Application of ilicin A in preparation of medicine for preventing and treating ischemic heart disease

Abstract

The invention discloses an application of ilicin A in preparation of a medicine for preventing and treating ischemic heart disease, and belongs to the technical field of ilicin A. An experiment for improving heart functions of a mouse I / R model by the ilicin A is carried out, and male C57BL / 6 mice (with the age of 6-8 weeks; according to the method, 70 mice (the weight is 20 + / -2g) are bred in a constant-temperature and constant-humidity environment, the illumination / darkness period is 12 hours and lasts for one week, food and water are obtained without limitation, all male mice are randomly grouped as follows: a false operation group (n = 10), an I / R group (n = 15), an ilicin A group (30 and 60 mg / kg) and a captopril group (20 mg / kg), the mice in the ilicin A group and the captopril group are subjected to intragastric administration every day five days before the model is established, and the mice in the captopril group are subjected to intragastric administration every day. And the isopyknic contrast solution is applied to the false operation group and the I / R group.

Description

Technical Field

[0001] The invention relates to an application of hollyl A, in particular to an application of hollyl A in preparing a medicine for preventing and treating ischemic heart disease, and belongs to the technical field of hollyl A application. Background Art

[0002] During the development of ischemic heart disease, various pathological factors stimulate myocardial cell death, which eventually leads to decreased cardiac pump function and ventricular remodeling. Currently, effective ingredients of traditional Chinese medicine have been widely used in the development of drugs for ischemic heart disease. For example, the alkaloid components in Trichosanthes kirilowii (CN111920808B) and Corydalis dwarfi (CN111228337A) have been applied for relevant patents for the preparation of drugs for the treatment of ischemic heart disease.

[0003] Ilex pubescens is one of the famous medicines in Lingnan and has been used to treat cardiovascular diseases. Preliminary studies of the present invention found that hollycin A, the main triterpenoid component of hollycin, can significantly inhibit myocardial cell damage caused by ischemia-reperfusion (I / R), thereby playing a role in treating ischemic heart disease. There has been no report on the study of the pharmacology of this component in ischemic heart disease. At present, clinical treatment measures for ischemic heart disease mainly include chemical drug intervention and revascularization surgery. However, long-term chemical drug treatment may cause side effects such as gastrointestinal bleeding, liver and kidney damage, headache or muscle pain; although interventional treatment is effective, there is also a risk of complications such as restenosis and thrombosis in the stent, and the treatment effect on diffuse three-vessel lesions is limited; surgical treatment faces the problems of large surgical trauma and long recovery time, as well as surgical risks such as infection and bleeding. In addition, there is also the possibility of re-stenosis or occlusion of the bridge blood vessels. For this reason, a method for using hollycin A in the preparation of drugs for the prevention and treatment of ischemic heart disease is designed to solve the above problems. Summary of the invention

[0004] The main purpose of the present invention is to provide an application of hollycin A in preparing medicine for preventing and treating ischemic heart disease.

[0005] The purpose of the present invention can be achieved by adopting the following technical solutions:

[0006] A use of hollycinol A in preparing a drug for preventing and treating ischemic heart disease comprises the following steps:

[0007] Step 1: To conduct an experiment on improving cardiac function of I / R model mice with hollycin A, 70 male C57BL / 6 mice (age 6-8 weeks; weight 20±2g) were selected and housed in a constant temperature and humidity environment with a light / dark cycle of 12 hours for one week with unlimited access to food and water;

[0008] Step 2: All male mice were randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), hollycin A group (30 and 60 mg / kg), and captopril group (20 mg / kg);

[0009] Step 3: Mice in the hollycin A group and the captopril group received daily oral administration five days before model establishment, while the sham operation group and the I / R group were given an equal volume of control solution.

[0010] Preferably, in step three, the myocardial ischemia-reperfusion injury model in mice is established by using anterior descending artery ligation / recanalization, and the cardiac function of each group of mice is detected by echocardiography 24 hours after reperfusion.

