A traditional Chinese medicine composition for preventing and treating cardiotoxicity caused by doxorubicin and its application

Abstract

This invention belongs to the field of traditional Chinese medicine, specifically relating to a traditional Chinese medicine composition for preventing and treating cardiotoxicity induced by doxorubicin and its application; the traditional Chinese medicine composition for preventing and treating cardiotoxicity induced by doxorubicin is composed of ginseng and salvia miltiorrhiza; this invention finds that the traditional Chinese medicine composition can promote the survival rate of doxorubicin-induced cardiomyocyte H9c2, reduce doxorubicin-induced apoptosis of cardiomyocyte H9c2, reduce doxorubicin-induced oxidative stress damage and cardiac damage, improve left ventricular function, and improve doxorubicin-induced cardiotoxicity, which is of great significance for the prevention and treatment of azithromycin-induced cardiotoxicity.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, specifically relating to a traditional Chinese medicine composition for preventing and treating cardiotoxicity caused by doxorubicin and its application. Background Technology

[0002] Doxorubicin (DOX) is one of the most effective and widely used drugs for treating various solid tumors and hematological malignancies. However, its dose-dependent cardiotoxicity greatly limits its clinical application. To date, the potential mechanisms of DOX-mediated cardiotoxicity have mainly focused on oxidative stress, mitochondrial dysfunction, and altered Ca2+ homeostasis. Among cancer patients receiving DOX treatment, 8% to 26% experience cardiotoxicity, exhibiting ST-T segment changes, decreased left ventricular ejection fraction, increased ventricular wall thickness, arrhythmias, and heart failure. This is attributed to oxidative damage, apoptosis, and autophagic death of cardiomyocytes, ultimately leading to abnormal cardiac function. Therefore, there is an urgent need to find more effective drugs to prevent DOX-induced cardiomyopathy and improve the quality of life for cancer survivors.

[0003] In the preliminary research of this invention, 53 traditional Chinese medicine compound formulas for treating cardiomyopathy were collected. Frequency analysis revealed that the top three most frequently used herbs were Danshen (Salvia miltiorrhiza), ginseng, and astragalus. Furthermore, through in vitro animal experiments combined with cell experiments, it was found that the ginseng, Danshen, and astragalus compound formula can reduce the cardiotoxicity of doxorubicin and can be used to prepare drugs for the prevention and treatment of doxorubicin-induced cardiomyopathy, showing promising application prospects. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a traditional Chinese medicine composition and its application for preventing and treating cardiotoxicity caused by doxorubicin, specifically including the following:

[0005] In a first aspect, the present invention provides a traditional Chinese medicine composition for preventing and treating cardiotoxicity caused by doxorubicin, the traditional Chinese medicine composition being composed of ginseng and salvia miltiorrhiza.

[0006] Preferably, the ratio of ginseng to salvia miltiorrhiza is 1-4:1-4.

[0007] Preferably, the ratio of ginseng to salvia miltiorrhiza is 1:2.

[0008] Preferably, the preparation method of the traditional Chinese medicine composition is as follows:

[0009] (1) Take the prescribed amounts of ginseng, salvia miltiorrhiza and astragalus respectively, add 12 times the amount of distilled water and soak for 60 minutes, then heat to boiling, simmer for 60 minutes, and then filter.

[0010] (2) Add 6 times the amount of distilled water to the filter residue and repeat step (1);

[0011] (3) Combine the two filtrates and concentrate them to 1 g / mL, which is equivalent to the amount of raw ginseng, salvia miltiorrhiza and astragalus. Prepare the solution according to the ratio of ginseng: salvia miltiorrhiza = 1:2.

[0012] Preferably, the traditional Chinese medicine composition is formulated into any pharmaceutically acceptable dosage form by adding a pharmaceutically acceptable carrier.

[0013] Preferably, the dosage form includes tablets, capsules, granules, powders, pills, tinctures, medicated wines, decoctions, and mixtures.

