Application of acacetin and coenzyme Q10 composition in medicine for preventing and treating adriamycin-induced cardiotoxicity

Abstract

The invention provides an application of an acacetin and coenzyme Q10 composition in a medicine for preventing doxorubicin-induced cardiotoxicity, and in doxorubicin-induced cardiotoxicity diseases, the acacetin and coenzyme Q10 composition can improve heart function indexes; myocardial cell apoptosis is inhibited, and myocardial tissue pathological changes are improved; according to the present invention, with the application of the traditional Chinese medicine composition, the effects of mitochondrial energy metabolism improving, active oxygen removing, Na < + >, K < + >-ATPase activity increasing and ATP content increasing can be provided, such that the impaired cardiac function improving effect can be provided, the new treatment strategy can be provided for the prevention and treatment of the adriamycin-induced cardiotoxic disease, and the good application prospect can be provided.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology. Specifically, it relates to the application of a composition of acacetin and coenzyme Q10 in a medicament for preventing adriamycin-induced myocardial toxicity. Background Art

[0002] Adriamycin (Dox) is an effective drug widely used in cancer chemotherapy, but it can cause serious cardiotoxic side effects. Acacetin (5,7-dihydroxy-4'-methoxyflavone) is widely present in various natural plants and has antioxidant, anti-inflammatory and anti-proliferative effects. However, due to the problem of low bioavailability, the concentration of acacetin in plasma and target tissues is usually low.

[0003] Coenzyme Q10 (or ubiquinone or ubiquinol) is an endogenous lipophilic substance present in many eukaryotic cells (mainly in mitochondria). As a key component of the mitochondrial electron transport chain, it can scavenge adriamycin-induced reactive oxygen species (ROS), maintain mitochondrial membrane potential, participate in the redox reaction of the electron transport chain, and produce ATP to increase cellular energy metabolism.

[0004] Acacetin provides an additional protective mechanism by regulating the expression of endogenous antioxidant enzymes such as SOD (superoxide dismutase), LDH (lactate dehydrogenase) and MDA (malondialdehyde) (see Acacetin Protects MyocardialCells against Hypoxia-Reoxygenation Injury throughActivation of Autophagy. JImmunol Res. 2021;2021:9979843. Published 2021 Jun 29. doi:10.1155 / 2021 / 9979843). This regulatory effect forms a second layer of defense barrier, which helps to reduce myocardial cell injury. The combination of the two not only helps to improve cardiac function, but also enhances the protective effect against adriamycin-induced myocardial toxicity. At present, there is no relevant report on the application of this composition in this field. Summary of the Invention

[0005] In view of this, the present invention proposes the application of a composition of acacetin and coenzyme Q10 in a medicament for preventing adriamycin-induced myocardial toxicity, so as to provide a new protective therapeutic drug for preventing and treating adriamycin-induced myocardial toxicity diseases. The present invention discloses the application of a composition of acacetin and coenzyme Q10 in a medicament for preventing adriamycin-induced myocardial toxicity.

[0006] Preferably, pretreatment administration of the composition of acacetin and coenzyme Q10 is carried out before adriamycin administration.

[0007] In this embodiment, the acacetin and coenzyme Q10 composition can achieve at least one of the following effects: improving the cardiac function indexes of adriamycin-induced myocardial toxicity; reducing adriamycin-induced myocardial injury; inhibiting cardiomyocyte apoptosis; enhancing cardiomyocyte energy metabolism; mediating the expression of proteins related to mitochondrial energy metabolism in cardiomyocytes.

[0008] Preferably, the acacetin and coenzyme Q10 composition can achieve at least one of the following effects: increasing the left ventricular ejection fraction and shortening rate; regulating the expression of cardiomyocyte apoptosis-related proteins Bcl-2, Bax, and Cleaved Caspase3; scavenging reactive oxygen species and increasing the Na + , K + -ATPase activity and ATP content in damaged cardiomyocytes; enhancing the expression of Mfn1 and Mfn2 proteins and reducing the expression of p-Drp protein.

