9apos; 9apos; carrying out apos; application of salvianolic acid B monomethyl ester in preparation of medicine for treating ischemic heart disease

Abstract

The invention discloses application of 9 ''-salvianolic acid B monomethyl ester in preparation of a medicine for treating ischemic heart disease, and belongs to the technical field of biological medicine. The CAS number of the 9 ''-salvianolic acid B monomethyl ester is 1167424-31-8, the ischemic heart disease refers to myocardial injury induced by heart ischemia reperfusion, and the invention further provides a pharmaceutical composition or a small molecule injection preparation. Animal experiments show that 9 ''-salvianolic acid B monomethyl ester is exogenously injected to relieve myocardial injury of a mouse and improve the heart function of the mouse, and it is proved that the medicine containing 9''-salvianolic acid B monomethyl ester can improve the ischemic heart disease, especially myocardial injury caused by heart ischemia reperfusion, and has a good application prospect. The invention provides potential drug selection and research direction for preventing and treating ischemic heart disease, and has important clinical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of 9"-salvianolic acid B monomethyl ester in preparation of a drug for treating ischemic heart disease. BACKGROUND

[0002] At present, the number of cardiovascular disease patients in China is about 330 million, among which about 11 million ischemic heart disease patients have myocardial infarction as a serious clinical outcome, and the morbidity and mortality thereof are still in the rising stage. In recent years, although the popularization of coronary intervention surgery and the optimization of drug thrombolytic therapy strategy restore blood flow of ischemic myocardial tissue to a certain extent, myocardial ischemia-reperfusion injury (MIR) still inevitably occurs in some patients, leading to poor clinical prognosis of ischemic heart disease, and bringing huge disease burden to patients and social development. Therefore, it is of great theoretical and clinical significance to find specific molecules and molecular pathways that block myocardial ischemia-reperfusion injury and to find drug targets for preventing and treating ischemic heart disease.

[0003] 9"-salvianolic acid B monomethyl ester (MSB) is a water-soluble phenolic acid compound extracted from traditional Chinese medicine Danshen (Salvia miltiorrhiza) or obtained by semi-synthetic modification, and belongs to Danshensu derivatives. The chemical structure of MSB is based on salvianolic acid B, retains the core phenylpropanoid skeleton and caffeic acid unit of salvianolic acid B, but introduces a methyl group at the C-9" position, which may improve the metabolic stability and bioavailability thereof. Previous studies have confirmed that the phenolic hydroxyl structure of salvianolic acid B can effectively neutralize ROS and RNS, directly scavenge free radicals, and also up-regulate the expression of HO-1, SOD and other antioxidant enzymes through the Nrf2 / ARE pathway, thereby playing an antioxidant role. In addition, salvianolic acid B can also inhibit the NF-κB signaling pathway, regulate NLRP3 inflammasome and macrophage polarization, and play an anti-inflammatory and immunoregulatory role. Therefore, salvianolic acid B has broad application prospects in the treatment of cardiovascular and cerebrovascular diseases, fibrotic diseases and neurodegenerative diseases. However, there is no related report on the role of the derivative 9"-salvianolic acid B monomethyl ester of salvianolic acid B in diseases. SUMMARY

[0004] Based on this, the purpose of the present application is to provide application of 9"-salvianolic acid B monomethyl ester in preparation of a drug for treating ischemic heart disease. Research has found that 9"-salvianolic acid B monomethyl ester can significantly improve myocardial injury induced by cardiac ischemia-reperfusion, thereby providing a potential drug selection and research direction for preventing and treating ischemic heart disease.

[0005] To achieve the above-mentioned purpose, the technical solutions adopted by the present application are as follows:

[0006] The present application provides the use of 9"-salvianolic acid B monomethyl ester or its pharmaceutically acceptable salt in the preparation of a medicament for treating ischemic heart disease, wherein the CAS number of 9"-salvianolic acid B monomethyl ester is 1167424-31-8, and the structural formula is:

[0007]

[0008] As preferred, the ischemic heart disease refers to myocardial injury induced by cardiac ischemia-reperfusion.

