Application of neryl acetate in preparing medicine for treating myocardial ischemia-reperfusion injury

Abstract

The invention relates to the application of neryl acetate in preparing a medicine for treating myocardial ischemia-reperfusion injury, and belongs to the field of medical technology. The object of the invention is to provide a new application of neryl acetate in treating myocardial ischemia-reperfusion injury, specifically the application of neryl acetate in preparing a medicine for treating myocardial ischemia-reperfusion injury. The invention makes full use of the advantages of traditional Chinese medicine with multiple components, multiple targets and few adverse reactions, uses neryl acetate to act on rat myocardial cells H9c2 and human myocardial cells AC16, and combines experiments to confirm that neryl acetate can inhibit apoptosis of H9c2 and AC16 cells, promote the increase of mitochondrial number, reduce the accumulation of intracellular ROS, protect mitochondrial damage and inhibit the decline of mitochondrial membrane potential, thereby providing a new application of neryl acetate in treating myocardial ischemia-reperfusion injury, and providing experimental basis and new strategy for clinical treatment of myocardial ischemia-reperfusion injury.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to application of neryl acetate in preparing medicines for treating myocardial ischemia-reperfusion injury. Background Art

[0002] Myocardial ischemia-reperfusion injury refers to the damage caused by excessive free radicals attacking cells in tissues that regain blood supply after blood pressure is restored during rescue and treatment after partial or complete acute blockage of the coronary artery. This damage is mainly caused by reactive oxygen free radicals, especially when the ability to synthesize antioxidant enzymes that scavenge free radicals in ischemic tissues is impaired, free radical damage to post-ischemic reperfusion tissues will be aggravated. Myocardial ischemia-reperfusion injury can be divided into three types: myocardial stunning, reperfusion arrhythmia and myocardial necrosis, among which myocardial cell necrosis and apoptosis are its main manifestations.

[0003] Nerolipoic acid is a natural active ingredient in traditional Chinese medicines such as Chuanxiong and Danshen. It is widely used in the preparation of daily chemical flavors due to its aroma. In addition, due to its unique psychological and physiological effects, it also plays a role in incense, mood regulation and health promotion. The patent "Application of Nerolipoic Acid in the Preparation of Antihypertensive Myocardial Fibrosis Drugs" with publication number CN108524484A discloses that nerolipoic acid can reduce body systolic and diastolic blood pressure, increase cardiac ejection fraction and short-axis contraction rate, reduce collagen content in the myocardium, and enhance body heart function. However, the prior art does not disclose the therapeutic effect of nerolipoic acid on myocardial ischemia-reperfusion injury. Summary of the invention

[0004] The invention aims to provide a new use of neryl acetate for treating myocardial ischemia-reperfusion injury, specifically the use of neryl acetate in preparing a medicine for treating myocardial ischemia-reperfusion injury.

[0005] The technical solution of the present invention:

[0006] Application of neryl acetate in preparing medicine for treating myocardial ischemia-reperfusion injury.

[0007] Furthermore, the drug for treating myocardial ischemia-reperfusion injury has neryl acetate as the only active ingredient or one of the active ingredients.

[0008] Furthermore, the content of neryl acetate in the drug for treating myocardial ischemia-reperfusion injury is 0.1wt%~99wt%.

[0009] Furthermore, the drug for treating myocardial ischemia-reperfusion injury also includes pharmaceutically acceptable adjuvants and / or carriers.

[0010] Furthermore, the drug for treating myocardial ischemia-reperfusion injury is in a dosage form for administration via the gastrointestinal tract.

[0011] Furthermore, the dosage form of the drug for treating myocardial ischemia-reperfusion injury is granules, tablets, capsules, pills or oral liquid preparations.