[0014] Secondly, the present invention provides a traditional Chinese medicine composition for preventing and treating cardiotoxicity caused by doxorubicin, the traditional Chinese medicine composition being composed of ginseng, salvia miltiorrhiza and astragalus membranaceus.

[0015] Preferably, the ratio of ginseng, salvia miltiorrhiza, and astragalus is 1-4:1-4:1-4.

[0016] Preferably, the ratio of ginseng, salvia miltiorrhiza, and astragalus is 1:2:1-3.

[0017] Preferably, the ratio of ginseng, salvia miltiorrhiza, and astragalus is 1:2:1.

[0018] Preferably, the preparation method of the traditional Chinese medicine composition is as follows:

[0019] (1) Take the prescribed amounts of ginseng, salvia miltiorrhiza and astragalus respectively, add 12 times the amount of distilled water and soak for 60 minutes, then heat to boiling, simmer for 60 minutes, and then filter.

[0020] (2) Add 6 times the amount of distilled water to the filter residue and repeat step (1);

[0021] (3) Combine the two filtrates and concentrate them to 1 g / mL, which is equivalent to the amount of raw ginseng, salvia miltiorrhiza and astragalus. Prepare the solution according to the ratio of ginseng: salvia miltiorrhiza: astragalus = 1:2:1.

[0022] Preferably, the traditional Chinese medicine composition is formulated into any pharmaceutically acceptable dosage form by adding a pharmaceutically acceptable carrier.

[0023] Preferably, the dosage form includes tablets, capsules, granules, powders, pills, tinctures, medicated wines, decoctions, and mixtures.

[0024] Thirdly, the present invention provides the application of the traditional Chinese medicine composition described in the first and second aspects above in the preparation of drugs for preventing and treating cardiotoxicity caused by doxorubicin.

[0025] Fourthly, the present invention provides the application of the traditional Chinese medicine composition described in the first and second aspects above in the preparation of a drug for treating cardiomyopathy.

[0026] The beneficial effects of this invention are: ① This invention provides a traditional Chinese medicine composition composed of ginseng, salvia miltiorrhiza, or ginseng, salvia miltiorrhiza, and astragalus for preventing and treating cardiotoxicity induced by doxorubicin; ② The traditional Chinese medicine composition can promote the survival rate of doxorubicin-induced cardiomyocyte H9c2, reduce doxorubicin-induced apoptosis of cardiomyocyte H9c2, reduce doxorubicin-induced oxidative stress damage and cardiac damage, improve left ventricular function, and improve doxorubicin-induced cardiotoxicity, which is of great significance for the prevention and treatment of azithromycin-induced cardiotoxicity. Attached Figure Description

[0027] Figure 1 The effect of different ginseng-danshen ratios on the survival rate of H9c2 cells, compared with Ctrl. * P<0.05, ** P<0.01;

[0028] Figure 2 The effects of different ginseng-tanshinone ratios on the survival rate of doxorubicin-induced H9c2 cells, compared with Ctrl. * P<0.05, ** P<0.01;

[0029] Figure 3 Results of doxorubicin and its effects on H9c2 cardiomyocyte nuclei and apoptosis after administration;

[0030] Figure 4 Doxorubicin and its effect on myocardial injury markers in H9c2 cardiomyocytes after administration. Note: Compared with Ctrl. * P<0.05, ** P<0.01;

[0031] Figure 5 The effects of ginseng-danshen on reducing DOX-induced in vivo myocardial dysfunction, where A represents the change in rat heart volume, B represents the change in rat body weight, and C represents the ratio of rat heart weight to body weight;

[0032] Figure 6 Results of echocardiography in rats;

[0033] Figure 7 Staining results of rat heart tissue, where A is HE staining and B is masson staining;

[0034] Figure 8 Results of detection of myocardial injury markers and oxidative stress indicators in rats, with comparisons to Ctrl. * P<0.05, ** P<0.01;

[0035] Figure 9Survival rates of H9C2 cells under different ratios and concentrations of ginseng, salvia miltiorrhiza, and astragalus, compared with Ctrl. * P<0.05, ** P<0.01;

