Use of chagmu polysaccharide in preparing drugs for preventing adriamycin-induced cardiac injury

Abstract

The application discloses application of chamaejasmine polysaccharide in preparation of a drug for preventing doxorubicin-induced cardiac injury, and belongs to the technical field of medicines.The application proves that chamaejasmine polysaccharide can significantly improve doxorubicin-induced cardiac function injury, reduce serum myocardial enzyme levels, reduce myocardial tissue pathological injury and fibrosis, and inhibit myocardial cell apoptosis by constructing a doxorubicin-induced acute cardiotoxicity mouse model.The mechanism of action is related to inhibition of abnormal activation of a ROS / MAPKs signal pathway and reduction of oxidative stress injury.The application provides a new natural drug source and experimental basis for prevention and treatment of doxorubicin chemotherapy-induced cardiotoxicity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Chamaegu polysaccharide in the preparation of drugs for preventing doxorubicin-induced cardiac damage. Background Technology

[0002] Doxorubicin (DOX), also known as doxorubicin, is a highly effective, broad-spectrum anthracycline antitumor antibiotic widely used clinically to treat breast cancer, lymphoma, leukemia, and various solid tumors. However, the clinical application of DOX is limited by its severe cardiotoxic side effects, mainly manifested as dose-dependent myocardial damage, arrhythmias, and even congestive heart failure. This cardiotoxicity not only seriously affects patients' quality of life and prognosis but also greatly limits the full use of DOX in cancer chemotherapy. Therefore, exploring safe and effective drugs to prevent and treat DOX cardiotoxicity has become a key research focus and hot topic in the field of cardio-oncology.

[0003] Current research suggests that the molecular mechanisms of DOX-induced cardiotoxicity involve multiple pathways, including oxidative stress, mitochondrial dysfunction, apoptosis, and ferroptosis. Among these, oxidative stress damage is considered one of the core mechanisms of DOX-induced cardiotoxicity. After DOX enters cardiomyocytes, it can generate large amounts of reactive oxygen species (ROS) through the redox cycle in the mitochondrial electron transport chain. When the amount of ROS generated exceeds the body's antioxidant system's clearance capacity, it triggers uncontrolled oxidative stress. Excessive ROS can directly damage the lipid membrane, proteins, and DNA of cardiomyocytes, thereby activating apoptosis signaling pathways, leading to massive cardiomyocyte apoptosis and progressive decline in cardiac function.

[0004] Mitogen-activated protein kinases (MAPKs) are important intracellular signal transduction molecules, mainly comprising three subfamilies: extracellular signal-regulated kinases (ERK1 / 2), c-Jun N-terminal kinases (JNK), and p38 MAPK. MAPKs are closely related to oxidative stress; abnormal accumulation of intracellular ROS can activate the MAPK signaling pathway, thereby regulating the expression of downstream apoptosis-related proteins and mediating cardiomyocyte apoptosis. Previous studies have confirmed that DOX can induce cardiomyocytes to produce large amounts of ROS, thereby abnormally activating the JNK and p38 MAPK pathways, amplifying oxidative stress damage, promoting apoptosis, and driving the occurrence and development of cardiotoxicity. Therefore, targeting the ROS / MAPK signaling axis and screening for natural active substances that can inhibit oxidative stress and block abnormal signal activation has become an important research direction for mitigating DOX cardiotoxicity.

[0005] Chamaegu, scientific name turnip ( Brassica rapa*Brassica oleracea* (L.), a biennial herb belonging to the genus *Brassica* in the Brassicaceae family, is a traditional medicinal and edible plant in Xinjiang, my country. The fleshy roots of *Brassica oleracea* are rich in polysaccharides, flavonoids, phenolic acids, glucosinolates, and other active ingredients, exhibiting significant pharmacological effects such as antioxidant, anti-inflammatory, anti-tumor, immunomodulatory, and cardioprotective properties. *Brassica oleracea* polysaccharides... Brassica rapa L. (BRPs) is one of the main active components extracted from *Chamaecyparis*. Modern pharmacological studies have shown that BRPs possess biological activities such as scavenging free radicals and enhancing the body's antioxidant capacity. Previous studies have confirmed that BRPs have a protective effect against DOX-induced H9c2 cardiomyocyte damage and can effectively inhibit intracellular ROS production. This mechanism may be related to regulating the Nrf2 / HO-1 pathway to antagonize oxidative stress. However, whether BRPs exert their anti-DOX cardiotoxicity effect by regulating the ROS / MAPK signaling pathway has not yet been reported.