[0012] Furthermore, the drug for treating myocardial ischemia-reperfusion injury can inhibit apoptosis of rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0013] Furthermore, the drug for treating myocardial ischemia-reperfusion injury has a protective effect on mitochondria in rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0014] Furthermore, the drug for treating myocardial ischemia-reperfusion injury can inhibit the decrease of mitochondrial membrane potential in rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0015] Furthermore, the drug for treating myocardial ischemia-reperfusion injury can reduce the ROS content in rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0016] Beneficial effects of the present invention:

[0017] The present invention makes full use of the advantages of traditional Chinese medicine with multiple components, multiple targets and few adverse reactions, uses neryl acetate to act on rat myocardial cells H9c2 and human myocardial cells AC16, and combines experiments to confirm that neryl acetate can inhibit apoptosis of H9c2 and AC16 cells, promote the increase of mitochondrial number, reduce the accumulation of intracellular ROS, protect mitochondrial damage and inhibit the decrease of mitochondrial membrane potential. Based on the efficacy of neryl acetate in alleviating myocardial damage caused by reperfusion therapy of myocardial ischemia, the present invention provides a new use of neryl acetate for treating myocardial ischemia-reperfusion injury, specifically the use of neryl acetate in the preparation of a drug for treating myocardial ischemia-reperfusion injury, which provides an experimental basis and a new strategy for clinical treatment of myocardial ischemia-reperfusion injury and improving the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The scatter plots of H9c2 cells and AC16 cells in each group detected by flow cytometry;

[0019] Figure 2 This is a comparison chart of the apoptosis rates of H9c2 cells in each group detected by flow cytometry;

[0020] Figure 3 This is a comparison chart of the apoptosis rates of AC16 cells in each group detected by flow cytometry;

[0021] Figure 4These are transmission electron microscopic photographs of each group of H9c2 cells and AC16 cells in Example 2 before and after intervention with neryl acetate;

[0022] Figure 5 This is a statistical diagram of the distribution of mitochondrial fluorescence intensity of H9c2 cells and AC16 cells in each group in the Mitotracker Deep Red staining experiment;

[0023] Figure 6 This is a comparison of the fluorescence intensity of H9c2 cells in each group in the Mitotracker Deep Red staining experiment;

[0024] Figure 7 This is a comparison of the fluorescence intensity of AC16 cells in each group in the Mitotracker Deep Red staining experiment;

[0025] Figure 8 Fluorescence images of H9c2 cells and AC16 cells in each group in ROS staining combined with laser confocal microscopy;

[0026] Fig. 9 This is a comparison of the average fluorescence intensity of H9c2 cells in each group in ROS staining combined with laser confocal microscopy;

[0027] Fig.10 This is a comparison of the average fluorescence intensity of AC16 cells in each group in ROS staining combined with laser confocal microscopy;

[0028] Fig.11 Fluorescence confocal microscopic images of H9c2 cells and AC16 cells in each group in Mitotracker Red staining combined with laser confocal microscopy experiments;

[0029] Fig.12 This is a comparison of the average fluorescence intensity of H9c2 cells in each group in Mitotracker Red staining combined with laser confocal microscopy;

[0030] Fig.13 This is a comparison of the mean fluorescence intensity of AC16 cells in each group in Mitotracker Red staining combined with laser confocal microscopy;

[0031] Fig.14 Fluorescence images of H9c2 cells in each group in JC-1 staining combined with laser confocal microscopy;

[0032] Fig.15 This is a comparison of JC-1 aggregates / monomers in each group of H9c2 cells in JC-1 staining combined with laser confocal microscopy;

[0033] Fig.16Fluorescence images of AC16 cells in each group in JC-1 staining combined with laser confocal microscopy;

[0034] Fig.17 This is a comparison of JC-1 aggregates / monomers in each group of AC16 cells in the JC-1 staining combined with laser confocal microscopy experiment. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0036] In the examples of the present invention, neryl acetate was purchased from Shanghai Yuanye Biotechnology Co., Ltd., DMEM medium was purchased from White Shark Biotechnology Co., Ltd., apoptosis kit was purchased from Aibixin Biotechnology Co., Ltd., fetal bovine serum, Mitotracker Deep Red, ROS, Mitotracker Red, JC1 dyes were purchased from Thermo Fisher Scientific.