[0009] Preferably, in the acacetin and coenzyme Q10 composition, the mass ratio of the two is (1-2):(1-2), and the administration doses of acacetin and coenzyme Q10 are 5-15 mg / kg and 5-15 mg / kg, respectively.

[0010] Preferably, in the acacetin and coenzyme Q10 composition, the mass ratio of the two is 1:1, and the administration doses of acacetin and coenzyme Q10 are both 5 mg / kg.

[0011] The present invention also discloses a drug for preventing adriamycin-induced myocardial toxicity, which uses the acacetin and coenzyme Q10 composition as the main ingredient.

[0012] The application of the acacetin and coenzyme Q10 composition provided by the present invention in the drug for preventing adriamycin-induced myocardial toxicity is to pre-administer a preset dose of the acacetin and coenzyme Q10 composition to rats by gavage. After a period of time, adriamycin is intraperitoneally injected into the rats to induce myocardial toxicity. It is found that the acacetin and coenzyme Q10 composition can significantly improve the cardiac function indexes; inhibit cardiomyocyte apoptosis and improve the histopathological changes of myocardial tissue; improve the energy metabolism of mitochondria, reduce the level of reactive oxygen species (ROS), and increase the Na + , K + -ATPase activity and ATP content, thereby playing a protective effect on improving the damaged heart function, providing a new treatment strategy for preventing and treating adriamycin-induced myocardial toxicity diseases, and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1HE staining results of rat heart tissue sections of the control group (rats that only received saline gavage and did not receive doxorubicin treatment), Dox (doxorubicin)-induced injury model group, acacetin pretreatment administration group, coenzyme Q10 pretreatment administration group, and acacetin + coenzyme Q10 pretreatment administration group in the embodiments of the present invention (magnification factors are 200x and 400x respectively); Figure 2 In the experiment of investigating the ability of the control group, Dox-induced group, acacetin pretreatment administration group, coenzyme Q10 pretreatment administration group, and acacetin and coenzyme Q10 combination pretreatment administration group to inhibit cardiomyocyte apoptosis in the embodiments of the present invention, the graphs of detecting cardiomyocyte apoptosis in H9C2 cells by TUNEL technology and flow cytometry; Figure 3 In the experiment of investigating the ability of the control group, Dox-induced group, acacetin pretreatment administration group, coenzyme Q10 pretreatment administration group, and acacetin and coenzyme Q10 combination pretreatment administration group to inhibit cardiomyocyte apoptosis in the embodiments of the present invention, the graphs of detecting the protein expressions of Bcl-2, Bax, and Cleaved Caspase3 by Western blot technology; Figure 4 In the experiment of investigating the ability of the control group, Dox-induced group, acacetin pretreatment administration group, coenzyme Q10 pretreatment administration group, and acacetin and coenzyme Q10 combination pretreatment administration group to inhibit cardiomyocyte apoptosis in the embodiments of the present invention, the bar graphs of the relative expressions of Bcl-2 / β-actin, Bax / β-actin, and Cleaved Caspase3 / β-actin; Figure 5 In the embodiments of the present invention, graphs of detecting the expression levels of ROS (reactive oxygen species) in H9C2 cardiomyocytes of the control group, Dox-induced injury model group, acacetin pretreatment administration group, coenzyme Q10 pretreatment administration group, and acacetin and coenzyme Q10 combination pretreatment administration group by fluorescence detection, where: A represents the bar graph of fluorescence intensity of different pretreatment administration groups; B represents the fluorescence graph of detecting ROS in H9C2 cardiomyocytes by fluorescence; C represents the bar graph of the influence of Dox induction on the ultra-trace ATP content in H9C2 cells; D represents the bar graph of the influence of Dox induction on the activity of + Na + -ATPase in H9C2 cells; Figure 6This is a diagram showing the effects of the control group, Dox-induced injury model group, acacetin pretreatment administration group, coenzyme Q10 pretreatment administration group, and acacetin and coenzyme Q10 combination pretreatment administration group on the expression of proteins related to mitochondrial energy metabolism in cardiomyocytes in the embodiments of the present invention. Among them: A represents the expression diagrams of mitochondrial energy metabolism Mfn1, Mfn2, and p-Drp protein blots in cardiomyocytes after treatment with each pretreatment administration group; B-D represent the bar graphs of the relative expression levels of Mfn1 / β-actin, Mfn2 / β-actin, and p-Drp / β-actin. Detailed implementation manners

[0014] The present invention provides an application of a composition of acacetin and coenzyme Q10 in a medicament for preventing and treating adriamycin-induced cardiotoxicity. In the present invention, no special limitation is imposed on the source of the acacetin. In the specific embodiments of the present invention, the acacetin is a commercially available acacetin with an HPLC content determination value exceeding 95%.