[0009] As preferred, the pharmaceutically acceptable salt of 9"-salvianolic acid B monomethyl ester is selected from one or more of the above combinations of hydrochloride, hydrobromide, sulfate, acetate, lactate, tartrate, tannate, citrate, trifluoroacetate, malate, maleate, succinate, p-toluenesulfonate or benzenesulfonate.

[0010] The present application also provides a pharmaceutical composition comprising a therapeutically effective amount of 9"-salvianolic acid B monomethyl ester or its pharmaceutically acceptable salt, wherein the CAS number of 9"-salvianolic acid B monomethyl ester is 1167424-31-8, and the structural formula is:

[0011]

[0012] As preferred, the concentration of 9"-salvianolic acid B monomethyl ester in the pharmaceutical composition is 1-100 μM.

[0013] As preferred, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and can be prepared into an injection, an emulsion, a tablet, a powder, a granule, an ointment, a liposome or an oral liquid.

[0014] The present application also provides a small molecule injection preparation comprising a therapeutically effective amount of 9"-salvianolic acid B monomethyl ester or its pharmaceutically acceptable salt, wherein the CAS number of 9"-salvianolic acid B monomethyl ester is 1167424-31-8, and the structural formula is:

[0015]

[0016] As preferred, 9"-salvianolic acid B monomethyl ester or its pharmaceutically acceptable salt is used as an active ingredient, and the concentration is 1-100 μM.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] 1、The application reduces myocardial injury of mice and improves heart function of mice by exogenous injection of 9"-salvianolic acid B monomethyl ester through animal experiments, thereby confirming that the drug containing 9"-salvianolic acid B monomethyl ester can improve ischemic heart disease, especially myocardial injury caused by cardiac ischemia-reperfusion, and provides potential drug selection and research direction for preventing and treating ischemic heart disease.

[0019] 2、Salvia miltiorrhiza has been safely used in traditional medicine for hundreds of years, and 9"-salvianolic acid B monomethyl ester, as a derivative thereof, has a low risk of side effects compared to synthetic drugs, is suitable for long-term medication, and has a low pharmaceutical cost due to its natural origin. Studies have confirmed that salvianolic acid B has therapeutic effects in various diseases, and 9"-salvianolic acid B monomethyl ester, as a derivative thereof, has higher metabolic stability, better lipid solubility and better pharmacokinetics, and has higher application value. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Experimental results for evaluating the effectiveness of MSB in in vitro cell experiments in the examples.

[0021] Figure 2 Experimental results for verifying MSB for treating ischemic heart disease in animal experiments in the examples. DETAILED DESCRIPTION

[0022] In order to more fully understand the technical solutions, objectives and advantages of the present application, the following will further describe the technical effects of the present application in conjunction with the drawings and specific examples. Obviously, the described examples are only a part of the embodiments of the present application, not all. It should be noted that for those skilled in the art, other embodiments obtained without departing from the concept of the present application all belong to the protection scope of the present application.

[0023] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0024] Example 1

[0025] This example verifies 9"-salvianolic acid B monomethyl ester for treating ischemic heart disease through animal experiments, wherein the mice involved are purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., and the animal experiments have been approved by the Ethics Committee of Zhongshan Hospital, Fudan University.

