Use of panaxadiol saponins IVa in the preparation of a drug for treating and improving adriamycin-induced myocardial injury

Abstract

The application discloses an application of panax notoginseng saponin IVa in preparation of a drug for treating and improving adriamycin-induced myocardial injury, and belongs to the technical field of medicines. As a traditional Chinese medicinal material, the active monomer panax notoginseng saponin IVa (triterpene saponin compound) has been proved to have the effects of anti-inflammation, anti-oxidation, anti-apoptosis and cardiovascular protection. The application first discovers and verifies the treatment effect of panax notoginseng saponin IVa on adriamycin-induced cardiotoxicity, proves that panax notoginseng saponin IVa realizes the heart protection by reducing myocardial apoptosis and relieving fibrosis, has the effects of treating and / or improving adriamycin-induced myocardial injury, cardiac dysfunction, myocardial inflammation and myocardial fibrosis, and can be used for preparing a drug for treating adriamycin-induced cardiotoxicity. The drug has clear efficacy, good safety and no adverse reaction in long-term taking.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of ginsenoside IVa in the preparation of drugs for treating and / or improving doxorubicin-induced myocardial injury. Background Technology

[0002] Doxorubicin (DOX), an anthracycline broad-spectrum chemotherapy drug, is widely used in the treatment of various malignant tumors such as leukemia, breast cancer, and hematological malignancies. However, this drug has dose-dependent cardiotoxicity, with an incidence of approximately 25%. The damage is progressive and irreversible, and even after discontinuation, it may still develop into ventricular dysfunction, dilated cardiomyopathy, and heart failure, severely limiting its long-term and high-dose clinical use. Currently, the mechanisms by which doxorubicin induces cardiomyopathy are known to be related to enhanced oxidative stress, increased cardiomyocyte apoptosis, excessive activation of the inflammatory response, and mitochondrial dysfunction, but there are no specific preventive or therapeutic methods in clinical practice.

[0003] As a traditional and precious Chinese medicinal herb, the active monomer of Panax notoginseng, Panax notoginseng saponin IVa (a triterpenoid saponin compound), has been proven to have anti-inflammatory, antioxidant, anti-apoptotic and cardiovascular protective effects, but there are no research reports on its use in the treatment of doxorubicin-induced cardiotoxicity.

[0004] Current clinical treatment for doxorubicin-induced cardiotoxicity primarily focuses on symptomatic and supportive care. Core medications include diuretics (such as furosemide), angiotensin-converting enzyme inhibitors (such as captopril), and beta-blockers (such as metoprolol). These drugs alleviate symptoms by improving cardiac load and regulating neuroendocrine function. Related treatment regimens are documented in *Internal Medicine (9th Edition)*, *Guidelines for the Prevention and Treatment of Adverse Drug Reactions in Clinical Oncology*, and numerous clinical research articles (such as the *Chinese Expert Consensus on the Prevention and Treatment of Anthracycline Cardiotoxicity*), and are currently recognized and widely used in clinical practice.

[0005] Existing symptomatic and supportive treatment regimens can only alleviate the surface symptoms of doxorubicin-induced cardiotoxicity, with limited therapeutic effects and still cannot reduce the incidence of cardiotoxicity. Furthermore, some drugs (such as beta-blockers) have adverse reactions such as low heart rate and decreased blood pressure, which limits their applicability to cancer patients with underlying cardiovascular diseases and makes it difficult to meet the clinical need for safe, effective and targeted treatment to improve myocardial damage. Summary of the Invention

[0006] To address the limitations of existing doxorubicin-induced cardiotoxicity treatments, which fail to target the core pathogenic mechanism and have limited therapeutic effects, this invention provides the application of ginsenoside IVa in this cardiotoxicity treatment, achieving targeted improvement of myocardial damage and protection of cardiac function.

[0007] This invention provides the application of ginsenoside IVa in the preparation of drugs for treating and / or improving doxorubicin-induced myocardial injury.

