Application of pentagalloylglucose in preparation of medicine for resisting doxorubicin cardiotoxicity

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically to the use of pentagalloylglucose in the preparation of drugs to resist doxorubicin cardiotoxicity. Background Art

[0002] Doxorubin (DOX) is one of the most effective broad-spectrum anticancer anthracycline antibiotics. It was first discovered in 1960 and is commonly used to treat solid tumors and hematological tumors. It can be used alone or in combination with other anticancer drugs. Doxorubicin can prolong the survival of cancer patients. However, doxorubicin treatment can cause a variety of side effects, the most common and serious of which is doxorubicin-induced cardiotoxicity (DIC). About 10% of patients with anthracycline-induced cardiotoxicity develop within 3.5 months after the final chemotherapy, and about 98% of patients develop cardiotoxicity one year after treatment. These cardiotoxic side effects significantly inhibit the use of anthracyclines, especially doxorubicin. The current treatment strategies for DIC patients are mainly anti-heart failure and anti-arrhythmic treatments, but their effects are limited. To date, there is still a lack of specific therapies that can prevent DIC in clinical practice, especially in heart transplantation. Therefore, exploring the mechanism of action of DIC and finding preventive mechanisms and possible treatment options are urgent issues that need to be addressed.

[0003] Pentagalloylglucose (PGG) is a natural hydrolyzable tannin that belongs to the gallotannin family, but also participates in the formation of ellagitannins. PGG is composed of five galloyl groups, with glucose as its core, and is widely found in a variety of natural plants and Chinese herbal medicines. PGG has attracted much attention due to its antibacterial, anti-inflammatory, anti-cancer, anti-diabetic, antioxidant and anti-angiogenic functions (Cristian Torres-L, et al. Pentagalloylglucose (PGG): A valuable phenolic compound with functional properties. Journal of Functional Foods 37, 176-189 (2017).). Studies have reported that PGG can act on multiple targets and inhibit several abnormal signaling pathways in cancer cells. The mechanisms by which PGG exerts its anti-cancer effects include increasing oxidative stress, preventing the proliferation of abnormally growing cells at cell cycle checkpoints, enhancing autophagy and apoptosis of cancer cells, and inhibiting angiogenesis and metastasis. The potential therapeutic targets of PGG are transcription factors, namely STAT3 and NF-кB, and growth factors, including vascular endothelial growth factor (VEGF). PGG has anti-inflammatory activity, and its main therapeutic targets are TNF-a, interferon, interleukin, and MCP-1. PGG regulates tumor growth by inhibiting several pro-angiogenic stimuli. It inhibits VEGF-induced proliferation of human umbilical vein endothelial cells and growth of immortalized human microvascular endothelial cells by inhibiting the binding of VEGF to its receptor. However, whether pentagalloylglucose can improve doxorubicin-induced cardiotoxicity and its related mechanisms are still unclear. Therefore, this study established a mouse model of doxorubicin-induced cardiotoxicity to explore whether pentagalloylglucose can improve doxorubicin-induced cardiotoxicity. Summary of the invention

[0004] The purpose of the present invention is to provide an application of pentagalloylglucose in the preparation of a drug for preventing and inhibiting myocardial toxicity caused by the anti-tumor chemotherapy drug adriamycin.

[0005] Furthermore, the structural formula of the pentagalloylglucose is as follows: .

[0006] Furthermore, the effective content of pentagalloylglucose in the drug is 0.1-100 μM.

[0007] Furthermore, pentagalloylglucose is used as the sole active ingredient or in combination with other drugs.

[0008] Furthermore, the drug is a drug mixture or a drug composition.

[0009] Furthermore, the mixture or pharmaceutical composition comprises pentagalloylglucose and doxorubicin.

[0010] A drug mixture or a drug composition, the active ingredient of which includes pentagalloylglucose, wherein the drug mixture or the drug composition has at least one of the following functions 1) to 5): 1) preventing and / or treating the myocardial toxicity of doxorubicin; 2) alleviating myocardial tissue inflammation caused by doxorubicin; 3) Reduce the increase of reactive oxygen free radicals caused by doxorubicin; 4) Alleviate the increase of myocardial fibrosis caused by doxorubicin; 5) Alleviate myocardial cell apoptosis caused by doxorubicin.

[0011] Furthermore, the drug mixture or drug composition is in any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions and solutions.

