Pharmaceutical composition for treating myocardial ischemia-reperfusion injury

Abstract

The invention relates to a pharmaceutical composition for treating myocardial ischemia-reperfusion injury, and belongs to the technical field of pharmaceutical compositions. In order to solve the problems that an existing myocardial ischemia-reperfusion injury treatment method is single in target spot and limited in mitochondrial function protection curative effect, the invention provides a pharmaceutical composition for treating myocardial ischemia-reperfusion injury, and the pharmaceutical composition comprises a component of glycyrrhiza spinosa chalcone derived from liquorice and a component of tanshinone IIA derived from salvia miltiorrhiza. In-vitro experiments prove that through compatibility of the licochalcone and the tanshinone IIA, the apoptosis of H9C2 cells after hypoxia reoxygenation injury can be relieved, the number of mitochondria in the H9C2 cells after hypoxia reoxygenation injury is effectively increased, the total ROS content and the mitochondria superoxide level in model cells are reduced, the mitochondria function and the mitochondria membrane potential in the model cells are effectively improved, and the effect of inhibiting hypoxia reoxygenation injury is achieved. Based on the effect of the composition on the aspect of protecting the mitochondrial function, clinical application of the composition on myocardial ischemia reperfusion injury can be further developed.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical compositions, and in particular relates to a pharmaceutical composition for treating myocardial ischemia-reperfusion injury. Background Art

[0002] Myocardial ischemia-reperfusion injury includes a series of damaging changes in myocardial ultrastructure, energy metabolism, cardiac function and electrophysiology caused by ischemia, which are more prominent after vascular reperfusion. Severe arrhythmias may occur and may even lead to sudden death of the patient.

[0003] There is a close relationship between myocardial ischemia-reperfusion injury and mitochondria. During myocardial ischemia, mitochondrial function is impaired, ATP production is reduced, and energy metabolism disorders occur. During reperfusion, although blood flow is restored, mitochondrial function does not recover immediately. Instead, it may be further damaged by reperfusion injury, aggravating energy metabolism disorders. During myocardial ischemia, mitochondria cannot obtain enough oxygen, resulting in incomplete reduction of oxygen and the production of more oxygen free radicals. At the same time, the cell's anti-free radical system is damaged during hypoxia and cannot effectively remove excess oxygen free radicals, resulting in their accumulation in cells. During reperfusion, a large amount of oxygen flows into the ischemic area, reacting with the accumulated oxygen free radicals to form more reactive oxygen species, further damaging mitochondria and other cellular components. During myocardial ischemia-reperfusion, the mitochondrial permeability transition pore may open, leading to the loss of mitochondrial membrane potential and the release of a variety of pro-apoptotic proteins, thereby inducing myocardial cell damage and apoptosis.

[0004] In summary, there is a close relationship between myocardial ischemia-reperfusion injury and mitochondria. However, existing treatments for myocardial ischemia-reperfusion injury have certain limitations in improving mitochondrial function. Most existing drugs target a single pathological link, such as anti-oxidation, anti-inflammatory, and anti-platelet, and single-target treatments cannot fully and effectively inhibit the injury process, especially for the protection of mitochondrial function. In terms of improving mitochondrial function, the efficacy of existing drugs is still limited. Summary of the invention

[0005] In order to solve the problem that the existing myocardial ischemia-reperfusion injury treatment methods have a single target and limited protective efficacy on mitochondrial function, the present invention provides a pharmaceutical composition for treating myocardial ischemia-reperfusion injury.

[0006] The technical solution of the present invention: A pharmaceutical composition for treating myocardial ischemia-reperfusion injury, comprising ingredients derived from liquorice and ingredients derived from salvia miltiorrhiza.

[0007] Furthermore, the component derived from licorice is Licorice chalcone, and the component derived from Salvia miltiorrhiza is Tanshinone IIA.

[0008] Furthermore, the molar ratio of the Licorice chalcone to Tanshinone IIA is 1:3 to 3:1.

[0009] Furthermore, the molar ratio of the Licorice chalcone to Tanshinone IIA is 1:1.