[0037] In each example, rat cardiomyocytes H9c2 and human cardiomyocytes AC16 were cultured in DMEM medium (10% fetal bovine serum, 1% double antibody), 5% CO2, and a 37°C cell culture incubator.

[0038] Example 1

[0039] This example uses flow cytometry to verify that neryl acetate can inhibit apoptosis of rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0040] In this example, the cells were divided into a blank CON group, a H2O2 group, and neryl acetate administration groups: CH-20 μM group and CH-40 μM group. Rat myocardial cells H9c2 and human myocardial cells AC16 were expressed at 10×10 4 Each well was inoculated into a 6-well plate and cultured at 37°C overnight. The next day, the CH-20μM group and the CH-40μM group were pre-medicated for 12 hours, with the CH-20μM group receiving 3.9258μg / ml, 2ml / well, and the CH-40μM group receiving 7.8516μg / ml, 2ml / well.

[0041] The H2O2 group was only given culture medium containing 400 μM H2O2 for intervention, while the CH-20 μM group and the CH-40 μM group were respectively given culture medium containing 400 μM H2O2 and corresponding concentrations of neryl acetate for intervention. After 2 hours of intervention, the cells in the four groups were digested and centrifuged, stained using the Annexin V-FITC / PI double staining method, and flow cytometry was used to detect cell apoptosis in the four groups.

[0042] Figure 1 The scatter plots of H9c2 cells and AC16 cells in each group detected by flow cytometry; Figure 2 and Figure 3 These are the comparison diagrams of the apoptosis rates of H9c2 cells and AC16 cells in each group detected by flow cytometry; the pictures show that compared with the CON group, the number of scattered points in the upper right quadrant and the lower right quadrant of the H2O2 group increased significantly, and the apoptosis rate also increased significantly; after the intervention of nerol acetate in the CH-20μM group and the CH-40μM group, the number of scattered points in the upper right quadrant and the lower right quadrant decreased significantly compared with the H2O2 group, and the apoptosis rate also decreased. This proves that nerol acetate can inhibit the apoptosis of rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0043] Example 2

[0044] In this example, transmission electron microscopy was used to observe the effect of neryl acetate on mitochondrial autophagy in rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0045] In this example, the cells were divided into a blank CON group, a H2O2 group, and neryl acetate administration groups: CH-20 μM group and CH-40 μM group. Rat myocardial cells H9c2 and human myocardial cells AC16 were expressed at 4×10 6 Each well was inoculated into a T25 culture flask and cultured at 37°C overnight. The next day, the CH-20μM group and the CH-40μM group were pre-medicated for 12 hours, with the CH-20μM group receiving 3.9258μg / ml, 5ml / bottle, and the CH-40μM group receiving 7.8516μg / ml, 5ml / bottle.

[0046] The H2O2 group was only given a medium containing 400μMH2O2 for intervention, while the CH-20μM group and the CH-40μM group were given a medium containing 400μMH2O2 and corresponding concentrations of neryl acetate for intervention. After 2 hours of intervention, the four groups of cells were digested and centrifuged, and the cells were washed with PBS buffer. After centrifugation, 1ml of electron microscopy fixative was slowly added along the tube wall of each group to fix the cells to keep their morphological structure unchanged. Dehydrated with isopropyl acetate, each group of cell samples was transferred to a transparent resin. After the resin was solidified, thin slices were prepared with an ultrathin slicer for electron microscopy observation.

[0047] Mitochondrial autophagy refers to the process in which cells, under the stimulation of reactive oxygen species, specifically remove dysfunctional mitochondria in the cytoplasm, thereby maintaining the integrity of mitochondrial function and cellular homeostasis.

