Use of pentagalloyl glucose in the preparation of an anti-doxorubicin cardiotoxicity drug

Abstract

The application discloses application of pentagalloyl glucose (PGG) in preparation of an anti-doxorubicin cardiotoxicity medicine. The process is as follows: a myocardial cell H9C2 injury model is induced and established by doxorubicin (DOX), and a minimum concentration of the myocardial cell injury induced by DOX is determined. After 20 muM of PGG is added to a treatment group, DOX-induced H9C2 cell apoptosis can be significantly improved. A mouse myocardial injury model is induced and established by DOX, compared with a doxorubicin group, the left ventricular systolic fraction (FS%) and the ejection fraction (EF%) of mice in the doxorubicin+pentagalloyl glucose group are significantly increased, the myocardial fiber structure is improved, cytoplasm vacuolization is reduced, the serum cTnT and BNP levels are reduced, and the ROS level is reduced. It is proved by the above experiments that the pentagalloyl glucose can significantly reduce DOX-induced myocardial toxicity.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the application of pentagalloyl glucose in the preparation of anti-doxorubicin cardiotoxic drugs. Background Technology

[0002] Doxorubin (DOX) is one of the most effective broad-spectrum anthracycline antibiotics for cancer treatment. First discovered in 1960, it is commonly used to treat solid tumors and hematologic malignancies, either alone or in combination with other anticancer drugs. Doxorubin can prolong the survival of cancer patients. However, doxorubicin treatment can cause various side effects, the most common and serious of which is doxorubicin-induced cardiotoxicity (DIC). Approximately 10% of patients experience DIC within 3.5 months after final chemotherapy, and about 98% develop DIC one year after treatment. These cardiotoxic side effects significantly inhibit the use of anthracyclines, especially doxorubicin. Current treatment strategies for DIC primarily involve anti-heart failure and antiarrhythmic therapies; however, their effectiveness is limited. To date, there is still a lack of specific therapies to prevent DIC, especially in heart transplantation. Therefore, exploring the mechanism of DIC and finding preventative mechanisms and possible treatment options are urgent problems that need to be addressed.

[0003] Pentagalloylglucose (PGG) is a naturally occurring, hydrolyzable tannin belonging to the gallotannin group, but it also participates in the formation of ellagitannins. PGG consists of five galloyl groups, with glucose at its core, and is widely found in various natural plants and traditional Chinese medicines. PGG has attracted considerable attention due to its antibacterial, anti-inflammatory, anticancer, antidiabetic, 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 anticancer effects include increasing oxidative stress, preventing the proliferation of abnormally growing cells at cell cycle checkpoints, enhancing autophagy and apoptosis in cancer cells, and inhibiting angiogenesis and metastasis. Potential therapeutic targets for PGG include transcription factors, namely STAT3 and NF-κB, and growth factors, including vascular endothelial growth factor (VEGF). PGG possesses anti-inflammatory activity, with its main therapeutic targets being TNF-α, 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 the growth of immortalized human microvascular endothelial cells by inhibiting the binding of VEGF to its receptor. However, whether pentamalloyl glucose can improve doxorubicin-induced cardiotoxicity and the underlying mechanisms remain unclear. Therefore, this study established a mouse model of doxorubicin-induced cardiotoxicity to investigate whether pentamalloyl glucose can improve doxorubicin-induced cardiotoxicity. Summary of the Invention

[0004] The purpose of this invention is to provide the application of pentagalloglucopyranoside in the preparation of a drug for preventing and inhibiting myocardial toxicity caused by the antitumor chemotherapy drug doxorubicin.

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

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

[0007] Furthermore, pentagalloglucopyranoside can be 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 includes pengaloyl glucose and doxorubicin.

[0010] A pharmaceutical mixture or composition having an active ingredient comprising pentagalloglucoside, said pharmaceutical mixture or composition having at least one of the following functions: 1) prevention and / or treatment of doxorubicin cardiotoxicity; 2) relief of doxorubicin-induced myocardial tissue inflammation;

[0011] 3) Reduces the increase of reactive oxygen species caused by doxorubicin; 4) Alleviates the increase of myocardial fibrosis caused by doxorubicin; 5) Alleviates myocardial cell apoptosis caused by doxorubicin.

[0012] Furthermore, the drug mixture or drug composition is any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions, and solutions.