[0015] Coenzyme Q10 is purchased from Shenzhou Biology&Technology Co.,Ltd or Kaneka Nutrients.

[0016] In the composition of acacetin and coenzyme Q10, the mass ratio of the two is (1-2):(1-2), and the administration doses of acacetin and coenzyme Q10 are 5-15 mg / kg and 5-15 mg / kg respectively.

[0017] Preferably, the mass ratio of acacetin to coenzyme Q10 in the composition of acacetin and coenzyme Q10 is 1:1, and the administration doses of acacetin and coenzyme Q10 are both 5 mg / kg.

[0018] In the embodiments of the present invention, the cardiotoxicity is caused by adriamycin.

[0019] In the embodiments of the present invention, pretreatment with a composition of acacetin and coenzyme Q10 (administering a set dose of the composition of acacetin and coenzyme Q10 before adriamycin administration) can improve the cardiac function indexes of adriamycin-induced cardiotoxicity and can reduce adriamycin-induced myocardial injury. The present invention uses the results of HE staining to evaluate the recovery of cardiomyocytes after pretreatment of rats with a composition of acacetin and coenzyme Q10.

[0020] Preferably, the composition of acacetin and coenzyme Q10 can increase the left ventricular ejection fraction and shortening rate.

[0021] Pretreatment with a composition of acacetin and coenzyme Q10 can inhibit cardiomyocyte apoptosis. The present invention detects and evaluates cardiomyocyte viability by TUNEL and flow cytometry methods.

[0022] Preferably, the acacetin and coenzyme Q10 composition can regulate the expression of apoptosis-related proteins Bcl-2, Bax, and Cleaved Caspase3 in cardiomyocytes.

[0023] Pretreatment with the acacetin and coenzyme Q10 composition can enhance the energy metabolism of cardiomyocytes. In the present invention, fluorescence detection is used to evaluate the effects of the acacetin and coenzyme Q10 composition on reactive oxygen species damage and energy metabolism in cardiomyocytes.

[0024] Preferably, the acacetin and coenzyme Q10 composition has the ability to scavenge reactive oxygen species, and can increase the Na + , K + -ATPase activity and ATP content in damaged cardiomyocytes.

[0025] Pretreatment with the acacetin and coenzyme Q10 composition can mediate the expression of proteins related to mitochondrial energy metabolism in cardiomyocytes. In the present invention, Western Blot is used to detect the expression levels of Mfn1 protein, p-Drp, and Mfn2 protein to evaluate the effects of the acacetin and coenzyme Q10 composition on the expression of proteins related to mitochondrial energy metabolism in cardiomyocytes.

[0026] Preferably, the acacetin and coenzyme Q10 composition can enhance the expression of Mfn1 protein and Mfn2 protein, and reduce the expression of p-Drp protein.

[0027] In the acacetin and coenzyme Q10 composition of the present invention, although acacetin has the problem of low bioavailability, the lipophilicity of coenzyme Q10 can promote its transmembrane transport, and the anti-inflammatory effect of acacetin can reduce the degradation of coenzyme Q10 in the inflammatory microenvironment, forming a delivery synergy. Therefore, the two can cooperate to reduce the accumulation of ROS in mitochondria, inhibit lipid peroxidation reactions, and further promote mitochondrial energy metabolism.

[0028] The present invention also provides a drug for preventing and treating adriamycin-induced myocardial toxicity, with the acacetin and coenzyme Q10 composition as the main ingredient.