[0010] A pharmaceutical composition for treating myocardial ischemia-reperfusion injury is used in the preparation of a drug for treating myocardial ischemia-reperfusion injury. The pharmaceutical composition for treating myocardial ischemia-reperfusion injury comprises a component derived from liquorice and a component derived from salvia miltiorrhiza.

[0011] Furthermore, the component derived from licorice is Licorice chalcone, and the component derived from Salvia miltiorrhiza is Tanshinone IIA.

[0012] Furthermore, the molar ratio of the Licorice chalcone to Tanshinone IIA is 1:3 to 3:1.

[0013] Furthermore, the molar ratio of the Licorice chalcone to Tanshinone IIA is 1:1.

[0014] Furthermore, the drug for treating myocardial ischemia-reperfusion injury is in an oral dosage form or a parenteral dosage form, and also includes a pharmaceutically acceptable carrier or excipient.

[0015] Furthermore, the oral dosage form is tablets, capsules, soft capsules, oral liquids, syrups, granules, pills, orodisintegrating tablets, sustained-release tablets, sustained-release capsules, controlled-release tablets or controlled-release capsules; the parenteral dosage form is water injection, lyophilized powder injection, sterile powder injection or infusion.

[0016] Beneficial effects of the present invention: The present invention uses a compound of licorice chalcone and tanshinone IIA for the treatment of myocardial ischemia-reperfusion injury, which has the advantages of multiple components and multiple targets. In vitro experiments have shown that the combination of licorice chalcone and tanshinone IIA can reduce the apoptosis of H9C2 cells after hypoxia-reoxygenation injury, effectively increase the number of mitochondria in H9C2 cells after hypoxia-reoxygenation injury, reduce the total ROS content and mitochondrial superoxide level in model cells, and effectively improve mitochondrial function and mitochondrial membrane potential in model cells. The present invention provides a basis for the application of the combination of licorice chalcone and tanshinone IIA in the treatment of myocardial ischemia-reperfusion injury. Based on the role of the combination in protecting mitochondrial function, its clinical application for myocardial ischemia-reperfusion injury can be further developed. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Electron microscope images of cells in each group in Example 6 at different magnifications; Figure 2 It is a scatter plot of each group of cells in flow cytometry of Example 6; Figure 3 This is a comparison chart of the apoptosis rates of cells in each group in the flow cytometer of Example 6; Figure 4 The staining images of each group of cells obtained by combining H2DCFDA probe with confocal microscopy in Example 6; Figure 5 The fluorescence intensity of cells measured under a confocal microscope after H2DCFDA probe labeling in Example 6, i.e., a comparison of ROS levels in cells of each group; Figure 6 The staining images of each group of cells obtained by combining Mito SOX stain with confocal microscopy in Example 6; Figure 7 The fluorescence intensity of cells under a confocal microscope after staining with Mito SOX dye in Example 6, i.e., a comparison of mitochondrial ROS levels in cells of each group; Figure 8 The staining images of each group of cells obtained by combining Mito Tracker Red dye with confocal microscopy in Example 6; Fig. 9 The fluorescence intensity of cells under a confocal microscope after staining with Mito Tracker Red dye in Example 6, i.e., a comparison of mitochondrial membrane potential in each group of cells; Fig.10 This is the mitochondrial fluorescence intensity distribution histogram of each group of cells obtained by combining MitoTracker Deep Red dye with flow cytometry in Example 6; Fig.11 This is a graph showing the fluorescence intensity of each group of cells detected by flow cytometry after staining with MitoTracker Deep Red dye in Example 6, i.e., a comparison of the changes in mitochondrial function in each group of cells. DETAILED DESCRIPTION

[0018] 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.

[0019] Example 1 This embodiment provides a pharmaceutical composition for treating myocardial ischemia-reperfusion injury, which is composed of Licorice chalcone and Tanshinone IIA in a molar ratio of 1:1.

[0020] Example 2 This embodiment provides a pharmaceutical composition for treating myocardial ischemia-reperfusion injury, which is composed of Licorice chalcone and Tanshinone IIA in a molar ratio of 1:2.