[0013] Doxorubicin has cardiotoxicity, and pentagalloglucopyranoside may improve the cardiotoxicity caused by doxorubicin. This may provide a new approach for using pentagalloglucopyranoside to prevent and treat doxorubicin-induced cardiotoxicity, providing a theoretical basis and new therapeutic targets for the subsequent development of drugs to alleviate dissipation of chronic obstructive inflammatory disease (DIC), promoting the development of new DIC treatments, and reducing the side effects of doxorubicin, which helps to expand the therapeutic window of doxorubicin and reduce the economic burden on patients and the healthcare system caused by DIC, thus having certain economic and social benefits. Attached Figure Description

[0014] Figure 1 The chemical structural formula of pentagalloyl glucose;

[0015] Figure 2 Figure 1 shows the effects of the control group, PGG group, DOX group, and DOX+PGG group on H9C2 cardiomyocytes: (A) MTT assay analysis of the effect of different concentrations of PGG on cardiomyocytes. (B) MTT assay analysis of the effect of PGG on DOX-treated cardiomyocytes. (C) PGG can reduce DOX-induced ROS levels, from left to right: DMSO, 20 μM PGG, 1 μM DOX, and 1 μM DOX+20 μM PGG. PGG: Pentagalloy glucose.

[0016] Figure 3Pentagalloylglucose alleviates 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 graph of left ventricular EF% and FS% in C57BL / 6 mice. FG. Detection of serum cTnT and BNP levels in mice using an ELISA kit. *, **, ***, and **** represent p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. PGG: Pentagalloylglucose.

[0017] Figure 4 To characterize the levels of cardiac inflammation, fibrosis, cardiomyocyte apoptosis, and fibrosis in C57BL / 6J mice under four different treatments, the myocardial tissues were stained with (A) H&E, (B) MASSON, and (C) WGA. (D) is the quantitative result of (C). PGG: Pentagalloylglucose. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention. The parameters, proportions, etc. of the embodiments can be selected according to local conditions without having a substantial impact on the results.

[0019] 1. Experimental Method:

[0020] 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.

[0021] 1.2 MTT Assay for Cytotoxicity: Logarithmic growth phase cells were collected, and the cell suspension concentration was adjusted. 100 μL of cell suspension was added to each well, and the cells were seeded to achieve a density of 5000 H9C2 cardiomyocytes / well. Cells were cultured at 37°C and 5% CO2. After adhesion, PGG was added for treatment, with final PGG concentrations in the cell suspension successively set to 0 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, and 100 μM. After approximately 24 hours, culture medium containing MTT was added, and the cells were incubated at 37°C for 2–4 hours. The MTT solution was then discarded, and 100 μL of DMSO was added to each well. The cells were then gently shaken on a shaker for 10 minutes to fully dissolve any crystals. The absorbance (OD) of each well was measured using a microplate reader. 490 nm, the analysis results are as follows Figure 2 As shown in Figure A.

[0022] 1.3 MTT Assay for Cell Proliferation: Cells in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted. 100 μL of cell suspension was added to each well, and the cells were seeded to achieve a density of 5000 H9C2 cardiomyocytes / well. Cells were cultured at 37°C and 5% CO2. After adhesion, DOX or PGG was added for treatment. The treatment groups were: DMSO, 20 μM PGG, 40 μM PGG, 1 μM DOX, 1 μM DOX + 20 μM DOX, and 1 μM DOX + 40 μM PGG. After approximately 24 hours, culture medium containing MTT was added, and the cells were incubated at 37°C for 2–4 hours. The MTT solution was then discarded, and 100 μL of DMSO was added to each well. The cells were then shaken gently for 10 minutes to dissolve any crystals. The absorbance (OD) of each well was measured using a microplate reader. 490 nm, analysis results.

[0023] 1.4 DIC Model Construction: Eight-week-old C57BL / 6J mice were used and divided into three groups: a blank control group (Control), a model group (DOX), a single-drug administration group (PGG), and a combined-drug treatment group (DOX+PGG). Except for the control group, which received intraperitoneal injection of saline, the model group received DOX via intraperitoneal injection at a dose of 18 mg / kg, administered once daily. The single-drug administration group received PGG via gavage at a dose of 20 mg / kg, once daily for one week. The combined-drug treatment group received a single intraperitoneal injection of DOX at a dose of 18 mg / kg, followed by PGG via gavage at a dose of 20 mg / kg, once daily for one week. After the last administration, thoracic echocardiography was performed on the mice using a Vevo 3100 instrument to obtain indicators such as ejection fraction (EF%) and fractional shortening (FS%).