[0006] To address the shortcomings of the existing technology, this invention provides a novel pharmaceutical application for Chaga polysaccharide: its use in the preparation of drugs to prevent doxorubicin-induced cardiac injury. This invention systematically evaluates the protective effect of BRPs against myocardial injury by constructing a DOX-induced acute cardiotoxicity mouse model and elucidates its molecular mechanism from the perspective of the ROS / MAPK signaling pathway. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide the application of Chaga polysaccharide in the preparation of drugs for preventing doxorubicin-induced cardiac damage. Animal experiments have demonstrated that Chaga polysaccharide can significantly improve doxorubicin-induced cardiac dysfunction in mice, reduce myocardial tissue pathological damage, lower serum myocardial enzyme levels, and inhibit cardiomyocyte apoptosis. Its mechanism is related to the inhibition of abnormal activation of the ROS / MAPK signaling pathway.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of Chamaegu polysaccharide in the preparation of drugs for preventing doxorubicin-induced cardiac damage.

[0009] In some embodiments of the present invention, the polysaccharide is obtained by extracting the polysaccharide fraction from the fleshy root of *Chamaegu* via water extraction and alcohol precipitation.

[0010] In some embodiments of the present invention, the quality index of the Chamaegu polysaccharide is that the polysaccharide content is not less than 50%.

[0011] In some embodiments of the present invention, the drug is an oral formulation or an injectable formulation.

[0012] In some embodiments of the present invention, the Chamaegu polysaccharide exerts its anti-doxorubicin cardiotoxicity effect by inhibiting the ROS / MAPK signaling pathway.

[0013] In some embodiments of the present invention, the Chamaegu polysaccharide inhibits the abnormal activation of the MAPK signaling pathway by downregulating the expression levels of p-p38MAPK and p-JNK proteins.

[0014] In some embodiments of the present invention, the Chamaegu polysaccharide alleviates oxidative stress damage by increasing the activity of superoxide dismutase and glutathione peroxidase in myocardial tissue and reducing malondialdehyde content.

[0015] This invention also provides the application of Chaga polysaccharide in the preparation of drugs that improve doxorubicin-induced myocardial oxidative stress damage.

[0016] It contains at least the following beneficial technical effects: This invention reveals for the first time the application of Chamaegu polysaccharide in the preparation of drugs to prevent doxorubicin-induced cardiac damage, expands the medicinal uses of Chamaegu polysaccharide, and provides a new natural drug source for the prevention and treatment of cardiotoxicity caused by doxorubicin chemotherapy.

[0017] This invention systematically evaluated the cardioprotective effect of Chamaegu polysaccharide from multiple levels, including cardiac function, histopathology, serum biochemistry, oxidative stress, and apoptosis, by constructing an doxorubicin-induced acute cardiotoxicity mouse model. The experimental evidence is sufficient and reliable.

[0018] This invention reveals for the first time the molecular mechanism by which Chaga polysaccharide exerts its anti-doxorubicin cardiotoxicity effect by inhibiting the ROS / MAPK signaling pathway, providing new scientific evidence for a deeper understanding of the pharmacological effects of Chaga polysaccharide.