[0029] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0030] 1. Experimental methods 1.1 Animal experiments Sixty healthy 3-month-old adult male Sprague-Dawley rats, weighing approximately 200-230 grams, were purchased from the Animal Experiment Center of Zhejiang University in April 2023 (Animal Experiment Certificate No. 20230412Abzz0100999573). From April 2023 to October 2023, they were housed in the Animal Laboratory of Taizhou Hospital of Zhejiang Province at a temperature of 22±2°C and a humidity of 50±10%. All experiments were carried out in accordance with the relevant regulations on the management of experimental animals and were approved by the Experimental Ethics Committee (TZY-2022097).

[0031] 1.1.1 Grouping of experimental animals The experiment was divided into a control group, a Dox-induced injury model group, an acacetin group, a coenzyme Q10 group, and an acacetin and coenzyme Q10 combination group, with 6 animals in each experimental group, among which: Control group: The rats were raised under normal breeding conditions without any drug treatment; Dox-induced injury model group: The rats were raised under normal breeding conditions for 12 days, and then a myocardial toxicity model was established by intraperitoneal injection of 7.5 mg / kg Dox on the 14th and 16th days; Acacetin 5 mg / kg pretreatment administration group: The rats were given 5 mg / kg acacetin by gavage every day for 12 consecutive days, and then 7.5 mg / kg Dox was intraperitoneally injected on the 14th and 16th days to induce myocardial toxicity; Acacetin 10 mg / kg pretreatment administration group: The rats were given 10 mg / kg acacetin by gavage every day for 12 consecutive days, and then 7.5 mg / kg Dox was intraperitoneally injected on the 14th and 16th days to induce myocardial toxicity; Acacetin 15 mg / kg pretreatment administration group: The rats were given 15 mg / kg acacetin by gavage every day for 12 consecutive days, and then 7.5 mg / kg Dox was intraperitoneally injected on the 14th and 16th days to induce myocardial toxicity; Coenzyme Q10 5 mg / kg pretreatment administration group: The rats were given 5 mg / kg coenzyme Q10 by gavage every day for 12 consecutive days, and then 7.5 mg / kg Dox was intraperitoneally injected on the 14th and 16th days to induce myocardial toxicity; Coenzyme Q10 10 mg / kg pretreatment administration group: The rats were given 10 mg / kg coenzyme Q10 by gavage every day for 12 consecutive days, and then 7.5 mg / kg Dox was intraperitoneally injected on the 14th and 16th days to induce myocardial toxicity; Coenzyme Q10 15 mg / kg pretreatment administration group: Rats received 15 mg / kg coenzyme Q10 by gavage every day for 12 consecutive days, and then on the 14th and 16th days, 7.5 mg / kg Dox was injected intraperitoneally to induce myocardial toxicity; Acacetin 5 mg / kg and coenzyme Q10 5 mg / kg combination pretreatment administration group: Rats received a mixed solution composed of 5 mg / kg acacetin and 5 mg / kg coenzyme Q10 by gavage every day for 12 consecutive days, and then on the 14th and 16th days, 7.5 mg / kg Dox was injected intraperitoneally to induce myocardial toxicity; Acacetin 10 mg / kg and coenzyme Q10 5 mg / kg combination pretreatment administration group: Rats received a mixed solution composed of 10 mg / kg acacetin and 5 mg / kg coenzyme Q10 by gavage every day for 12 consecutive days, and then on the 14th and 16th days, 7.5 mg / kg Dox was injected intraperitoneally to induce myocardial toxicity; Acacetin 5 mg / kg and coenzyme Q10 10 mg / kg combination pretreatment administration group: Rats received a mixed solution composed of 5 mg / kg acacetin and 10 mg / kg coenzyme Q10 by gavage every day for 12 consecutive days, and then on the 14th and 16th days, 7.5 mg / kg Dox was injected intraperitoneally to induce myocardial toxicity.

[0032] 1.1.2. Cardiac function assessment to screen the administration dose After anesthetizing the experimental rats in each group by intraperitoneal injection of chloral hydrate, they were fixed in a supine position on a rat board, and then a Doppler diagnostic instrument was used to detect the fractional shortening (FS) of the left ventricular axis and the left ventricular ejection fraction (LVEF) of the rats, and the data were recorded.