[0011] Preferably, the experiment of improving cardiac structural damage in I / R model mice by hollycin A comprises the following steps:

[0012] S11: A total of 70 male C57BL / 6 mice (age 6-8 weeks; weight 20 ± 2 g) were housed in a constant temperature and humidity environment with a 12-h light / dark cycle for one week with unrestricted access to food and water;

[0013] S12: All male mice were randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), hollycin A group (30 and 60 mg / kg), and captopril group (20 mg / kg);

[0014] S13: Mice in the hollycin A group and captopril group received daily gavage for five days before model establishment, while the sham operation group and I / R group were given an equal volume of control solution.

[0015] Preferably, the myocardial ischemia-reperfusion injury model in mice is established by using anterior descending artery ligation / recanalization in S13, and samples are collected 24 hours after reperfusion for H&E and Ly6G staining to evaluate the changes in cardiac structure in each group of mice.

[0016] Preferably, the experiment of reducing myocardial infarction area in I / R model mice by using hollycin A comprises the following steps:

[0017] S21: A total of 70 male C57BL / 6 mice (age 6-8 weeks; weight 20 ± 2 g) were housed in a constant temperature and humidity environment with a 12-h light / dark cycle for one week with unrestricted access to food and water;

[0018] S22: All male mice were randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), hollycin A group (30 and 60 mg / kg), and captopril group (20 mg / kg);

[0019] S23: Mice in the hollycin A group and the captopril group received daily oral administration five days before model establishment, while the sham operation group and the I / R group were given an equal volume of control solution.

[0020] Preferably, the myocardial ischemia-reperfusion injury model in mice is established by using the anterior descending artery ligation / recanalization technique in S23, and samples are collected 24 hours after reperfusion for TTC staining to evaluate the myocardial infarction area in each group of mice.

[0021] Preferably, the experiment of reducing serum myocardial necrosis marker levels in I / R model mice by using hollycin A comprises the following steps:

[0022] S31: A total of 70 male C57BL / 6 mice (age 6-8 weeks; weight 20 ± 2 g) were housed in a constant temperature and humidity environment with a 12-h light / dark cycle for one week with unrestricted access to food and water;

[0023] S32: All male mice were randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), hollycin A group (30 and 60 mg / kg), and captopril group (20 mg / kg);

[0024] S33: Mice in the hollycin A group and the captopril group received daily oral administration five days before model establishment, while the sham operation group and the I / R group were given an equal volume of control solution.

[0025] Preferably, in S33, anterior descending artery ligation / recanalization is used to construct a mouse myocardial ischemia-reperfusion injury model, and after 24 hours of reperfusion, eye blood is collected to prepare serum, and Elisa assay is performed to evaluate the levels of myocardial necrosis markers in each group of mice.

[0026] Preferably, the experiment of improving the viability of primary cardiomyocytes in neonatal rats in the (OGD / R) model by hollytin A comprises the following steps:

[0027] S41: Neonatal rat primary cardiomyocytes were treated with 10-100 μM hollycin A for 2 hours and then 2 , 5% CO 2 and 1% O 2 Cardiomyocytes were cultured in serum- and glucose-free cell culture medium for 3 h in a controlled atmosphere to induce oxygen and glucose deprivation;

[0028] S42: The culture medium was replaced with a medium containing normal serum and high glucose, and the cells were incubated under normal conditions for 4 hours to simulate myocardial reperfusion. After the time was up, the cell viability was measured.

[0029] Beneficial technical effects of the present invention:

[0030] The invention provides an application of hollyl A in the preparation of a drug for preventing and treating ischemic heart disease, which clarifies that hollyl A can protect the heart function decline and myocardial structure damage caused by ischemia-reperfusion injury, and reduce myocardial cell death, which provides a basis for the future development of drugs, health products, foods and other biological products such as hollyl A for preventing and treating ischemic heart disease. Compared with chemical drugs, it has lower side effects and rich pharmacological activities: current studies have shown that IA has multiple pharmacological activities and is non-invasive. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A preferred embodiment of the use of hollycin A in the preparation of a drug for preventing and treating ischemic heart disease according to the present invention is the effect of hollycin A on cardiac function in a C57BL / 6 mouse ischemia-reperfusion (I / R) model, wherein (A) is a representative echocardiographic image, and (B) from left to right are left ventricular ejection fraction (LVEF), left ventricular diastolic fraction (LVFS), left ventricular end-systolic diameter (LVDs) and left ventricular end-diastolic diameter (LVDd), and * in the figure represents significant difference;

