Application of acacetin and coenzyme Q10 composition in drugs for preventing and treating adriamycin-induced myocardial toxicity

Abstract

The present invention provides an application of a composition of acacetin and coenzyme Q10 in a medicament for preventing doxorubicin-induced myocardial toxicity. In doxorubicin-induced myocardial toxicity diseases, the composition of acacetin and coenzyme Q10 can improve cardiac function indexes, inhibit cardiomyocyte apoptosis, improve myocardial tissue pathological changes, improve mitochondrial energy metabolism, scavenge reactive oxygen species, increase the activities of Na+, K+-ATPase and the content of ATP, thereby achieving the effect of improving the damaged cardiac function, and can provide a new treatment strategy for preventing and treating doxorubicin-induced myocardial toxicity diseases, having good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and more particularly, to the use of an acacetin and coenzyme Q10 composition 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 a variety of natural plants and has antioxidant, anti-inflammatory and anti-proliferative effects. However, affected by the problem of low bioavailability, the concentration of acacetin in plasma and target tissues is usually low.

[0003] Coenzyme Q10 (or ubiquinone or ubidecarenone) is an endogenous lipophilic substance present in many eukaryotic cells (mainly present in mitochondria). As a key component of the mitochondrial electron transport chain, it can scavenge adriamycin-induced reactive oxygen species (ROS), maintain the 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. Currently, there are no related reports on the application of this composition in this field. Summary of the Invention

[0005] In view of this, the present invention provides the use of an acacetin and coenzyme Q10 composition 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.

[0006] The present invention discloses the use of an acacetin and coenzyme Q10 composition in a medicament for preventing adriamycin-induced myocardial toxicity.

[0007] Preferably, pretreatment administration of the acacetin and coenzyme Q10 composition is carried out before doxorubicin administration.

[0008] In this embodiment, the acacetin and coenzyme Q10 composition can achieve at least one of the following effects: improving the cardiac function indexes of doxorubicin-induced myocardial toxicity; reducing doxorubicin-induced myocardial injury; inhibiting cardiomyocyte apoptosis; enhancing cardiomyocyte energy metabolism; mediating the expression of proteins related to mitochondrial energy metabolism in cardiomyocytes.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] The present invention also discloses a drug for preventing doxorubicin-induced myocardial toxicity, which uses the acacetin and coenzyme Q10 composition as the main component.

[0013] The application of the acacetin and coenzyme Q10 composition provided by the present invention in the drug for preventing doxorubicin-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, doxorubicin 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 the myocardium; 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 cardiac function, providing a new treatment strategy for preventing and treating doxorubicin-induced myocardial toxicity diseases, and having good application prospects. Description of the Drawings

[0014] Figure 1HE staining results of cardiac tissue sections of rats in 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 multiples are 200x and 400x respectively);

[0015] Figure 2 In the experiment for 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 figure showing the apoptosis of myocardial H9C2 cells detected by TUNEL technology and flow cytometry;

[0016] Figure 3 In the experiment for 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 figure showing the protein expression of Bcl-2, Bax, and Cleaved Caspase3 detected by Western blot technology;

[0017] Figure 4 In the experiment for 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 graph of the relative expression levels of Bcl-2 / β-actin, Bax / β-actin, and Cleaved Caspase3 / β-actin;

[0018] Figure 5 In the embodiments of the present invention, the figure showing the ROS (reactive oxygen species) expression levels in H9C2 cardiomyocytes detected by fluorescence in 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. Among them: A represents the bar graph of the fluorescence intensity of different pretreatment administration groups; B represents the fluorescence image of ROS in H9C2 cardiomyocytes detected by fluorescence; C represents the bar graph of the effect of Dox induction on the ultra-trace ATP content in H9C2 cells; D represents the bar graph of the effect of Dox induction on the activity of + Na + , K

[0019] 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

[0020] The present invention provides an application of an acacetin and coenzyme Q10 combination in a medicament for preventing and treating adriamycin-induced cardiotoxicity. In the present invention, there is no special limitation 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%.

