECs was examined to confirm the effects of ATO and TAX on cell death.

[0093] AC16 cells were randomly divided into different groups:

[0094] (a) Control group: culture medium treatment for 24 h;

[0095] (b) TAX group: treated with 100 μM TAX for 24 h;

[0096] (c) ATO group: treated with 5 μM ATO for 24 h;

[0097] (d) ATO+TAX group: Pretreated with 100 μM TAX for 12 h, then incubated with 5 μM ATO for 24 h.

[0098] HUVECs cells were randomly divided into different groups:

[0099] (e) Control group: culture medium treatment for 24 h;

[0100] (f) TAX group: treated with 100 μM TAX for 24 h;

[0101] (g) ATO group: treated with 8 μATO for 24 h;

[0102] (h) ATO+TAX group: Pretreated with 100 μM TAX for 12 h, then incubated with 8 μM ATO for 24 h.

[0103] After drug treatment, the cells were washed twice with PBS, then 100 μL of TUNEL assay solution was added and incubated at 37°C in the dark for 60 min. The cells were then washed three times with PBS, stained with DAPI for 5 min, and observed under a fluorescence microscope.

[0104] Figure 5 and Figure 6 These are representative fluorescence images of AC16 cells and HUVECs cells from different treatment groups in this embodiment, stained with TUNEL. Figure 7 and Figure 8The bar graphs show the number of TUNEL-positive cells in different groups of AC16 cells and HUVECs cells, respectively. The values are the mean ± SD of each group. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3.

[0105] Results are shown in Figures 5 to 8 Compared with the control group, the number of TUNEL-positive cells in the ATO group was significantly increased (P < 0.001), and a large number of apoptotic cells were found in the ATO-treated cells. Compared with the ATO group, the number of TUNEL-positive cells in the ATO+TAX group was significantly reduced, showing a strong therapeutic effect (P < 0.001 or P < 0.01). In addition, compared with the control group, the number of TUNEL-positive cells in the TAX group alone did not change. This indicates that ATO induces cell apoptosis, while TAX can alleviate ATO-induced cell death.

[0106] Example 4

[0107] This example investigates the ability of dihydroquercetin TAX to improve apoptosis caused by ATO exposure.

[0108] Apoptosis is an important process of ATO-induced cardiotoxicity, and ATO activates apoptosis through multiple mechanisms. In this example, Western blot was used to detect the expression levels of proteins related to the mitochondrial apoptosis pathway.

[0109] AC16 cells were randomly divided into different groups:

[0110] (a) Control group: treated with medium for 24 h;

[0111] (b) TAX group: treated with 100 μM TAX for 24 h;

[0112] (c) ATO group: treated with 5 μM ATO for 24 h;

[0113] (d) ATO+TAX group: pretreated with 100 μM TAX for 12 h, and then incubated with 5 μM ATO for 24 h.

[0114] HUVECs cells were randomly divided into different groups:

[0115] (e) Control group: treated with medium for 24 h;

[0116] (f) TAX group: treated with 100 μM TAX for 24 h;

[0117] (g) ATO group: treated with 8 μM ATO for 24 h;

[0118] (h) ATO+TAX group: pre-treated with 100 mM TAX for 12 h, and then incubated with 8 mM ATO for 24 h.

[0119] AC16 cells and HUVECs cells in each treatment group were lysed in protease inhibitor and RIPA lysis buffer. After determining the protein concentration by BCA kit, the protein samples were denatured in 5x loading buffer. The protein samples were added to 10% SDS-PAGE, and then transferred to PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 h, and then incubated with the following primary antibodies at 4°C overnight: Bax (1: 1000, abways), Bcl-2 (1: 1000, abways), Caspase-3 (1: 1000, abways) and β-actin (1:2000, Affinity Biosciences). After incubation with the primary antibodies, the membrane was washed with PBST for 3 times, and then incubated with the corresponding secondary antibodies (1:2000, Invitrogen) for 1 h. The PVDF membrane was scanned by gel imaging system, and the protein bands were quantitatively analyzed by Image J software. β-actin was used as the internal reference.

[0120] Figure 9 and Figure 10 are the comparison diagrams of the protein expression levels of Caspase-3, Bcl-2 and Bax in AC16 cells and HUVECs cells in different treatment groups in this example, respectively; Figure 11 and Figure 12 are the comparison diagrams of the immunoblotting quantitative analysis of Caspase3 / β-actin, Bcl-2 / β-actin and Bax / β-actin in AC16 cells and HUVECs cells in different treatment groups in this example, respectively; the values are the average value ± SD of each group. *p<0.05, **p<0.01, ***p<0.001, n>3.