[0012] Doxorubicin is cardiotoxic, and pentagalloylglucose may improve the cardiotoxicity caused by doxorubicin. This may provide a new idea for the use of pentagalloylglucose to prevent and treat doxorubicin-induced cardiotoxicity, provide a theoretical basis and new therapeutic targets for the subsequent development of drugs to relieve DIC, promote the development of new DIC treatment methods, and reduce the side effects of doxorubicin, which will help expand the doxorubicin treatment window and reduce the economic burden on patients and the medical system caused by DIC, with certain economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the chemical structural formula of pentagalloylglucose; Figure 2 The results of the control group, PGG group, DOX group and DOX+PGG group on cardiomyocytes H9C2: (A) MTT assay to analyze the effects of different concentrations of PGG on cardiomyocytes. (B) MTT assay to analyze the effects of PGG on DOX-treated cardiomyocytes. (C) PGG can reduce the level of ROS induced by DOX, from left to right: DMSO, 20μM PGG, 1μM DOX and 1μM DOX+20μM PGG. PGG: Pentagalloylglucose.

[0014] Figure 3Pentagalloylglucose alleviated DOX-induced cardiotoxicity in C57BL / 6 mice: A, Schematic diagram of animal experiments. B, Representative echocardiogram of C57BL / 6 mice. C, Quantitative results of heart weight / tibia length. DE, Statistical graphs of left ventricular EF% and FS% in C57BL / 6 mice. FG, ELISA kits were used to detect the levels of cTnT and BNP in mouse serum. *, **, *** and **** indicate p<0.05, p<0.01, p<0.001 and p<0.0001, respectively. PGG: Pentagalloylglucose.

[0015] Figure 4 The myocardial tissues of C57BL / 6J mice treated with four different methods were subjected to (A) H&E staining; (B) MASSON staining; (C) WGA staining to characterize the levels of cardiac inflammation, fibrosis, cardiomyocyte apoptosis and fibrosis in mice. (D) is the quantitative result of (C). PGG: Pentagalloylglucose. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below in conjunction with the embodiments of the drawings. It should be understood that the embodiments described in this specification are only for explaining the present invention, not for limiting the present invention, and the parameters, proportions, etc. of the embodiments can be selected according to local conditions without substantial impact on the results.

[0017] 1. Experimental methods: 1.1 Cell culture and treatment: H9C2 cells were cultured in DMEM medium (containing 10% FBS and penicillin-streptomycin) at 37°C and 5% CO2.

[0018] 1.2 MTT cytotoxicity test: Collect logarithmic phase cells, adjust the concentration of cell suspension, add 100uL cell suspension to each well, plate the H9C2 cardiomyocytes to be tested to a density of 5000 cells / well, and culture at 37°C and 5% CO2. After the cells adhere to the wall, add PGG for drug treatment, so that the final concentration of PGG in the cell suspension is 0 μM, 0.1μM, 0.5 μM, 1μM, 5 μM, 10 μM, 20 μM, 40μM, 60μM, and 100 μM; after about 24 hours, add culture medium containing MTT, incubate at 37°C for 2~4 hours, discard the MTT solution, add 100μL DMSO to each well, place on a shaker and shake for 10 minutes to fully dissolve the crystals, and use an enzyme marker to measure the absorbance value of each well, i.e. OD 490 nm, the analysis results are as follows Figure 2 As shown in A.

[0019] 1.3 MTT method for cell proliferation assay: Collect cells in the logarithmic phase, adjust the concentration of the cell suspension, add 100uL of cell suspension to each well, plate the H9C2 cardiomyocytes to be tested to a density of 5000 cells / well, and culture at 37°C and 5% CO2. After the cells adhere to the wall, add DOX or PGG for drug treatment, and divide the drug addition groups into DMSO, 20μM PGG, 40μM PGG, 1μM DOX, 1μM DOX+20μM PGG, and 1μM DOX+40μM PGG; after about 24h, add culture medium containing MTT, incubate at 37°C for 2~4h, then discard the MTT solution, add 100μL DMSO to each well, place on a shaker for 10min to fully dissolve the crystals, and use an enzyme marker to measure the absorbance value of each well, i.e., OD 490 nm, analysis results.

[0020] 1.4 DIC model construction: 8-week-old C57BL / 6J mice were used and divided into groups: blank control group (Control), model group (DOX), single-dose group (PGG) and combined-dose treatment group (DOX+PGG). Except for the control group, which was intraperitoneally injected with normal saline, the model group was intraperitoneally injected with DOX at a dose of 18 mg / kg, once; the single-dose group was intragastrically administered with PGG at a dose of 20 mg / kg, once a day, for one week; the combined-dose treatment group DOX+PGG was first intraperitoneally injected with DOX at a dose of 18 mg / kg, and then PGG was administered by gavage at a dose of 20 mg / kg, once a day, for one week. After the last dose, the mice were examined by chest echocardiography using the Vevo 3100 instrument to obtain indicators such as ejection fraction (EF%) and shortening fraction (FS%).