[0019] Chamaegu is a traditional medicinal and edible plant in Xinjiang, my country. It is abundant and safe. This invention lays the foundation for the in-depth development and utilization of Xinjiang's unique resources and has significant economic and social value. Attached Figure Description

[0020] Figure 1 The following are the echocardiographic results of the mice in each group. A: Representative echocardiographic images of the mice in each group; B: Ejection fraction (EF%) of the heart in each group; C: Short axis shortening rate (FS%) of the heart in each group; D: Left ventricular end-diastolic diameter (LVIDd) of the left ventricle in each group; E: Left ventricular end-systolic diameter (LVIDs) of the left ventricle in each group.

[0021] Figure 2 The effect of Chaga polysaccharide on doxorubicin-induced cardiac weight index in mice. A: Schematic diagram of animal model construction and drug administration time; B: Mouse body weight change curve; CE: Mouse organ index.

[0022] Figure 3 The results of hematoxylin-eosin (HE) staining of myocardial tissue from each group of mice (magnification: ×200; scale bar: 100μm).

[0023] Figure 4 Masson staining results of myocardial tissue from mice in each group (magnification: ×200; scale bar: 100 μm).

[0024] Figure 5 The effect of Chaga polysaccharide on the serum myocardial enzyme profile in doxorubicin-induced mice. A: Serum AST level; B: Serum LDH level; C: Serum CK level; D: Serum CK-MB level.

[0025] Figure 6 The effect of Chaga polysaccharide on doxorubicin-induced oxidative stress indices in mouse myocardial tissue. A: SOD activity in myocardial tissue; B: MDA content in myocardial tissue; C: GSH-Px activity in myocardial tissue.

[0026] Figure 7 TUNEL staining results of myocardial tissue from mice in each group (magnification: ×200; scale bar: 125 μm).

[0027] Figure 8 The effect of Chaga polysaccharide on the expression of apoptosis-related proteins in the cardiac tissue of mice with doxorubicin injury. A: Western blot bands of Caspase-3 and Caspase-9 proteins; B: Quantitative analysis of Caspase-3 and Caspase-9 protein expression.

[0028] Figure 9 The effect of Chaga polysaccharide on the expression of MAPK pathway-related proteins in the heart tissue of mice with doxorubicin injury. A: Western blot bands of p-p38MAPK, p38MAPK, p-JNK, and JNK proteins; B: Quantitative analysis of the expression of p-p38MAPK, p38MAPK, p-JNK, and JNK proteins. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific embodiments, and are not intended to limit the present invention. In addition, for any numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range, as well as any other stated value or intermediate value within the stated range, is also encompassed in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the cited documents. In the event of conflict with any incorporated document, the content of the present specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations can be made to the specific embodiments of the specification of the present invention without departing from the scope or spirit of the present invention. Other embodiments derived from the specification of the present invention will be apparent to those skilled in the art. The description and examples of the present application are merely exemplary.

[0033] As used herein, the terms "comprising", "including", "having", "containing" and the like are all open-ended terms, meaning including but not limited to.

[0034] The "room temperature" and "normal temperature" described in the present invention, unless otherwise specified, are all calculated as 25±2°C.

[0035] Unless otherwise specified, all raw materials or instruments used in the following examples of the present invention are commercially available.

[0036] Example 1 I. Materials and Methods 1. Animals and cells: SPF grade C57BL / 6J mice, with a body weight of 18-22 g, 10-week-old males, were purchased from the Animal Experiment Center of Xinjiang Medical University, with the license number SCXK (Xin) 2023-0002. The mice were kept in a barrier environment of the Animal Center of Xinjiang Institute of Materia Medica, with controlled lighting, temperature and humidity. This experiment was approved by the Animal Ethics Committee of Xinjiang Uygur Autonomous Region Institute of Materia Medica (Approval No.: XJIMM-20240201).