[0008] Bamboo joint ginsenoside IVa is derived from the traditional Chinese medicine bamboo joint ginseng ( Panax japonicus Oleanane-type triterpenoid saponins extracted from CA Mey. have no recorded obvious toxic side effects. Their structural formula is as follows: .

[0009] This invention provides the application of ginsenoside IVa in the preparation of drugs for treating doxorubicin-induced myocardial injury.

[0010] This invention provides the application of ginsenoside IVa in the preparation of drugs that improve doxorubicin-induced myocardial injury.

[0011] This invention provides the application of ginsenoside IVa in the preparation of drugs that improve doxorubicin-induced cardiac dysfunction.

[0012] This invention provides the application of ginsenoside IVa in the preparation of drugs that improve doxorubicin-induced myocardial inflammation.

[0013] This invention provides the application of ginsenoside IVa in the preparation of drugs that improve doxorubicin-induced myocardial fibrosis.

[0014] As described above, the drug comprises ginsenoside IVa and pharmaceutically acceptable excipients or auxiliary ingredients.

[0015] As described above, the drug includes oral preparations, which are granules, pills, tablets, capsules, powders, or oral liquids.

[0016] Due to the adoption of the above technical solution, the technical effects achieved by this application are as follows: This application is the first to discover and verify the therapeutic effect of ginsenoside IVa on doxorubicin-induced cardiotoxicity, and establishes a technical system of "doxorubicin modeling - ginsenoside IVa intervention - multi-dimensional detection and verification".

[0017] This application has clarified the effective dose range (5~20 mg / kg, for mice) and preferred administration method (gavage, 0.1 mL / 10 g body weight) of ginsenoside IVa through animal experiments, providing data support for subsequent clinical translation.

[0018] This application confirms that ginsenoside IVa achieves cardioprotection by reducing myocardial apoptosis and alleviating fibrosis, and elucidates its mechanism of action and its specific correlation with the core mechanism of doxorubicin-induced cardiotoxicity.

[0019] This application confirms that ginsenoside IVa has a protective effect on cardiac function. Echocardiography showed that medium and high doses of ginsenoside IVa can significantly increase doxorubicin-induced decreased ejection fraction (EF) and heart failure (FS), directly improving cardiac contractile function. Compared with existing drugs that only relieve symptoms, it can protect the heart from a functional level and reduce the risk of heart failure.

[0020] Bamboo ginsenoside IVa has the effect of reducing cardiac fibrosis. Masson staining confirmed that bamboo ginsenoside IVa can significantly reduce doxorubicin-induced perivascular and interstitial collagen fiber deposition, prevent the progression of myocardial fibrosis, and avoid further deterioration of cardiac structure. There are currently no reports of this in the existing technology.

[0021] Bamboo joint ginsenoside IVa is derived from the traditional Chinese medicine bamboo joint ginseng. There are no records of obvious toxic side effects. Compared with the potential adverse reactions of existing drugs, it is safer. Moreover, Chinese medicine resources are abundant, which facilitates subsequent development and application. Attached Figure Description

[0022] Figure 1 This refers to the effect of CSIVA on the specific gravity of the heart and body. Figure 2 It is the effect of CSIVA on cardiac function indicators; Figure 3 It is the effect of CSIVa on the expression of myocardial injury markers; Figure 4 The effect of CSIVa on the expression of apoptosis-related genes; Figure 5 Masson staining was used to observe the effect of CSIVa on the morphology and structure of mouse heart tissue. A is a representative morphological image of perivascular fibrosis in mouse myocardium, and B is the effect of CSIVa on the area of ​​perivascular fibrosis induced by doxorubicin. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention provides the application of ginsenoside IVa in the preparation of a therapeutic agent for doxorubicin-induced cardiotoxicity, wherein the structural formula of ginsenoside IVa is as follows:

[0025] Example 1 This embodiment experimentally verified the therapeutic effect of ginsenoside IVa on doxorubicin-induced cardiotoxicity. The specific experimental contents are as follows.