[0021] Coenzyme Q10 is purchased from Shenzhou Biology&Technology Co., Ltd or Kaneka Nutrients.

[0022] In the acacetin and coenzyme Q10 combination, 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.

[0023] Preferably, the mass ratio of acacetin to coenzyme Q10 in the acacetin and coenzyme Q10 combination is 1:1, and the administration doses of both acacetin and coenzyme Q10 are 5 mg / kg.

[0024] In the embodiments of the present invention, the cardiotoxicity is caused by adriamycin.

[0025] In the embodiments of the present invention, pretreatment with the acacetin and coenzyme Q10 combination (administering a set dose of the acacetin and coenzyme Q10 combination 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 HE staining results to evaluate the recovery of cardiomyocytes after pretreatment of rats with the acacetin and coenzyme Q10 combination.

[0026] Preferably, the acacetin and coenzyme Q10 combination can increase the left ventricular ejection fraction and shortening rate.

[0027] Pretreatment with the acacetin and coenzyme Q10 combination can inhibit cardiomyocyte apoptosis. The present invention detects and evaluates cardiomyocyte viability by TUNEL and flow cytometry methods.

[0028] Preferably, the acacetin and coenzyme Q10 composition can regulate the expression of apoptosis-related proteins Bcl-2, Bax, and Cleaved Caspase3 in cardiomyocytes.

[0029] Pretreatment with the acacetin and coenzyme Q10 composition can enhance the energy metabolism of cardiomyocytes. The present invention evaluates the effects of the acacetin and coenzyme Q10 composition on reactive oxygen species damage and energy metabolism in cardiomyocytes by fluorescence detection.

[0030] Preferably, the acacetin and coenzyme Q10 composition has the ability to scavenge reactive oxygen species and increase the Na + , K + -ATPase activity and ATP content in damaged cardiomyocytes.

[0031] Pretreatment with the acacetin and coenzyme Q10 composition can mediate the expression of proteins related to mitochondrial energy metabolism in cardiomyocytes. The present invention evaluates the effects of the acacetin and coenzyme Q10 composition on the expression of proteins related to mitochondrial energy metabolism in cardiomyocytes by detecting the expression levels of Mfn1 protein, p-Drp, and Mfn2 protein using Western Blot.

[0032] 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.

[0033] 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.

[0034] 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 component.

[0035] 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 creative efforts shall fall within the protection scope of the present invention.

[0036] 1. Experimental methods

[0037] 1.1 Animal experiments

[0038] Sixty healthy 3-month-old adult male Sprague-Dawley (SD) 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 in Zhejiang Province at a temperature of 22±2°C and a humidity of 50±10%. All experiments were conducted in accordance with the relevant regulations for the management of experimental animals and were approved by the Experimental Ethics Committee (TZY-2022097).

[0039] 1.1.1 Grouping of experimental animals

[0040] 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 them:

[0041] Control group: The rats were housed under normal breeding conditions without any drug treatment;

[0042] Dox-induced injury model group: The rats were housed 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;

[0043] Acacetin 5 mg / kg pretreatment 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 injected intraperitoneally on the 14th and 16th days to induce myocardial toxicity;

[0044] Acacetin 10 mg / kg pretreatment 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 injected intraperitoneally on the 14th and 16th days to induce myocardial toxicity;

[0045] Acacetin 15 mg / kg pretreatment 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 injected intraperitoneally on the 14th and 16th days to induce myocardial toxicity;

[0046] Coenzyme Q10 5 mg / kg pretreatment 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 injected intraperitoneally on the 14th and 16th days to induce myocardial toxicity;

[0047] Coenzyme Q10 10 mg / kg pretreatment administration group: Rats received 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;

[0048] Coenzyme Q10 15mg / 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;

[0049] 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;

[0050] 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;

[0051] Acacetin 5 mg / kg and coenzyme Q10 10mg / 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.