[0026] (I) Animal experiments

[0027] 1. Isolation of adult mouse myocardial cells

[0028] Mice were anesthetized and the heart was exposed. Then, 7 mL of EDTA buffer (130 mM NaCl, 5 mM KCl, 0.5 mM Na2HPO4, 10 mM HEPES, 10 mM taurine, 10 mM D-glucose, 10 mmol / L BDM, 5 mM EDTA, pH 7.8) was infused steadily into the right ventricle. After the descending aorta was cut, the heart was excised and sequentially perfused with 10 mL of EDTA buffer, 3 mL of perfusion buffer (130 mM NaCl, 5 mM KCl, 0.5 mM Na2HPO4, 10 mM HEPES, 10 mM taurine, 10 mM D-glucose, 10 mmol / L BDM, 1 mM MgCl2, pH 7.8), and 30 mL of pre-warmed collagenase buffer (perfusate buffer supplemented with 0.5 mg / mL collagenase II, 0.5 mg / mL collagenase IV, and 0.05 mg / mL protease XIV), all of which were filtered through a 0.22 μm filter before use. The ventricle was further dissociated into fragments in collagenase buffer, and after the digestion was terminated with 5 mL of stop buffer (perfusate buffer containing 5% FBS), the cell suspension was filtered through a 100 μm filter into a 50 mL tube, and a pellet was formed by gravity sedimentation for 15-20 min. The pellet was the cardiac myocyte fraction, and the supernatant was collected, centrifuged at 1000 g for 10 min, and the non-cardiomyocyte fraction was obtained.

[0029] 2. Hypoxia-reoxygenation treatment of adult mouse cardiomyocytes

[0030] Adult mouse ventricular cardiomyocytes were isolated and plated on laminin-coated dishes, and then incubated in a standard cell culture incubator for 24 h to promote cell attachment. Subsequently, the cardiomyocytes were transferred to ischemic buffer (118 mmol / L NaCl, 24 mmol / L NaHCO3, 1.0 mmol / L NaH2PO4, 2.5 mmol / L CaCl2-2H2O, 1.2 mmol / L MgCl2, 20 mmol / L sodium lactate, 16 mmol / L KCl, and 10 mmol / L 2-deoxyglucose, pH adjusted to 6.2), and exposed to hypoxic conditions (1% O2, 94% N2, 5% CO2) in a hypoxia chamber for 30 min. After the hypoxic treatment, the ischemic buffer was replaced with fresh medium, and the cells were transferred back to the standard cell culture incubator for the indicated reoxygenation treatment. Specifically, the isolated adult mouse primary cardiomyocytes were treated with 1 uM MSB and then subjected to hypoxia-reoxygenation (H / R) treatment. After the reoxygenation treatment, the cells and their supernatants were collected for further analysis.

[0031] 3. Microscopic imaging of cell death

[0032] In the dark, PI (Invitrogen, P3566, 1:1000 dilution) and Hoechst (Biological Industries, C1028, 1:100 dilution) dyes were added to freshly isolated hypoxic cardiomyocytes. The Bio Tek live cell workstation was started and the gas valve was opened to maintain a constant temperature of 37°C, with a mixture of 21% O2, 5% CO2 and 74% N2. Then, the 6-well plate containing the cardiomyocytes was placed in the workstation, which was started after adjusting the focal length and setting the shooting parameters (including randomly selecting 6 fields of view per well and taking pictures every 30 minutes). The workstation automatically obtained the images of the cardiomyocyte state and PI / Hoechst fluorescence at the designated time points according to the preset program.

[0033] 4. Construction of myocardial ischemia reperfusion model (MIR model)

[0034] The mice were anesthetized with isoflurane gas. An oblique skin incision was made along the lower edge of the left pectoral muscle to the left upper limb of the mouse, and then the lower edge of the pectoral muscle was clamped with forceps to find the pectoral muscle near the sternum. The pectoral muscle was slightly separated with a blunt straight forceps. After the intercostal space was poked open with a sharp blood vessel clamp, the heart was squeezed out of the chest cavity. The needle was inserted from the right side of the heart below the left atrial appendage, and the anterior descending branch was accurately ligated without penetrating the ventricle. An active knot was made at the ligation site. After successful ligation of the anterior descending branch, ischemia was observed by naked eye after ligation, and the color of the area became white. After the anterior descending branch was ligated, the heart was gently clamped with a sharp hemostat and inserted back into the chest cavity. The thread left outside the incision was retained. After 30 minutes of ligation, the thread left outside the chest cavity was used to pull the active knot to restore blood flow and start myocardial reperfusion. The muscle and skin were simply sutured, the wound was disinfected with iodophor, and then the mouse was placed on a warming table for recovery. After waking up, the mouse was returned to the cage. The sham operation group was treated in the same way, but only the thread was passed without ligation.