[0032] Figure 2 A preferred embodiment of the use of hollycin A in the preparation of a drug for preventing and treating ischemic heart disease according to the present invention is the effect of hollycin A on cardiac structural damage in a mouse I / R model. From top to bottom are representative images of H&E and Ly6G staining of cardiac sections. * in the figure indicates significant difference;

[0033] Figure 3 A preferred embodiment of the use of hollycin A in the preparation of a drug for preventing and treating ischemic heart disease according to the present invention is the effect of hollycin A on the infarct size of myocardial infarction in a mouse I / R model. Representative images (left) and data analysis diagrams (right) of TTC staining of cardiac tissue are shown. * in the figure indicates significant difference;

[0034] Figure 4 A preferred embodiment of the use of hollyl A in the preparation of a drug for preventing and treating ischemic heart disease according to the present invention is the effect of hollyl A on the levels of serum myocardial necrosis markers in a mouse I / R model. From left to right, they are creatine kinase isoenzyme (CK-MB), cardiac troponin T (cTnT), and lactate dehydrogenase (LDH). * in the figure represents significant difference;

[0035] Figure 5 A preferred embodiment of the use of hollyl A in the preparation of a drug for preventing and treating ischemic heart disease according to the present invention is the effect of hollyl A on the cell viability of the primary cardiomyocytes of neonatal rats in the oxygen-glucose deprivation / reoxygenation (OGD / R) model, and a bar graph is used to show the cell viability. In the figure, * represents a significant difference. DETAILED DESCRIPTION

[0036] In order to make the technical solution of the present invention more clear and specific to those skilled in the art, the present invention is further described in detail below in conjunction with embodiments and drawings, but the implementation manner of the present invention is not limited thereto.

[0037] Example 1

[0038] Experiment on improving cardiac function in I / R model of mice.

[0039] A total of 70 male C57BL / 6 mice (age 6-8 weeks; weight 20±2g) were housed in a constant temperature and humidity environment with a 12-hour light / dark cycle for one week, with unlimited access to food and water. All male mice were then randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), holly A group (30 and 60 mg / kg) and captopril group (20 mg / kg). Mice in the holly A group and captopril group received daily gavage for five days before model establishment, while the sham operation group and I / R group were given an equal volume of control solution.

[0040] The myocardial ischemia-reperfusion injury model was established in mice by ligation / recanalization of the anterior descending artery. After 24 hours of reperfusion, echocardiography was performed to detect the cardiac function of mice in each group. Figure 1 As shown in the results, the left ventricular ejection fraction (LVEF), left ventricular diastolic fraction (LVFS) and left ventricular end-systolic diameter (LVDs) of the I / R group were significantly reduced, while the left ventricular end-diastolic diameter (LVDd) did not change significantly, indicating that the normal contraction and relaxation function of the mouse heart was affected after acute I / R. Hollysin A improved the first three indicators in a dose-dependent manner, suggesting that it can protect against I / R-induced cardiac function damage.

[0041] Example 2

[0042] Hollyl A improves cardiac structural damage in I / R model of mice.

[0043] A total of 70 male C57BL / 6 mice (age 6-8 weeks; weight 20±2g) were housed in a constant temperature and humidity environment with a 12-hour light / dark cycle for one week, with unlimited access to food and water. All male mice were then randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), holly A group (30 and 60 mg / kg) and captopril group (20 mg / kg). Mice in the holly A group and captopril group received daily gavage for five days before model establishment, while the sham operation group and I / R group were given an equal volume of control solution.

[0044] The myocardial ischemia-reperfusion injury model was established in mice by ligation / recanalization of the anterior descending artery. After 24 hours of reperfusion, samples were collected for H&E and Ly6G staining to evaluate the changes in cardiac structure in each group of mice. Figure 2 As shown in the figure, myocardial cells in the I / R group were significantly shrunk, disordered, and had enlarged intercellular spaces. Ly6G staining further showed that there was a large amount of neutrophil infiltration in the I / R group. However, compared with the I / R group, hollycin A significantly improved myocardial tissue structural damage and reduced inflammatory cell infiltration, indicating that hollycin A has a protective effect on I / R-induced cardiac structural damage.