[0052] 1.1.2. Cardiac function assessment to screen the administration dose

[0053] After anesthetizing the experimental rats in each group by intraperitoneal injection of chloral hydrate, they were fixed supine on the 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.

[0054] 1.1.3 Pathological observation

[0055] The myocardial tissues of the experimental rats in each group were taken for 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.

[0056] 1.2 Cell experiments

[0057] 1.2.1 Cell culture, grouping and model establishment

[0058] H9C2 cells were cultured in high-glucose DMEM medium at 37 °C and 5% CO2. When the growth confluence of H9C2 cells reached 80% - 90%, the cells were digested and passaged with 0.25% trypsin, and cells in the logarithmic growth phase were used for the experiment. To verify the pharmacological effects of the acacetin and coenzyme Q10 combination, 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 combination 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.

[0059] 1.2.2 Detection of cell apoptosis

[0060] The TUNEL kit was used to detect cell apoptosis to evaluate the viability of cardiomyocytes. The cardiomyocytes were co-cultured with Dox (adriamycin) and different concentrations of acacetin, coenzyme Q10 and the combination of acacetin and coenzyme Q10 for 48 hours, and then the TUNEL reagent was added. After incubation for 12 hours, the cell apoptosis was observed under a microscope.

[0061] 1.2.3 Detection of protein expression

[0062] The expression of related proteins in cardiomyocytes was detected by Western blot. After extracting the total proteins 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 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 was performed.

[0063] 1.2.4 Determination of reactive oxygen species

[0064] Four hours after the completion of the cardiomyocyte model establishment by treating with H2O2, the determination of reactive oxygen species (ROS) in cardiomyocytes was carried out. The specific steps were as follows: The culture medium in each well of the cell culture plate was removed, and the cells were rinsed 3 times with serum-free culture medium. 0.5 ml of 10 μmol / l DCFH-DA dye (diluted with PBS) was added to each well, and the cells were incubated at 37 °C for 20 min. After the incubation, the cells were rinsed 3 times again with PBS, and then 200 μl of PBS was added to each well; Images were taken using a fluorescence microscope at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, or the cells were transferred to a black transparent bottom plate by trypsin digestion, and the fluorescence data were read and the mean fluorescence intensity (MFI) was determined. The DCF fluorescence intensity is positively correlated with the ROS level.

[0065] 1.2.5 Determination of ultra-trace intracellular Na + , K + -ATPase and ATP contents

[0066] Four hours after the completion of the cardiomyocyte model establishment by treating with H2O2, the culture medium in each well of the cell culture plate was discarded, and the cells were rinsed 1 - 2 times with normal saline. 200 μl of NP-40 lysis buffer was added, and the cells were lysed on ice bath. According to the kit instructions, the intracellular Na + , K + -ATPase enzyme activity was detected by the phosphorus determination method and the protein content was determined by the BCA method.

[0067] The ATP content was determined by the BCA method for protein content, and the intracellular ATP content was detected by chemiluminescence method.

[0068] 1.3 Statistical analysis

[0069] Statistical analysis was performed using SPSS 13.0 software, and the results were expressed as mean ± standard deviation (Mean ± SD). For data with normal distribution and homogeneous variance, one-way analysis of variance was used, and SNK-q test was used for pairwise comparison between groups. When the results showed statistically significant differences, the statistical significance was set at P < 0.05. The results were as follows Figure 1-6 .