[0121] The results are shown in Figures 9 to 12 ATO significantly reduced the expression of anti-apoptotic protein Bcl-2 in both AC16 and HUVECs cells (P<0.001), while up-regulated the expression of pro-apoptotic proteins Bax and Caspase-3 (P<0.001). Compared with the ATO group, the expression of Bcl-2 was significantly up-regulated in the ATO+TAX group (P<0.001), and the levels of Bax and Caspase-3 were significantly down-regulated (P<0.001). This indicates that ATO activates apoptosis, while TAX can alleviate the apoptosis induced by ATO.

[0122] Example 5

[0123] This example investigates the improvement ability of dihydroquercetin TAX on the ferroptosis caused by ATO exposure.

[0124] Ferroptosis is a form of non-apoptotic, non-necrotic cell death. Studies have shown that ferroptosis is related to the occurrence and development of various cardiovascular diseases, including drug-induced cardiotoxicity, myocardial ischemia / reperfusion injury, heart failure, heart transplantation, etc. In order to explore the role of ferroptosis in ATO-induced toxicity of different cardiac cells, Western blot was used to detect the effect of ATO on the expression level of ferroptosis-related proteins in different cells.

[0125] AC16 cells were randomly divided into different groups:

[0126] (a) Control group: treated with culture medium for 24 h;

[0127] (b) TAX group: treated with 100 μM TAX for 24 h;

[0128] (c) ATO group: treated with 5 μM ATO for 24 h;

[0129] (d) ATO+TAX group: pretreated with 100 μM TAX for 12 h, and then incubated with 5 μM ATO for 24 h.

[0130] HUVECs cells were randomly divided into different groups:

[0131] (e) Control group: treated with culture medium for 24 h;

[0132] (f) TAX group: treated with 100 μM TAX for 24 h;

[0133] (g) ATO group: treated with 8 μM ATO for 24 h;

[0134] (h) ATO+TAX group: pretreated with 100 μM TAX for 12 h, and then incubated with 8 μM ATO for 24 h.

[0135] AC16 cells and HUVECs cells were lysed in protease inhibitor and RIPA lysis buffer. After determining the protein concentration by BCA kit, the protein samples were denatured in 5x loading buffer. The protein samples were added to 10% SDS-PAGE, and then transferred to PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 h, and then incubated with the following primary antibodies at 4°C overnight: GPX4 (1:1000, abways), xCT (1:1000, abways), and β-actin (1:2000, Affinity Biosciences). After incubation with the primary antibodies, the membrane was washed with PBST for 3 times, and then incubated with the corresponding secondary antibodies (1:2000, Invitrogen) for 1 h. The PVDF membrane was scanned by gel imaging system, and the protein bands were quantitatively analyzed by Image J software. β-actin was used as an internal reference.

[0136] Figure 13 and Figure 14 are the comparison charts of protein expression levels of xCT and GPX4 in AC16 cells and HUVECs cells in different treatment groups in this example, respectively; Figure 15 and Figure 16 are the comparison charts of immunoblotting quantitative analysis of xCT / β-actin and GPX4 / β-actin in AC16 cells and HUVECs cells in different treatment groups in this example, respectively; the numerical value is the average value ± SD of each group. *p<0.05, **p<0.01, ***p<0.001, n>3.

[0137] The results are shown in Figures 13 to 16 ATO significantly reduced the expression levels of GPX4 and xCT in AC16 and HUVECs cells (P<0.001 or P<0.01). This indicates that ATO can cause ferroptosis in the two types of cells. Compared with the ATO group, the expression levels of GPX4 and xCT in different cells in the ATO+TAX group were significantly increased (P<0.5 or P<0.001). This indicates that ATO activates ferroptosis, and TAX can significantly alleviate the ferroptosis induced by ATO.

[0138] Example 6

[0139] This example investigates the improvement ability of dihydroquercetin TAX on oxidative stress damage caused by ATO exposure.

[0140] Arsenic-induced oxidative stress plays a central role in cardiac injury. GSH, SOD, MDA, and ROS are closely related to oxidative stress response and can reflect the level of oxidative stress.

[0141] AC16 cells were randomly divided into different groups:

[0142] (a) Control group: treated with culture medium for 24 h;

[0143] (b) TAX group: treated with 100 μM TAX for 24 h;

[0144] (c) ATO group: treated with 5 μM ATO for 24 h;

[0145] (d) ATO+TAX group: pretreated with 100 μM TAX for 12 h, and then incubated with 5 μM ATO for 24 h.