[0048] Figure 4 The transmission electron microscope photos of each group of H9c2 cells and AC16 cells in Example 2 before and after the intervention of neryl acetate; the pictures show that H2O2 group H9c2 cells and AC16 cells have mitochondrial fragments degraded in lysosomes; compared with the H2O2 group, CH-20μM group and CH-40μM group H9c2 cells and AC16 cells under the protection of neryl acetate, mitochondria maintain a good morphology, clear structure, intact membrane, no obvious signs of damage, less mitochondrial autophagosomes, and the number of mitochondria is relatively stable, without significant reduction. This proves that neryl acetate effectively reduces the level of reactive oxygen species, reduces the damage to mitochondria, and thus reduces the occurrence of mitochondrial autophagy.

[0049] Example 3

[0050] In this example, Mitotracker Deep Red staining experiments were used to verify the protective effect of neryl acetate on mitochondria in rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0051] In this example, the cells were divided into a blank CON group, a H2O2 group, and neryl acetate administration groups: CH-20 μM group and CH-40 μM group. Rat myocardial cells H9c2 and human myocardial cells AC16 were expressed at 10×10 4 Each well was inoculated into a 6-well plate and cultured at 37°C overnight. The next day, the CH-20μM group and the CH-40μM group were pre-medicated for 12 hours, with the CH-20μM group receiving 3.9258μg / ml, 2ml / well, and the CH-40μM group receiving 7.8516μg / ml, 2ml / well.

[0052] The H2O2 group was only given the culture medium containing 400μM H2O2 for intervention, while the CH-20μM group and the CH-40μM group were respectively given the culture medium containing 400μM H2O2 and the corresponding concentration of neryl acetate for intervention. After 2h of intervention, a 10μM Mitotracker Deep Red dye solution was prepared, 2ml was added to each well and placed in a 37℃ cell culture incubator for staining for 30min. Then the cells were digested and centrifuged, and the fluorescence intensity of the four groups of cells was detected by flow cytometry.

[0053] Figure 5 The distribution statistics of mitochondrial fluorescence intensity of H9c2 cells and AC16 cells in each group in Mitotracker Deep Red staining experiment, the average fluorescence intensity shown in the histogram is shown in Table 1; Figure 6 and Figure 7 These are the fluorescence intensity comparison diagrams of H9c2 cells and AC16 cells in each group in the Mitotracker Deep Red staining experiment.

[0054] Table 1

[0055]

[0056] The results of the pictures and tables show that the fluorescence intensity of H9c2 cells and AC16 cells in the H2O2 group decreased compared with the CON group, indicating that H2O2 caused a certain degree of damage to the cell mitochondria and affected the activity of mitochondria. After the intervention of neryl acetate in the CH-20μM group and the CH-40μM group, the mitochondrial fluorescence intensity of H9c2 cells and AC16 cells was better than that in the H2O2 group. This proves that neryl acetate has a protective effect on the mitochondria of rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0057] Example 4

[0058] This example uses ROS staining combined with laser confocal microscopy to verify that neryl acetate can reduce the ROS content in rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0059] In this example, the cells were divided into a blank CON group, a H2O2 group, and neryl acetate administration groups: CH-20 μM group and CH-40 μM group. Rat myocardial cells H9c2 and human myocardial cells AC16 were expressed at 8×10 4 Cells were inoculated into confocal microplates and cultured at 37°C overnight. The next day, CH-20μM and CH-40μM groups were pre-medicated for 12 hours, with the CH-20μM group receiving 3.9258μg / ml, 1ml / dish, and the CH-40μM group receiving 7.8516μg / ml, 1ml / dish.

[0060] The H2O2 group was only given culture medium containing 400μM H2O2 for intervention, while the CH-20μM group and the CH-40μM group were respectively given culture medium containing 400μM H2O2 and corresponding concentrations of neryl acetate for intervention. After 2h of intervention, ROS and Hoechst dye solutions with concentrations of 10μM were prepared, 1.5ml was added to each well and placed in a 37℃ cell culture incubator for staining for 30min. Laser confocal microscopy was used to detect the fluorescence intensity of the four groups of cells.