[0036] Figure 10 The effects of different ratios of ginseng, salvia miltiorrhiza, and astragalus on the survival rate of H9c2 cells induced by doxorubicin, compared with Ctrl. * P<0.05, ** P<0.01;

[0037] Figure 11 The effects of serum containing different concentrations and doses of ginseng, salvia miltiorrhiza, and astragalus on the recovery of three types of cardiomyocytes under DOX treatment;

[0038] Figure 12 Ginseng, danshen, and astragalus improved cardiac function in mice with doxorubicin-induced myocardial injury (n=6, ±s);

[0039] Figure 13 The effects of serum containing different concentrations and doses of ginseng, salvia miltiorrhiza, and astragalus on the recovery of three types of cardiomyocytes under DOX treatment;

[0040] Figure 14 The effects of different Chinese herbal medicines and combinations on the survival rate of H9c2 cells induced by doxorubicin were investigated. In the figure, ## indicates comparison with the blank group, P<0.01; ** indicates comparison with the model group, P<0.01. Detailed Implementation

[0041] The scope of protection of the present invention will be described in detail below with reference to specific embodiments. However, it should be noted that the scope of protection of the present invention is not limited to the following embodiments. Any technical solutions derived by those skilled in the art based on the concept of the present invention through logical analysis, deduction, and experimentation in the prior art are all within the scope of protection claimed by the present invention.

[0042] The experimental animals described in the following examples were: ginseng, salvia miltiorrhiza, and astragalus, all purchased from Huirentang Pharmaceutical Lanxin Store; specific pathogen-free (SPF) grade male SD rats, weighing (200±20g), purchased from Speford (Beijing) Biotechnology Co., Ltd., and housed in the SPF-grade animal facility of Gansu University of Traditional Chinese Medicine. They were randomly grouped, given free access to food and water, and kept under natural circadian lighting.

[0043] Example 1: Protective effect of ginseng and danshen against doxorubicin-induced cardiac injury

[0044] 1. Preparation of Ginseng and Salvia miltiorrhiza Combination

[0045] Ginseng and salvia miltiorrhiza were soaked in 12 times their volume of distilled water for 60 minutes. Then, the herbs and water were poured into a decoction pot and heated to a boil over high heat. After a slight boil, the decoction was simmered over low heat for 60 minutes. The decoction was then filtered, and 6 times their volume of distilled water was added. The decoction was filtered again, and the two filtrates were combined and concentrated to a concentration equivalent to 1 g / mL of raw ginseng and salvia miltiorrhiza. The decoction was prepared by mixing ginseng and salvia miltiorrhiza in ratios of 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, and 4:1 and stored at -20°C.

[0046] 2. Cell experiments

[0047] 2.1 Experimental Methods

[0048] 2.1.1 Culture and inoculation of H9C2 cells

[0049] Adjust the H9C2 cell concentration to 5 × 10⁻⁶. 4 / ml; seed the cell suspension into 96-well plates, 100μl per well, with 6 replicates per group; place the 96-well plates in a cell culture incubator for further culture; after the cells adhere, add the corresponding concentration of drug according to the intervention method for each group.

[0050] The model group cells were treated with 2 μM DOX at a dose of 10% of the total culture medium. The experimental groups included a blank control group, a DOX model group (DOX 2 μM), and different proportions and concentrations of ginseng and danshen (Salvia miltiorrhiza) treatment groups. The blank group received an equal volume of culture medium, while the other treatment groups received different concentrations of the drug solution at 10% of the total culture medium. Cells were collected after 24 hours, and MTT was added after cell treatment to calculate cell viability.

[0051] 2.1.2 Hoechst 33258 staining method to observe the effect of RD on DOX-induced H9c2 cell morphology and nucleus.

[0052] H9c2 cells were seeded in 6-well plates and drug was administered after cell adhesion. Experimental model grouping was performed as described in section 2.1.1, and other procedures were performed according to the Hoechst 33258 staining solution instructions. Hoechst staining was then performed for observation.