[0021] 1.5 HE staining: Fix the wax block on the slicer and set the slice thickness to 5µm in advance. After cutting the appropriate slice, carefully float it on the water with a brush, and pick it up with a clean slide after it is fully stretched out and dry it; finally, put the slide into the slide box, bake it in a 37℃ oven overnight, and store it at room temperature. Then, HE stain the paraffin sections of the experimental mouse heart tissue: first dewax with xylene, then rehydrate the paraffin tissue with different proportions of ethanol, use hematoxylin to stain for about 2 minutes, differentiate with differentiation solution for about 3-5s, rinse with ddH2O for 10 minutes, stain with eosin for 1 minute, rinse with tap water for 10 minutes, dehydrate with 95% EtOH (2min) and 100% EtOH (2min) in turn, seal the slide, and take pictures to count the results.

[0022] 1.6 Masson staining: The dewaxed experimental mouse heart sections were stained with Masson trichrome staining kit (Solarbio, GI1340) and Weigert iron hematoxylin for 5-10 minutes, the excess stain was washed off with distilled water, acidic ethanol differentiation solution was added, washed with water, blued with Masson blueing solution for 3-5 minutes, washed with distilled water for 30 seconds, stained with Ponceau fuchsin staining solution for 5-10 minutes, washed with weak acid working solution for 30 seconds, the excess liquid was poured off, phosphomolybdic acid solution was added for 1-2 minutes, the excess liquid was poured off, aniline blue staining solution was added for 1-2 minutes, washed with weak acid working solution for 30 seconds, dehydrated with 95% ethanol and anhydrous ethanol in turn, transparentized with xylene twice, 1-2 dishes each time, sealed with neutral glue and photographed and recorded.

[0023] 1.7 WGA staining: H9C2 cells were fixed with 4% formaldehyde and 100 µL iFluor 647-WGA working solution was added. The cells were incubated with WGA working solution for 30 minutes at room temperature, then washed twice with HHBS buffer and photographed on a fluorescence microscope using a Cy5 filter set.

[0024] 1.8 Reactive oxygen species (ROS) detection: H9C2 cardiomyocytes in the logarithmic growth phase were collected, DCFH-DA was diluted at a ratio of 1:1000 using serum-free culture medium, and the collected cells were resuspended in the diluted DCFH-DA working solution to a cell density of 1.0×10 7 cells / mL, and then placed in a 37℃ cell culture incubator in the dark for 20 minutes, inverted and mixed every 5 minutes to allow the probe and cells to fully contact. Then wash the cells three times with serum-free culture medium, suspend the cells in serum-free culture medium, divide them into several equal parts, and then stimulate with drugs, incubate them in a 37℃ cell culture incubator in the dark for 20 minutes, and finally observe and record them with a laser confocal microscope.

[0025] 1.9 ELISA experiment: Take the mouse serum supernatant and use the ELISA kit to detect the levels of cardiac injury markers such as cTnT and BNP: first dilute the standard sample in a gradient concentration, and then set up blank wells (blank control wells do not contain samples, enzyme-labeled reagents, and biotin-labeled antibodies, and the rest of the steps are the same) and test sample wells. First add 40μL of sample to the test sample well on the enzyme-labeled plate, and then add 10μL of biotin-labeled antibody (add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the wall of the well, and gently shake to mix). Add 50μL of enzyme-labeled reagent to each well, seal the plate with a sealing film, and incubate at 37°C for 30 minutes. Discard the supernatant, add the colorimetric solution and incubate for 10 minutes, and finally add the stop solution to measure the OD 450 nm and analyze the results.

[0026] 2. Experimental results: (1) Pentagalloylglucose can alleviate DOX-induced cardiotoxicity in cardiomyocytes In order to clarify the pentagalloylglucose (structural formula Figure 1 As shown in Figure 2, the potential mechanism of reducing DOX-induced cardiomyocyte injury was studied in this application. This application conducted a cell experiment. This application conducted an MTT experiment to detect the killing effect of pentagalloyl glucose on H9C2 cells. Figure 2 As shown in A, pentagalloylglucose does not affect the growth of H9C2, indicating that pentagalloylglucose has low toxicity to cells. Next, the present application uses MTT experiments to determine the combined killing effect of DOX and pentagalloylglucose in H9C2 cardiomyocytes. The results show that pentagalloylglucose can indeed reverse DOX-induced cardiomyocyte damage ( Figure 2 A). In addition, the present application also detected the level of reactive oxygen species (ROS) in cells. The results showed that compared with the control group, DOX (1 μM) could significantly increase the level of ROS in cardiomyocytes, while the combined administration of pentagalloylglucose (20 μM) and DOX (1 μM) could significantly reverse this phenomenon ( Figure 2 C). These results suggest that pentagalloylglucose can alleviate DOX-induced cardiotoxicity in cardiomyocytes.