[0033] 1.1.3 Pathological observation Myocardial tissues of the experimental rats in each group were subjected to HE staining to observe the pathological changes of the myocardial tissues. The specimens were fixed in 10% formalin, dehydrated, cleared and sectioned, and finally observed and analyzed under a microscope.

[0034] 1.2 Cell experiment 1.2.1 Cell culture, grouping and model establishment H9C2 cells were cultured in high-glucose DMEM medium at 37 °C, 5% CO 2Cultured under the conditions, when the growth confluence of H9C2 cells reached 80% - 90%, the cells were digested with 0.25% trypsin and passaged, and cells in the logarithmic growth phase were used for the experiment. To verify the efficacy of the acacetin and coenzyme Q10 composition, H9C2 cells were divided into a control group (cells without any treatment), a Dox-induced injury model group (induced with 1 μmol / ml Dox (adriamycin) for 24 h); an acacetin 10 μmol / ml pretreatment administration group: acacetin was pretreated and administered for 24 hours, and then induced with 1 μmol / ml Dox (adriamycin) for 24 h; a coenzyme Q10 10 μmol / ml pretreatment administration group: coenzyme Q10 was pretreated and administered for 24 hours, and then induced with 1 μmol / ml Dox (adriamycin) for 24 h; an acacetin 5 μmol / ml and coenzyme Q10 5 μmol / ml composition pretreatment administration group: a mixed solution composed of acacetin 5 μmol / ml and coenzyme Q10 5 μmol / ml was pretreated and administered for 24 hours, and then induced with 1 μmol / ml Dox (adriamycin) for 24 h.

[0035] 1.2.2 Apoptosis detection The TUNEL kit was used to detect apoptosis to evaluate the viability of cardiomyocytes. Cardiomyocytes were co-cultured with Dox (adriamycin) and different concentrations of acacetin, coenzyme Q10, and the composition of acacetin and coenzyme Q10 for 48 hours. Subsequently, the TUNEL reagent was added, and after incubation for 12 hours, the apoptosis of cells was observed under a microscope.

[0036] 1.2.3 Protein expression detection The Western blot method was used to detect the expression of related proteins in cardiomyocytes. After extracting the total protein of cardiomyocytes, SDS-PAGE electrophoresis was used for separation, and then the separated proteins were transferred to a membrane. Primary antibodies with a dilution of 1:1000 were used to detect the Bcl-2, Bax, Cleaved Caspase-3, p-Drp, Mfn1, and Mfn2 proteins in cardiomyocytes respectively. The primary antibodies were incubated overnight (about 12 - 16 h) at 4°C to ensure sufficient binding to the target proteins; then appropriate secondary antibodies were used for incubation and colorimetric analysis.

[0037] 1.2.4 Reactive oxygen species determination In using H 2 O 2At 4 h after the completion of cardiomyocyte modeling, the determination of reactive oxygen species (ROS) in cardiomyocytes was carried out. The specific steps were as follows: Remove the culture medium in each well of the cell culture plate, wash it 3 times with serum-free culture medium, add 0.5 ml of 10 μmol / l DCFH-DA dye (diluted with PBS) to each well, incubate at 37 °C for 20 min. After the incubation, wash the cells 3 times again with PBS, and then add 200 μl of PBS to each well; At an excitation wavelength of 488 nm and an emission wavelength of 525 nm, use a fluorescence microscope to take images, or transfer the cells to a black transparent bottom plate by trypsin digestion, read the fluorescence data and determine the mean fluorescence intensity (MFI). The DCF fluorescence intensity is positively correlated with the ROS level.

[0038] 1.2.5 Ultra-trace Na + , K + -ATPase and ATP content determination After 4 h of treating cardiomyocytes after the completion of modeling with H 2 O 2 , discard the culture medium in each well of the cell culture plate, wash it 1 - 2 times with normal saline, add 200 μl of NP-40 lysis buffer, lyse the cells on ice bath, operate according to the kit instructions, and use the phosphorus determination method to detect the intracellular Na + , K + -ATPase enzyme activity and use the BCA method to determine the protein content.

[0039] The ATP content is determined by the BCA method for protein content, and the intracellular ATP content is detected by chemiluminescence method.