[0053] 2.1.3 Effect of the kit on serum enzyme indices in detecting DOX-induced myocardial injury

[0054] Prepare the necessary reagents, samples, and standards according to the instructions of each kit. Add the prepared samples and standards to the ELISA plate. Incubate at 37°C for 30 minutes. After the reaction, wash the plate 5 times, add the enzyme-labeled reagent, and incubate at 37°C for 30 minutes. After the reaction, wash the plate 5 times, add chromogenic solutions A and B, and incubate at 37°C for 10 minutes. Add the stop solution and detect the results within 15 minutes.

[0055] 2.2 Results

[0056] (1) Effects of different ginseng-danshen ratios on the survival rate of H9c2 cells

[0057] The effects of different ginseng-tanshinone ratios on the survival rate of H9c2 cells are as follows: Figure 1 As shown, after administration of different ratios of ginseng and danshen, cell activity increased with increasing concentration at low concentrations, until it decreased at 8 μmol / L. Therefore, subsequent studies used different ratios of ginseng and danshen with a maximum concentration not exceeding 4 μmol / L to jointly administer the drugs to H9c2 cardiomyocytes (P<0.05).

[0058] (2) Effects of different ginseng-tanshinone ratios on the survival rate of doxorubicin-induced H9c2 cells

[0059] Depend on Figure 2 As shown, the cell viability of different ratios of ginseng and danshen combined with doxorubicin was detected by the MTT assay. The results showed that the 1:2 ratio of ginseng and danshen had the lowest EC50 value, indicating that the cell viability was optimal. Subsequent studies used the 1:2 ratio of ginseng and danshen to administer the drug to H9c2 cardiomyocytes (P<0.05).

[0060] (3) Effects of different concentrations of doxorubicin and ginseng-tanshinone in a 1:2 ratio (RD) on nuclei and apoptosis of H9c2 cardiomyocytes.

[0061] Hoechst 33258 is a blue fluorescent dye that can penetrate cell membranes and has low cytotoxicity, often used to detect apoptosis. When cells undergo apoptosis, the nuclei of apoptotic cells appear densely stained or fragmented with dense staining. Figure 3 It can be seen that after modeling with doxorubicin at a concentration of 2 μmol / L, the cell morphology changed significantly, exhibiting an irregular shape, cell nucleus rupture, cell vacuolization, cell lysis, and a large number of apoptosis. After administration, the cell state gradually improved, and the number of apoptosis was significantly reduced in the 1 μmol / L and 2 μmol / L dosage groups. When the dosage was 2 μmol / L, the morphology gradually returned to spindle shape. (4) Effect of different concentrations of ginseng-tanshinone 1:2 ratio (RD) on serum enzyme index of DOX-induced myocardial injury

[0062] The measurement of CK-MB, TN-I, and LDH activities can be used as an auxiliary diagnostic tool for myocardial diseases and reflects the degree of cardiac damage under pathological conditions. Elevated serum CK-MB levels indicate loss of cardiomyocyte structural integrity, while changes in MDA and SOD activities can reflect the degree of oxidative stress damage to myocardial tissue. Results are as follows... Figure 4As shown, compared with the normal group, the serum CK-MB, LDH, and MDA activities in the model group were significantly increased, while SOD activity was decreased. Serum CK-MB activity, LDH, and MDA levels were significantly decreased in all RD dose groups and the Dex group. SOD activity was significantly increased in all RD dose groups and the Dex group. These results indicate that DOX can induce cardiac injury and oxidative stress damage in rats. RD and dexzosen can reduce oxidative stress damage and alleviate DOX-induced cardiac injury.

[0063] 3. Animal experiments

[0064] 3.1 Experimental Methods

[0065] 3.1.1 Preparation of RD aqueous decoction

[0066] Ginseng and salvia miltiorrhiza were soaked in 12 times their volume of distilled water for 60 minutes. Then, the herbs and water were poured into a decoction pot and heated to a boil over high heat. After a slight boil, the decoction was simmered over low heat for another 60 minutes. The decoction was then filtered, and 6 times their volume of distilled water was added. The decoction was filtered again. Finally, the two filtrates were combined and concentrated to a concentration equivalent to 1 g / mL of the raw herb of the RD herb pair. The solution was then stored at -20°C.

