Use of angelica sinensis lactone A in preparation of products for resisting myocardial ischemic injury

Abstract

The application discloses application of Angelica acutiloba Nakai lactone A in preparation of a product for resisting myocardial ischemic injury, and the myocardial ischemic injury is an increase in cell membrane permeability of myocardial cells or apoptosis, necrosis and fragmentation of the myocardial cells, and the product is a drug or a pharmaceutical preparation. In the application, the Angelica acutiloba Nakai lactone A has low toxicity to normal cells, is derived from traditional Chinese medicines of Angelica and Chuanxiong, is safe, has low preparation cost, and can be used as a promising drug for protecting myocardial cells from injury caused by ischemia.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of angelica lactone A in the preparation of products for treating myocardial ischemia injury. Background Technology

[0002] Myocardial ischemia, as a pathophysiological state, is caused by insufficient blood and oxygen supply to the heart due to conditions such as coronary artery stenosis, spasm, or embolism. High-risk groups include patients with coronary heart disease, arrhythmia, and other diseases. If it develops into the ischemic heart disease stage, the severity of the coronary heart disease will be considered.

[0003] The etiology and pathological processes of myocardial ischemia are complex, and current treatment primarily relies on conservative drug therapy. The key to drug treatment of myocardial ischemia is restoring the balance between oxygen supply and consumption. Treatment methods can be mainly divided into two categories: reducing myocardial oxygen consumption and improving coronary blood flow. Currently, various anti-myocardial ischemia drugs are used clinically, commonly including nitrates, beta-blockers, and myocardial metabolic drugs. Although these drugs have certain effects, myocardial cell damage caused by myocardial ischemia is difficult to avoid, especially since dead myocardial cells cannot regenerate. Therefore, the hospitalization and mortality rates of chronic heart failure remain high, and it is one of the leading causes of death in humans.

[0004] Traditional Chinese medicine (TCM) has the natural advantage of being effective and having few toxic side effects in the prevention and treatment of cardiovascular diseases. Angelica lactone A is one of the main active ingredients of the TCM herbs Angelica sinensis and Ligusticum chuanxiong. Angelica sinensis and Ligusticum chuanxiong are the most common blood-activating TCM herbs in clinical practice, and current reports indicate that they have cardiovascular protective effects, anti-fibrotic effects, and effects in treating ventricular remodeling after myocardial infarction.

[0005] Current research suggests that angelica lactone A can inhibit the proliferation of myocardial fibroblasts after myocardial infarction, thus suggesting its potential use in preventing ventricular remodeling after myocardial infarction. However, this invention proposes that angelica lactone A can also directly protect cardiomyocytes from damage caused by ischemia, which undoubtedly has significant theoretical and practical value for preventing myocardial ischemia-induced injury. Summary of the Invention

[0006] The purpose of this invention is to propose the application of angelicin A in the preparation of products for treating myocardial ischemia injury.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The application of angelicin A, as shown in Formula 1, in the preparation of products for treating myocardial ischemia injury:

[0009]

[0010] Myocardial ischemia injury is caused by increased cell membrane permeability of cardiomyocytes or by cardiomyocyte apoptosis, necrosis, and fragmentation.

[0011] Preferably, the product is a drug or a drug preparation.

[0012] A method for preparing a drug that promotes cell proliferation and reduces cell damage includes adding angelicin A.

[0013] A method for preparing a drug to improve H2O2-damaged cells includes adding angelicin A.

[0014] A method for preparing a drug that lowers LDH and increases SOD includes adding angelicin A.

[0015] A method for preparing a drug that improves H9C2 cardiomyocyte injury includes adding angelicin A.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] Angelica lactone A has low toxicity to normal cells. It is derived from traditional Chinese medicines such as Angelica sinensis and Ligusticum chuanxiong. It is safe and has a low preparation cost, making it a promising drug to protect cardiomyocytes from ischemic damage. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the toxic and proliferative effects of angelica lactone A on H9C2 in the application of angelica lactone A proposed in this invention in the preparation of products for treating myocardial ischemia injury.

[0019] Figure 2 This is a schematic diagram illustrating the application of angelica lactone A proposed in this invention in the preparation of products for treating myocardial ischemia injury, showing how angelica lactone A improves the viability of cells damaged by H2O2.

[0020] Figure 3 This is a schematic diagram illustrating the application of angelica lactone A proposed in this invention in the preparation of products for treating myocardial ischemia injury, showing that angelica lactone A can reduce LDH and increase SOD content.