[0070] 2. Experimental results

[0071] 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

[0072] The detection results of rat cardiac function indexes showed that compared with the Dox (adriamycin) induction group and the control group, the cardiac function indexes of the left ventricular ejection fraction (LVEF) and shortening fraction (FS) of rats in each dose group of the acacetin pretreatment administration group, coenzyme Q10 pretreatment administration group, and the combination of acacetin and coenzyme Q10 pretreatment administration group were significantly improved (P < 0.05). It can be seen from Table 1 that the improvement of cardiac function indexes in the pretreatment administration group of the combination of acacetin (5 mg / kg) and coenzyme Q10 (5 mg / kg) was significantly better than that of other experimental groups. In addition, it can be seen from Table 1 that when acacetin was used alone, the optimal administration dose was 10 mg / kg; when coenzyme Q10 was used alone, the optimal administration dose was 10 mg / kg; the mixed solution of acacetin and coenzyme Q10 with an administration dose of 5 mg / kg each was the best combination.

[0073] Table 1

[0074] 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**# Combination of acacetin (5 mg / kg) and coenzyme Q10 (5 mg / kg) 43.19 ± 3.12** 74.43 ± 3.07** Combination of acacetin (5 mg / kg) and coenzyme Q10 (10 mg / kg) 41.89 ± 2.12** 69.31 ± 4.81** Combination of acacetin (10 mg / kg) and coenzyme Q10 (5 mg / kg) 38.12 ± 3.15** 68.23± 3.21**

[0075] 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).

[0076] The following were selected as the control groups: the acacetin pretreatment administration group with an administration dose of 10 mg / kg and the coenzyme Q10 pretreatment administration group with an administration dose of 10 mg / kg, and the following detection studies were carried out on the myocardial protection effect of the combination of acacetin and coenzyme Q10 with an administration dose of 5 mg / kg each

[0077] 2.2 Pathological changes in rat myocardial tissue

[0078] 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.

[0079] 2.3 Detection of cardiomyocyte viability

[0080] In the adriamycin-induced cardiotoxicity model, cardiomyocyte apoptosis is one of its hallmark features. The results are shown in Figures 2-4 :from Figure 2 The results of TUNEL and flow cytometry showed that compared with the control group, the apoptosis of cardiomyocytes treated with doxorubicin increased, while the apoptosis of cardiomyocytes pretreated with acacetin, coenzyme Q10, and acacetin and coenzyme Q10 combination showed a decrease in cardiomyocyte apoptosis. Among them, the acacetin and coenzyme Q10 combination pretreated group had the best improvement effect. In addition, the Western blot test results showed that Figure 3 : Acacetin pretreatment, coenzyme Q10 pretreatment, and acacetin and coenzyme Q10 combination pretreatment of cardiomyocytes enhanced the expression of Bcl-2 protein and reduced the expression of Bax and Cleaved Caspase3 proteins (P<0.05); among them, the acacetin and coenzyme Q10 combination pretreatment group had a more obvious effect on regulating the expression of related proteins; Figure 4 It can be seen that the combination of acacetin and coenzyme Q10 at a dosage of 5 mg / kg can inhibit cardiomyocyte apoptosis by significantly and effectively regulating the expression of apoptosis-related proteins Bcl-2, Bax, and Cleaved Caspase3.