[0067] 3.1.2 Establishment of animal models, experimental grouping and drug administration

[0068] SD rats were randomly divided into 6 groups (n=9): a blank control group (administered with an equal volume of physiological saline), a DOX model group (DOX 15 mg / kg), a low-dose RD + DOX group (3.3 g / kg), a high-dose RD + DOX group (6.6 g / kg), and a dexrazoxane group (DZR; 150 mg / kg). RD was administered by gavage one week before and two weeks after DOX administration. DOX was administered intraperitoneally in three divided doses (days 8, 13, and 18) within two weeks after administration, with each model group receiving the same dose (5 mg / kg), for a cumulative dose of 15 mg / kg. The DOX group received an intraperitoneal injection of DZR (150 mg / kg, with a dose ratio of 10:1 to DOX according to the instructions) 30 minutes before the DOX injection. The control group received an equal volume of physiological saline intraperitoneally.

[0069] 3.1.3 Echocardiography

[0070] After the last injection, rats were anesthetized by inhalation of isoflurane (induction with 1% oxygen and 5% isoflurane, maintenance with 1% oxygen and 2% isoflurane). The 3100LT ultra-high resolution small animal ultrasound imaging system was used to assess left ventricular function in rats. Left ventricular end-systolic diameter (LVDS), end-diastolic diameter (LVDS), left ventricular ejection fraction (EF%), and left ventricular shortening fraction (FS%) were measured. At the end of the experiment, rats were anesthetized with sodium pentobarbital, and blood was collected under sterile conditions. Serum was separated at 3000 rpm for 10 minutes. The hearts were then dissected and stored separately in a -80°C freezer and 4% paraformaldehyde.

[0071] 3.1.4 Collection of serum and organ samples

[0072] At the end of the experiment, rats were anesthetized with sodium pentobarbital. Blood was collected from the abdominal aorta under sterile conditions using vacuum blood collection tubes. The blood was centrifuged at 3500 rpm and 4℃ for 10 min. The supernatant was then collected to obtain serum. The obtained serum was aliquoted and stored in a -80℃ refrigerator.

[0073] 3.1.5 Myocardial Histopathology

[0074] After rinsing with physiological saline, cardiac tissue was fixed with 4% paraformaldehyde for at least 24 hours. The tissue was then trimmed, dehydrated, paraffin-embedded, embedded, sectioned, and stored at room temperature for later use, followed by Masson staining. CK-MB, LDH, MDA, and SOD activity assays were performed strictly according to the kit instructions. Positive stained areas were analyzed using ImageJ software to assess cardiac morphology and the degree of myocardial fibrosis.

[0075] 3.2 Results

[0076] (1) Body weight and heart weight of rats

[0077] Rats were administered DOX at a cumulative dose of 15 mg / kg to simulate DOX-induced subacute myocardial injury. DZR was used as a positive control. Figure 5 As shown, the control group rats showed no trend of weight loss. At 10 days, the DOX group showed a trend of weight loss and a decrease in heart weight. The DZR group and the RD treatment group rats showed a slow increase in weight and a certain degree of recovery in heart weight loss.

[0078] (2) Cardiac function testing

[0079] Echocardiographic data such as Figure 6 As shown, the LVEDD and LVESD values ​​of mice in the DOX group were significantly higher than those in the control group. After RD treatment, both of these indicators decreased. The FS and EF of the DOX group were significantly reduced, indicating decreased myocardial contractility. The FS significantly increased after RD administration, indicating that RD treatment improved left ventricular function.

[0080] (3) Histopathological changes

[0081] Myocardial tissue pathological changes results as follows Figure 7 As shown, the myocardial tissue in the control group was neatly arranged. Significant myocardial fiber breakage was observed in the DOX group, while RD treatment mediated the recovery of myocardial fiber damage. In Massen staining, the hearts of DOX-treated mice showed a significant increase in collagen fibers compared to the control group. These results indicate that DOX can induce significant cardiac dysfunction in mice. Elevated serum CK-MB levels indicate loss of myocardial cell structural integrity.