[0040] 1.3 Statistical analysis SPSS 13.0 software was used for statistical analysis. The results were expressed as mean ± standard deviation (Mean ± SD). For data with normal distribution and homogeneous variance, one-way ANOVA was used, and the SNK-q test was used for pairwise comparison between groups. When the results showed statistical significance, the statistical significance was set as P < 0.05. The results were as Figure 1 - 6 .

[0041] 2. Experimental results 2.1 Screening of the administration doses of acacetin, coenzyme Q10, and the combination of acacetin and coenzyme Q10 and their effects on improving the cardiac function of rats with myocardial toxicity The results of the test of cardiac function indexes of rats showed that compared with the Dox (adriamycin) induced group and the control group, the cardiac function indexes of the acacetin pretreatment group, the coenzyme Q10 pretreatment group, and the acacetin and coenzyme Q10 combination pretreatment group were significantly improved (P<0.05) in the rat left ventricular ejection fraction (LVEF) and shortening rate (FS) of the rats. From Table 1, it can be seen that the improvement of cardiac function indexes in the acacetin (5 mg / kg) and coenzyme Q10 (5 mg / kg) combination pretreatment group was significantly better than that in the other experimental groups. In addition, from Table 1, it can be seen that when acacetin is used alone, the optimal dosage is 10 mg / kg; when coenzyme Q10 is used alone, the optimal dosage is 10 mg / kg; the mixed solution of acacetin and coenzyme Q10 with a dosage of 5 mg / kg is the best combination.

[0042] Table 1 Group FS (%) LVEF (%) Control group 46.08 ± 3.07 79.13 ± 4.21 Dox 21.73 ± 1.24* 40.23 ± 3.42* Acacetin (5 mg / kg) 29.97 ± 2.17** 50.18 ± 3.89** Acacetin (10 mg / kg) 38.12 ± 1.21** 66.25±4.18** Acacetin (15 mg / kg) 32.49 ± 2.14**# 58.21±3.91**# Coenzyme Q10 (5 mg / kg) 28.73 ± 1.98** 52.11 ± 3.98** Coenzyme Q10 (10 mg / kg) 35.19 ± 2.63** 60.25 ± 4.18** Coenzyme Q10 (15 mg / kg) 30.29 ± 1.13**# 58.17 ± 2.44**# Acacetin (5 mg / kg) and Coenzyme Q10 (5 mg / kg) combination 43.19 ± 3.12** 74.43 ± 3.07** Acacetin (5 mg / kg) and Coenzyme Q10 (10 mg / kg) combination 41.89 ± 2.12** 69.31 ± 4.81** Acacetin (10 mg / kg) and Coenzyme Q10 (5 mg / kg) combination 38.12 ± 3.15** 68.23± 3.21** Note: FS: fractional shortening; LVEF: left ventricular ejection fraction; *P<0.05 compared with the control group; **P<0.05 compared with the Dox-induced injury model group; #P<0.05 compared with the combination of acacetin (5 mg / kg) and Coenzyme Q10 (10 mg / kg) or acacetin (10 mg / kg) and Coenzyme Q10 (5 mg / kg).

[0043] The following selected the 10 mg / kg acacetin pretreatment administration group and the 10 mg / kg coenzyme Q10 pretreatment administration group as comparative examples, and conducted the following detection study on the myocardial protective effect of the 5 mg / kg acacetin and coenzyme Q10 combination pretreatment administration groups: 2.2 Pathological changes of rat myocardial tissue After the treatment of each experimental group in the present invention, the pathological changes of the rat myocardial tissue were shown by HE staining results. In the myocardial tissue of the rats in the control group, the myocardial cells were arranged neatly and evenly, and no obvious myocardial fibrosis was observed. In contrast, the myocardial tissue of the rats in the Dox-induced injury model group showed disordered myocardial cell arrangement, accompanied by obvious myocardial fibrosis; the results of the acacetin, coenzyme Q10, acacetin and coenzyme Q10 combination pretreatment groups showed that the pathological changes of the rat myocardial tissue were improved (see Figure 1 ), among which, the pathological improvement of the group pretreated with acacetin and coenzyme Q10 combination was more significant.