[0021] Example 3 This embodiment provides a pharmaceutical composition for treating myocardial ischemia-reperfusion injury, which is composed of Licorice chalcone and Tanshinone IIA in a molar ratio of 2:1.

[0022] Example 4 This embodiment provides a pharmaceutical composition for treating myocardial ischemia-reperfusion injury, which is composed of Licorice chalcone and Tanshinone IIA in a molar ratio of 1:3.

[0023] Example 5 This embodiment provides a pharmaceutical composition for treating myocardial ischemia-reperfusion injury, which is composed of Licorice chalcone and Tanshinone IIA in a molar ratio of 3:1.

[0024] Example 6 This example investigates the effects of the composition of Glycyrrhiza uralensis chalcone and Tanshinone IIA in a molar ratio of 1:1 provided in Example 1 on cardiomyocytes and their mitochondria through a cell model experiment.

[0025] 1.1 Cell model construction method 1. Experimental cells: rat myocardial H9C2 cell line.

[0026] 2. Experimental materials: Echinatin and Tanshinone IIA were purchased from Shanghai Yuanye Biotechnology Co., Ltd., DMEM culture medium was purchased from White Shark Biotechnology Co., Ltd., apoptosis kit was purchased from Aibixin Biotechnology Co., Ltd., H2DCFDA probe was purchased from MCE Biotechnology Co., Ltd., MitoSOX, MitoTracker Red, and MitoTracker Deep Red dyes were purchased from Thermo Fisher Scientific.

[0027] 3. Instruments: flow cytometer (Luminex, Guava easyCyte), microscope (Thermo Fisher Scientific, EVOS), centrifuge (Shanghai Anting Scientific Instrument Factory, TGL-16c), confocal scanning microscope (Zeiss LSM980).

[0028] 4. Cell culture: H9C2 cells were cultured in DMEM medium (10% fetal bovine serum, 1% double antibody), 5% CO2, 37℃ cell culture incubator.

[0029] (II) Cell modeling and grouped drug administration methods: The experiment was divided into blank group, model group, low-dose group and high-dose group. H9C2 cardiomyocytes in the logarithmic growth period were taken and administered after passage and attachment. The molar ratio of Licorice chalcone to Tanshinone IIA was 1:1. 24 hours after administration, the blank group was replaced with ordinary culture medium, the model group was replaced with culture medium containing 400 μM H2O2, the low-dose group was replaced with culture medium containing 400 μM H2O2 and 20 μM of the combination of Licorice chalcone and Tanshinone IIA, and the high-dose group was replaced with culture medium containing 400 μM H2O2 and 40 μM of the combination of Licorice chalcone and Tanshinone IIA. After replacing the culture medium and incubating in the dark for 1 hour, the model was completed.

[0030] The number of mitochondria in each group of cells was observed under an electron microscope. Figure 1 shown.

[0031] Figure 1 The results showed that the number of mitochondria in the blank group was usually within the normal range, with normal morphology, complete structure and orderly arrangement. The mitochondrial membrane was clear, indicating that the mitochondrial function was normal; the number of mitochondria in the model group was significantly reduced, indicating that the mitochondrial function was impaired; the line in the low-dose group was restored, and the mitochondrial morphology and structure were improved; the number of mitochondria in the high-dose group was restored to a near normal level, and the mitochondrial morphology and structure were basically normal, with clear membranes, indicating that the combination of Licorice Chalcone and Tanshinone IIA can increase the number of mitochondria in myocardial cells damaged by hypoxia and reoxygenation, and has a strong protective effect on mitochondria.

[0032] 3. Flow cytometry detection of cell apoptosis H9C2 cells were collected at 1×10 5 Cells were inoculated into 6-well plates and cultured at 37°C overnight. The next day, the drugs were administered and the model was established according to the method in (II). After the model was established, the cells were digested and centrifuged, and stained using the Annexin V-FITC / PI (Annexin V-fluorescein isothiocyanate / propidium iodide) double staining method. Flow cytometry was used to detect cell apoptosis in each group. The results are shown in the figure. Figure 2 and Figure 3 shown.