[0024] 1.5 HE staining: The paraffin block was fixed on the microtome, and the section thickness was set to 5µm in advance. After cutting a suitable section, it was carefully floated on water with a brush until it was fully extended. Then, it was lifted out with a clean glass slide and dried. Finally, the slide was placed in a slide box and dried overnight in an oven at 37℃, and stored at room temperature. HE staining was then performed on paraffin sections of mouse heart tissue: First, xylene was used for dewaxing, followed by rehydration with ethanol of different proportions. Hematoxylin was used for staining for about 2 minutes, differentiation was performed with differentiation solution for about 3-5 seconds, rinsing with ddH2O for 10 minutes, followed by eosin staining for 1 minute, rinsing with tap water for 10 minutes, and dehydration was performed sequentially with 95% EtOH (2 min) and 100% EtOH (2 min). The slides were then mounted, photographed, and the results were analyzed.

[0025] 1.6 Masson staining: For dewaxed mouse heart sections, stain with Weigert iron hematoxylin using the Masson trichrome staining kit (Solarbio, GI1340) for 5-10 min. Wash away excess staining solution with distilled water, add acidic ethanol differentiation solution, wash with water, re-blue with Masson blue solution for 3-5 min, wash with distilled water for 30 s, stain with Ponceau S and fuchsin for 5-10 min, wash with weak acid working solution for 30 s, descaling off excess liquid, adding phosphomolybdic acid solution for 1-2 min, descaling off excess liquid, adding aniline blue staining solution for 1-2 min, washing with weak acid working solution for 30 s, dehydrating with 95% ethanol and anhydrous ethanol sequentially, clearing twice with xylene, 1-2 dishes each time, mounting with neutral glue, and then photographing and recording.

[0026] 1.7 WGA staining: H9C2 cells were fixed with 4% formaldehyde and then incubated with 100 µL of iFluor 647-WGA working solution. Cells were incubated with WGA working solution at room temperature for 30 minutes, followed by washing twice with HHBS buffer. Cells were then photographed and recorded using a fluorescence microscope with a Cy5 filter set.

[0027] 1.8 Reactive Oxygen Species (ROS) Detection: H9C2 cardiomyocytes in logarithmic growth phase were collected, and DCFH-DA was diluted 1:1000 with serum-free culture medium. The collected cells were resuspended in the diluted DCFH-DA working solution to achieve a cell density of 1.0 × 10⁶ cells / year. 7 Cells / mL were collected and incubated in a 37°C cell culture incubator in the dark for 20 min, inverting the incubator every 5 min to ensure full contact between the probe and cells. Cells were then washed three times with serum-free culture medium, suspended in serum-free culture medium, divided into several aliquots, and then stimulated with the drug. After incubation in a 37°C cell culture incubator in the dark for 20 min, the cells were observed and recorded using a laser confocal microscope.

[0028] 1.9 ELISA Experiment: Mouse serum supernatant was collected, and the levels of cardiac injury markers such as cTnT and BNP were detected using an ELISA kit. First, the standard concentrations were diluted in a gradient. Then, blank wells (blank control wells did not contain sample, enzyme-labeled reagent, or biotin-labeled antibody; all other steps were the same) and sample wells were prepared. 40 μL of sample was added to each sample well on the ELISA plate, followed by 10 μL of biotin-labeled antibody (the sample was added to the bottom of the well, avoiding contact with the well walls, and gently shaken to mix). 50 μL of enzyme-labeled reagent was added to each well. The plate was sealed with sealing film and incubated at 37°C for 30 minutes. The supernatant was discarded, and the chromogenic solution was added and incubated for 10 minutes. Finally, the stop solution was added, and the OD was measured. 450 nm and analyze the results.

[0029] 2. Experimental Results:

[0030] (1) Pengalloglucoside can alleviate DOX-induced cardiotoxicity in cardiomyocytes.