[0146] HUVECs cells were randomly divided into different groups:

[0147] (e) Control group: treated with culture medium for 24 h;

[0148] (f) TAX group: treated with 100 μM TAX for 24 h;

[0149] (g) ATO group: treated with 8 mM ATO for 24 h;

[0150] (h) ATO+TAX group: pretreated with 100 mM TAX for 12 h, then incubated with 8 mM ATO for 24 h.

[0151] In this example, the levels of ROS, MDA, GSH and SOD in cells were determined by spectrophotometry using a kit. According to the kit instructions, the level of ROS in cells was detected by adding DCFH-DA 10 mM to the cells after washing with PBS, incubating at 37°C for 30 min in the dark, and observing under a fluorescence microscope.

[0152] Figure 17 and Figure 18 are the comparison charts of the levels of MDA, GSH and SOD in AC16 cells and HUVECs in different treatment groups in this example; Figure 19 is a representative fluorescence image of ROS staining in AC16 cells and HUVECs in different treatment groups in Example 6; Figure 20 and Figure 21 are the comparison charts of the fluorescence intensity of ROS in AC16 cells in different treatment groups in this example. The numerical values are the mean ± SD of each group. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3.

[0153] The results are shown in Figures 17 to 21 Compared with the control group, the level of MDA in the ATO group was significantly increased (P < 0.001), and the levels of GSH and SOD were significantly decreased (P < 0.001) in both types of cells. Compared with the ATO group, the combined group could significantly reduce the level of MDA (P < 0.001), and increase the activity of GSH and SOD (P < 0.001 or P < 0.01). At the same time, the level of ROS in cells was evaluated by fluorescence staining, and the intensity of green fluorescence image represented the level of ROS. Compared with the control group, the cells in the ATO group showed obvious green fluorescence, and the fluorescence area was larger and brighter, i.e. the level of ROS was significantly increased (P < 0.001). TAX could significantly reduce the fluorescence intensity, suggesting a decrease in ROS level (P < 0.001). The above results showed that TAX could alleviate the oxidative stress damage induced by ATO by reducing the levels of ROS and MDA, and up-regulating the activity of GSH and SOD.

[0154] Example 7

[0155] This example investigated the ability of dihydroquercetin TAX to improve DNA damage caused by ATO exposure.

[0156] ATO, as a clastogenic compound, can induce DNA damage or mutation, mitotic arrest and apoptosis by changing mitochondrial membrane potential. γ-H2AX is a marker of DNA double-strand breaks in myocardium. Therefore, the expression of γ-H2AX was evaluated by immunofluorescence analysis to assess DNA damage in this example.

[0157] AC16 cells were randomly divided into different groups:

[0158] (a) Control group: treated with medium for 24 h;

[0159] (b) TAX group: treated with 100 μM TAX for 24 h;

[0160] (c) ATO group: treated with 5 μM ATO for 24 h;

[0161] (d) ATO+TAX group: pretreated with 100 μM TAX for 12 h, and then incubated with 5 μM ATO for 24 h.

[0162] HUVECs cells were randomly divided into different groups:

[0163] (e) Control group: treated with medium for 24 h;

[0164] (f) TAX group: treated with 100 μM TAX for 24 h;

[0165] (g) ATO group: treated with 8 μM ATO for 24 h;

[0166] (h) ATO+TAX group: pretreated with 100 μM TAX for 12 h, and then incubated with 8 μM ATO for 24 h.

[0167] The expression of DNA damage marker γ-H2AX in cells was detected by immunofluorescence analysis. Cells treated with drugs were washed with PBS at 37 °C for 1 min, fixed with 4% formaldehyde solution for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with goat serum for 1 h. Then, the cells were incubated with γ-H2AX primary antibody (1:200, a11097, ABclonal, USA) at 4 °C for 12 h. After PBST washing, the cells were incubated with Alexa Fluor 647-labeled goat anti-rabbit IgG secondary antibody (1:200, A0468) at room temperature for 1 h, and stained with DAPI to label the cell nucleus. Image acquisition was performed using a fluorescence microscope.

[0168] Figure 22 and Figure 23 are representative immunofluorescence images of H2AX staining of AC16 cells and HUVECs cells in different treatment groups in this example, respectively, scale bar = 50 μm, red represents H2AX, and blue represents the cell nucleus.Figure 24 and Figure 25 Figure 4 is a graph showing the quantification of H2AX fluorescence staining in AC16 cells and HUVECs cells treated with different treatments in this example; the values are the mean ± SD of each group. *p<0.05, **p<0.01, ***p<0.001, n=3.