[0035] 5. Evan's blue and TTC staining of mice

[0036] Evan's Blue dye was diluted. After 24 hours of reperfusion, the mouse was anesthetized with an appropriate anesthetic, the ligation thread left on the heart was re-ligated, and then Evan's Blue solution was injected through the aorta. After the Evan's Blue solution was fully injected into the heart through the coronary system, the heart was sectioned for subsequent TTC staining experiments. The heart tissue was removed, frozen at -80°C for 15 minutes, and then cut into five 1.2 mm thick cross sections.

[0037] TTC staining is a step following Evan's Blue staining. 2,3,5-Triphenyltetrazolium chloride (TTC) is dissolved in physiological saline. Afterwards, Evans Blue-stained heart sections are placed in a 1% TTC solution and incubated in a 37°C waterbath in the dark for 30 minutes. Excess dye is then washed away with phosphate buffered saline. The stained areas are then observed. Blue areas represent normal areas, red areas represent areas at risk (AAR), and white areas represent infarcted areas (IA). Images are then taken to detect I / R injury. The IA / AAR ratio is calculated and used to compare differences between groups to assess ischemia-reperfusion injury.

[0038] 6. TUNEL staining

[0039] Mouse heart tissue was embedded in OCT and serially sectioned at 6 μm thickness. Adult mouse cardiomyocytes and heart sections were fixed with 4% paraformaldehyde, blocked with 3% BSA, and permeabilized with 0.3% Triton-X100. Apoptosis was detected using a one-step TUNEL apoptosis detection kit (Beyotime, Shanghai, China) according to the manufacturer's instructions: after fixation, the samples were incubated with the TUNEL reaction solution, counterstained with DAPI for nuclear localization, and imaged under a Zeiss fluorescence microscope.

[0040] 7. Mouse Heart Ultrasound

[0041] After depilation, the mice were anesthetized with isoflurane mask at a dose of 1%-2%, and then fixed on a heating pad. The isoflurane flow was carefully controlled to maintain a heart rate of about 500 beats / min.

[0042] Two-dimensional (2-D) guided M-mode echocardiography (VisualSonics Vevo2100, Canada) equipped with a 30-mHz linear transducer was used to assess cardiac geometry and function. Parasternal long-axis views were obtained while heart rate was recorded. Cardiac geometry and functional indices including ejection fraction (EF) and fractional shortening (FS) were obtained. Each measurement was averaged over at least five consecutive cardiac cycles.

[0043] 8. Masson's trichrome staining

[0044] Tissue processing: Paraffin sections were dewaxed with xylene and rehydrated with graded ethanol to distilled water.

[0045] Nuclear staining: Stain with Weigert iron hematoxylin solution for 5-10 minutes, then rinse with running water to return to blue.

[0046] Cytoplasmic staining: stain with Ponceau-acid fuchsin solution for 5 minutes, followed by differentiation with 1% glacial acetic acid.

[0047] Collagen staining: after 5 minutes differentiation with phosphomolybdic acid solution, 1-2 minutes staining with aniline blue, acetic acid rinsing.

[0048] Dehydration and mounting: fast gradient ethanol dehydration, xylene transparency, neutral gum fixation. Collagen fibers are blue, muscle fibers are red, and cell nuclei are dark brown.