[0037] 2. Drugs and Reagents: BRPs were identified by the New Drug Research Center of Xinjiang Institute of Materia Medica (content 55.13%, batch number: 20231211); Doxorubicin (batch number S1208) was purchased from Selleck, USA; malondialdehyde (MDA) assay kit, superoxide dismutase (SOD) assay kit, and glutathione peroxidase (GSH-PX) assay kit (batch numbers A003-4-1, A001-3, A005-1-2) were purchased from Nanjing Jiancheng Bioengineering Institute; TUNEL in situ apoptosis detection kit (batch number E-CK-A321) was purchased from Wuhan Yilairuit Biotechnology Co., Ltd.; Masson's trichrome staining kit (batch number G1340) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; aspartate transaminase (AST) assay kit, lactate dehydrogenase (LDH) assay kit, and lactate dehydrogenase (LDH) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. LDH (Lactal-Desenylopeptide) kit, creatine kinase (CK) kit, and creatine kinase isoenzyme (CK-MB) kit (lot numbers 140225007, 142723003, 142512006, 142623013) were purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd.; anti-GAPDH antibody (lot number 60004-1-lg) was purchased from Proteintech Co., Ltd.; anti-p-p38 MAPK antibody, anti-p38 mitogen-activated protein kinase (p38 MAPK) antibody, anti-p-JNK antibody, anti-JNK antibody, anti-Caspase-3 antibody, and anti-Caspase-9 antibody (lot numbers 4511, 8690, 4668, 9252, 9662, 9504) Purchased from CST Corporation; horseradish enzyme-labeled goat anti-mouse IgG and horseradish enzyme-labeled goat anti-rabbit IgG (batch numbers ZB-2305 and ZB-2301) were purchased from China Zhongshan Jinqiao Company.

[0038] 3. Instruments: Optical microscope (OLYMPUS CX21, Japan); SpeedMill PLUS homogenizer (Jena, Germany); Flow cytometer (Coulter EPICS XL, Beckman, USA); Echocardiometer (Philips HD11XE); Multifunctional microplate reader (TECAN SPARK, Switzerland); Multifunctional imaging system (FUSION FX6, VILBER, France); Benchtop high-speed refrigerated centrifuge (TGL-16K, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); Microscope (DM3000, LEICA Camera AG, Germany); Microtome (RM2016, LEICA Camera AG, Germany); Intelligent tissue dehydrator (TZT-12M, Wuhan Tianzhirui Medical Technology Co., Ltd.). Biological tissue slide warmer (Shanghai Precision Instruments Co., Ltd., YD-AB); biological tissue embedding machine (Changzhou Zhongwei Electronic Instruments Co., Ltd., BMJ-A); freezing stage (Xiaogan Hongye Medical Instruments Co., Ltd., BM-C2); fully automated biochemical analyzer (Mindray, BS-420).

[0039] 4. Animal grouping, drug administration, and model establishment: Ten-week-old male C57BL / 6J mice were randomly divided into 6 groups after one week of acclimatization feeding: normal group, DOX group, DOX combined with BRPs administration group (low, medium, and high doses), and positive control group (15 mg / kg). -1 ·d -1 The dosage was determined based on previous work (0.5, 1.0, 2.0 g / kg). -1 ·d -1 Six animals were placed in each group. The BRPs group and the positive control group received oral BRPs and berberine daily for two weeks. The control group received normal saline, and began intraperitoneal injection of DOX (10 mg / kg) at the beginning of the second week. -1 Or an equal volume of normal saline, with a second DOX injection 3 days later (the cumulative DOX dose is 20 mg / kg). -1 ).

[0040] 5. Cardiac Function Testing: At the end of the experiment, the patient was lightly anesthetized with 1% isoflurane until the heart rate stabilized at 400-500 beats / minute. Echocardiography was performed, and left ventricular function was assessed using two-dimensional M-mode and B-mode echocardiography. Indicators included: left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), ejection fraction (EF), and fractional shortening (FS). FS% and EF% were calculated.

[0041] FS%=[(LVIDd-LVIDs) / LVIDd]×100% EF%=[(LVEDV-LVESV) / LVEDV]×100% 6. Effects on mouse body weight and heart weight Observe the growth of each group of mice daily and record their weight. At the end of the experiment, remove the mouse heart, weigh it, and calculate the heart weight index (heartweight / body weight, HW / BW) according to the following formula. At the end of the experiment, weigh the mouse heart weight (HW) and body weight (BW) and calculate HW / BW.