[0082] (4) Results of the influence of serum enzyme indices on myocardial injury

[0083] The results are as follows Figure 8 As shown, compared with the normal group, the serum CK-MB, LDH, and MDA activities in the model group were significantly increased, while SOD activity was decreased. Serum CK-MB activity, LDH, and MDA levels were significantly decreased in all RD dose groups and the DZR group. SOD activity was significantly increased in all RD dose groups and the DZR group. These results indicate that DOX can induce cardiac injury and oxidative stress damage in rats. RD and DZR can significantly alleviate cardiac injury and oxidative stress damage, and can reduce DOX-induced cardiac dysfunction.

[0084] In summary, the above studies demonstrate that the ginseng and danshen combination can improve DOX-induced cardiotoxicity by alleviating DOX-induced cardiac dysfunction, oxidative damage, and apoptosis at different levels, both in vivo and in vitro, which is of great significance for the prevention and treatment of DOX-induced cardiotoxicity.

[0085] Example 2: Protective effect of ginseng, salvia miltiorrhiza, and astragalus combination against doxorubicin-induced cardiac injury.

[0086] 1. Preparation of Ginseng, Salvia miltiorrhiza, and Astragalus membranaceus Combination (RDH)

[0087] Take ginseng, salvia miltiorrhiza, and astragalus membranaceus, add 12 times their volume of distilled water and soak for 60 minutes, heat to boiling, simmer for 60 minutes, filter the decoction, add 6 times their volume of distilled water, repeat the above steps, filter the decoction, combine the two filtrates and concentrate to the equivalent of 1 g / mL of each herb's raw weight. Based on the example, with ginseng:salvia miltiorrhiza = 1:2 as the base, add astragalus membranaceus, prepare the decoction according to the ginseng:salvia miltiorrhiza:astragalus ratio of 1:2:1, 1:2:2, and 1:2:3, and store at -20℃.

[0088] 2. Cell experiments

[0089] 2.1 Experimental Methods

[0090] 2.1.1 Culture and inoculation of H9C2 cells

[0091] Adjust the H9C2 cell concentration to 5 × 10⁻⁶. 4 / ml; seed the cell suspension into 96-well plates, 100μl per well, with 6 replicates per group; place the 96-well plates in a cell culture incubator for further culture; after the cells adhere, add the corresponding concentration of drug according to the intervention method for each group.

[0092] The model group cells were treated with 2 μM DOX at a dose of 10% of the total culture medium. The experimental groups included a blank control group, a DOX model group (DOX 2 μM), and groups treated with different proportions and concentrations of ginseng, danshen, and astragalus. The blank control group received an equal volume of culture medium, while the other treatment groups received different concentrations of the drug solution at 10% of the total culture medium. Cells were collected after 24 hours, and MTT was added after cell treatment to calculate cell viability.

[0093] 2.2 Results

[0094] (1) Effects of different ratios of ginseng, salvia miltiorrhiza, and astragalus on the survival rate of H9c2 cells

[0095] The results are as follows Figure 9 As shown, when the drug concentration was between 0.5 and 4 mg / ml, the survival rate of H9C2 rat cardiomyocytes was not affected compared to the control group. However, when the drug concentration exceeded 4 mg / ml, cell survival was significantly inhibited. Therefore, in subsequent experiments, 0.5–2 mg / ml of ginseng, danshen, and astragalus was selected as the drug concentration.

[0096] (2) Effects of different ratios of ginseng, salvia miltiorrhiza, and astragalus on the survival rate of H9c2 cells induced by doxorubicin

[0097] Depend on Figure 10 As shown, after determining the dosing concentration as 0.5–2 mg / ml, cell viability was detected under different drug solution ratios in combination with DOX administration, and EC50 was calculated. 50 Value. When ginseng: danshen: astragalus = 1:2:1, EC... 50 When the EC value is 1.07, and the ratio of ginseng: salvia miltiorrhiza: astragalus is 1:2:2, the EC value is 1.07. 50 When the EC value is 1.24, and the ratio of ginseng: salvia miltiorrhiza: astragalus is 1:2:3, the EC value is 1.24. 50 The value was 6.24. In conclusion, in subsequent experiments, the optimal ratio of ginseng: danshen: astragalus was selected for administration.