[0081] 2.4 Analysis of cardiomyocyte energy metabolism and cell damage

[0082] Reactive oxygen species (ROS) are key mediators of cell damage, and their excessive accumulation can lead to myocardial cell dysfunction. In this example, fluorescence detection was performed on each experimental group, and the results were as follows: Figure 5 A in (according to Figure 5From left to right in the fluorescence intensity calculated for B in () are the control group, the Dox-induced injury model group, the acacetin pretreatment administration group, the coenzyme Q10 pretreatment administration group, and the acacetin and coenzyme Q10 combination pretreatment administration group. It can be seen that: the fluorescence intensity of the Dox-induced injury model group is significantly higher than that of the control group, while the fluorescence intensity of myocardial cells in the acacetin pretreatment administration group, the coenzyme Q10 pretreatment administration group, and the acacetin and coenzyme Q10 combination pretreatment administration group is significantly decreased. Among them, the decrease amplitude of the fluorescence intensity of myocardial cells in the acacetin and coenzyme Q10 combination pretreatment administration group is significantly greater than that in the acacetin pretreatment administration group and the coenzyme Q10 pretreatment administration group; Figure 5 C in () shows the effect of Dox on the ultra-trace ATP activity in H9C2 cells: the ATP activity significantly decreases after Dox treatment, while the ATP activities in the acacetin pretreatment administration group, the coenzyme Q10 pretreatment administration group, and the acacetin and coenzyme Q10 combination pretreatment administration group are all increased. Among them, the active content of myocardial cells in the acacetin and coenzyme Q10 combination pretreatment administration group is 23.1 μg / ml, which is 1.84 times that of the acacetin pretreatment administration group (23.2 μg / ml) and 4.11 times that of the coenzyme Q10 pretreatment administration group (5.6 μg / ml); Figure 5 D in () shows the effect of Dox on the ultra-trace Na⁺, K⁺-ATPase activity in H9C2 cells: the Na⁺, K⁺-ATPase activity significantly decreases after Dox treatment, while the Na⁺, K⁺-ATPase activities in the acacetin pretreatment administration group, the coenzyme Q10 pretreatment administration group, and the acacetin and coenzyme Q10 combination pretreatment administration group are significantly increased. Among them, the Na⁺, K⁺-ATPase active content of myocardial cells in the acacetin and coenzyme Q10 combination pretreatment administration group is 32.5 μg / ml, which significantly exceeds the Na⁺, K⁺-ATPase 30.3 μg / ml (active content) of the acacetin pretreatment administration group and the Na⁺, K⁺-ATPase 25.2 μg / ml (active content) of the coenzyme Q10 pretreatment administration group.

[0083] It can be seen from the above that: through fluorescence detection in myocardial cells and analysis of Na + , K + -ATPase activity and ATP content, the acacetin pretreatment administration group, the coenzyme Q10 pretreatment administration group, and the acacetin and coenzyme Q10 combination pretreatment administration group can all improve the damage caused by Dox. Among them, the improvement effect of the acacetin and coenzyme Q10 combination pretreatment administration group is significantly better than that of other pretreatment administration groups.

[0084] 2.5 Detection of the expression of proteins related to mitochondrial energy metabolism in myocardial cells

[0085] 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 6 BD in the figure represents the relative expression of Mfn1 / β-action, Mfn2 / β-action and p-Drp / β-action in myocardial cells after treatment in each pretreatment and medication group. From the above figures, it can be concluded that in the doxorubicin-induced myocardial cell injury model, the acacetin pretreatment group, the coenzyme Q10 pretreatment group and the acacetin and coenzyme Q10 combination pretreatment group can significantly enhance the expression of Mfn1 and Mfn2 proteins and reduce the expression of p-Drp protein; among them, the acacetin and coenzyme Q10 combination pretreatment group has the most significant regulatory effect. Therefore, it can be concluded that the acacetin and coenzyme Q10 combination can effectively regulate the expression of proteins related to mitochondrial energy metabolism in myocardial cells, thereby protecting mitochondrial function and alleviating myocardial cell injury.

[0086] In summary, the acacetin and coenzyme Q10 composition provided by the present invention has important clinical application prospects as a potential drug for treating doxorubicin-induced myocardial toxicity. It can not only improve cardiac function indicators, but also effectively protect myocardial cells from damage, providing a new protection measure for doxorubicin users.

[0087] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a composition of acacetin and coenzyme Q10 in the preparation of a drug for preventing doxorubicin-induced cardiotoxicity, characterized in that, In the acacetin and coenzyme Q10 composition, the mass ratio of the two is 1:

1.

2. A drug for preventing and treating adriamycin-induced myocardial toxicity, characterized in that, Taking the acacetin and coenzyme Q10 composition as the main component, in the acacetin and coenzyme Q10 composition, the mass ratio of the two is 1:1.

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