[0045] Example 3

[0046] Hollyl A reduces myocardial infarction area in I / R model of mice.

[0047] A total of 70 male C57BL / 6 mice (age 6-8 weeks; weight 20±2g) were housed in a constant temperature and humidity environment with a 12-hour light / dark cycle for one week, with unlimited access to food and water. All male mice were then randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), holly A group (30 and 60 mg / kg) and captopril group (20 mg / kg). Mice in the holly A group and captopril group received daily gavage for five days before model establishment, while the sham operation group and I / R group were given an equal volume of control solution.

[0048] The myocardial ischemia-reperfusion injury model was established in mice by ligation / recanalization of the anterior descending artery. After 24 hours of reperfusion, samples were collected for TTC staining to evaluate the infarct size of each group of mice. Figure 3 As shown in the figure, the infarct area (white) in the I / R group was significantly larger than that in the sham operation group, while the infarct area in the mice of the hollyin A group was reduced. This shows that hollyin A can reduce myocardial damage caused by ischemia-reperfusion and improve myocardial cell death.

[0049] Example 4

[0050] Experiment on reducing serum levels of myocardial necrosis markers in I / R model of mice.

[0051] A total of 70 male C57BL / 6 mice (age 6-8 weeks; weight 20±2g) were housed in a constant temperature and humidity environment with a 12-hour light / dark cycle for one week, with unlimited access to food and water. All male mice were then randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), holly A group (30 and 60 mg / kg) and captopril group (20 mg / kg). Mice in the holly A group and captopril group received daily gavage for five days before model establishment, while the sham operation group and I / R group were given an equal volume of control solution.

[0052] The myocardial ischemia-reperfusion injury model was established in mice by ligation / recanalization of the anterior descending artery. After 24 hours of reperfusion, blood was collected from the eyeball to prepare serum, and the levels of myocardial necrosis markers in each group of mice were evaluated by Elisa. Figure 4 As shown in the results, after I / R, the levels of serum cardiac troponin T (cTnT), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH) in mice increased significantly, while the levels of the above indicators in the hollyvin A group decreased significantly, which once again showed that hollyvin A can protect myocardial tissue damage caused by I / R.

[0053] Example 5

[0054] Holly A improves the viability of primary cardiomyocytes in neonatal rats with (OGD / R) model.

[0055] After treating neonatal rat primary cardiomyocytes with 10-100 μM hollycin A for 2 hours, the cardiomyocytes were cultured in a cell culture medium without serum and glucose for 3 hours in a controlled gas environment of 94% N2, 5% CO2 and 1% O2 to induce oxygen and glucose deprivation. Subsequently, the culture medium was replaced with a medium containing normal serum and high glucose, and the cells were incubated under normal conditions for 4 hours to simulate myocardial reperfusion. After the time was up, the cell viability was measured. The results are shown in Figure 5 As shown, 10-100 μM hollyhock A can improve glucose-oxygen deprivation / reoxygenation-induced cardiomyocyte death in vitro, confirming that hollyhock A has a protective effect on cardiomyocytes.

[0056] The above description is only a further embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and concepts of the present invention within the scope disclosed by the present invention, which belong to the protection scope of the present invention.

Claims

1. A use of hollycinol A in the preparation of a drug for preventing and treating ischemic heart disease, characterized in that: The steps include: Step 1: To conduct an experiment on improving cardiac function of I / R model mice with hollycin A, 70 male C57BL / 6 mice (age 6-8 weeks; weight 20±2g) were selected and housed in a constant temperature and humidity environment with a light / dark cycle of 12 hours for one week with unlimited access to food and water; Step 2: All male mice were randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), hollycin A group (30 and 60 mg / kg), and captopril group (20 mg / kg); Step 3: Mice in the hollycin A group and the captopril group received daily oral administration five days before model establishment, while the sham operation group and the I / R group were given an equal volume of control solution.

2. The use of hollycinol A according to claim 1 in the preparation of a drug for preventing and treating ischemic heart disease, characterized in that: In step three, the myocardial ischemia-reperfusion injury model in mice was established by ligation / recanalization of the anterior descending artery. After 24 hours of reperfusion, echocardiography was performed to detect the cardiac function of mice in each group.