[0098] (3) Protective effects of ginseng, salvia miltiorrhiza, and astragalus (RDH) on different species of myocardial cells under DOX treatment

[0099] Three different cell lines—H9C2 rat cardiomyocytes, HL-1 mouse cardiomyocytes, and AC16 human cardiomyocytes—were selected. Serum containing ginseng, danshen, and astragalus was used to administer the drugs, and the protective effect of the drugs against doxorubicin-induced myocardial damage was verified. The experimental results are as follows: Figure 11As shown, serum at different concentrations and doses had no damaging effect on any of the three types of cardiomyocytes. After combined DOX administration, 10% serum showed the best recovery effect on cardiomyocyte damage induced by HL-1 and AC16 doxorubicin; 2.5% serum showed the best recovery effect on H9C2 cardiomyocyte damage.

[0100] 3. Animal experiments

[0101] 3.1 Experimental Methods

[0102] 3.1.2 Establishment of animal models, experimental grouping and drug administration

[0103] SD rats were randomly divided into 6 groups (n=9): a blank control group (administered with an equal volume of physiological saline), a DOX model group (DOX 15 mg / kg), a low-dose RDH + DOX group (3.3 g / kg), a high-dose RDH + DOX group (6.6 g / kg), and a dexrazoxane group (DZR; 150 mg / kg). RDH was administered by gavage one week before and two weeks after DOX administration. DOX was administered intraperitoneally in three divided doses (days 8, 13, and 18) within two weeks after administration, with each model group receiving the same dose (5 mg / kg), for a cumulative dose of 15 mg / kg. The DOX group received an intraperitoneal injection of DZR (150 mg / kg, with a dose ratio of 10:1 to DOX according to the instructions) 30 minutes before the DOX injection. The control group received an equal volume of physiological saline intraperitoneally.

[0104] 3.1.3 Echocardiography

[0105] After the last injection, rats were anesthetized by inhalation of isoflurane (induction with 1% oxygen and 5% isoflurane, maintenance with 1% oxygen and 2% isoflurane). The 3100LT ultra-high resolution small animal ultrasound imaging system was used to assess left ventricular function in rats. Left ventricular end-systolic diameter (LVDS), end-diastolic diameter (LVDS), left ventricular ejection fraction (EF%), and left ventricular shortening fraction (FS%) were measured. At the end of the experiment, rats were anesthetized with sodium pentobarbital, and blood was collected under sterile conditions at 3000 rpm for 10 minutes to separate serum. The hearts were then dissected and stored separately in a -80°C freezer and 4% paraformaldehyde.

[0106] 3.1.4 Collection of serum and organ samples

[0107] At the end of the experiment, rats were anesthetized with sodium pentobarbital, and blood was collected from the abdominal aorta under sterile conditions using vacuum blood collection tubes. The blood was centrifuged at 3500 rpm and 4°C for 10 min, and the supernatant was collected to obtain serum. The serum was aliquoted and stored at -80°C. After blood collection, the heart was dissected, and the wet weight was measured and recorded after absorbing water with filter paper.

[0108] 3.1.5 Myocardial Histopathology

[0109] After rinsing with physiological saline, cardiac tissue was fixed with 4% paraformaldehyde for at least 24 hours. The tissue was then trimmed, dehydrated, paraffin-embedded, embedded, sectioned, and stored at room temperature for later use, followed by Masson staining. CK-MB, LDH, MDA, and SOD activity assays were performed strictly according to the kit instructions. Positive stained areas were analyzed using ImageJ software to assess cardiac morphology and the degree of myocardial fibrosis.