[0033] Figure 2 and Figure 3 The results showed that compared with the blank control group, the cell apoptosis rate in the model group was significantly increased, indicating that the mitochondria were severely damaged; the cell apoptosis rates in the low-dose group and the high-dose group were significantly lower than those in the model group, indicating that the combination of Licorice Chalcone and Tanshinone IIA can reduce the apoptosis of myocardial cells damaged by hypoxia and reoxygenation.

[0034] 4. Confocal microscopy to detect changes in total intracellular ROS and superoxide levels in mitochondria H9C2 cells were collected at 1×10 6Each cell was inoculated into a confocal microplate and cultured at 37°C overnight. The next day, the drug was administered according to the method in (II). 24 hours after administration, the cells were stained with H2DCFDA (2',7'-dichlorofluorescein diacetate) probe and MitoSOX (mitochondrial superoxide) indicator, respectively. After staining, the cell nuclei were counterstained with Hoechst (Hoechst fluorescent dye) 33342 and then detected. The images were analyzed using ZEISS ZEN software. The results are shown in Figure 2. Figure 4 and Figure 5 shown.

[0035] Figure 4 and Figure 5 The results showed that compared with the blank control group, the fluorescence intensity of the cells in the model group was significantly enhanced after staining with the H2DCFDA probe, indicating that the intracellular ROS level was increased and the mitochondrial function might be damaged. The fluorescence intensity of the low-dose group and the high-dose group was lower than that of the model group, indicating that the combination of Licorice Chalcone and Tanshinone IIA reduced the total intracellular ROS content and had a certain protective effect on the mitochondria of myocardial cells damaged by hypoxia and reoxygenation.

[0036] Figure 6 and Figure 7 The results showed that compared with the blank control group, the fluorescence intensity of the mitochondria in the model group was significantly enhanced after staining with MitoSOX probe, indicating that the level of superoxide in the mitochondria was increased and there was an obvious oxidative stress response. After staining with MitoSOX probe, the fluorescence intensity of the mitochondria in the low-dose group and the high-dose group was lower than that in the model group, indicating that the combination of Licorice Chalcone and Tanshinone IIA reduced the level of superoxide in the mitochondria of myocardial cells damaged by hypoxia and reoxygenation, and had a significant protective effect on mitochondria.

[0037] 5. Confocal microscopy to detect changes in mitochondrial function in cells H9C2 cells were collected at 5×10 3 The cells were inoculated into a 24-well plate with a round glass slide and cultured at 37°C overnight. The next day, the drug was administered according to the method in (II). 24 hours after administration, the cells were stained with MitoTracker Red (chromosome red fluorescence) dye, and then sealed with DAPI anti-fading sealing medium. After drying in the dark, the cells were photographed using a confocal microscope. The images were analyzed using ZEISS ZEN software. The results are shown in Figure 2. Figure 8 and Fig. 9 shown.

[0038] Figure 8 and Fig. 9The results showed that compared with the blank control group, the cells in the model group stained with Mito Tracker Red showed abnormal phenomena such as weakened mitochondrial fluorescence intensity and uneven fluorescence intensity, indicating that the number of mitochondria was reduced, the morphological structure was damaged, and the function was reduced. After the cells in the low-dose group and the high-dose group were stained with Mito Tracker Red, the mitochondrial fluorescence intensity was enhanced compared with the model group, and the distribution was more uniform, indicating that the combination of Licorice chalcone and Tanshinone IIA has a significant protective effect on the mitochondria of myocardial cells damaged by hypoxia and reoxygenation, and can effectively improve mitochondrial function.

[0039] (VI) Flow cytometry detection of changes in mitochondrial membrane potential of cells in each group H9C2 cells were collected at 1×10 5 The cells were inoculated into 6-well plates and cultured at 37°C overnight. The next day, the drugs were administered and the model was established according to the method in (II). After the model was established, the cells were digested and centrifuged, and stained with MitoTracker Deep Red (mitochondrial deep red fluorescent probe). The changes in cell membrane potential of each group were detected by flow cytometry. The results are shown in the figure. Fig.10 and Fig.11 shown.