[0042] 7. Specimen Collection: Blood was collected from the eyeballs of each group of animals after 2 weeks. Hearts were harvested, weighed, and subsequent weight changes were analyzed. One portion of the heart samples was quickly soaked in absorbent paper, wrapped in aluminum foil, and flash-frozen in liquid nitrogen. Another portion of the hearts was perfused with approximately 15 mL of 4% paraformaldehyde to fix the tissue for later use.

[0043] Hematoxylin-eosin (HE) staining was used to observe the pathological morphology of mouse myocardial tissue: Mouse heart tissue was taken and fixed in 4% paraformaldehyde for 2-3 days, then placed in an embedding cassette. After dehydration, clearing, paraffin embedding, and preparation of paraffin sections, HE staining was performed, and the sections were mounted with neutral resin. After the sections were allowed to air dry, the pathological changes of mouse heart tissue were observed under an optical microscope.

[0044] Masson's trichrome staining (Masson staining) was used to observe the degree of cardiac fibrosis: Heart tissue from mice in each group was fixed in paraformaldehyde solution, dehydrated, embedded in paraffin, sectioned, dewaxed, and stained with Masson's trichrome. The degree of cardiac fibrosis was observed under a microscope, and the images were saved.

[0045] Serum biochemical marker detection: When the myocardium is injured, corresponding myocardial enzymes are released in the peripheral blood. Myocardial enzymes such as AST, LDH, CK, and CK-MB are considered core biomarkers for assessing damage to various organs, including the heart. The levels of ALT, LDH, CK, and CK-MB in mouse serum were detected using a fully automated biochemical analyzer.

[0046] Detection of oxidative stress indicators in tissues: Heart tissue was homogenized, the supernatant was collected after centrifugation, and the results were analyzed according to the kit instructions.

[0047] TUNEL assay for intracellular apoptosis: Paraffin sections of myocardial tissue from each group were stained with TUNEL. The specific procedures were performed according to the instructions of the apoptosis detection kit. The tissues were observed and photographed under a confocal microscope. The excitation wavelengths were 495 nm (TUNEL green fluorescence) and 519 nm (DAPI blue fluorescence). The apoptosis rate (%) was calculated as: (Number of TUNEL-positive cells / Total number of cells) × 100%.

[0048] Western blot detection of protein expression in cardiac tissue: Small pieces of cardiac tissue were taken, minced, and lysed with an appropriate amount of RIPA lysis buffer containing 1× protease inhibitor and phosphatase inhibitor. The tissue was homogenized on ice for 30 s, lysed on ice for 30 min, and then centrifuged at 16000×g for 30 min at 4°C. The supernatant was quantified by BCA protein assay, separated by SDS-PAGE, and wet-blown onto a PVDF membrane. The membrane was blocked with 5% skim milk powder at room temperature for 2 h. The membrane was washed three times with TBST, and the corresponding primary antibodies (p38 MAPK, p-p38 MAPK, JNK, p-JNK) were added, respectively. The membrane was incubated overnight at 4°C. After washing three times with TBST, the corresponding secondary antibodies were added and incubated for another 2 h. The membrane was developed with ECL chemiluminescence buffer, and images were taken using a multi-functional imaging system. ImageJ analysis was used to determine the grayscale values ​​of the protein bands.

[0049] Statistical methods: GraphPad Prism 10.1 software was used for data analysis and graphing. All results were presented using statistical methods. x ± s This indicates that one-way ANOVA was used for comparisons among multiple groups. P A value <0.05 is considered statistically significant.