[0021] Figure 4 This is a schematic diagram illustrating the application of angelica lactone A proposed in this invention in the preparation of products for treating myocardial ischemia-reperfusion injury, showing that angelica lactone A can improve H9C2 cardiomyocyte apoptosis. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] This invention provides the application of angelicin A in the preparation of products for treating myocardial ischemia injury.

[0024] Among them, the structural formula of angelica lactone A is shown in Formula 1:

[0025]

[0026] The pharmaceutical uses of angelocinolone A provided by this invention include:

[0027] ① The application of angelicin A in the preparation of drugs that promote cell proliferation;

[0028] ② Application of angelica lactone A in the preparation of drugs to improve H2O2-damaged cells;

[0029] ③ The application of angelica lactone A in the preparation of drugs that lower LDH and increase SOD;

[0030] ④ Application of angelica lactone A (LSA) in the preparation of drugs that improve H9C2 cardiomyocytes.

[0031] Application ①: Detection of the toxicity and proliferative effects of angelica lactone A on H9C2 cells.

[0032] Reference Appendix Figure 1 In the study of A, B, and C, the cytotoxicity of different concentrations and treatment times of angelica lactone A on H9C2 cells was detected when the cell seeding density or growth fusion density reached 80%-90%.

[0033] Reference Appendix Figure 1 Compared with the Control group, treatment with 1 nM, 10 nM, 100 nM, 1 μM and 10 μM angelica lactone A for 2 h and 24 h had virtually no effect on the morphology and density of H9C2.

[0034] Reference Appendix Figure 1 In addition to LSA treatment with 100 μM and 1 mM for 2 h and 24 h, cells not only showed hypertrophy and deformation and varying degrees of density reduction, but also precipitated angelica lactone A drug crystals.

[0035] Reference Appendix Figure 1In the B group, in terms of cell viability detection, after 2 hours of treatment, compared with the Control group, there were basically no significant differences among the other groups except for a slight increase in the 100 nM group and an abnormal increase in the 10 mM group.

[0036] Reference Appendix Figure 1 Similarly, after 24 hours of treatment, compared with the Control group, there were no significant differences in cell viability in the 1mM and 10mM groups.

[0037] In summary, the cytotoxicity results of the drug showed that low concentrations of angelica lactone A (1 nM, 10 nM, 100 nM, 1 μM and 10 μM) did not affect cell morphology, density and viability, while high concentrations of angelica lactone A (100 μM, 1 mM and 10 mM) damaged cell morphology and density. However, the abnormal increase in cell viability was due to the increased absorbance caused by the precipitation of drug crystals.

[0038] Reference Appendix Figure 1 In sections D and E, based on the cytotoxicity results of the drug, the proliferative effect of the optimized angelica lactone A concentration on H9C2 cells at different treatment times was detected when the cell seeding or growth density was 50%-60%.

[0039] Reference Appendix Figure 1 CCK8 cell viability assays showed that, compared to the Control group, there were no significant differences among the groups treated with 10 nM, 100 nM, 1 μM and 10 μM angelica lactone A for 2 h.

[0040] Reference Appendix Figure 1 In the treatment of E, after 24 hours, cell viability increased significantly in the other three groups except for the 100 nM group, where there was no difference in the increase.

[0041] This indicates that low concentrations of angelica lactone A, after prolonged exposure, can promote the proliferation of H9C2 cells. Based on the results of cytotoxicity and proliferation tests, we initially set the treatment time and concentration gradient of angelica lactone A as 24h and 10nM, 100nM, 1μM and 10μM.

[0042] Regarding the appendix Figure 1 It needs to be emphasized that:

[0043] A: Toxicity observation of different concentrations and treatment times of angelica lactone A on H9C2 under a 10x microscope, Bar = 100 μm;

[0044] B, C: Toxicity test of angelica lactone A, in order of cell viability of H9C2 cells after 2 hours and 24 hours of treatment with different concentrations of angelica lactone A;

[0045] D, E: Detection of the proliferative effect of angelica lactone A, in order of cell viability of H9C2 cells after 2 hours and 24 hours of treatment with different concentrations of angelica lactone A;

[0046] ns: no difference; *: P value < 0.05; **: P value < 0.01; ****: P value < 0.0001.

[0047] Application ② was used to detect the effect of angelica lactone A on improving H2O2-damaged cells:

[0048] Reference Appendix Figure 2 In the H2O2 model group, patients were treated with 10 nM, 100 nM, 1 μM and 10 μM angelica lactone A for 24 h. Compared with the control group and the treatment group, the H9C2 cells in the H2O2 model group showed decreased cell density, abnormal morphological hypertrophy and increased dead light spots.