[0026] 1. Experimental Methods Experimental materials include: Fifty SPF-grade male 6-week-old C57 / 6J mice (provided by the Animal Center of Three Gorges University, Animal Ethics No.: 2025060X), weighing 20–22 g, were selected as experimental animals. The mice were housed at the Animal Center of Three Gorges University, with free access to food, and the environment was maintained at a temperature of 22–24℃, humidity <60%, and a 12-hour light-dark cycle. All animal husbandry and experimental procedures strictly adhered to relevant regulations on laboratory animal management and animal welfare.

[0027] Bamboo joint ginsenoside IVa was purchased from Chengdu Pusi Technology Biotechnology Co., Ltd., and its purity was 98% as determined by HPLC.

[0028] Experimental grouping and model building 1.1 Animal Model Making: A myocardial injury model was established by multiple intraperitoneal injections of doxorubicin (cumulative dose 20 mg / kg) according to the method described in "AIG1 protects against doxorubicin-induced cardiomyocyte ferroptosis and cardiotoxicity by promoting ubiquitination-mediated p53 degradation". The injection was given once a week at a dose of 5 mg / kg for four weeks. The intraperitoneal injection site was located in the right lower abdomen of the mouse. The injection should be performed gently, and aspiration should be performed before injection. At the same time, care should be taken to avoid the organs and major blood vessels in the abdominal cavity.

[0029] 1.2 Grouping Mice were randomly assigned to control groups (n=10), model groups (n=10), and low-, medium-, and high-dose treatment groups (DOX+CSIVa-L, n=10), DOX+CSIVa-M, n=10, and DOX+CSIVa-H, n=10) according to a completely randomized design. The mice in the model and treatment groups were the mice modeled in section 1.1. Mice in the control group were administered an equal volume of CMC-Na solution. The DOX+CSIVa-L, DOX+CSIVa-M, and DOX+CSIVa-H groups were administered ginsenoside IVa via gavage at doses of 5 mg / kg, 10 mg / kg, and 20 mg / kg, respectively, prepared as a 0.1 mL / 10g CMC-Na solution.

[0030] 1.3 Statistical Methods All data in this section are expressed as mean ± standard deviation. x ± s) represents the data. SPSS 22.0 was used for statistical analysis, and GraphPad Prism 7.0 was used for graphing. The data conformed to a normal distribution, and t-tests or one-way analysis of variance (ANOVA) were used. Bonferroni test was used for post-hoc comparisons between groups.

[0031] 2. Experimental Procedure and Results 2.1 Ratio of mouse heart weight to body weight (HW / BW) Experimental Methods: After animal modeling and drug administration, mice were anesthetized with 1% isoflurane, and their weight was measured. Blood was collected through the eyeballs, and whole blood was collected in enzyme-free EP tubes. The mouse heart was then perfused with physiological saline. After thorough perfusion, the heart was removed and placed in ice-cold physiological saline to allow residual blood to be pumped out. The moisture was blotted with filter paper, and the heart was photographed and weighed.

[0032] Calculation formula: Specific gravity of heart = weight of heart tissue (mg) / weight of mouse (g); Mouse heart specificity is a core quantitative indicator in the field of laboratory animals (especially in cardiovascular disease research). Essentially, it is used to assess the size relationship of the mouse heart relative to the overall body by calculating the ratio of "mouse heart weight" to "mouse body weight". It is used to determine whether laboratory mice have cardiac hypertrophy, atrophy or structural and functional abnormalities. It is a key basis for validating cardiovascular disease models (such as hypertensive heart disease and myocardial infarction models) and assessing the effects of drugs on the heart.

[0033] Experimental results: The specific gravity of the heart is as follows Figure 1 The results showed that the heart weight to body weight ratio (HW / BW) in the DOX group mice was lower than that in the normal group (Con.). P <0.01); and compared with the DOX group, the heart-to-body ratio of mice in the low, medium and high dose groups of bamboo rhizome saponin IVa was significantly increased, and the differences were statistically significant. P <0.01)( P <0.01)( P <0.05), among which CSIVa-M showed the best therapeutic effect. The above experimental results indicate that the DOX-induced myocardial injury model was successfully established, and that ginsenoside IVa (CSIVa) can reverse DOX-induced myocardial injury.