[0044] 2.3 Detection of cardiomyocyte viability In the adriamycin-induced cardiotoxicity model, cardiomyocyte apoptosis is one of its hallmark features. The results are shown inFigures 2 - 4 : It was found from the TUNEL and flow cytometry assays of Figure 2 that compared with the control group, the apoptosis of cardiomyocytes treated with doxorubicin increased, while the cardiomyocytes pretreated with acacetin, coenzyme Q10, and the combination of acacetin and coenzyme Q10 showed a decrease in cardiomyocyte apoptosis. Among them, the group pretreated with the combination of acacetin and coenzyme Q10 had the best improvement effect. In addition, the Western blot results showed as Figure 3 : The cardiomyocytes pretreated with acacetin, coenzyme Q10, and the combination of acacetin and coenzyme Q10 enhanced the expression of Bcl-2 protein and decreased the expression of Bax and Cleaved Caspase3 proteins (P<0.05); among them, the group pretreated with the combination of acacetin and coenzyme Q10 had a more obvious effect on regulating the expression of related proteins; it can be seen from Figure 4 that the combination of acacetin and coenzyme Q10 with a dosage of 5 mg / kg could inhibit cardiomyocyte apoptosis by significantly and effectively regulating the expression of apoptosis-related proteins Bcl-2, Bax, and Cleaved Caspase3.

[0045] 2.4 Analysis of cardiomyocyte energy metabolism and cell damage Reactive oxygen species (ROS) are key mediators of cell damage, and their excessive accumulation can lead to cardiomyocyte dysfunction. In this example, fluorescence detection was performed on each experimental group, and the results are as Figure 5 in A (fluorescence intensity calculated according to B in Figure 5 ), from left to right are the control group, Dox-induced injury model group, acacetin-pretreated group, coenzyme Q10-pretreated group, and the combination of acacetin and coenzyme Q10-pretreated group. It can be seen that the fluorescence intensity of the Dox-induced injury model group was significantly higher than that of the control group, while the fluorescence intensity of cardiomyocytes in the acacetin-pretreated group, coenzyme Q10-pretreated group, and the combination of acacetin and coenzyme Q10-pretreated group decreased significantly. Among them, the decrease in fluorescence intensity of cardiomyocytes in the combination of acacetin and coenzyme Q10-pretreated group was significantly greater than that in the acacetin-pretreated group and coenzyme Q10-pretreated group; Figure 5 ​​​​​​​C in the figure shows the effect of Dox on the ultra-trace ATP activity in H9C2 cells: after Dox treatment, the ATP activity decreased significantly, while the ATP activity of the acacetin pretreatment group, the coenzyme Q10 pretreatment group, and the acacetin and coenzyme Q10 combination pretreatment group increased, among which: the activity content of the cardiomyocytes in the acacetin and coenzyme Q10 combination pretreatment group was 23.1 μg / ml, which was 1.84 times that of the acacetin pretreatment group (23.2 μg / ml) and 4.11 times that of the coenzyme Q10 pretreatment group (5.6 μg / ml); Figure 5 D in the figure shows the effect of Dox on the activity of ultra-trace Na⁺, K⁺-ATPase in H9C2 cells: after Dox treatment, the activity of Na⁺, K⁺-ATPase decreased significantly, while the activity of Na⁺, K⁺-ATPase in the acacetin pretreatment group, the coenzyme Q10 pretreatment group, and the acacetin and coenzyme Q10 combination pretreatment group was significantly increased. Among them, the Na⁺, K⁺-ATPase activity content of myocardial cells in the acacetin and coenzyme Q10 combination pretreatment group was 32.5μg / ml, which was significantly higher than the Na⁺, K⁺-ATPase 30.3μg / ml (active content) of the acacetin pretreatment group and the Na⁺, K⁺-ATPase 25.2μmg / ml (active content) of the coenzyme Q10 pretreatment group.

[0046] From the above, we can see that through fluorescence detection and Na + , K + -ATPase activity and ATP content analysis showed that the acacetin pretreatment group, the coenzyme Q10 pretreatment group, and the acacetin and coenzyme Q10 combination pretreatment group were all able to improve the damage caused by Dox, among which the improvement effect of the acacetin and coenzyme Q10 combination pretreatment group was significantly better than that of the other pretreatment groups.