[0049] Reference Appendix Figure 2 In the control group, the cell viability of the H2O2 model group was significantly decreased.

[0050] Except for the 10 nM group, all groups treated with angelica lactone A gradient therapy showed significant improvement in cell viability damaged by H2O2.

[0051] Regarding the appendix Figure 2 It needs to be emphasized that:

[0052] A: Cell growth status under 10x magnification after 24 hours of treatment with angelica lactone A concentration gradient on the basis of H2O2 modeling, Bar=100μm;

[0053] B: Cell viability after 24 hours of treatment with angelica lactone A concentration gradient;

[0054] ns: no difference; *: P value < 0.05; **: P value < 0.01; ***: P value < 0.001.

[0055] Regarding the effects of application ③ in detecting the drug angelica lactone A on lowering LDH and increasing SOD levels:

[0056] Lactate dehydrogenase (LDH) is a glycolytic enzyme found in the cytoplasm of all cells in the body. LDH is an important biochemical indicator in human blood and is often used to assist in the diagnosis of coronary heart disease, myocardial infarction, liver disease, and hematological disorders. When the myocardium is inflamed or damaged, the permeability of the myocardial cell membrane increases, or myocardial cells undergo apoptosis, necrosis, or fragmentation, leading to the release of LDH into the extracellular space and causing an increase in extracellular LDH levels. Additionally, superoxide dismutase (SOD) is an active substance derived from living organisms that can remove harmful substances produced during metabolism. SOD is an important antioxidant enzyme in the body, capable of scavenging superoxide and free radicals, counteracting or blocking the damage caused by oxygen free radicals to cells, and promptly repairing damaged cells.

[0057] Therefore, based on H2O2-induced damage to H9C2 cardiomyocytes, we treated them with different concentrations of angelica lactone A (10 nM, 100 nM, 1 μM and 10 μM). The degree of cardiomyocyte damage and the antioxidant effect of the drug were reflected by detecting the LDH and SOD activities in the cell supernatant.

[0058] Reference Appendix Figure 3 In the study, the results showed that compared with the control group, the H2O2 model group caused a significant increase in LDH. Treatment with angelica lactone A significantly reduced the increase in LDH caused by H2O2, except for the 10nM group which showed no difference.

[0059] Reference Appendix Figure 3 In addition, the H2O2 group significantly reduced SOD content, while the angelica lactone A dosage groups, except for the 10nM group, significantly increased SOD content.

[0060] Regarding the appendix Figure 3 It needs to be emphasized that:

[0061] A: LDH activity statistics chart;

[0062] B: SOD activity statistics chart;

[0063] ns: no difference; *: P value < 0.05; **: P value < 0.01; ***: P value < 0.001.

[0064] Application ④ was used to detect the effect of angelica lactone A on improving H9C2 cardiomyocytes:

[0065] Reference Appendix Figure 4In order to observe the damage and apoptosis of H9C2 cardiomyocytes, we detected TUNEL apoptosis staining of cardiomyocytes in groups A and B. The results of fluorescence staining showed that, compared with the control group, the H2O2 group had significantly increased apoptosis fluorescence and significantly increased relative TUNEL fluorescence intensity.

[0066] Reference Appendix Figure 4 Compared with the H2O2 group, there was no statistically significant difference in the relative fluorescence intensity of the 10 nM group treated with angelica lactone A, while the 100 nM, 1 μM and 10 μM groups all significantly reduced the fluorescence intensity of apoptosis.

[0067] Regarding the appendix Figure 4 It needs to be emphasized that:

[0068] A: TUNEL fluorescent staining, Bar = 100 μm;

[0069] B: Statistical graph of relative fluorescence intensity of TUNEL / DAPI;

[0070] ns: no difference; *: P value < 0.05; **: P value < 0.01; ****: P value < 0.0001.

[0071] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of angelica lactone A (as shown in Formula 1) in the preparation of products for reducing LDH and increasing SOD in cardiomyocytes: ； The product mentioned, which reduces LDH and increases SOD in cardiomyocytes, is used to combat myocardial ischemia-induced injury; myocardial ischemia-induced injury is characterized by increased cell membrane permeability of cardiomyocytes or myocardial cell apoptosis, necrosis, and fragmentation; and The products that reduce LDH and increase SOD in myocardial cells are products that protect myocardial cells from damage caused by ischemia.

2. The application according to claim 1, characterized in that, The product is a drug or a drug preparation.

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