3. The use of hollycinol A according to claim 2 in the preparation of a drug for preventing and treating ischemic heart disease, characterized in that: The experiment of using hollycin A to improve cardiac structural damage in the I / R model of mice includes the following steps: S11: A total of 70 male C57BL / 6 mice (age 6-8 weeks; weight 20 ± 2 g) were housed in a constant temperature and humidity environment with a 12-h light / dark cycle for one week with unrestricted access to food and water; S12: All male mice were randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), hollycin A group (30 and 60 mg / kg), and captopril group (20 mg / kg); S13: Mice in the hollycin A group and captopril group received daily gavage for five days before model establishment, while the sham operation group and I / R group were given an equal volume of control solution.

4. The use of hollycinol A according to claim 3 in the preparation of a drug for preventing and treating ischemic heart disease, characterized in that: The myocardial ischemia-reperfusion injury model was established in mice using the anterior descending artery ligation / recanalization in S13. After 24 hours of reperfusion, samples were collected for H&E and Ly6G staining to evaluate the changes in cardiac structure in each group of mice.

5. The use of hollycinol A according to claim 4 in the preparation of a drug for preventing and treating ischemic heart disease, characterized in that: The experiment of reducing myocardial infarction area in I / R model mice with hollycin A includes the following steps: S21: A total of 70 male C57BL / 6 mice (age 6-8 weeks; weight 20 ± 2 g) were housed in a constant temperature and humidity environment with a 12-h light / dark cycle for one week with unrestricted access to food and water; S22: All male mice were randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), hollycin A group (30 and 60 mg / kg), and captopril group (20 mg / kg); S23: Mice in the hollycin A group and the captopril group received daily oral administration five days before model establishment, while the sham operation group and the I / R group were given an equal volume of control solution.

6. The use of hollycinol A according to claim 5 in the preparation of a drug for preventing and treating ischemic heart disease, characterized in that: The myocardial ischemia-reperfusion injury model was established in mice by ligation / recanalization of the anterior descending artery in S23. TTC staining was performed to evaluate the myocardial infarction area in each group of mice 24 hours after reperfusion.

7. The use of hollycinol A according to claim 6 in the preparation of a drug for preventing and treating ischemic heart disease, characterized in that: The experiment of reducing serum myocardial necrosis marker levels in I / R model mice by hollytin A included the following steps: S31: A total of 70 male C57BL / 6 mice (age 6-8 weeks; weight 20 ± 2 g) were housed in a constant temperature and humidity environment with a 12-h light / dark cycle for one week with unrestricted access to food and water; S32: All male mice were randomly divided into the following groups: sham operation group (n=10), I / R group (n=15), hollycin A group (30 and 60 mg / kg), and captopril group (20 mg / kg); S33: Mice in the hollycin A group and the captopril group received daily oral administration five days before model establishment, while the sham operation group and the I / R group were given an equal volume of control solution.

8. The use of hollycinol A according to claim 7 in the preparation of a drug for preventing and treating ischemic heart disease, characterized in that: The myocardial ischemia-reperfusion injury model was established in mice by ligation / recanalization of the anterior descending artery in S33. After 24 hours of reperfusion, blood was collected from the eyeball to prepare serum, and then ELISA test was performed to evaluate the levels of myocardial necrosis markers in each group of mice.

9. The use of hollycinol A in the preparation of a drug for preventing and treating ischemic heart disease according to claim 8, characterized in that: The experiment of improving the viability of primary cardiomyocytes in neonatal rats in the (OGD / R) model by hollytin A includes the following steps: S41: After neonatal rat primary cardiomyocytes were treated with 10-100 μM hollycin A for 2 hours, they were cultured in serum- and glucose-free cell culture medium for 3 hours in a controlled gas environment of 94% N2, 5% CO2, and 1% O2 to induce oxygen and glucose deprivation; S42: The culture medium was replaced with a medium containing normal serum and high glucose, and the cells were incubated under normal conditions for 4 hours to simulate myocardial reperfusion. After the time was up, the cell viability was measured.

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