[0061] Figure 8 Fluorescence images of H9c2 cells and AC16 cells in each group in ROS staining combined with laser confocal microscopy; Fig. 9 and Fig.10These are the comparison diagrams of the mean fluorescence intensity of H9c2 cells and AC16 cells in each group in the ROS staining combined with laser confocal experiment; the pictures show that compared with the CON group, the distribution of ROS in the H2O2 group increased significantly, and the mean fluorescence intensity of the cells also increased significantly; after the intervention of nerol acetate in the CH-20μM group and the CH-40μM group, the distribution of ROS was significantly reduced compared with the H2O2 group, and the mean fluorescence intensity of the cells also decreased, and the mean fluorescence intensity of H9c2 cells was close to that of the control group. This proves that nerol acetate can reduce the content of ROS in rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0062] Example 5

[0063] In this example, Mitotracker Red staining combined with laser confocal microscopy was used to verify the protective effect of neryl acetate on mitochondria in rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0064] In this example, the cells were divided into a blank CON group, a H2O2 group, and neryl acetate administration groups: CH-20 μM group and CH-40 μM group. Rat myocardial cells H9c2 and human myocardial cells AC16 were expressed at 8×10 4 Cells were inoculated into confocal microplates and cultured at 37°C overnight. The next day, CH-20μM and CH-40μM groups were pre-medicated for 12 hours, with the CH-20μM group receiving 3.9258μg / ml, 1ml / dish, and the CH-40μM group receiving 7.8516μg / ml, 1ml / dish.

[0065] The H2O2 group was only given culture medium containing 400μM H2O2 for intervention, while the CH-20μM group and the CH-40μM group were respectively given culture medium containing 400μM H2O2 and corresponding concentrations of neryl acetate for intervention. After 2 hours of intervention, Mitotracker Red and Hoechst dye solutions with concentrations of 10μM were prepared, 1.5ml was added to each well and placed in a 37℃ cell culture incubator for staining for 30 minutes, and then the fluorescence intensity of the four groups of cells was detected using a laser confocal microscope.

[0066] Fig.11 Fluorescence confocal microscopic images of H9c2 cells and AC16 cells in each group in Mitotracker Red staining combined with laser confocal microscopy experiments; Fig.12 and Fig.13The comparison of the mean fluorescence intensity of H9c2 cells and AC16 cells in each group in Mitotracker Red staining combined with laser confocal experiment; the mitochondria in the figure showed red fluorescence, and the picture showed that the number of mitochondria in the H2O2 group was significantly reduced compared with the CON group, and the mean fluorescence intensity of the cells also weakened; after the intervention of neryl acetate, the number of mitochondria in the CH-20μM group and the CH-40μM group was better than that in the H2O2 group. This proves that neryl acetate has a protective effect on the mitochondria of rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0067] Example 6

[0068] This example uses JC-1 staining combined with laser confocal microscopy to verify that neryl acetate can inhibit the decrease of mitochondrial membrane potential in rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

[0069] In this example, the cells were divided into a blank CON group, a H2O2 group, and neryl acetate administration groups: CH-20 μM group and CH-40 μM group. Rat myocardial cells H9c2 and human myocardial cells AC16 were expressed at 8×10 4 Cells were inoculated into confocal microplates and cultured at 37°C overnight. The next day, CH-20μM and CH-40μM groups were pre-medicated for 12 hours, with the CH-20μM group receiving 3.9258μg / ml, 1ml / dish, and the CH-40μM group receiving 7.8516μg / ml, 1ml / dish.

[0070] The H2O2 group was only given the culture medium containing 400μM H2O2 for intervention, while the CH-20μM group and the CH-40μM group were given the culture medium containing 400μM H2O2 and the corresponding concentration of neryl acetate for intervention, respectively. After 2h of intervention, JC-1 and Hoechst dye solutions with concentrations of 10μM were prepared, 1.5ml was added to each well and placed in a 37℃ cell culture incubator for staining for 30min. At the same time, CCCP was diluted to 10μM and the cells were treated for 20min. The fluorescence intensity of the four groups of cells was detected using a laser confocal microscope.