[0027] (2) Pentagalloylglucose can alleviate DOX-induced cardiotoxicity in mice.

[0028] The present application studies the effect of pentagalloyl glucose on DIC. To this end, the present application constructed a DIC model using C57BL / 6 male mice, such as Figure 3 As shown in A. As expected, the ratio of ventricular weight to tibia length in the DOX-treated group decreased significantly, while the ratio of ventricular weight to tibia length in the pentagalloyl glucose co-administration group increased significantly, indicating that pentagalloyl glucose alleviated DOX-induced cardiotoxicity ( Figure 3 C). Echocardiography showed ( Figure 3 B) When treated with DOX, the left ventricular contraction fraction (FS%) and ejection fraction (EF%) of mice were significantly reduced ( Figure 3 DE), indicating that the cardiac function of mice was significantly reduced; while the left ventricular contraction fraction (FS%) and ejection fraction (EF%) of mice in the pentagalloylglucose combined treatment group increased significantly. In addition, compared with the DOX group, the levels of myocardial injury markers (i.e., cTnT and BNP) in the myocardial tissue homogenate of C57BL / 6 mice treated with pentagalloylglucose were also significantly reduced ( Figure 3 FG). In summary, pentagalloylglucose alleviated DOX-induced cardiotoxicity in mice.

[0029] (3) Histological analysis showed that pentagalloylglucose alleviated DOX-induced myocardial toxicity in mice The present application also found through histological analysis that H&E staining results showed that DOX enhanced myocardial cell inflammatory infiltration, while the addition of pentagalloylglucose alleviated this phenomenon ( Figure 4 A); MASSON staining results showed that the level of myocardial inflammation in DOX-treated myocardial tissue was significantly upregulated, and pentagalloylglucose could significantly downregulate the level of DOX-induced myocardial inflammation ( Figure 4 B); The cardiomyocytes of DIC mice shrank, and the administration of PGG also significantly restored the size of cardiomyocytes ( Figure 4 CD). The above results indicate that pentagalloylglucose can alleviate DOX-induced cardiotoxicity in mice.

[0030] Apoptosis and oxidative stress damage are one of the main mechanisms of doxorubicin-induced cardiotoxicity. PGG can induce multiple types of cell death, including apoptosis and autophagy. In the present application, by comparing with the control group, the present application found that the level of oxidative stress factor ROS and the proportion of apoptosis in mice in the doxorubicin group were significantly increased. This phenomenon shows that doxorubicin can significantly enhance the apoptosis rate and oxidative stress damage of cardiomyocytes. After the intervention of pentagalloylglucose, the ROS level and apoptosis level of mice were significantly reduced, indicating that pentagalloylglucose can effectively reduce the apoptosis and oxidative stress damage of cardiomyocytes caused by doxorubicin. Therefore, the research results of the present application further support that pentagalloylglucose can be used as a potential protective agent to counteract the cardiotoxicity caused by doxorubicin.

Claims

1. Application of pentagalloylglucose in the preparation of drugs for preventing and inhibiting myocardial toxicity caused by the anti-tumor chemotherapy drug doxorubicin.

2. The use according to claim 1, characterized in that: The structural formula of the pentagalloylglucose is as follows: .

3. The use according to claim 1, characterized in that: The effective content of pentagalloylglucose in the drug is 0.1-100 μM.

4. The use according to claim 1, characterized in that: Pentagalloylglucose is used as the sole active ingredient or in combination with other drugs.

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

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

7. A drug mixture or a drug composition, characterized in that: The active ingredient includes pentagalloylglucose, and the drug mixture or drug composition has at least one of the following functions 1) to 5): 1) To prevent and / or treat doxorubicin-induced cardiotoxicity; 2) Alleviate myocardial tissue inflammation caused by doxorubicin; 3) Reduce the increase of reactive oxygen free radicals caused by doxorubicin; 4) Alleviate the increase in myocardial fibrosis caused by doxorubicin; 5) Alleviate myocardial cell apoptosis induced by doxorubicin.

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

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