[0034] 2.2 Functional detection of mouse cardiac ultrasound Experimental Methods: After modeling, mice underwent hair removal in the precordial region using cotton swabs dipped in depilatory cream. Anesthesia was induced with isoflurane, and the mice were placed supine on a temperature-controlled operating table with their limbs fixed to electrodes for electrocardiogram (ECG) measurement. Anesthesia was maintained at approximately 1% isoflurane concentration to stabilize the mouse's heart rate. Coupling agent was applied to the precordial region of the mice. The angle of the ultrasound probe was adjusted, and in B-mode ultrasound, the probe was placed above the left parasternal line of the mouse. The probe was stopped when the heart appeared in the field of view. The probe was slightly adjusted to make the apex of the heart pointed, ensuring the apex was at the same level as the outflow tract and that the outflow tract was patent. After finding the standard parasternal long-axis view, the B-mode ultrasound data was saved. Switching to M-mode ultrasound, the sampling line was placed behind the papillary muscle, and the data was saved. The short-axis image was obtained by rotating clockwise based on the long-axis view, and the B-mode and M-mode ultrasound images were saved sequentially. The ejection fraction (EF) and fractional shortening (FS) were measured and calculated using ultrasound analysis software.

[0035] Experimental results: Echocardiographic examination of mice after modeling showed the following results. Figure 2 As shown, compared with the control group, the ejection fraction (EF) of mice in the DOX group was significantly reduced. P <0.01), while the EF of mice in the medium and high doses of bamboo rhizome saponin IVa treatment groups increased, showing statistically significant differences ( P <0.001)( P <0.001). Compared with the control group, the ejection fraction shortening (FS) of mice in the DOX group was significantly reduced ( P <0.001), while the FS of mice in the medium and high doses of bamboo rhizome saponin IVa treatment groups increased, showing statistically significant differences ( P <0.001)( P <0.01). The above experimental results indicate that ginsenoside IVa effectively inhibited DOX-induced cardiac dysfunction in mice.

[0036] 2.3 Detection of myocardial injury markers in cardiac tissue Experimental Methods: Heart tissue was weighed, RNA was extracted, and reverse transcribed into cDNA. Using cDNA as a template, polymerase, primers, and RNase-free ultrapure water were added, and amplification was performed using a real-time quantitative PCR instrument. Reaction conditions are shown in Tables 1 and 2. The melting curve was gradually increased from 60ºC to 95ºC, with readings recorded every 0.5ºC. β-actin was used as an internal control, and the relative mRNA expression level of the target gene was calculated using the 2-ΔΔCT method. The expression of ANP and BNP myocardial injury markers was detected using real-time PCR.

[0037] Table 1 Real-time PCR reaction system

[0038] Table 2 Real-time PCR reaction conditions

[0039] Experimental results: such as Figure 3 Real-time P The CR results showed that, compared with the Con group, the AN in the heart tissue of DOX group mice was lower. P Increased expression, the difference was statistically significant. P <0.0001); and compared with the DOX group, the AN of the CSIVA-L, CSIVA-M, and CSIVA-H groups was lower than that of the DOX group. P Decreased mRNA expression, the difference was statistically significant. P <0.01)( P <0.0001)( P <0.0001). Compared with the Con group, the BN in the heart tissue of DOX group mice was significantly lower. P Increased mRNA expression, the difference was statistically significant. P <0.01); and compared with the DOX group, the BN of the CSIVA-M and CSIVA-H groups was lower. P Decreased mRNA expression, the difference was statistically significant. P <0.01)( P <0.05). These results indicate that ginsenoside IVa can improve DOX-induced myocardial damage in vivo.

[0040] 2.4 Detection of apoptosis factors in cardiac tissue Experimental Methods: Heart tissue was weighed, RNA was extracted, and reverse transcribed into cDNA. Using cDNA as a template, polymerase, primers, and RNase-free ultrapure water were added, and amplification was performed using a real-time quantitative PCR instrument. Reaction conditions are shown in Tables 1 and 2. The melting curve was gradually increased from 60ºC to 95ºC, with readings recorded every 0.5ºC. β-actin was used as an internal control, and the relative mRNA expression level of the target gene was calculated using the 2-ΔΔCT method. The expression of BAX and BCL2 was detected using real-time PCR.