[0050] 8. Experimental Results: 1. BRPs improve DOX-induced cardiac dysfunction The results of echocardiography in mice showed that ( Figure 1 Compared with the control group, the LVEF and LVFS of mice in the DOX group were significantly decreased. P <0.000 1), LVEDd and LVEDs increased significantly ( P <0.000 1), indicating impaired cardiac function in mice, indicating successful model establishment; compared with the DOX group, LVEF and LVFS were significantly increased in different BRPs dosage groups ( P <0.00 1), LVEDd and LVEDs were significantly reduced ( P The result was <0.01, indicating that BRPs can improve DOX-induced cardiac function impairment in mice, with the BRPs-H group and the Ber group showing the best effects.

[0051] 2. Effects of BRPs on improving the heart weight coefficient in DOX-induced mice The heart weight coefficient is a key parameter for monitoring cardiotoxicity in mice. This parameter changes significantly when the heart shrinks or becomes congested and edematous. Figure 2 A is a schematic diagram of the model construction method, and the mouse weight results are shown ( Figure 2 Compared with the control group, the BW, HW, and HW / BW ratio of mice in the DOX group were all decreased (BE). P <0.01); Compared with the DOX group, the HW values ​​of mice in the BRPs-H group and the Ber group were significantly increased after treatment ( P <0.01). This indicates that BRPs have a mitigating effect on DOX-induced cardiotoxicity.

[0052] 3. BRPs alleviate DOX-induced pathological symptoms in mouse cardiac tissue. HE staining showed that cardiac fibroblasts in the control group mice were regularly arranged and morphologically normal; compared with the control group, cardiac fibroblasts in the DOX group mice were irregularly arranged, structurally altered, and unevenly distributed; cardiac fibroblasts in the BRPs-H group and the Ber group mice showed restored regular arrangement and less muscle fiber breakage. Figure 3 Compared with the control group, the cardiac tissue damage score of mice in the DOX group was significantly increased. P <0.000 1); Compared with the DOX group, the cardiac tissue damage scores of the BRPs group and the Ber group were significantly reduced in a dose-dependent manner. P <0.01).

[0053] 4. BRPs ameliorate DOX-induced myocardial fibrosis in mice. Masson staining results showed that ( Figure 4 In the control group, no myocardial fiber damage was observed; compared with the control group, the DOX group mice showed significant cardiac fibrosis and a significantly increased CVF score. P <0.001); Compared with the DOX group, the BRPs group and the Ber group significantly reduced the area of ​​myocardial fibrosis and significantly reduced the CVF score ( P <0.1). The results showed that DOX could disrupt the normal morphological structure of mouse myocardial tissue, and BRPs could reverse the damaging effects of DOX on the morphological structure of mouse myocardial tissue to a certain extent.

[0054] 5. BRPs alleviate abnormal serum myocardial enzymes in mice When the myocardium is injured, corresponding myocardial enzymes are released in the peripheral blood. AST, LDH, CK, and CK-MB are considered core biomarkers for assessing damage to various organs, including the heart. The levels of AST, LDH, CK, and CK-MB were measured to investigate the cardioprotective effect of BRPs. Results ( Figure 5 As shown in the figure, compared with the control group, myocardial injury in the DOX group led to the release of AST, LDH, CK, and CK-MB. P <0.001), while compared with the DOX group, the indicators in both the BRPs group and the Ber group were reduced, indicating that BRPs can alleviate DOX-induced cardiotoxicity in mice and improve cardiac function in DOX-damaged mice.

[0055] 6. BRPs reduce oxidative stress in DOX-induced cardiac tissue in mice. The results of the oxidative stress index content determination showed that ( Figure 6 After DOX modeling, the levels of SOD and GSH-PX in mouse heart tissue decreased. P <0.000 1), MDA content increased ( P <0.000 1); Compared with the DOX group, the BRPs dosage groups and the Ber group significantly increased the content of SOD and GSH-PX in mouse heart tissue ( P <0.001), reducing MDA content ( P <0.01), and showed a dose-dependent effect.