[0047] 2.5 Detection of protein expression related to mitochondrial energy metabolism in cardiomyocytes Mitochondrial energy metabolism Mfn1, Mfn2 and p-Drp proteins can reflect one of the hallmark characteristics of cardiomyocyte energy metabolism. Figure 6 A in the figure indicates the expression of Mfn1, Mfn2 and p-Drp protein blot in cardiomyocytes after treatment in each pretreatment and drug administration group; Figure 6The B-D in it represents the relative expression levels of Mfn1 / β-action, Mfn2 / β-action, and p-Drp / β-action in cardiomyocytes after treatment with each pretreatment administration group. It can be concluded from the above figures that in the adriamycin-induced cardiomyocyte injury model, the acacetin pretreatment group, coenzyme Q10 pretreatment group, and the combination of acacetin and coenzyme Q10 pretreatment group can all significantly enhance the expression of Mfn1 and Mfn2 proteins and reduce the expression of p-Drp protein. Among them, the regulatory effect of the combination of acacetin and coenzyme Q10 pretreatment group is the most significant. Therefore, it can be concluded that the combination of acacetin and coenzyme Q10 can effectively regulate the expression of proteins related to mitochondrial energy metabolism in cardiomyocytes, thereby playing a role in protecting mitochondrial function and alleviating cardiomyocyte injury.

[0048] In summary, the combination of acacetin and coenzyme Q10 provided by the present invention, as a potential drug for treating adriamycin-induced cardiotoxicity, has important clinical application prospects. It can not only improve cardiac function indicators but also effectively protect cardiomyocytes from damage, providing a new protective measure for adriamycin users.

[0049] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. Use of a combination of acacetin and coenzyme Q10 in a drug for preventing doxorubicin-induced cardiotoxicity.

2. Use of the acacetin and coenzyme Q10 composition according to claim 1 in a drug for preventing doxorubicin-induced myocardial toxicity, characterized in that: Acacetin and coenzyme Q10 combination were administered as pretreatment before doxorubicin administration.

3. Use of the acacetin and coenzyme Q10 composition according to claim 1 in a drug for preventing doxorubicin-induced myocardial toxicity, characterized in that: The acacetin and coenzyme Q10 composition can achieve at least one of the following effects: improving cardiac function indicators of adriamycin-induced myocardial toxicity; inhibiting myocardial cell apoptosis; enhancing myocardial cell energy metabolism; and mediating the expression of proteins related to myocardial cell mitochondrial energy metabolism.

4. Use of the acacetin and coenzyme Q10 composition according to claim 3 in a drug for preventing doxorubicin-induced myocardial toxicity, characterized in that: The acacetin and coenzyme Q10 composition can achieve at least one of the following effects: improving left ventricular ejection fraction and shortening rate; regulating the expression of myocardial cell apoptosis-related proteins Bcl-2, Bax, and CleavedCaspase3; removing reactive oxygen species and increasing Na in damaged myocardial cells. + , K + -ATPase activity and ATP content; enhanced the expression of Mfn1 and Mfn2 proteins, and decreased the expression of p-Drp protein.

5. Use of the acacetin and coenzyme Q10 composition according to claim 1 in a drug for preventing doxorubicin-induced myocardial toxicity, characterized in that: In the composition of acacetin and coenzyme Q10, the mass ratio of the two is (1-2): (1-2), and the dosages of acacetin and coenzyme Q10 are 5-15 mg / kg and 5-15 mg / kg, respectively.

6. Use of the acacetin and coenzyme Q10 composition according to claim 5 in a drug for preventing doxorubicin-induced myocardial toxicity, characterized in that: In the composition of acacetin and coenzyme Q10, the mass ratio of the two is 1:1, and the dosage of acacetin and coenzyme Q10 is 5 mg / kg.

7. A drug for preventing and treating doxorubicin-induced myocardial toxicity, characterized in that: The main ingredients are acacetin and coenzyme Q10 combination.

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