[0169] Results are shown in Figure 4. Figures 22 to 25 Compared with the control group, ATO significantly increased the expression of H2AX in AC16 and HUVECs cells (P<0.001). Compared with ATO, ATO+TAX group significantly reduced the expression of H2AX (P<0.01). This indicates that ATO significantly increases the expression of H2AX, and TAX can inhibit the DNA damage induced by ATO.

[0170] Example 8

[0171] This example investigates the ability of dihydroquercetin TAX to improve mitochondrial membrane dysfunction caused by ATO exposure.

[0172] Mitochondrial membrane potential (MMP) is a sensitive parameter for detecting mitochondrial dysfunction, and the maintenance of MMP is crucial for cell survival. In this example, the mitochondrial membrane potential detection kit (JC-1) was used to detect MMP.

[0173] AC16 cells were randomly divided into different groups:

[0174] (a) Control group: treated with culture medium for 24h;

[0175] (b) TAX group: treated with 100 μM TAX for 24h;

[0176] (c) ATO group: treated with 5 μM ATO for 24h;

[0177] (d) ATO+TAX group: first pretreated with 100 μM TAX for 12h, then incubated with 5 μM ATO for 24h.

[0178] HUVECs cells were randomly divided into different groups:

[0179] (e) Control group: treated with culture medium for 24h;

[0180] (f) TAX group: treated with 100 μM TAX for 24h;

[0181] (g) ATO group: treated with 8 μM ATO for 24h;

[0182] (h) ATO+TAX group: first pretreated with 100 μM TAX for 12h, then incubated with 8 μM ATO for 24h.

[0183] After drug treatment, cells were washed twice with PBS, then incubated with JC-1 working solution at 37℃ for 30 min in the dark, washed twice with JC-1 buffer, and photographed using a fluorescence microscope. A decrease in the relative fluorescence (red / green fluorescence intensity ratio) level indicates a decrease in MMP.

[0184] Figure 26 and Figure 27 are representative fluorescence images of JC-1 staining of AC16 cells and HUVECs cells in different treatment groups in this example, scale bar = 50 μm. Figure 28 and Figure 29 are comparative graphs of the quantitative analysis of the relative fluorescence values of JC-1 staining in AC16 cells and HUVECs cells in different treatment groups in this example; the numerical values are the mean ± SD of each group. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3.

[0185] The results are shown in Figures 26 to 29 Compared with the control group, the ATO group significantly reduced the red / green fluorescence intensity ratio in AC16 and HUVECs cells (P < 0.001), indicating that ATO significantly reduced MMP; after pretreatment with TAX, the ATO-induced decrease in MMP was significantly alleviated (P < 0.001 or P < 0.05). This indicates that ATO significantly reduced MMP, and TAX can alleviate the ATO-induced decrease in MMP.

Claims

1. Use of dihydroquercetin for the preparation of a medicament for the prevention and treatment of cardiac toxicity induced by arsenic trioxide, characterized in that, The content of dihydroquercetin in the medicine for preventing and treating cardiac toxicity induced by arsenic trioxide is 0.1wt%-99wt%.

2. Use of Dihydroquercetin according to claim 1 for the preparation of a medicament for the prevention and treatment of cardiac toxicity induced by arsenic trioxide, characterized in that, The medicine for preventing and treating cardiac toxicity induced by arsenic trioxide takes dihydroquercetin as the only active ingredient.

3. Use of Dihydroquercetin according to claim 2 for the preparation of a medicament for the prevention and treatment of cardiac toxicity induced by arsenic trioxide, characterized in that, The medicine for preventing and treating cardiac toxicity induced by arsenic trioxide further comprises pharmaceutically acceptable adjuvants and / or carriers.

4. Use of Dihydroquercetin according to claim 3 for the preparation of a medicament for the prevention and treatment of cardiac toxicity induced by arsenic trioxide, characterized in that, The medicine for preventing and treating cardiac toxicity induced by arsenic trioxide is a gastrointestinal administration dosage form.

5. Use of Dihydroquercetin according to claim 4 for the preparation of a medicament for the prevention and treatment of cardiac toxicity induced by arsenic trioxide, characterized in that, The gastrointestinal administration dosage form comprises tablets, granules, capsules, solutions, powders, sustained-release preparations, effervescent preparations, emulsions, suspensions, syrups, drops or chewable preparations.

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