[0056] 7. BRPs reduce DOX-induced apoptosis in mouse cardiomyocytes. Tunel staining results of mouse myocardial tissue as follows Figure 7 As shown, the green fluorescence is TUNEL staining, which reacts with broken DNA in the cell nucleus, reflecting the degree of cardiomyocyte apoptosis. The apoptosis rate was calculated using ImageJ software. Compared with the control group, the apoptosis rate of cardiomyocytes in the DOX group was significantly increased. P <0.000 1); Compared with the DOX group, the apoptosis rate of cardiomyocytes was significantly reduced in all BRPs dosage groups and the Ber group ( P The result (<0.001) indicates that BRPs can effectively antagonize DOX-induced cardiomyocyte apoptosis, thereby reducing myocardial damage.

[0057] 8. BRPs mitigate the effects of DOX on Caspase-3 and Caspase-9 expression. Figure 8 The results showed that, compared with the control group, the expression levels of Caspase-3 and Caspase-9 proteins in the cardiac tissue of the DOX group were higher than those in the control group. P <0.001); Compared with the DOX group, the BRPs dosage groups and the Ber group reduced the expression of Caspase-3 and Caspase-9 proteins in tissues after DOX injury. P<0.01), which suggests that BRPs may antagonize DOX-induced cardiomyocyte apoptosis by downregulating the protein expression of apoptosis-related proteins (Caspase-3 / 9).

[0058] 9. Effects of BRPs on the expression of MAPK pathway-related proteins in DOX-injured mouse heart tissue Figure 9 The results showed that, compared with the control group, DOX injury upregulated the protein expression levels of p-p38MAPK and p-JNK in mouse myocardial tissue. P <0.001), compared with the DOX group, the expression levels of p-p38MAPK and p-JNK proteins in the myocardial tissue of mice in each BRPs dose group and the Ber group were reduced.

[0059] Chamaegu is a widely cultivated medicinal and edible plant in Xinjiang. Its fleshy root is both a traditional Uyghur food and a medicinal ingredient, possessing nutritional and health benefits and earning the reputation of "longevity fruit." BRPs, a polysaccharide fraction obtained from Chamaegu in Keping County, Xinjiang through water extraction and alcohol precipitation, possess significant characteristics of traditional ethnic medicine. Its preparation process is mature, reliable, and suitable for large-scale production, and is protected by patents. Previous studies have confirmed that BRPs not only possess pharmacological effects such as anti-tumor and immunomodulatory effects, but also effectively scavenge DPPH free radicals and reduce DOX-induced ROS accumulation in H9c2 cardiomyocytes, exhibiting significant antioxidant protective effects. Based on these findings, this study, by constructing a DOX-induced acute cardiotoxicity model in mice, delves into the protective effects of BRPs on myocardial injury and its potential molecular mechanisms.

[0060] Currently, extensive research has been conducted both domestically and internationally on the molecular mechanisms of DOX cardiotoxicity, achieving significant progress. After entering the body, DOX can induce cardiomyocytes to produce large amounts of ROS through mitochondrial metabolic disorders and enzyme system abnormalities. When the amount of ROS produced exceeds the body's antioxidant system's clearance capacity, it triggers uncontrolled oxidative stress. Excessive ROS directly damages the proteins and cell membrane structure of cardiomyocytes, exacerbating myocardial tissue inflammation and infiltration, thereby activating apoptosis pathways, leading to massive cardiomyocyte apoptosis, worsening cardiac function damage, and in severe cases, progressing to heart failure. This study established an acute cardiotoxicity mouse model of DOX through intraperitoneal injection. The results showed that DOX caused a decline in cardiac function in mice, manifested as increased LVIDs and decreased EF% and FS%. After administration of BRPs, EF% and FS% increased, and LVIDs returned to normal values. HE and Masson staining results showed that the DOX-treated mice exhibited significantly increased cardiomyocyte arrangement disorder, structural damage, and myocardial tissue fibrosis. Serum AST, LDH, CK, and CK-MB levels were significantly elevated, indicating the successful establishment of the myocardial injury model. After intervention with BRPs, echocardiographic parameters, myocardial pathological morphology, and myocardial enzyme levels all showed significant improvement, indicating that BRPs can effectively reduce DOX-induced cardiotoxicity. SOD and GSH-PX are key antioxidant enzymes for scavenging oxygen free radicals, and MDA reflects the degree of lipid peroxidation; all three are classic biomarkers for evaluating myocardial oxidative stress damage and are used to assess the intervention effect of cardioprotective drugs. In the DOX group, myocardial MDA content was significantly increased, while SOD and GSH-PX activities were significantly decreased, suggesting a significantly enhanced level of myocardial oxidative stress. After BRPs intervention, MDA levels decreased, and SOD and GSH-PX activities increased, indicating that BRPs can significantly reduce DOX-induced myocardial oxidative stress, maintain the balance of the body's oxidation-antioxidant system, and thus exert a cardioprotective effect.