[0071] Fig.14 and 16 These are the fluorescence images of H9c2 cells and AC16 cells in each group in JC-1 staining combined with laser confocal microscopy; Fig.15 and Fig.17 These are comparison pictures of JC-1 aggregates / monomers of H9c2 cells and AC16 cells in each group in JC-1 staining combined with laser confocal microscopy.

[0072] In healthy cells with high mitochondrial ΔΨm, JC-1 forms J-aggregates that fluoresce red / orange. This color indicates that the mitochondrial membrane potential is high and the cell is in good condition. If JC-1 is mainly present in monomeric form and fluoresces green, it may indicate that the mitochondrial membrane potential is low and the cell may be stressed or damaged.

[0073] The pictures show that compared with the CON group, the proportion of J-aggregates in the H2O2 group decreased and the proportion of JC-1 monomers increased, indicating that the mitochondria of H9c2 cells and AC16 cells were damaged and the mitochondrial membrane potential was low. After intervention with nerolidin acetate in the CH-20μM group and the CH-40μM group, the proportion of J-aggregates increased and the proportion of JC-1 monomers decreased. This proves that nerolidin acetate can inhibit the decrease of mitochondrial membrane potential in rat cardiomyocytes H9c2 and human cardiomyocytes AC16.

Claims

1. The application of neryl acetate in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

2. The use of neryl acetate in the preparation of a medicine for treating myocardial ischemia-reperfusion injury according to claim 1, characterized in that: The medicine for treating myocardial ischemia-reperfusion injury uses neryl acetate as the only active ingredient or one of the active ingredients.

3. The use of neryl acetate in the preparation of a medicine for treating myocardial ischemia-reperfusion injury according to claim 2, characterized in that: The content of neryl acetate in the medicine for treating myocardial ischemia-reperfusion injury is 0.1wt%-99wt%.

4. The use of neryl acetate in the preparation of a medicament for treating myocardial ischemia-reperfusion injury according to claim 3, characterized in that: The medicine for treating myocardial ischemia-reperfusion injury also includes pharmaceutically acceptable adjuvants.

5. The use of neryl acetate in the preparation of a medicament for treating myocardial ischemia-reperfusion injury according to claim 4, characterized in that: The medicine for treating myocardial ischemia-reperfusion injury is in the form of a gastrointestinal tract administration dosage form.

6. The use of neryl acetate in the preparation of a medicament for treating myocardial ischemia-reperfusion injury according to claim 5, characterized in that: The dosage form of the medicine for treating myocardial ischemia-reperfusion injury is granules, tablets, capsules, pills or oral liquid preparations.

7. The use of neryl acetate in the preparation of a medicament for treating myocardial ischemia-reperfusion injury according to claim 6, characterized in that: The drug for treating myocardial ischemia-reperfusion injury can inhibit apoptosis of rat myocardial cells H9c2 and human myocardial cells AC16.

8. The use of neryl acetate in the preparation of a medicament for treating myocardial ischemia-reperfusion injury according to claim 7, characterized in that: The drug for treating myocardial ischemia-reperfusion injury has a protective effect on mitochondria in rat myocardial cells H9c2 and human myocardial cells AC16.

9. The use of neryl acetate in the preparation of a medicament for treating myocardial ischemia-reperfusion injury according to claim 8, characterized in that: The drug for treating myocardial ischemia-reperfusion injury can inhibit the decrease of mitochondrial membrane potential in rat myocardial cells H9c2 and human myocardial cells AC16.

10. The use of neryl acetate according to claim 9 in the preparation of a medicament for treating myocardial ischemia-reperfusion injury, characterized in that: The drug for treating myocardial ischemia-reperfusion injury can reduce the ROS content in rat myocardial cells H9c2 and human myocardial cells AC16.

Images