[0049] (ii) Results

[0050] 1. Evaluate the effectiveness of MSB by in vitro cell experiment. Isolate adult mouse primary cardiomyocytes, give 1 uM MSB treatment, then perform hypoxia-reoxygenation (H / R) treatment, and detect by live cell workstation. The results show that MSB effectively reduces the PI fluorescence intensity of cardiomyocytes Figure 1 A), while reducing the lactate dehydrogenase (LDH) content in cell supernatant Figure 1 B), and the Caspase 3 activity of cardiomyocytes Figure 1 C), indicating that MSB can reduce cardiomyocyte necrosis and apoptosis caused by hypoxia-reoxygenation.

[0051] 2. Randomly divide C57BL / 6 mice into groups, give 5 mg / kg / day MSB to the treatment group, intraperitoneally, once a day, and give the control group MSB solvent, with the same injection method and frequency as the experimental group, for 7 consecutive days. Randomly divide again after the first injection, and perform surgery modeling 2 hours later. The I / R group constructs a heart ischemia-reperfusion injury model, and the Sham group only threads without ligation Figure 2 A).

[0052] 3. Measure the ratio of infarct area to risk area after Evans blue and TTC staining to evaluate the severity of myocardial infarction. When cardiomyocytes are damaged, lactate dehydrogenase leaks from the cell into the blood. Measure the lactate dehydrogenase content in the serum. The results show that injecting MSB can reduce the severity of myocardial infarction in mice with myocardial ischemia-reperfusion and reduce the degree of myocardial damage Figure 2 B-C).

[0053] 4. TUNEL staining can be used to evaluate the degree of apoptosis. The results show that injecting MSB can reduce cardiomyocyte apoptosis in mice with myocardial ischemia-reperfusion Figure 2 D).

[0054] 5. Measure the left ventricular ejection fraction (EF%) of mice by echocardiography to evaluate the heart function of mice in different intervention groups. The results show that compared with the control group, injecting MSB can significantly reduce the deterioration of heart function caused by MIR and increase the EF value Figure 2 E-F).

[0055] 6. Myocardial fibrosis was evaluated by Masson's trichrome staining in different treatment groups. The results showed that injection of MSB significantly reduced MIR-induced myocardial fibrosis compared with the control group Figure 2 G).

[0056] The above only is the preferred embodiment of the present application, and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of 9″-salvianolic acid B monomethyl ester or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating ischemic heart disease, wherein the CAS number of 9″-salvianolic acid B monomethyl ester is 1167424-31-8, and the structural formula is:

2. The use according to claim 1, characterized in that The ischemic heart disease refers to myocardial damage induced by cardiac ischemia-reperfusion.

3. The use according to claim 1, characterized in that The pharmaceutically acceptable salt of 9″-salvianolic acid B monomethyl ester is selected from one or more combinations of hydrochloride, hydrobromide, sulfate, acetate, lactate, tartrate, tannate, citrate, trifluoroacetate, malate, maleate, succinate, p-toluenesulfonic acid or benzenesulfonic acid.

4. A pharmaceutical composition, characterized in that It contains a therapeutically effective amount of 9"-salvianolic acid B monomethyl ester or a pharmaceutically acceptable salt thereof, wherein the CAS number of 9"-salvianolic acid B monomethyl ester is 1167424-31-8, and the structural formula is:

5. The pharmaceutical composition according to claim 4, characterized in that The concentration of 9″-salvianolic acid B monomethyl ester in the pharmaceutical composition is 1-100 μM.

6. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and can be prepared into an injection, emulsion, tablet, powder, granule, ointment, liposome or oral solution.

7. A small molecule injection preparation comprising a therapeutically effective amount of 9″-salvianolic acid B monomethyl ester or a pharmaceutically acceptable salt thereof, wherein the CAS number of 9″-salvianolic acid B monomethyl ester is 1167424-31-8 and the structural formula is:

8. The small molecule injection preparation according to claim 7, characterized in that: 9"-salvianolic acid B monomethyl ester or a pharmaceutically acceptable salt thereof is used as an active ingredient, and its concentration is 1-100 μM.

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