[0031] To clarify pentagalloyl glucose (structural formula as shown) Figure 1 (As shown) To investigate the potential mechanisms by which DOX-induced cardiomyocyte damage is mitigated, this application conducted cell experiments. This application performed an MTT assay to detect the cytotoxic effect of pentagalloglucopyranoside on H9C2 cells. (See figure) Figure 2 As shown in Figure A, pentagalloglucopyranoside did not affect the growth of H9C2 cells, indicating that pentagalloglucopyranoside has low cytotoxicity. Next, this application used an MTT assay to determine the combined killing effect of DOX and pentagalloglucopyranoside in H9C2 cardiomyocytes. The results showed that pentagalloglucopyranoside can indeed reverse DOX-induced cardiomyocyte damage. Figure 2 A). In addition, this application also examined intracellular reactive oxygen species (ROS) levels, and the results showed that, compared with the control group, DOX (1 μM) significantly increased intracellular ROS levels in cardiomyocytes, while the combined administration of pentalgaloyl glucose (20 μM) and DOX (1 μM) significantly reversed this phenomenon. Figure 2 C). These results indicate that pentagalloglucopyranoside can alleviate DOX-induced cardiotoxicity in cardiomyocytes.

[0032] (2) Pengalloglucoside can reduce DOX-induced cardiotoxicity in mice.

[0033] This application investigated the effects of pentagalloglucopyranoside on disseminated intravascular coagulation (DIC). To this end, a DIC model was constructed using male C57BL / 6 mice, as follows: Figure 3 As shown in Figure A, as expected, the ratio of ventricular weight to tibial length significantly decreased in the DOX treatment group, while the ratio significantly increased in the pentamalloyl glucose plus adjuvant group, indicating that pentamalloyl glucose alleviated DOX-induced cardiotoxicity. Figure 3 C). Echocardiography showed ( Figure 3 B), when mice received DOX intervention, the left ventricular systolic fraction (FS%) and ejection fraction (EF%) were significantly reduced. Figure 3 DE (destructive echocardiography) indicated a significant decrease in cardiac function in mice; while the left ventricular systolic fraction (FS%) and ejection fraction (EF%) were significantly increased in the pentaloloyl glucose combined with other drugs group. Furthermore, compared to 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 pentaloloyl glucose were also significantly reduced. Figure 3 (FG). In summary, pentagalloglucoside alleviated DOX-induced cardiotoxicity in mice.

[0034] (3) Histological analysis showed that pentagalloglucoside alleviated DOX-induced cardiotoxicity in mice.

[0035] This application also found through histological analysis that H&E staining results showed that DOX enhanced inflammatory infiltration of cardiomyocytes, while the addition of pentagalloglucopyranoside alleviated this phenomenon. Figure 4 A); MASSON staining results showed that DOX-treated myocardial tissue inflammation levels were significantly upregulated, while pentagalloglucopyranoside significantly downregulated DOX-induced myocardial tissue inflammation levels. Figure 4 B); Cardiac cardiomyocytes in DIC mice shrank, while administration of PGG significantly restored the size of cardiomyocytes (B). Figure 4 CD). The above results indicate that pentagalloglucopyranoside can alleviate DOX-induced cardiotoxicity in mice.

[0036] Apoptosis and oxidative stress damage are among the main mechanisms by which doxorubicin induces cardiotoxicity. PGG can induce various types of cell death, including apoptosis and autophagy. In this application, by comparing with the control group, we found that the levels of the oxidative stress factor ROS and the proportion of apoptotic cells were significantly increased in the doxorubicin group. This phenomenon indicates that doxorubicin can significantly enhance the apoptosis rate and the degree of oxidative stress damage in cardiomyocytes. However, after intervention with pengalloglucopyranoside, the ROS levels and apoptosis levels in mice were significantly reduced, indicating that pengalloglucopyranoside can effectively alleviate doxorubicin-induced cardiomyocyte apoptosis and oxidative stress damage. Therefore, the results of this application further support the view that pengalloglucopyranoside is a potential protective agent against doxorubicin-induced cardiotoxicity.

Claims

1. The application of pentagalloglucopyranoside in the preparation of drugs for preventing and inhibiting myocardial toxicity induced by the antitumor chemotherapy drug doxorubicin, characterized in that, The pentagalloglucoside has at least one of the following functions (1) to (5): 1) Prevention and / or treatment of doxorubicin cardiotoxicity; 2) Relieves myocardial tissue inflammation caused by doxorubicin; 3) Reduces the increase in reactive oxygen species induced by doxorubicin; 4) Relieves the increase in myocardial fibrosis caused by doxorubicin; 5) Alleviates doxorubicin-induced cardiomyocyte apoptosis; The structural formula of the pentagalloglucoside is as follows: 。 2. The application according to claim 1, characterized in that, The effective content of pentagalloglucopyranoside in the drug is 0.1~100 μM.

3. The application according to claim 1, characterized in that, Pengalloyl glucose is the sole active ingredient.

4. The application according to claim 1, characterized in that, The drug is a pharmaceutical composition.

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