[0061] Based on previous research, our group found that BRPs have a significant protective effect on DOX-damaged rat H9c2 cardiomyocytes and can effectively inhibit ROS generation. Therefore, we hypothesize that BRPs may antagonize DOX-induced cardiotoxicity by regulating the ROS / MAPK signaling pathway. The MAPK family mainly includes subtypes such as extracellular signal-regulated kinases 1 / 2 (ERK1 / 2), JNK, and p38 MAPK. Among them, JNK and p38 MAPK, as key signaling molecules in cellular responses to oxidative stress and other stimuli, play important regulatory roles in stress-induced apoptosis. Previous studies have confirmed that the ROS / MAPK signaling pathway plays a crucial role in DOX-induced cardiotoxicity. Excessive ROS accumulation can activate the JNK and p38 MAPK pathways, exacerbating oxidative stress damage and cardiomyocyte apoptosis; while scavenging ROS can inhibit the activation of these pathways, thereby reducing myocardial damage and improving cardiac function. The results of this study show that DOX induction significantly promotes ROS production and the expression of p38MAPK and JNK proteins, while BRP treatment significantly inhibits ROS release, reduces p38MAPK and JNK protein expression, improves mitochondrial function, and inhibits apoptosis. By blocking the abnormal activation of the ROS / MAPK signaling pathway, BRP treatment ultimately antagonizes DOX-induced cardiotoxicity, thus effectively mitigating DOX-induced cardiotoxicity.

[0062] In summary, BRPs have a significant protective effect against DOX-induced acute cardiotoxicity in mice. The mechanism may be related to the regulation of the ROS / MAPK signaling pathway, mitigation of DOX-induced oxidative stress damage, and apoptosis. These findings provide experimental evidence for the application of BRPs, a food and medicinal homology substance from Xinjiang, in the prevention and treatment of DOX-induced cardiotoxicity, and also lay the foundation for the in-depth development and utilization of Chamaegu, a unique resource from Xinjiang.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of Chamaegu polysaccharide in the preparation of drugs for preventing doxorubicin-induced cardiac damage.

2. The application according to claim 1, characterized in that, The polysaccharide from Chamaegu was obtained by water extraction and alcohol precipitation from the fleshy root of Chamaegu.

3. The application according to claim 1 or 2, characterized in that, The polysaccharide content of the *Chamaegu* polysaccharide is not less than 50%.

4. The application according to claim 1, characterized in that, The drug is an oral or injectable formulation.

5. The application according to claim 1, characterized in that, The polysaccharide from Chamaegu exerts its anti-doxorubicin cardiotoxicity effect by inhibiting the ROS / MAPK signaling pathway.

6. The application according to claim 5, characterized in that, The chamaegu polysaccharide inhibits the abnormal activation of the MAPK signaling pathway by downregulating the expression levels of p-p38MAPK and p-JNK proteins.

7. Application of Chamaegu polysaccharide in the preparation of drugs that improve doxorubicin-induced myocardial oxidative stress damage.

8. The application according to claim 7, characterized in that, The polysaccharide from Chamaegu alleviates oxidative stress damage by increasing the activity of superoxide dismutase and glutathione peroxidase in myocardial tissue and reducing malondialdehyde content.

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