[0040] Fig.10 The results showed that compared with the blank control group, the mitochondrial fluorescence intensity distribution histogram obtained by flow cytometry showed a significant shift after the cells in the model group were stained with MitoTracker Deep Red dye. The peak shifted to the left, indicating that mitochondrial function was reduced. After the cells in the low-dose group and the high-dose group were stained with MitoTracker Deep Red dye, the mitochondrial fluorescence intensity distribution histogram obtained by flow cytometry showed a normal distribution shape similar to that of the blank group, and the peak was closer to the level of the blank group, indicating that mitochondrial function was restored.

[0041] Fig.11 The results showed that compared with the blank control group, the fluorescence intensity of the model group decreased, indicating that the accumulation of MitoTracker Deep Red dye in cells decreased, indicating that the mitochondrial membrane potential decreased. The fluorescence intensity of the low-dose group and the high-dose group increased compared with the model group, indicating that the combination of Licorice Chalcone and Tanshinone IIA can effectively improve the mitochondrial membrane potential of myocardial cells damaged by hypoxia and reoxygenation.

Claims

1. A pharmaceutical composition for treating myocardial ischemia-reperfusion injury, characterized in that: The pharmaceutical composition comprises components derived from liquorice and components derived from salvia miltiorrhiza.

2. A pharmaceutical composition for treating myocardial ischemia-reperfusion injury according to claim 1, characterized in that: The component derived from licorice is licorice chalcone, and the component derived from salvia miltiorrhiza is tanshinone IIA.

3. A pharmaceutical composition for treating myocardial ischemia-reperfusion injury according to claim 2, characterized in that: The molar ratio of the licorice chalcone to tanshinone IIA is 1:3-3:

1.

4. A pharmaceutical composition for treating myocardial ischemia-reperfusion injury according to claim 3, characterized in that: The molar ratio of the licorice chalcone to tanshinone IIA is 1:

1.

5. Use of a pharmaceutical composition for treating myocardial ischemia-reperfusion injury in the preparation of a drug for treating myocardial ischemia-reperfusion injury, characterized in that: The pharmaceutical composition for treating myocardial ischemia-reperfusion injury comprises components derived from liquorice and components derived from salvia miltiorrhiza.

6. Use of the pharmaceutical composition for treating myocardial ischemia-reperfusion injury according to claim 5 in preparing a drug for treating myocardial ischemia-reperfusion injury, characterized in that: The component derived from licorice is licorice chalcone, and the component derived from salvia miltiorrhiza is tanshinone IIA.

7. Use of the pharmaceutical composition for treating myocardial ischemia-reperfusion injury according to claim 6 in preparing a drug for treating myocardial ischemia-reperfusion injury, characterized in that: The molar ratio of the licorice chalcone to tanshinone IIA is 1:3-3:

1.

8. Use of the pharmaceutical composition for treating myocardial ischemia-reperfusion injury according to claim 7 in preparing a drug for treating myocardial ischemia-reperfusion injury, characterized in that: The molar ratio of the licorice chalcone to tanshinone IIA is 1:

1.

9. Use of the pharmaceutical composition for treating myocardial ischemia-reperfusion injury according to claim 8 in preparing a drug for treating myocardial ischemia-reperfusion injury, characterized in that: The drug for treating myocardial ischemia-reperfusion injury is in an oral dosage form or a parenteral dosage form, and further comprises a pharmaceutically acceptable carrier or excipient.

10. Use of the pharmaceutical composition for treating myocardial ischemia-reperfusion injury according to claim 9 in preparing a drug for treating myocardial ischemia-reperfusion injury, characterized in that: The oral dosage form is tablets, capsules, soft capsules, oral liquids, syrups, granules, dripping pills, orodisintegrating tablets, sustained-release tablets, sustained-release capsules, controlled-release tablets or controlled-release capsules; the parenteral dosage form is water injection, lyophilized powder injection, sterile powder injection or infusion.

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