[0041] Experimental results: such as Figure 4 As shown, compared with the Con group, the expression of Bax mRNA in the heart tissue of DOX group mice was increased, and the difference was statistically significant. P <0.001); and compared with the DOX group, Bax mRNA expression was decreased in the CSIVA-M group, with a statistically significant difference ( P <0.01)( P<0.01. Compared with the Con group, the expression of BCL2 mRNA in the heart tissue of DOX group mice was decreased, and the difference was statistically significant. P <0.01); and compared with the DOX group, BCL2 mRNA expression increased in the CSIVa-L and CSIVa-M groups, with statistically significant differences. P <0.01)( P <0.01). These results indicate that ginsenoside IVa can inhibit apoptosis in DOX-induced myocardial injury mice in vivo.

[0042] 2.5 Masson staining to observe cardiac tissue fibrosis and structural morphological changes Experimental Methods: Dewaxing of paraffin sections of the heart. Prepare Weigert iron hematoxylin staining solution and evenly add it to the stained sections for 5-10 min. After rinsing the sections with distilled water, add acidic ethanol differentiation solution for 5-10 s, then rinse with distilled water for 30 s. Use Masson's blue solution for 3-5 min to restore blue color, then rinse with distilled water for 30 s, and then stain with virgin red and fuchsin solution for 5-10 min. Prepare a weak acid working solution by mixing distilled water and a weak acid solution in a 2:1 ratio, and add it to the section and wash for 30 s. Remove any residual liquid from the sections, add phosphomolybdic acid solution for 1-2 min, then wash with the weak acid working solution for 30 s. Remove any residual liquid from the sections, add aniline blue staining solution for 1-2 min, then wash with the weak acid working solution for 30 s. Rapidly dehydrate with 95% anhydrous ethanol for 2-3 s, repeating twice with anhydrous ethanol for 5-10 s each time. Clear the sections twice with xylene for 1-2 min each time, and then mount with neutral resin mounting medium.

[0043] Experimental results: such as Figure 5 The results showed that, compared with the control group, the number of cardiomyocytes in the DOX group mice was significantly reduced and the deposition of collagen fibers in the heart tissue was significantly increased. P <0.01)( P <0.01); while all three doses of bamboo-root saponin IVa significantly reduced perivascular collagen fiber deposition ( P <0.001)( P <0.001)( P <0.01); The above experimental results show that ginsenoside IVa effectively inhibited the morphology and structure of mouse heart tissue induced by DOX.

[0044] In summary, this invention confirms that Panax japonicus saponin IVa has a therapeutic effect on doxorubicin-induced cardiotoxicity, with a clear efficacy. Panax japonicus saponin IVa is an oleanane-type triterpenoid saponin extracted from the traditional Chinese medicine Panax japonicus. It has no recorded significant toxic side effects and is safer than existing drugs that may have adverse reactions. Furthermore, it is derived from abundant traditional Chinese medicine resources, which facilitates its subsequent development and application.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of ginsenoside IVa in the preparation of drugs for treating doxorubicin-induced myocardial injury.

2. Application of ginsenoside IVa in the preparation of drugs to improve doxorubicin-induced myocardial injury.

3. Application of ginsenoside IVa in the preparation of drugs to improve doxorubicin-induced cardiac dysfunction.

4. Application of ginsenoside IVa in the preparation of drugs to improve doxorubicin-induced myocardial inflammation.

5. Application of ginsenoside IVa in the preparation of drugs to improve doxorubicin-induced myocardial fibrosis.

6. The application according to claims 1-5, characterized in that: The drug includes ginsenoside IVa and pharmaceutically acceptable excipients or auxiliary ingredients.

7. The application according to claim 6, characterized in that: The drug includes oral preparations, which are granules, pills, tablets, capsules, powders, or oral liquids.

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