[0110] 3.2 Results

[0111] The results are as follows Figure 12 As shown, daily changes in rat body weight were recorded, and the hearts of rats in each group were weighed. The body weight of rats in the model group decreased daily, and the heart weight / body weight ratio increased significantly after modeling, indicating cardiac hypertrophy. The drug-treated groups significantly alleviated cardiac hypertrophy. Ultrasound analysis showed that, compared with the normal group, the model group rats had significantly increased ejection fraction (EF) and fractional shortening (FS), and significantly decreased left ventricular end-systolic diameter, left ventricular end-diastolic diameter, left ventricular end-systolic volume, and left ventricular end-diastolic volume. Furthermore, after administration of different doses of ginseng, danshen, and astragalus, the related cardiac dysfunction in rats was significantly improved.

[0112] The results of ginseng, danshen, and astragalus in alleviating doxorubicin-induced myocardial tissue damage and oxidative stress are as follows: Figure 13 As shown in the Massen staining, the hearts of DOX-treated mice showed a significant increase in collagen fibers compared to the control group. Treatment with ginseng, danshen, and astragalus alleviated DOX-induced cardiac dysfunction, manifested as tissue structure recovery, reduced fibrosis area, and decreased serum CK-MB, LDH, and TN-I levels.

[0113] In summary, the above studies demonstrate that the combination of ginseng, salvia miltiorrhiza, and astragalus can improve DOX-induced cardiotoxicity by alleviating DOX-induced cardiac dysfunction, oxidative damage, and apoptosis at different levels, both in vivo and in vitro. This is of great significance for the prevention and treatment of DOX-induced cardiotoxicity.

[0114] Example 3: Control Experiment

[0115] Adjust the H9C2 cell concentration to 5 × 10⁻⁶. 4 / ml; seed the cell suspension into 96-well plates, 100μl per well, with 6 replicates per group; place the 96-well plates in a cell culture incubator for further culture; after the cells adhere, add the corresponding concentration of drug according to the intervention method for each group.

[0116] The model group cells were treated with 2 μM DOX at a dose of 10% of the total culture medium. The control group received the same amount of culture medium, while the other treatment groups received different concentrations of DOX at 10% of the total culture medium (Ginseng 1 received 1 mg / ml of ginseng solution, and Ginseng 2 received 2 mg / ml of ginseng solution; the same applies thereafter). Cells were collected after 24 hours, and MTT was added after cell treatment to calculate cell viability.

[0117] The results are as follows Figure 14 As shown, compared with the blank group, the activity of H9C2 cells in the model group was significantly reduced. However, compared with the model group, ginseng, salvia miltiorrhiza, and astragalus alone did not increase the activity of H9C2 cells. In contrast, the ginseng-salvia miltiorrhiza combination and the ginseng-salvia miltiorrhiza-astragalus combination described in this application significantly improved the DOX-induced activity of H9C2 cells.

[0118] The above results indicate that the composition of ginseng, salvia miltiorrhiza, or ginseng, salvia miltiorrhiza, and astragalus described in this application can prevent and treat cardiotoxicity induced by doxorubicin, promote the survival rate of doxorubicin-induced cardiomyocyte H9c2, reduce doxorubicin-induced apoptosis of cardiomyocyte H9c2, reduce doxorubicin-induced oxidative stress damage and cardiac damage, improve left ventricular function, and improve doxorubicin-induced cardiotoxicity. This is of great significance for the prevention and treatment of azithromycin-induced cardiotoxicity.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of drugs for preventing and treating cardiotoxicity caused by doxorubicin; characterized in that, The traditional Chinese medicine composition consists of ginseng and salvia miltiorrhiza; the ratio of ginseng to salvia miltiorrhiza is 1:

2.

2. The application of a traditional Chinese medicine composition in the preparation of drugs for preventing and treating cardiotoxicity caused by doxorubicin; characterized in that, The traditional Chinese medicine composition consists of ginseng, salvia miltiorrhiza, and astragalus, and the ratio of ginseng, salvia miltiorrhiza, and astragalus is 1:2:

1.

3. The application as described in any one of claims 1-2, characterized in that, The traditional Chinese medicine composition is formulated into any pharmaceutically acceptable dosage form by adding a pharmaceutically acceptable carrier.

Images